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Editor in chiefM. Y. Taher

Founder Editors Hilmy AbazaSeham Abdel-Reheem

Co-Editors Ahmed ShawkyFathalla SidkyMaher Osman Mohamed Sharaf El-Din

International Advisory Board JP Galmiche FranceA Sandeberg SwedenX Rogiers BelgiumA Kruse DenmarkDes Verrannes France Antonio Ascione ItalyS Brauno ItalyP Almasio Italy

National Advisory BoardMostafa El-HenawyAmira Shams El-DinNabil Abdel-BakyHoda El-AgganM. Essam Moussa Ahmed BassiounySaeid El-KayalAbdel-Fattah HannoKhaled MadboulyEzzat Aly

ContentsAlexandria Journal of Hepatogastroenterology,Volume (XXV) - April 2018--------------------------------------------------Manuscript Submission: For information and to submit manuscripts please contact the editors by e-mail at:dryousrytaher@yahoo.comfathallas@yahoo.comDisclaimer: The Publisher, the Egyptian Society of Hepatology Gastroenterology and Infectious Diseases in Alexandria, and Editors cannot be held responsible for errors or any consequences arising from the use of information contained in this journal; the views and opinions expressed do not necessarily reflect the those of the Publisher, The Egyptian Society of Hepatology Gastroenterology & Infectious Diseases in Alexandria, Editors, neither dose the publication of advertisementsconstitute any endorsement by the Publisher, society, and editors of the products advertised.

Original Article

Epigenetics in HCV Related Liver Cirrhosis and HepatocellularCarcinoma in Egyptian PatientsDina Nour, Mustafa Nemat Alla, Mona Arafa, and Mahmoud El-Bendary----------------------------------------------Original Article

Role of Dynamic Susceptibility Contrast (DSC) Perfusion Magnetic Resonance Imaging in the Differentiation between Recurrent Brain Tumor and Radiation NecrosisHeba M. Soliman, Ahmed A. ElBeheiry, Amr A. Abdel-Kerim, Ahmed H. Farhoud, M. IhabReda----------------------------------------------Original Article

Role of Echocardiography and Pro-BNP in Detection of Pulmonary Hypertension in ESRD Patients on Maintaince HaemodialysisAmr Mohamed Ebaid, Amel Fouad Mohamed Ketat,Eman Mohamed Elsharkawy, Yasmine Salah Naga,Asmaa Abdelkader Abdelal Eissa----------------------------------------------Original Article

Serum Endoglin (CD105) in Cirrhotic Hepatitis C Virus and Hepatitis B Virus Patients with and without Hepatocellular CarcinomaKhaled Mohiedeen, Ali El-Kady, Akram Deghady,Rania Abou Youssef, Mohamed Samy----------------------------------------------Original Article

Serum Thioredoxin in Cirrhotic Hepatitis C Virus Patients with and without Hepatocellular CarcinomaAyman El-Shayeb, Soraya Hamouda, Akram Deghady,Doaa El-Wazzan, Rafik Mehanna----------------------------------------------Original Article

The Effects of Common House Fly (Musca Domestica) Larvae-Derived Substances on Wound Healing in Mice ModelDoaa Elsaid Said Ahmed, Hoda Mahmoud Khalifa,Radwa GalalDiab, Lamiaa Moustafa Abdel Samad,Marwa Ahmed Meheissen, Hala Elsayed Diab Basiony

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Original Article

Epigenetics in HCV Related Liver Cirrhosis and HepatocellularCarcinoma in Egyptian Patients

Dina Nour, Mustafa Nemat Alla2, Mona Arafa1, and Mahmoud El-Bendary1; 1Department of Tropical Medicine, 2Department of Medical Biochemistry, Mansoura University, Mansoura, Egypt

ABSTRACTRecently there has been considerable interest in the field of epigenetics as a part of the process of carcinogenesis. Molecular evidence of aberrant methylation of cytosine in promoter CpG islands causing transcriptional silencing of energetic tumor suppressor genes in cancer cells. DNA methylation markers deliver an exclusive combination of specificity, sensitivity and high information content in diagnosis of cancer. Recognition of circulating DNA can be useful for the diagnosis of several cancers, including the hepatocellular carcinoma (HCC). Runt-related transcription factor 3 (RUNX3) geneis tumor suppressor genewhich could be inactivated by hypermethylation in many tumors including HCC, causing down regulation of gene expression. Aim of the work: In this study, the methylation of RUNX3 gene was studied in the bloodof patients with HCV related liver cirrhosis and patients with HCC as diagnostic biomarkers for HCC.Patients and Methods: DNA extracted from the peripheral blood of 90 HCV with liver cirrhosis (LC)patients and 90 HCV with HCC patients. Routine laboratory investigations, assessment of serum AFP and detection of circulating hypermethylated RUNX3 gene by methylation-specific PCR were done for all patients. Results: highly significant hypermethylated RUNX3 gene was found in HCC group compared to LC (OR= 3.1169) (95% CI=1.5202 to 6.3906) with p-value=0.0019. Conclusion: The presence of hypermethylated RUNX3 gene in serum may be useful biomarkers for HCC early diagnosis in HCV patients with LC.

IntroductionAlthough it has been evident for years that somatic mutations can enhance the function of oncogenes or down-regulate the activity of tumor suppressor genes resulting in the development of cancer, researches have shown the presence of epigenetic changes resulting in the development of tumors from the earliest to the latest stages (1).Molecular evidence of aberrant methylation of cytosine in promoter CpG islands causing transcriptional silencing of energetic tumor suppressor genes in cancer cells. (2, 3).Despite the fact that HCV is a RNA virus without a DNA there were studies demonstrate that HCV viral proteins may participate in epigenetic regulation of hepatic cancer and induce HCC-specific epigenetic changes.HCV infection was related to aberrant methylation on multiple genes. Other epigenetic alterations included histone proteins, chromatin remodeling, and noncoding RNAs also were described before. Uncovering the epigenetic alterations of HCV-induced HCC carcinogenesis could highlight a new strategy for understanding

the mechanism of HCC tumorigenesis and development, as well as a potential diagnostic advantage (4). DNA methylationmarkers provide a unique combination of specificity, sensitivity, and high information content in diagnosis of cancer. Methylation markers are particularly suited for situations where sensitive detection is necessary, such as when tumor DNA is either scarce or diluted by excess normal DNA. One of the most widely used methods for evaluatingmethylation levels is methylation-specific polymerase chain reaction (MSP). MSP requires only a small quantity of DNA and is sensitive to 0.1% methylated alleles of a certain CpG island locus (5). PCR-based methylation assays have been applied to the detection of tumor DNA in a variety of tissues and body fluids including serum, plasma, urine, sputum, stool, bile juice and lavage fluids (6).As present policies for cancer recognition are often costly, invasive and not well-defined, there remains a need to develop a reliable non-invasive test, rather asimple peripheral blood sample, for diagnostic purposes as well as for possible

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therapeutic monitoring. Therefore, using serum for this purpose is ideal. The presence of tumor DNA in serum was reported more than two decades ago(7). Methylation of several genes occurred not only in HCC andits precursor lesions, but also in chronic hepatitis and liver cirrhosis, suggesting that these changes are early events during HCC progression (8). In this study, we sought to determine the feasibility and clinical associations of detecting gene promoter hypermethylation utilizing the serum of patients with HCV related liver cirrhosis and patients with HCC. Mainly focusing on unstudied tumor suppressor gene RUNX3 in Egyptian patients, by detecting the methylation status of candidate gene by methylation specific PCR (MSP) technique.

Patients and MethodsWe recruited 90HCV patients with liver cirrhosis and 90 HCV patients with HCCattending the Clinics of Tropical Medicine Department, Mansoura University and obtained their informed consent. Ethics approval was obtained from the institution’s human research committee in Mansoura University. Methods: Patients with hepatitis B virus infection (HBV), other types of tumors or liver metastasis as proved by spiral CT were excluded from this study. All Groups Were Subjected To: • Full history taking and Clinical examination. • Radiological investigations: Abdominal ultrasonography U/S and spiral CT for detection of hepatic focal lesions, liver cirrhosis, hepatosplenomegaly and presenceof ascites. Complete blood count (CBC), prothrombin time and concentration. • Liver function tests, Kidney function tests. • Special Investigations (HBs Ag, and HCV antibodies, serum AFP). • Molecular study: detection of hyper methylated RUNX3 geneby methylation specific PCR. Collection of blood samples: Eight ml blood sample was taken from each patient. Four ml were delivered to k2EDTA vacutainer tubes for DNA host analysis. The EDTA blood was then aliquoted and stored at -70 oC for epigenetic analysis (until DNA extraction and methylation specific PCR done).The

residual blood sample was delivered into plain tubes centrifuged at 4000 rpm for 10 min. Then the serum was separated and aliquoted into 250 µl volume and kept at - 50until used for biochemical and serological testing. Serological markers: Anti-CHC Abs was assessed using an ELIZA immunoassay technique (Abbott Laboratories, Abbott Park, IL, USA) and PCR. Biochemical analysis:The lab investigations included: Bilirubin (Total & Direct), Aspartate aminotransferase (AST/GOT), Alanine aminotransferase (ALT/GPT), Alkaline phosphatase (ALP), and Albumin in the serum. Serum AFP concentration was measured by micro particle enzyme immunoassay (Abbott Laboratories, AXSYM, USA). Methylation specific PCR (MSP) technique: a-DNAExtraction: DNA was extracted using QIAamp® DNA Blood Mini kit (Qiagen) following manufacturer’s instructions. Extracted DNA was quantified using Nanodrop spectrophotometer (Thermoscientific 2000) to calculate the purity and concentration. d- Standard Bisulfite conversion: Sodium bisulfite conversion of genomic DNA extracted from peripheral blood was carried out using Epi Tect Bisulfite kit (Qiagen) as per manufacturer’s protocol. Purified bisulfite converted DNAwas stored at -20°C until using Primers and TaqMan probes (9) Followed by MSP.DNA methylation of CpG islands for RUNX3 gene was determined using specific primers for methylated (M) and unmethylated (UM) DNA.PCR products were analyzed onethidium bromide stained agarose gels and visualized under ultraviolet illumination.Semi quantitative methylation Calculation:Standard curve establishment and evaluation of quantitative analysis of DNA methylation were performed as previously described. The relative amount of methylation (%) was calculated in each sample according to the formula [M/ (U + M)] ×100. Concentrations of methylated (M) and unmethylated (U) portions were determined from simultaneously amplified standard curves for each gene. Methylation levels up to 0.5% were considered to be the background of this sensitive quantitative method. The

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cumulative methylation index (CMI) was calculated as the sum of percentage methylation for all evaluated genes. For RUNX3 gene, CMI of 500 was themaximum value of methylation.

Statistical AnalysisStatistical analysis was run on SPSS Statistics 17.0 (SPSS Inc., Chicago, Ill, USA). Quantitative non-parametric data were presented as median and percentiles (25th-75th).Qualitative data were presented as number (percent) and compared using2x2 Contingency Table for Odds ratio, 95 % Confidence interval, and Fisher exact P values using Medcalc software. The provided P values were adjusted using Chi-square test and Bonferroni formula to eliminate any false results. Receiver operator characteristic (ROC) curve was constructed for RUNX3 with determination of sensitivity, specificity, positive predictive value “PPV”, Negative Predictive Value” NPV” and accuracy at different cut-off levels. All tests are statistically significant at a P value of < 0.05.

ResultsBaseline Socio-demographic and clinical data distribution within studied groups in thisstudy were illustrated in (Table 1). Group 1: Comprised 90 HCV with liver cirrhosis, their age ranged from 44-69 years, 66 (74.16%)were males and 23 (25.8%) were females.There are 1 failed sample to examine methylation ofRUNX3 gene. Group 2: Comprised 90 HCC patients on top of HCV infection, their age ranged from (30-75) years. This group comprised of 63(72.41%) males and 24(27.59%) females. There are 3failed sample to examine methylation of RUNX3 gene. Association between methylation of candidate gene within the studied groups: The number of hypermethylated RUNX3 samples were 14of 89 and 32 of 87 in LC, and HCC groupsrespectively. The percentage of methylated examined RUNX3 were 15.7% and 36.8% inLC and HCC groups respectively. Dataillustrated in (table 2). The risk of methylation of RUNX3 gene on

development of HCC. Also, it was observed an association of methylation of RUNX3gene with development of HCC. The odds patients with methylated RUNX3 gene was significantly higher in HCC group compared to that of LC group (OR= 3.1169, 95 % CI =1.5202 to 6.3906, P = 0.0019) (table.3).Association between methylation of RUNX3 gene in relation to focal hepatic lesions size within HCC group. The tumor size in HCC group ranged from 2 to 9 cm and divided into two groups either <5 cm or > 5cm. A significant association was found betweensmaller tumor size (<5 cm) andhypermethylated RUNX3 gene in HCC group (P=0.0355). (table.4). Diagnostic performance of hyper methylated RUNX 3 gene in discrimination between HCC and LC. Performance of the hyper methylatedRUNX 3 genes as new biomarker for prediction of HCC on top of HCV from peripheral blood DNA by MSP revealed that at a cut off level of >0.23 IU l ml the sensitivity was 76.19% and specificity was 58.6% (figure1).

DiscussionMany studies have published on the prognostic value of DNA methylation in HCC specially from 2003 to 2017 (10); (11); (12);

(13), (14). This study analyzed promoter DNA methylation of RUNX 3 gene in a group of Egyptian HCC patients and HCV cirrhotic patients. HCC cases in this study were positive for HCV infection and negative for HBV infection. This may be explained by the fact that HCV is more prevalent in Egypt than HBV. As previous Egyptian studies revealed that HBV, having a prevalence of 2–8% in Egypt (15), while the overall prevalence of anti-HCV antibodies in Egypt was found to be 20% (16). This study detected a significant elevation in the median level of hyper-methylated RUNX 3 in sera of HCC patients, this finding was in agreement with the study done by Tanetal., According to their results, promoter hyper-methylation of RUNX3 was detected in the serum of 7/8 (88%) liver carcinoma (17).Additionally, in another study, RUNX3 hyper-methylation is also seen in chronic hepatitis and non-

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cancerous liver tissues of both HCV-positive and HCV-negative HCC cases, but it is absent in normal liver tissues(14, 18).Westudied the association of the hypermethylation of RUNX3 gene with clinical characteristics from all the HCC patients. There was no significant association between the methylation status of each gene and clinical parameters including age, gender, distant metastases, number of the focal lesions and smoking history apart of the size of the focal lesions as there is a significant association was found between smaller tumor size (<5 cm) and hypermethylated RUNX3 gene in HCC group. In conclusion, detection of hyper-methylated RUNX 3 gene in serum can be a tool for early detection of HCC. However this study has several limitations as being a

cross- sectional study in a single center and including relatively small number of patients. Still more work needs to be done to establish the importance of this marker as a routine laboratory investigation for high risk patients. It would also be motivating to study the variant circulating tumor DNA methylation levels in relation to management and to associate the post treatment level with the patient’s prognosis. Ethical approval and informed consent: All procedures performed in this study involving human participants were in accordance with the ethical standards of clinical and chemical research committee. Informed consent was taken from all participants prior to enrolment in this study and the study Ethics approval was obtained from the institution’s human research committee in Mansoura University.

Table.1: Baseline Socio-demographic and clinical data distribution within studied groupsLC(n=89) HCC(n=87)

Age: Median (min.-max.)

56(30-75)

57(44-69)

Sex:Female n. (%) 23 (25.84) 24(27.59)Male n. (%) 66(74.16) 63(72.41)OCP: (among females)Yes n. (%) 12(52.2) 5(20.8)No n. (%) 11(47.8) 19(79.2)Ascites:No n. (%) 14(15.73) 17(19.54)Yes n.(%) 75(84.27) 70(80.46)History of hepatic encephalopathy:No n. (%) 57(64.04) 24(27.59)Yes n. (%) 32(35.96) 63(72.41)Jaundice:Yes n. (%) 42(47.19) 62(71.26)No n. (%) 47(52,81) 25(28.74)Portal hypertension:Yes n. (%) 76(85.39) 67(77.01)No n. (%) 13(14.61) 20(22.99)

ALT=Alanine transaminase, AST= aspartate transaminase, CHC=chronic hepatitis C. LC= liver cirrhosis.HCC= hepatocellular carcinoma, OCP= oral contraceptive pills.

Table2: Association between methylation of candidate gene within the studied groupsLCNo.(%) HCCNo.(%) p-value

RUNX 3 n=89 n=87P = 0.001Hypo 75(84.3) 55(63.2)

Hyper 14(15.7) 32(36.8)Chi-square test. LC= liver cirrhosis. HCC= hepatocellular carcinoma. RUNX3 =Runt-related transcription factor

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Table 3: show that the risk of methylation of different genes on development of. HCC.HCC vs LC

RUNX3OR 3.116995% CI 1.5202 to 6.3906z statistic 3.103P 0.0019

LC= liver cirrhosis. HCC= hepatocellular carcinoma. RUNX3 =Runt-related transcription factor, CI= confidence interval, OR= odds ratio.

Table 4. Association between methylation of candidate genes in relationto focal hepatic lesions size within HCC group

Focal hepatic lesions size within HCC group No. (row%) p-valueNo. ≤5 >5

RUNX 3 Hypo 55 25(45.5) 30(54.5) 0.0355hyper 32 22(68.8) 10(31.2)

Chi-square test. LC= liver cirrhosis. HCC= hepatocellular carcinoma. RUNX3 =Runt-related transcription factor.

Figure1: ROC curve illustrates validity of RUNX1 within HCC vs LC

References1. Herman, J.G. and S.B. Baylin, Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med, 2003. 349(21): p. 2042-54.2. Jones, P.A. and S.B. Baylin, The fundamental role of epigenetic events in cancer. Nat Rev Genet, 2002. 3(6): p. 415-28.3. Esteller, M., CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene, 2002. 21(35): p. 5427-40.4. Rongrui, L., et al., Epigenetic Mechanism Involved in the HBV/HCV-Related Hepatocellular Carcinoma Tumorigenesis. Current Pharmaceutical Design, 2014. 20(11): p. 1715-1725.

5. Herman, J.G., et al., Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proceedings of the National Academy of Sciences of the United States of America, 1996. 93(18): p. 9821-9826.6. Cottrell, S.E. and P.W. Laird, Sensitive detection of DNA methylation. Ann N Y Acad Sci, 2003. 983: p. 120-30.7. Leon, S.A., et al., Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res, 1977. 37(3): p. 646-50.8. Chiba, T., et al., Identification and investigation of methylated genes in hepatoma. Eur J Cancer, 2005. 41(8): p. 1185-94.9. Dammann, R., G. Yang, and G.P. Pfeifer, Hypermethylation of the cpG island of Ras association domain family 1A (RASSF1A), a putative tumor suppressor gene from the 3p21.3

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locus, occurs in a large percentage of human breast cancers. Cancer Res, 2001. 61(7): p. 3105-9.10. Gao, W., et al., Variable DNA methylation patterns associated with progression of disease in hepatocellular carcinomas. Carcinogenesis, 2008. 29(10): p. 1901-10.11. Hernandez-Vargas, H., et al., Hepatocellular carcinoma displays distinct DNA methylation signatures with potential as clinical predictors. PLoS One, 2010. 5(3): p. e9749.12. Song, M.-A., et al., Elucidating the Landscape of Aberrant DNA Methylation in Hepatocellular Carcinoma. PLOS ONE, 2013. 8(2): p. e55761.13. Dong, X., et al., Combination of serum RASSF1A methylation and AFP is a promising non-invasive biomarker for HCC patient with chronic HBV infection. Diagnostic Pathology, 2015. 10(1): p. 133.14. Mansour, L.A., et al. Circulating Hypermethylated RASSF1A as a Molecular Biomarker for Diagnosis of Hepatocellular Carcinoma. Asian Pacific journal of cancer prevention : APJCP, 2017. 18, 1637-1643.

15. El-Zayadi, A.-R., et al., Hepatocellular carcinoma in Egypt: A single center study over a decade. World Journal of Gastroenterology : WJG, 2005. 11(33): p. 5193-5198.16. Arafa, N., et al., Changing pattern of hepatitis C virus spread in rural areas of Egypt. J Hepatol, 2005. 43(3): p. 418-24.17. Tan, S.H., et al., Detection of promoter hypermethylation in serum samples of cancer patients by methylation-specific polymerase chain reaction for tumour suppressor genes including RUNX3. Oncol Rep, 2007. 18(5): p. 1225-30.18. Nishida, N., et al., Aberrant methylation of multiple tumor suppressor genes in aging liver, chronic hepatitis, and hepatocellular carcinoma. Hepatology, 2008. 47(3): p. 908-918.

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Original Article

Role of Dynamic Susceptibility Contrast (DSC) Perfusion Magnetic Resonance Imaging in the Differentiation between Recurrent Brain Tumor and Radiation Necrosis

Heba M. Soliman1, Ahmed A. ElBeheiry1, Amr A. Abdel-Kerim1, Ahmed H. Farhoud2,M.IhabReda1;1Department of Radiodiagnosis, 2Department of Neurosurgery, Faculty of Medicine, University of Alexandria, Egypt

ABSTRACTDSC-perfusion MRI is a valuable non-invasive tool besides conventional MRI Aim of the work: To evaluate the role of dynamic susceptibility contrast (DSC) perfusion magnetic resonance imaging in thedifferentiation between recurrent brain tumors and radiation necrosis. Patients and Methods: Twenty patients with a history of operated primary brain tumors and postoperative radiotherapy with or without chemotherapy were enrolled in this prospective study having conventional MRI findings of enhancing lesion suspicious of being recurrence or radiation necrosis. All patients were examined by DSC-perfusion MRI.Definitive diagnosis was reached through subsequent surgical biopsy. Results: Fifteen patients (75%) were diagnosed as tumor recurrence and 5 patients as radiation necrosis (25%).The relative cerebral blood volume (rCBV) and relative peak height (rPH) were significantly higher (P <0.05) in recurrent tumors than in radiation necrosis lesions. The rCBV and rPHthresholds in differentiating between them were 1.8 and 1.22 respectively with 87%, 93% sensitivity and 100% specificity for each respectively. Conclusions: DSC-perfusion MRI is a valuable non-invasive tool besides conventional MRI whenever available to differentiate between radiation injury changes and tumor recurrence.

IntroductionThe current standard management for high-grade gliomas, especially glioblastoma,comprises surgical resection followed by adjunctive chemoradiotherapy. These adjuvant treatments improve the adequacy of tumor therapy, nonetheless, it may also increase the risk of radiation necrosis.(1)The distinction between radiation necrosis andtumor recurrence presents usually a diagnostic dilemma as they usually showsimilar conventional MRI features (contrast-enhancing lesions with mass effect). (2)

Correct diagnosis of both entities is critical and would largely affect the managementplan. Radiation necrosis usually requiresconservative management with steroids, where as tumor recurrence implies a change in the chemotherapy protocol and possible surgical re-intervention.(3)Currently, surgical biopsy or excision is the only definitive discriminative method. This surgery would be beneficial for patients with recurrent tumors, however, it would lead to further damage to adjacent normal cerebral parenchyma in cases of radiation necrosis in

deep-seated or eloquent cortical locations.(4)Elevated microvascular density and elevated capillary permeability are pathologic features that distinguish neoplastic lesions from irradiated brain tissue. Perfusion MR imaging is currently a promising tool to quantify a tumor’s hemodynamic properties andmicrovasculature.(5) DSC MRI technique entails the rapid intravenous injection of a contrast agent and the serial measurement of signal loss during the bolus passage through the tissue, using T2*-weighted images. The signal drop correlates with the contrast agent concentration and can be used to measure the hemodynamic parameters. The hemodynamic characteristics of the tissue could be quantified by several parameters, the relative cerebral blood volume (rCBV)and relative peak height (rPH) are of our concern.(6)We herein try to evaluate the usefulness of the DSC perfusion to discriminate between tumoral recurrence and radiation necrosis.

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Patients and MethodsThe study was approved by our institutional ethical committee. All patients signed informed consent before the MRI study. Patients: Between January 2016 and February 2017, we included patients referred to the MRI unit of our university hospital forimaging of the brain, presenting with ahistory of operated brain tumors followed by radiotherapy with or without chemotherapyand who showed suspicious enhancing lesion at their initial follow up conventional MRI.We ended with 20 patients presenting with 20 enhancing brain lesions who were enrolled in this prospective study. MRI Imaging protocol: MRI examinations were obtained with a1.5 T closed MRI scanner (Siemens, Avanto, Germany) using an 8-channel head coil. The contrast material used in the study was Gadolinium diethylene triaminepentacetic acid (Gad-DTPA) (Dotarem) in a dose of 0.1 mmol/kg body weight.MR images were acquired with the following protocols: (a)Pre-contrast series included axial and sagittal T1-weighted spin echo (repetition time msec/echo time msec, 600/15), axial and coronal T2-weighted turbo spin echo (4000/100), axial FLAIR(repetition time msec/echo time msec/ inversion time msec, 11000/140/2200), Diffusion-weighted imaging (DWI)(TR=5072; number of sections=16-22),(b)Dynamic Susceptibility Weighted contrast-enhanced (DSC) gradient echo echo-planar imaging (TR/TE;1250 ms/54 ms, flip angle: 35°).A series of gradient-echo echo-planar images were taken immediately before, during, and after bolus injection of the contrast agent.(c)Post-contrast series included axial, coronal and sagittal T1-weighted spin echo. Image processing and evaluation: The DSC and the anatomic data were transferred to an off-line workstation, T2*- weighted signal intensity–time curves were derived on a voxel by voxel basis. ROI analysis for CBV was performed. The control CBVNAWM was initially placed in the normal appearing white matter contralateral to the enhancing lesion. Next, four small (approximately 0.5 mm2) circular ROIs were drawn in the

enhancing lesion, transferred to the CBV map, adjusted as necessary to target the areas with the visually highest CBV. The ROI with the maximal CBV abnormality was measured and selected as the CBV lesion. The CBV lesion was divided by the CBVNAWM to yield the relative CBVlesion (rCBVlesion).Then these values were used to calculate the relative peak height (rPH) by the followingequation: rPH =[S0(lesion)-Smin(lesion)]/[S0(NAWM)-Smin(NAWM)].The final diagnosis:The final diagnosis of the examined brainlesions was established by histopathological analysis of the surgically excised lesions;where there current tumor was defined as any amount of tumor (ie, pure tumor and tumor admixed with necrotizing treatment effects).

Statistical AnalysisData were fed to the computer using IBM SPSS (Statistical Package for the Social Sciences) software package, V20 (SPSS Inc., Chicago, USA). Comparison between different groups regarding categorical variables was tested using Mann Whitney test. Quantitative data were described using mean, standard deviation (SD), median, minimum and maximum values. Agreement of the different predictions with the outcome was used and was expressed in sensitivity and specificity. Receiver operating characteristic curve (ROC) was plotted to analyze a recommended cutoff, the area under the ROC curve denotes the diagnostic performance of the test. Significance test results are quoted as two-tailed probabilities. The significance of the obtained results was judged at the 5% level.

ResultsAmong 20 examined brain lesions, 15 (75%) showed to be due to tumor recurrence while 5 (15%) were due to radiation necrosis. DSC hyper perfusion was seen in 14 out of 15 recurrent lesions (93.3 %). Hypoperfusion was only seen in a single recurrent lesion (6.7% [1 out of 15]) and in all 5 lesions with radiation necrosis (figures no.1).We found the measured hemodynamic parameters (rCBV and rPH) are significantly higher in

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recurrent tumor when compared with those of radiation necrosis (P<0.001).In recurrentlesions, the relative CBV (rCBV) ranged from 0.2 to 9.92 with a mean of 3.66 while the relative peak height (rPH) ranged from 0.52 to 6.5 with a mean of 2.44. While in lesions with radiation necrosis rCBV ranged from 0.2 to 0.88 with a mean of 0.24 and r PH from 0.27 to 1.2 with a mean of 0.64.(Table I). rCBV and rPH Cutoff values: Wefound the measured hemodynamic

parameters (rCBV and rPH) significantly higher in recurrent tumor lesions whencompared with those of radiation necrosis (P<0.001). At the generated ROC curve of rCBV, the best cutoff value to discriminate between both groups was 1.8 with 87% sensitivity and 100% specificity. On the other hand, ROC curve of rPH showed the best cutoff value to be 1.22 with 93.3% sensitivity and 100% specificity.

Table (I): Relative CBV and relative PH in recurrent and radiation necrosis lesionDiagnosis MW P

Recurrence(n = 15)

Radiation necrosis(n = 5)

CBVMin. – Max. 0.2 – 9.92 0.20 – 0.88Mean ± SD. 3.66 ± 2.52 0.41 ± 0.30 3.467* 0.001*

Median 2.51 0.24PHMin. – Max. 0.52 – 6.50 0.27 – 1.2Mean ± SD. 3.01 ± 1.88 0.64 ± 0.38 3.464* 0.001*

Median 2.30 0.46

DiscussionGliomas are the most common primary tumors of the central nervous system and represent about one-third of all intracranial tumors in adults.(2)Tumor recurrence and radiation necrosis present with similar clinical and conventional MRI characteristics, nonetheless the suitable therapeutic strategy is completely different. (7).Recurrent tumors are known to produce distorted and permeable blood vessel in their vicinity than in normal brain parenchyma. This would result into higher rCBV, in contrast, radiation necrosis that hinders the blood flow and consequently lowers the rCBV values.(4, 7, 8).rPH is a quantifiable measure of tumor vasculature and has been reported to strongly correlate with rCBV.Higher rPH values are expected to be associated with tumor recurrence because progressing tumors have greater vasculature than does radiation necrosis. rPH has been used in together with rCBV to differentiate between tumor progression and treatment necrosis.(4, 7, 8)In the current series, the degree of vascularity correlated significantly with the final diagnosis. All of the lesions sequel

to radiation necrosis were hypoperfused and 93.3% of the proven recurrent tumors were hyperperfused in comparison to the contralateral normal-appearing white matter (NAWM). Only one recurrent lesion showed hypo perfusion and was therefore wrongly diagnosed as radiation necrosis. This could be attributed to an early process of recurrence, where no prominent vascularity had been developed to be adequately depicted with MR perfusion. More over,petechial hemorrhage produces susceptibility artifact decreasing the r CBV ratio when it occurs at the tumor site, and this was also seen in our case where multiple hemorrhagic foci were detected by SWI sequence. These justifications agreed with those of Da Cruz et al. review article.(9)Mean r CBV was 3.66 ± 2.52 versus 0.41 ± 0.30 for the recurrent lesions and the radiation necrosis,and therPH was 3.01 ± 1.88. versus 0.64 ± 0.38 respectively. These results came in good agreement with other published studies. Mean rCBV varied between 2.38±0.87 and 3.33±1.16 for the recurrent lesions andranged between 1.57±0.67 and 1.82± 0.79 for the radiation necrosis in the studies of

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Barajas et al (4) and Matsusue et al. (10)respectively. Similarly, Barajas et al(4)showed mean rPH was 2.07± 0.69 for the recurrent lesions versus 1.25±0.42 for the radiation necrosis. The optimal thresholds of rCBV and rPH determined in this study are similar to those proposed in the current literature.(4)(11)(10)(12)We proposed a cutoff value of 1.8 for the rCBV and 1.22 for the rPH to discriminate between recurrent tumors and radiation necrosis. At these values, rPH was more sensitive than rCBV (93.3% versus 82%) , and this was agreed with the results of Barajas et al.(4) andYoung et al.(13) who showed that the rPH was the most accurate predictor with a sensitivity of 100% in their studies. The specificity of both r CBV and rPH in our study as well as in those two later series was also 100%. Other published series had suggested a nearly similar cut off values for both parameters. Cut off values of rCBV varied between 1.75 (sensitivity 78%, specificity 71%) and 1.8 (sensitivity 71%, andspecificity 95% ) respectively in the studies of Barajas et al(4)and Gasparetto etal(11)performed on 57 and 30patients respectively. The reported cut off value of rPH varied between 1.37 and 1.38 in the studies of Martínez-Martínez et al. andBarajas et al.(4)who showed consequentsensitivity of 88% and 89% and specificity of 82% and 81%.The difference in cutoff values between our study and others could be attributed to variations the in

magnetic field strength, type and amount of contrast, the use of preload contrast dosing,post processing methods (comparison to white matter vs. gray matter) and the difference in sample size. The limitations of this study include the small patient population and the unavailability of histopathologic diagnosis in all patients. As many of them were not a candidate for reoperation. So the clinical and imaging follow-up data had to be used as surrogate markers for the identity of the lesion. The prolonged follow-up of these lesions would minimize any possibility of misclassification. It is unlikely that a denovo tumor recurrence shows prolonged stability or regression, or the patient improves clinically; thus, it is highly unlikely that the five patients classified as radiation necrosis on this basis actually had tumor recurrence. Similarly, continued progression in the size and the extent of the lesion is a strong evidence of tumor recurrence.

ConclusionsDSC perfusion MR imaging issuggested as acomplementary yet essential technique to assess the lesional vascular density andhence to discriminate between tumor recurrence and radiation necrosis. Hyper perfusion usually denotes recurrent lesions,while hypoperfusion is the rule in radiation necrosis. rCBV and rPH cutoff values of 1.8 and 1.22 respectively could be proposed to differentiate between the two entities.

Figure no. (1)

A 43-year old male patient with history of surgically removed left parietal GBM followed by chemo and radiotherapy; routine follow-up MRI showed;(A)Axial T1 post-GAD showing heterogeneously enhancing

lesion with central non-enhancing breaking down.(B)CBV color map: the lesion is hyperperfused in comparison to the contralateral NAWM with rCBV= 6.44 .(C)T2* signal intensity time curve: the

A B C

12

lesion is hyperperfused in comparison to the contralateral NAWM with rPH=2.9 (lesion:solid red line, contralateral NAWM:yellowdashed line) Features of operative bed tumor recurrence that was confirmed by histopathology.

References1. Sanghvi DA. Recent advances in imaging of brain tumors. Indian J Cancer. 2009;46(2):82-7.2. Wan B, Wang S, Tu M, Wu B, Han P, Xu H. The diagnostic performance of perfusion MRI for differentiating glioma recurrence from pseudoprogression: A meta-analysis. Medicine (Baltimore). 2017;96(11):e6333.3. Siu A, Wind JJ, Iorgulescu JB, Chan TA, Yamada Y, Sherman JH. Radiation necrosis following treatment of high grade glioma--areview of the literature and current understanding. Acta Neurochir (Wien). 2012;154(2):191-201; discussion 4. Barajas RF, Jr., Chang JS, Segal MR, Parsa AT, McDermott MW, Berger MS, et al. Differentiation of recurrent glioblastoma multiforme from radiation necrosis after external beam radiation therapy with dynamic susceptibility-weighted contrast-enhanced perfusion MR imaging. Radiology. 2009;253(2):486-96.5. Paulson ES, Schmainda KM. Comparison of dynamic susceptibility-weighted contrast-enhanced MR methods: recommendations for measuring relative cerebral blood volume in brain tumors. Radiology. 2008;249(2):601-13.6. Jahng GH, Li KL, Ostergaard L, Calamante F. Perfusion magnetic resonance imaging: a comprehensive update on principles and techniques. Korean J Radiol. 2014;15(5):554-77.7. Wang S, Martinez-Lage M, Sakai Y, Chawla S, Kim SG, Alonso-Basanta M, et al. Differentiating Tumor Progression from Pseudoprogression in Patients with Glioblastomas Using Diffusion Tensor Imaging and Dynamic Susceptibility Contrast MRI. AJNR Am J Neuroradiol. 2016;37(1):28-36.

8. Kim YH, Oh SW, Lim YJ, Park CK, Lee SH, Kang KW, et al. Differentiating radiation necrosis from tumor recurrence in high-grade gliomas: assessing the efficacy of 18F-FDG PET, 11C-methionine PET and perfusion MRI. Clin Neurol Neurosurg. 2010;112(9):758-65.9. Da Cruz LH, Rodriguez I, Domingues R, Gasparetto E, Sorensen A. Pseudoprogression and pseudoresponse: imaging challenges in the assessment of posttreatment glioma. American Journal of Neuroradiology. 2011;32(11):1978-85.10. Matsusue E, Fink JR, Rockhill JK, Ogawa T, Maravilla KR. Distinction between glioma progression and post-radiation change by combined physiologic MR imaging. Neuroradiology. 2010;52(4):297-306.11. Gasparetto EL, Pawlak MA, Patel SH, Huse J, Woo JH, Krejza J, et al. Posttreatment recurrence of malignant brain neoplasm: accuracy of relative cerebral blood volume fraction in discriminating low from high malignant histologic volume fraction. Radiology. 2009;250(3):887-96.12. Mitsuya K, Nakasu Y, Horiguchi S, Harada H, Nishimura T, Bando E, et al. Perfusion weighted magnetic resonance imaging to distinguish the recurrence of metastatic brain tumors from radiation necrosis after stereotactic radiosurgery. J Neurooncol. 2010;99(1):81-8.13. Young RJ, Gupta A, Shah AD, Graber JJ, Chan TA, Zhang Z, et al. MRI perfusion in determining pseudoprogression in patients with glioblastoma. Clin Imaging. 2013;37(1):41-9.

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Original Article

Role of Echocardiography and Pro-BNP in Detection of Pulmonary Hypertension in ESRD Patients on Maintaince Haemodialysis

Amr Mohamed Ebaid1, Amel Fouad Mohamed Ketat2, Eman Mohamed Elsharkawy3, Yasmine Salah Naga1, Asmaa Abdelkader Abdelal Eissa1; 1Internal medicine Department, 2Medical Biochemistry Department, 3Cadiology Department Alexandria university.

ABSTRACTChronic glomerulonephritis and interstitial nephritis are currently the principal causes of chronic kidney disease in developing countries The aim of the work was to assess the presence of pulmonary hypertension in ESRD patients on maintenance haemodialysis and the role of pro-BNP in its prediction. Patients and Methods The present study was conducted on: 1. Thirty ESRD patients on haemodialysis for more than 6 months in the Alexandria Main University Hospital Dialysis unit. 2. Ten age and sex matched healthy volunteers. All participants gave a written informed consent. PASP was significantly higher in patients with pulmonary hypertension (p= 0.05), yet EF and RV were not significantly different Pro-BNP was significantly higher in the patients group (P<0.001). Conclusions: Pulmonary hypertension is common in HD patients, yet it is underdiagnosed. In hemodialysis patents, pro-BNP may have a role in predicting pulmonary hypertension

IntroductionChronic kidney disease (CKD) has significantly increased in developing countries such as Egypt. Chronic glomerulonephritis and interstitial nephritis are currently the principal causes of chronic kidney disease in developing countries, reflecting the high prevalence of bacterial, viral, and parasitic infections but also the incidence of CKD secondary to diabetes and hypertension is rising in developing countries. (1,2) The National Kidney Foundation classifies CKD according to glomerular filtration rate and albumin: creatinine ratio (ACR). The severest stage in this classification is end stage renal disease (ESRD) where the GFR falls below 15ml/min/1.73m2. When patients reach this stage, they need renal replacement therapy in the form of hemodialysis (HD), peritoneal dialysis, or kidney transplantation to sustainlife. (3,4) Dialysis initiation should be considered when GFR is below 10mL/min/1.73m2. Studies suggest that the well-selected patient without overt uremic symptoms may wait to initiate dialysis until GFR is closer to 7 mL/min/1.73 m2. Recent KDIGO and KDOQI guidelines recommend referral of all individuals with GFR below 30 mL/min/1.73 m2 to a nephrologist, stressing that timely nephrology referral maximizes the likelihood of adequate planning for RRT

to optimize decision making and outcomes.The decision to initiate maintenance dialysis in patients who choose to do so should be based primarily upon an assessment of signs and/or symptoms associated with uremia, evidence of protein-energy wasting, and the ability to safely manage metabolic abnormalities and/or volume overload with medical therapy rather than on a specific level of kidney function in the absence of such signs and symptoms.(5,6) The most common cause of death in ESRD patients on haemodialysis is cardiac disease (43%).Cardiac arrest secondary to ischemic heart disease, acute coronary syndromeandchronic coronary artery disease were responsible for almost half of these deaths. Other causes include infection, cerebrovascular disease, and malignancy.(7-9)

Pulmonary hypertension is a complex problem characterized by pathologic elevation in pulmonary arterial pressure. Normal pulmonary artery systolic pressure at rest is 15–30 mm Hg, with a mean pressure ranging between 10 mm Hg and 18 mm Hg. The World Health Organization currently classifies pulmonary hypertension based on similarities in pathologic mechanisms into the following five groups: group 1: pulmonary arterial hypertension secondary to various disorders, group 2: secondary to left heart disease, group 3: secondary to lung

14

disease or hypoxemia, group 4: secondary to chronic thromboembolism, group 5: secondary to hematologic, systemic, metabolic, or miscellaneous causes. This last category includes ESRD patients on dialysis. (10-12) There are several potential explanations for the development of pulmonary hypertension in patients with ESRD. Hormonal and metabolic derangement associated with ESRD might lead to pulmonary arterial vasoconstriction and an increase of the pulmonary vascular resistance. Pulmonary arterial pressure may be further increased by high cardiac output resulting from the arteriovenous access itself, worsened by commonly occurring anemia and fluid overload. (13, 14) Diagnosis of pulmonary hypertension is achieved by echocardiography which is a pivotal screening test in symptomatic patients at risk for pulmonary arterial hypertension (PAH). It has the advantage of being widely available, cost effective, and safe. It also plays an important role in assessing outcomes, monitoring the efficiency of specific therapeutic interventions for PAH, and detecting the preclinical stages of disease. (15, 16) Plasma levels of brain natriuretic peptide (BNP) are increased in patients with left heart failure. One of the major contributing factors for the markedly elevated BNP level in this population is the very high prevalence of LV structural and functional abnormalities. BNP level is strongly associated with LV hypertrophy and systolic dysfunction in patients who have ESRD and are on maintenance HD. (17-19)

Serial monitoring of BNP level may be useful in identifying patients who have ESRD and are at increased cardiovascular and mortality risk. Little is known about BNP secretion in right ventricular failure in pulmonary hypertension.

The Aim of the Workwas to assess the presence of pulmonary hypertension in ESRD patients on maintenance haemodialysis and the role of pro-BNP in its prediction.

Patients and MethodsThe present study was conducted on: 1.Thirty ESRD patients on haemodialysis for more than 6 months in the Alexandria Main

University Hospital Dialysis unit. 2. Ten age and sex matched healthy volunteers. All participants gave a written informed consent after explanation of the nature and the aim of the study. Patients suffering from following diseases were excluded: Significant valvular heart disease, ischemic heart disease, significant arrhythmia, LVEF <40%, known vasculitis and known hepatic disease. All subjects included in the study were subjected to the following: 1. Complete history taking including the duration of haemodialysis, original kidney disease, and history of hepatic and cardiac dysfunction. 2. Thorough clinical examination. 3. Laboratory investigation including: I. Routine Lab Investigation: -Complete blood count. -Serum calcium. -Serum phosphorus. -Parathyroid hormone. -Serum Alkaline phosphatase. -Lipid profile: serum triglyceride, serum cholesterol. II. Special Biochemical Investigation: -Estimation of serum Pro-BNP (by ELISA) (19)4.Echocardiography. With special emphasis on pulmonary pressure and right ventricle.Analysis of NT-proBNP: Blood samples were drawn before a mid-week dialysis session. Samples were collected in prechilled tubes containing EDTA, immediately placed on ice, and promptly centrifuged at 4°C. After separation, plasma was stored at -80°C. NT-proBNP measurements were done using an ELISA - two step sandwich assay with streptavidin coated microtitre plates. This assay does not require sample extraction and there is no detectable cross reactivity with ANP, NT-proANP, BNP, or urodilatin.

Echocardiography: Echocardiography was done with special emphasis on pulmonary pressure and right ventricle after a mid-week dialysis session by a single investigator using a Hewlett Packard Image point, model M2410A (Andover, Massachusetts, USA). LVEF was estimated according to Teich holzand colleagues in all subjects with a homogeneous contraction pattern.(15)

Pulmonary hypertension is a hemodynamic state defined by a resting mean pulmonary artery pressure at or above 25 mm Hg.

15

ResultsThe present study was conducted on two groups: Patients group: Thirty ESRD patients on haemodialysis for more than 6 months. Control group: Ten age and sex matched healthy volunteers. The demographic, clinical and laboratory data are summarized in table (1). There was no statisticallysignificant difference between the two studied groups regarding demographic data. There was a statistically significant difference between the two studied groups regarding blood picture (Hg, WBCs, Plt) (P < 0.05). There was statistically significant difference between the two studied groups

regarding lipid profile (S.cholesterol, S.TG) (P < 0.05). Serum calcium was significantly lower in the patients group (P=0.038), while serum phosphorus was significantly higher (P=0.001). Creatinine and urea were significantly higher in patients group than control group, (P < 0.05).Table (2) shows comparison between Pro-BNP in the study groups. In patients group, Pro-BNP ranged from 7780-34240 with mean value 16427.33±7365.20 pg/ml, and in control group, it ranged from 5170-6700 with mean value 6041.0±526.04pg/ml.Pro-BNP was significantly higher in the patients group (P<0.001)

Table (1): Comparison between the two studied groups regarding demographic data.Patients Control P

Age (years)RangeMeanS.D.

35-6649.939.63

36-6652.2011.22 0.27

SexMale Female

No % No %0.42917

1356.743.3

64

6040

BMI (kg/m2)

RangeMeanS.D.

18.8-31.524.933.5138

20.9-31.226.932.97

0.06

BMI Category Under weight Normal weight Over weight

41610

13.353.333.3

055

050.050.0

Hg (g/dl)RangeMeanS.D.

5.5-10.58.341.44

12-13.512.650.58

0.001*

WBCs (x109)RangeMeanS.D.

4.4-137.342.25

4.2-5.44.790.41 0.001*

Plt (x109)RangeMeanS.D.

150-385257.1367.09

166-250197.8030.43

0.005*

S.cholesterol (mg/L)RangeMeanS.D.

160-450254.8374.98

140-156148.606.19

0.001*

S.TG (mg/L)RangeMeanS.D.

120-300173.5048.37

100-120107.908.09 0.001*

S.calcium (mg)RangeMeanS.D.

6.7-10.78.900.82

8.9-109.400.47

0.038*

S.Phosphorous (mg)RangeMeanS.D.

2.7-7.55.091.26

2.5-3.52.910.43 0.001*

Creatinine (mg/L)RangeMeanS.D.

2-8.75.262.07

0.6-10.740.16 0.001*

Urea (mg/L)RangeMeanS.D.

38-184.899.6542.01

12-2116.503.54 0.001*

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Table (2): Comparison between the two studied groups regarding Pro-BNP.Patients Control P

Pro-BNP (pg/ml)RangeMeanS.D.

7780-3424016427.337365.20

5170-67006041.00526.04

0.001*

Table (3) shows the correlation between pro-BNP and different studied variables in patients group. There was a statisticallysignificant positive correlation regarding with the duration of hemodialysis, WBCs,

Plt, S.cholesterol, S.T G, Urea and Creatinine (P< 0.05), while there was a significant negative correlation with Hg, S.calcium and PTH.

Table (3): Correlation between pro-BNP and different studied variables in patients group.Pro-BNP Pearson correlation (r) P valueduration of hemodialysis .856** 0.0001RV 0.055 0.774PASP 0.111 0.558EF -.163- 0.390Hg -.540-** 0.0001WBCs .634** 0.0001Plt .598** 0.0001S.CHOLESTEROL .324* 0.041S.TG .373* 0.018S.calcium -.213- 0.187S.Phosphorous 0.25 0.119ALK 0.198 0.294PTH -.125- 0.510Creatinine .547** 0.0001Urea .567** 0.0001

Table (4) shows the comparison between those with and without pulmonary hypertension as regard the echo parameters, in patients group. PASP was significantly higher in patients with pulmonary hypertension (p= 0.05), yet EF and RV were not significantly different.Table (5) shows comparison between patients with and

without pulmonary hypertension asregards the studied variables, in patients group.Patients with pulmonary hypertension had significantly higher S.cholesterol, while there was no statistical significant relation between Hg, WBCs, Plt, S.TG, S.calcium, S.Phosphorous, ALK, PTH, Creatinine and Urea to pulmonary HTN (P > 0.05).

Table (4): Relation between RV, PASP and EF to Pulmonary HTN in patients groupPulmonary HTN

pNo YesRV RangeMeanS.D.

24.0032.00

26.14291.955

23.0029.00

25.87501.784

.154

.698

PASP RangeMeanS.D.

20.0040.00

24.28575.283

40.0075.00

53.125010.53803

85.715.000

EFRangeMeanS.D.

41.0079.0060.78

10.123

40.0080.0053.3110.39

3.953.057

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Table (5): Relation between laboratory findings in relation to Pulmonary HTN in patients groupPulmonary HTN p

No YesHg RangeMeanS.D.

41.0-79.0060.7810.12

40.0-80.0053.3125

10.39691

3.953.057

WBCs RangeMeanS.D.

4.40-13.007.752.81

4.4-9.506.97

1.622

.896

.352

PltRangeMeanS.D.

150.0-385.00285.071.13

156.0-341.00232.7554.38

5.182.031

S.CHOLESTEROLRangeMeanS.D.

180.0-300.00214.2835.67

160.0-450.00290.3182.99

10.081.004

S.TGRangeMeanS.D.

120.0-250.00155.7136.52

140.0-300.00189.0653.047

3.904.058

S.calciumRangeMeanS.D.

6.70-10.108.72.792

7.10-10.709.05.839

1.252.273

S.PhosphorousRangeMeanS.D.

3.30-7.505.081.39

2.7-7.305.1001.180

.001

.976

ALKRangeMeanS.D.

70.0-150.00106.0725.95

50.0-200.0114.0042.261

.370

.548

PTHRangeMeanS.D.

120.0-950.00566.42

294.921

19.0-1300.00534.87416.12

.056

.815

CreatinineRangeMeanS.D.

2.30-8.705.13

2.138

2.00-8.405.3688

2.07677

.091

.765

UreaRangeMeanS.D.

41.40-157.7093.6138.37

38.00-184.80104.9345.510

.534

.471

DiscussionIn the present study pro-BNP levels were significantly higher in the patients group compared to the control group (16427.33±7365.20vs6041.0±526.04. P=0.001). There was statistically significant differences between the two studied groups regarding Pro-BNP. There was a statistically significant positive correlation between pro BNP and duration of hemodialysis, WBCs, Plt, s.cholesterol, S.TG, urea and creatinine

(P< 0.05), while there was a negative correlation between Pro BN Pand Hg, S.calcium and PTH. In agreement with our study, Jacques, et al., (2017)(20) found that hemodialysis (HD) patients exhibited high levels of NT-proBNP, Horland Jourdain, et al., (21,22) found the same in haemodialysis patients, NT-proBNP was associated with left ventricular ejection fraction, independently of age, SBP, diabetes and hsCRP in hemodialysis patients.(20)In

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addition , Jacques, et al., (2017) consideredNT-proBNP values ≥ 6243 pg/ mL (75th percentile) as the highest NT-proBNP levels, since therewas no threshold-consensus forHDpatients. In a recent study in 238 Japanese HD patients, NT-proBNP values ≥ 5760 pg/mL (higher tertile) were considered as the higher values [22]. Predialysis median NT-proBNP levels were previously found markedly elevated than in HD patients, 4079 pg/ml [1893–15076] [14], compared to median population-based normal values, 20 pg/ml.(20-25)Also, Jacques, et al., demonstrated that, patients with high NT-proBNP levels were more likely to have higher dialysis vintage, higher frequencies of weight loss, low BMI (≤23 Kg/m2), low serum albumin levels (≤38 g/L), low serum creatinine levels (≤818 𝜇𝜇mol/L), low nPCR (≤0.8 g/kg/d), lower mean hemoglobin rate, higher frequencies of hsCRP> 5 (mg/L).(4)Given that BNP and NT-pro-BNP are secreted in response to increases in myocardial wall stretch, it is tempting to hypothesize that circulating BNP and NT-pro-BNP levels are useful markers of volume status. Indeed, an earlier but very small study of HD patients suggested an association between plasma BNP and extracellular water estimated by bioimpedance;(22) however, subsequent studies of HD and PD patients that compared the use of BNP and NT-pro-BNP with measurements of extracellular water by bioimpedance or inferior vena cava diameter to assess volume status have so far yielded disappointing results and failed to confirm a consistent link between BNP and NT-pro-BNP with extracellular water. A more recent study demonstrated that NT-pro-BNP levels showed small decrements with HD and ultrafiltration; however, the decrements had no correlation with volume removal or interdialytic weight gain.(23,24)Another study showed a significant relationship between serum NT-pro-BNP and extracellular water/body weight ratio only in HD patients with LV systolic dysfunction but not in those without systolic dysfunction.(25)In our study, there was statistically significant relation between Pro-BNP to Pulmonary HTN in patients group.

Pro-BNP was significantly higher in patients with pulmonary hypertension. In this study, the level of Pro-BNP predicted the presence of pulmonary hypertension at a cut off value of Pro-BNP with a sensitivity, 96.0, specificity of 98.0and accuracy (97.0%).In our study, the prevalence of abnormal left ventricular function (LVEF < 60%) was not high (13.8%) and only 1.9% had a LVEF lower than 40%. In addition, there was no significant correlation between pro-BNP and EF, suggesting that factors other than LVEFcardiac status may have an impact on NT-proBNP concentrations in hemodialysis patients. Jaque et al., further noted that inagreement with previous literature results, the patients’ serum levels of NT-proBNP inversely correlated with GFR and were significantly higher in patients with renal insufficiency. Regardless of renal function, NT-proBNP levels also correlated with hemodynamic parameters. At patient follow-ups, which occurred after a mean time of 20.6 months, 46% of patients showed clinical worsening of their pulmonary hypertension. When the retrospective NT-proBNP values were used to predict this worsening, the researchers needed to define a higher cutoff value in renal insufficiency patients (1660 ng/L, sensitivity 68%,specificity 73%) as compared to patients with normal renal function (1292 ng/L, sensitivity 50%, specificity 83%). Patients with NT-proBNP levels higher than these cutoffs were 4.8 times more likely to have clinical worsening. The authors concluded that NT-proBNP can be used to predict pre-capillary pulmonary hypertension, but renal dysfunction must be taken into account.(20) Acommunity-based study in patients with HF demonstrated that pulmonary artery systolic pressure (PASP) estimated by echocardiography strongly predicted all-cause and cardiovascular mortality independently of known predictors of outcome. While numerous studies have consistently shown an inverse correlation between PH and survival, a combination of elevated PAP and reduced RV systolic function was particularly associated with an unfavorable outcome in HFrEF.13 In HFpEF,

19

studies demonstrated that the presence of PH was also strongly associated with mortality.(28-30) Furthermore, HFpEF patients commonly display RV dysfunction, which is associated with elevated PAP, occurs at more advanced stages, and represents a strong predictor of death.(26-32)

ConclusionPulmonary hypertension is common in HD patients, yet it is underdiagnosed. In hemodialysis patents, pro-BNP may have a role in predicting pulmonary hypertension, but more research is needed to define its role and to examine factors that may affect its level in hemodialysis patients.

References1. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 2004; 27:1047-53.2. Cooper BA. A randomized, controlled trial of early versus late initiation of dialysis. N Engl J Med 2010; 7:363:609.3. National Kidney Foundation. K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. American Journal of Kidney Diseases 2002; 39:1-266.4. National Collaborating Centre for Chronic Conditions, Chronic kidney disease: National clinical guideline for early identification and management in adults in primary and secondary care. Royal College of Physicians: London 2008.5. Inker LA, Astor BC, Fox CH. KDOQI US commentary on the 2012 KDIGO clinical practice guideline for the evaluation and management of CKD. Am J Kidney Dis 2014; 63(5):713-35.6. O'Hare AM, Batten A, Burrows NR, Pavkov ME, Taylor L, Gupta I, et al. Trajectories of kidney function decline in the 2 years before initiation of long-term dialysis. Am J Kidney Dis 2012; 59(4):513-22.7. Rahul Sakhuja, Ashok J. Shah, SwapnilHiremath, Ranjan K. Thakur, End-Stage Renal Disease and Sudden Cardiac Death, Card ElectrophysiolClin 1 2009; 61-77.8. Davison R. Prognosis and management of chronic kidney disease (CKD) at the end of life. Postgrad Med J 2014; 90:98-105.9. James PA, Oparil S, Carter BL, Cushman WC, Dennison-Himmelfrab C4, Handler J, et al.

Evidence-based guideline for the management of high blood pressure in adults: report from the panel members appointed to the Eighth JointNational Committee (JNC 8). JAMA 2014; 311:507-20. 10. Turner JM. Treatment of chronic kidney disease. Kidney Int 2012; 81:351-62. 11. Hassoun PM. Update in pulmonary vascular diseases 2011. Am J RespirCrit Care Med 2012; 185:1177-82.12. Lourenço AP. Current pathophysiological concepts and management of pulmonary hypertension. Int J Cardiol 2012; 155:350-61. 13. Michel RP, Hakim TS, Hanson RE, Dobell AR, Keith F, Drinkwater D. Distribution of lung resistance after chronic systemic-to-pulmonary shunts. Am Physiol 1985; 249:106-1314. Okura H, Takatsu Y. High output failure as cause of pulmonary hypertension. Intern Med 1994; 33:363-5.15. Milan A, Magnino C, Veglio F. Echocardiographic indexes for the noninvasive evaluation of pulmonary hemodynamics. J Am SocEchocardiogr 2010; 23:225-39.16. Rudski LG, Lai WW, Afilalo J, Hua L, Handschumacher MD, Chandrasekaran K, et al. Guidelines for the echocardiographic assessment of the right heart in adults: a report from the American Society of Echocardiography endorsed by the European Association of J AM SocEchocardiogr 2010; 23(7):685-713.17. Gibelin A. Epidemiology and etiology of Wegener granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome and Goodpasture syndrome: vasculitides with frequent lung involvement. SeminRespirCrit Care Med 2011; 32(3):264-73. 18. Saggi SJ, Allon M, Bernardini J, Kalantar-Zadeh K, Shaffer R, Mehrotra R. Considerations in the optimal preparation of patients for dialysis. Nat Rev Nephrol 2012; 8:381-9.19. Porstmann T, Kiessing ST. Enzyme Immunoassay techniques. An overview. J Immunological Methods 1992; 150:5-12.20. Jacques Ducros, Laurent Larifla, Henri Merault, and Lydia Foucan. NT-proBNP, Cardiometabolic Risk Factors, and Nutritional Status in Hemodialysis Patients. International Journal of Nephrology. 2017; 1: 1-8.21. [20] H¨orl W. H., “Natriuretic peptides in acute and chronic kidney disease and during renal replacement therapy,” Journal of Investigative Medicine, 2005; 53(7): 366–370.22. [21] Jourdain P., G. Lefevre, C. Oddoze et al., “NT-proBNP in practice: from chemistry to

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medicine,” Annales de Biologie Clinique. 2009; 67; 255–271.23. Fagugli RM, Palumbo B, Ricciardi D, Pasini P, Santirosi P, Vecchi L, Pasticci F, Palumbo R: Association between brain natriuretic peptide and extracellular water in hemodialysis patients. Nephron ClinPract. 2003; 95 : c60 –c66.24. Lee SW, Song JH, Kim GA, Lim HJ, Kim MJ: Plasma brain natriuretic peptide concentration on assessment of hydration status in hemodialysis patient. Am J Kidney Dis. 2003; 41 : 1257 –1266.25. David S, Kumpers P, Seidler V, Biertz F, Haller H, Fliser D: Diagnostic value of N-terminal pro-B-type natriuretic peptide (NT-ProBNP) for left ventricular dysfunction in patients with chronic kidney disease stage 5 on haemodialysis. Nephrol Dial Transplant December. 2007; 18. 26. 13. Ghio S, Gavazzi A, Campana C, Inserra C, Klersy C, Sebastiani R, Arbustini E, Recusani F, Tavazzi L. Independent and additive prognostic value of right ventricular systolic function and pulmonary artery pressure in patients with chronic heart failure. J Am CollCardiol 2001; 37: 183–188. [PubMed]27. 14. Tampakakis E, Leary PJ, Selby VN, De Marco T, Cappola TP, Felker GM, Russell SD, Kasper EK, Tedford RJ. The diagnostic pulmonary gradient does not predict survival in patients with pulmonary hypertension due to left heart disease. JACC Heart Fail 2015; 3: 9–16. [PMC free article] [PubMed]28. 15. Lam CS, Roger VL, Rodeheffer RJ, Borlaug BA, Enders FT, Redfield MM. Pulmonary hypertension in heart failure with preserved ejection fraction. A community-based study. J Am CollCardiol 2009; 53: 1119–1126. [PMC free article] [PubMed]29. 16. Leung CC, Moondra V, Catherwood E, Andrus BW. Prevalence and risk factors of pulmonary hypertension in patients with elevated pulmonary venous pressure and preserved ejection fraction. Am J Cardiol 2010; 106: 284–286. [PubMed]30. 17. Shah AM, Shah SJ, Annand IS, Sweitzer NK, O'Meara E, Heitner JF, Sopko G, Li G, Assmann SF, McKinlay SM, Pitt B, Pfeffer MA, Solomon SD; TOPCAT Investigators. Cardiacstructure and function in heart failure with preserved ejection fraction: Baseline findings from the echocardiographic study of the treatment of preserved cardiac function heart failure with an aldosterone antagonist trial (TOPCAT). Circ Heart Fail 2014; 7: 104–115.

31. 19. Melenovsky V, Hwang SJ, Lin G, Redfield MM, Borlaug BA. Right heart dysfunction in heart failure with preserved ejection fraction. Eur Heart J 2014; 35: 3452–3462. [PMC free article] [PubMed]32. 20. Mohammed SF, Hussain I, AbouEzzeddine OF, Takahama H, Kwon SH, Forfia P, Roger VL, Redfield MM. Right ventricular function in heart failure with preserved ejection fraction: a community-based study. Circulation 2014; 130: 2310–2320.33. 11. Vachiery JL, Adir Y, Barbera JA, Champion H, Coghlan JG, Cottin V, De Marco T, Galiè N, Ghio S, Gibbs JS, Martinez F, Semigran M, Simonneau G, Wells A, Seeger W. Pulmonary hypertension due to left heart diseases. J Am CollCardiol 2013; 62(Suppl D): D100–D108.34. 26. Melenovsky V, Hwang SJ, Redfield MM, Zakeri R, Lin G, Borlaug BA. Left atrial remodeling and function in advanced heart failure with preserved or reduced ejection fraction. Circ Heart Fail 2015; 8: 295–303. [PubMed]35. 27. Sanchis L, Gabrielli L, Andrea R, Falces C, Duchateau N, Perez-Villa F, Bijnens B, Sitges M. Left atrial dysfunction relates to symptom onset in patients with heart failure with preserved ejection fraction. Eur Heart J Cardiovasc Imaging 2015; 16: 62–67.

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Original Article

Serum Endoglin (CD105) in Cirrhotic Hepatitis C Virus and Hepatitis B Virus Patients with and without Hepatocellular Carcinoma

Khaled Mohiedeen1, Ali El-Kady1, Akram Deghady2, Rania Abou Youssef 1, Mohamed Samy1;1Tropical Medicine Department, 2Clinical Pathology Department, Faculty of Medicine, Alexandria University.

ABSTRACTHepatocellular carcinoma (HCC) is a common disorder worldwide and ranks 2nd and 6th most common cancer among men and women in Egypt respectively. HCC has a rising incidence in Egypt mostly due to high prevalence of viral hepatitis B&C and their complications. AFP is early serum marker of HCC but with low sensitivity. So, there is a need for new serum markers of HCC. Endoglin (CD105) is a promising serum marker for HCC which may be more sensitive and specific than AFP. Aim of the work: The aim of this study was to assess the role of Endoglin (CD105) as a potential marker for HCC in cirrhotic HCV and HBV patients. Patients and methods: The study was conducted on eighty patients classified into two groups: group I consisted of 30 patients of HCV and HBV induced liver cirrhosis without HCC; group II included 50patients with HCC on top of HCV and HBV induced liver cirrhosis. Serum Endoglin was measured by ELISA. Results: Serum Endoglin was significantly higher in HCC (group II) than cirrhotic patients (group I)(p<0.001). Moreover, significant positive correlation was found between serum Endoglin and Child Pugh score, BCLC, portal vein thrombosis and tumor number in cases of HCC.(P<0.001, P<0.001, P<0.040,P<0.001 respectively) Diagnosis of HCC among patients with HCV and HBV cirrhosis could be suggested when serum Endoglin is detected at a cutoff value of 5.1 ng/ml. Conclusion: Serum Endoglinmight be used as a valuable serum marker of HCC development in cirrhotic HCV and HBV patientsparticularly in those with normal AFP level.

IntroductionHepatitis C virus (HCV) is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma, as well as the most common indication for liver transplantation in many countries. The annual risk of developing HCC is approximately 1% in those without cirrhosis and 3-10% in those with cirrhosis, depending on the stage of cirrhosis and presence of etiological cofactors. (1) Chronic hepatitis B (HBV) infection is a major risk factor for (HCC). Most HCCs complicate the evolution of active or inactive cirrhosis. However, some tumors occur on livers with minimal histological changes. (2) In Egypt, hepatocellular carcinoma (HCC) is the second most common cancer in men and the 6th most common cancers in women. (3)

Hospital-based studies from Egypt have reported an overall increase in the relativefrequency of all liver-related cancers in Egypt, from approximately 4% in 1993 to 7.3% in 2003. (4) This rising incidence (5) may be due to high prevalence of hepatitis C virus

(HCV) and its complications (6) and the fact that people born 20 years ago or earlier in Egypt has not been vaccinated against hepatitis B virus (HBV).(7) AFP is an α1-globulin normally present in high concentrations in fetal serum but in only minute amounts thereafter. Reappearance of high serum levels of AFP strongly suggests the presence of HCC. (8) AFP has been utilized as a marker for HCC, despite its low sensitivity and positive predictive value. Moreover, false-positive and false-negative results are obtained when AFP is used as a serum marker for HCC,(8) so the search for an ideal marker is needed. Endoglin (CD 105) is a homodimeric membrane glycoprotein expressed on endothelial cells that can bind to transforming growth factor -ß1 and transforming growth factor-ß3.(9)CD105 is only weakly expressed in normal tissues, but it is strongly expressed in tumor endothelia. Recent studies have suggested that CD 105 is a proliferation-associated marker of endothelial cells. (9, 10)

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Aim of the WorkThe aim of the work was to assess the value of serum endoglin level as a marker for HCC. Furthermore, the correlation of serum Endoglin with serum AFP and GGT were also evaluated in HCV and HBV induced cirrhotic patients.

Patients and MethodsAll patients were selected from Tropical Medicine ward, Faculty of Medicine, Alexandria University. This study was carried out on 80 patients classified into two groups. Group I consisted 30 patients with HCV& HBV induced liver cirrhosis. Group II included 50 patients of HCV and HBV induced liver cirrhosis with HCC. Exclusion Criteria: Patients with pulmonary disease,alcoholics, those suffering from fever,patients with autoimmune hepatitis or any other form of chronic liver disease other than chronic viral infection or other malignancies were excluded from the study. Ethical approval: The ethical committee of Faculty of Medicine, Alexandria University, approved this study. Written informed consent was obtained from all patients.Methods: All patients were subjected to thorough history taking and proper clinical examination. Laboratory investigations including: complete blood count (CBC);Complete urine and stool analysis; liver function tests; renal function tests, viral hepatitis markers, serum antinuclear antibody (ANA), Anti-smooth muscle antibody (ASMA), serum anti liver kidney microsomal antibody (LKMA) and Alpha-fetoprotein. Serum Endoglin was measured by ELISA. Abdominal imaging including: Ultrasound examination of the abdomen and triphasic C.T scan (was done for patients with U/S positive hepatic focal lesions).

ResultsAs regards cirrhosis group, it contained 6 patients Child A, 10 patients Child B and 14 patients Child C. While HCC group contained 5 patients with Child score A, 17 patients with Child score B and 28 patients with Child score C. There was no significant difference regarding number of patients of

both groups (P value 0.432).Among studied HCC patients, 5 (10%) patients were BCLC A, 9 (18%) patients were BCLC B, 8(16%) patient was BCLC C, and 28(56%) patients were BCLC D. 24 (48%) patients had one lesion, 6 (12%) patients had 2 lesions, 10 (20%) patients had 3 lesions and 10(20%) patient had more than 3 lesions. Tumor size ranged between1.50 – 15.10with mean±S.D. 6.35 ± 3.92. 18 (36%) out of the patients hadinfiltrative HCC lesions, 10 (20%) out of the patients had lymph node metastases. As regards to GGT, in cirrhosis group it ranged between 10.0 – 95.0 (u/l) with mean±S.D. 62.97 ± 24.58 (u/l) while in HCC group it ranged between 55.0 – 140.0 (u/l) with mean±S.D. 84.16 ± 21.85 (u/l). There was statistically significant difference between the two groups where (P value 0.002) as it was higher in HCC group than in cirrhotic group. As regards to AFP, in cirrhosis group it ranged between 4.0 - 95.0 (ng/dl) with mean±S.D. 36.82 ± 30.09 (ng/dl) while in HCC group it ranged between 15.0 –39000.0 (ng/dl) with mean±S.D1142.30 ± 5490.09 (ng/dl). There was statistically significant difference between the two groups where (P value <0.001*) as it was higher in HCC group than in cirrhotic group.As regards to Endoglin, in cirrhosis group it ranged between 0.40 – 6.90 (ng/ml) with mean±S.D. 3.62 ± 2.10 (ng/ml) while in HCC group it ranged between 4.0 – 17.20(ng/ml) with mean±S.D 8.86 ± 3.74 (ng/ml). There was statistically significant difference between the two groups where (P value <0.001*), as it was higher in HCC group than in cirrhosis group.(Figure 1)

Figure (1): Comparison between the two studied groups according to serum Endoglin (ng/ml)

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In both HCC and cirrhosis group, there was significant correlation between serum Endoglin and Child Pugh score in which (P value equal <0.001*) as it was higher in Child Pugh C than in B and A in both group.In HCC group, there was significant correlation between serum Endoglin and BCLC in which (P value equal <0.001*).Itwas higher in BCLC D than in C, B and A.Furthermore, there was also a significant correlation between serum Endoglin and tumor number in which (P value equal <0.001*) as it was higher in patients with multiple focal lesions than those with single focal lesion. In HCC group, there was significant correlation between serum Endoglin and portal vein thrombosis in which (P value equal 0.040*) as it was higher in the patient with PVT than in those without PVT. However, there was no significant correlation between serum Endoglin and lymph node metastasis and tumor size in which (P value equal 0.923),(P value equal <0.879) respectively. Receiver operating characteristic curves (ROC curves) were done to estimate the cutoff points discriminating cirrhosis from HCC of serum Endoglin, AFP as well as GGT. Tables (I -IV), Figures (2-5). 1-Serum Endoglin: The ROC curve for serum Endoglin was

significant (p.001*) and showed that the cutoff point discriminating cirrhosis from HCC cases was 5.1, with sensitivity of 88.0%, specificity of 73.33%, positive predictive value of 84.6 % and negative predictive value of 78.6%.2-Alphafetoprotein (AFP):The ROC curve for alpha fetoprotein was significant (p.0.001*), showed that at a cutoff point discriminating cirrhosis from HCC cases was 69, with sensitivity of 80%, specificity of 70%, positive predictive value of 81.6 % and negative predictive value 0f 67.7 %.Thus, at these cut off points, serum Endoglin was found to have better diagnostic sensitivity and larger AUC (0.912*) than serum alphafetoprtein (0.856*) as regards differentiating cirrhosis from HCC cases.3-Gamma glutamyl transferase (GGT): The ROC curve for Serum GGT was significant (p.002*) and showed that the cutoff point discriminating cirrhosis from HCC cases was 69, with sensitivity of 64.0%, specificity of 53.33%, positive predictive value of 69.6 % and negative predictive value of 47.1%.Thus, at these cut off points, serum Endoglin wasfound to have better diagnostic sensitivity and larger AUC (0.912*) than serum GGT (0.709*) as regards differentiating cirrhosis from HCC cases.

Figure (2): ROC curve for different parameters to predict HCC cases vs cirrhosis

Table (I): Agreement (sensitivity, specificity) for different parameters to predict HCC cases vs cirrhosis

AUC p95% C.I

Cut off Sensitivity Specificity PPV NPVLL ULGGT (u/l) 0.709* 0.002* 0.596 0.823 >69 64.0 53.33 69.6 47.1

Serum AFP (ng/ml) 0.856* <0.001* 0.778 0.935 >48 80.0 70.0 81.6 67.7Serum Endoglin

(ng/ml) 0.912* <0.001* 0.852 0.972 >5.1 88.0 73.33 84.6 78.6

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Figure (3): ROC curve for Serum Endoglin + Serum AFP to predict HCC cases vs cirrhosis

Table (II): Agreement (sensitivity, specificity) for Serum Endoglin + Serum AFPto predict HCC cases vs cirrhosis

AUC p 95% C.I Cut off Sensitivity Specificity PPV NPVLL UL

Serum Endoglin (ng/ml) + Serum AFP

(ng/ml)0.964* <0.001* 0.931 0.997 >48/ >5.1 90.0 86.67 91.84 83.87

Combined use of serum Endoglin and AFP with a cutoff value of 5.1 (ng/ml), and 48 (ng/ml), respectively was significant (p value <0.001*) with sensitivity of 90% and

specificity 86.67, positive predictive value of 91.84 % and negative predictive value of 83.87%.

Figure (4): ROC curve for Serum endoglin + GGT to predict HCC cases vs cirrhosis

Table (III): Agreement (sensitivity, specificity) for Serum Endoglin + GGT to predict HCC cases vs cirrhosis

AUC p95% C.I

Cut off Sensitivity Specificity PPV NPVLL ULSerum Endoglin (ng/ml)

+ GGT 0.939* <0.001* 0.890 0.987 >69/>5.1 88.0 76.67 86.27 79.31

Combined use of serum Endoglin and GGT with a cutoff value of 5.1(ng/ml), and 69 (u/l),respectively was significant (p value <0.001*) with sensitivity of 88% and

specificity 76.67, positive predictive value of 86.27 % and negative predictive value of 79.31%.

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Figure (5): ROC curve for Serum Endoglin + GGT + AFP to predict HCC cases vs cirrhosis

Table (IV): Agreement (sensitivity, specificity) for Serum Endoglin + AFP + GGTto predict HCC cases vs cirrhosis

AUC p95% C.I

Cut off Sensitivity Specificity PPV NPVLL UL

Serum Endoglin (ng/ml) + GGT+ Serum AFP 0.969* <0.001* 0.940 0.999

>69/>48/>5.1

90.0 83.33 90.0 83.33

Combined use of serum Endoglin, AFP and GGT with a cutoff value of 5.1(ng/ml),48 (ng/ml), and 69 (u/l), respectively was significant (p value <0.001*) with sensitivity

of 90% and specificity 83.33, positive predictive value of 90.0 % and negative predictive value of 83.33%.

DiscussionThe diagnosis of HCC particularly in the early stage of the disease is a challenging issue particularly with the use of AFP only as a predictor of HCC development which has a low sensitivity.(11)One candidate marker for the diagnosis, progression and prognosis of multiple malignant tumors is serum Endoglin which is a homodimeric membrane glycoprotein expressed on endothelial cells that can bind to transforming growth factor -ß1 and transforming growth factor-ß3.(9)Endoglin (CD 105) CD 105 is only weakly expressed in normal tissues, but it is strongly expressed in tumor endothelia. Recent studies have suggested that CD 105 is a proliferation-associated marker of endothelial cells.(9,10)

Endoglin is a component of the transforming growth factor-β (TGF-β) receptor complex as it binds TGF- β1 and TGF- β3 with high affinity. Endoglin has been reported as expressed by endothelial cells of proliferating capillaries.(12) It is expressed

with marked tissue-specificity, predominantly in vascular endothelial cells of tissues undergoing active angiogenesis such as regenerating tissue and tumoral stroma.(13)Thus, Endoglin has been attracted considerable attention, not only as a biological marker of tumor growth but also as a target molecule for diagnostic and therapeutic application against cancer.(14) In addition, expression of Endoglin along hepatic sinusoids has been reported. (15)Clinical studies first uncovered high levels of Endoglin (CD 105) in the sera of patients with liver disease, particularly HCC.(15, 16)

Compared with AFP, the most commonly used serum marker for over four decades, Endoglin serum levels appear to be more sensitive for early HCC.(16)Therefore, the present work was held to study the changes in serum Endoglin (CD 105) in HCV and HBV induced liver cirrhosis and HCC and to evaluate its role as an emerging novel biomarker of HCC in comparison to AFP and GGT. Currently, the measurement of AFP has been routinely used as a serum

26

marker for detecting and monitoring HCC. AFP can be reex pressed in adults by the tumor cells with respect to their differentiation. AFP has high sensitivity in detecting advanced stages of HCC; however, its sensitivity decreases significantly for the detection of small tumors as stated by Debruyne and Delanghe. (17)In the present study, serum AFP was found to be significantly higher in the HCC group in comparison to cirrhosis group, which is in agreement with Jun Li et al study. (18)In the present study, the cut off for serum AFP to diagnose HCC was 48 ng/ml, with sensitivity of 80 % and specificity of 70%, positive predictive value of 81.6 % and negative predictive value 0f 67.7 % and the ROCcurve for it was significant with (p value <0.001). Jun Li et al study, (18) showed the cut off for serum AFP to diagnose HCC was 18.5ng/ml, with sensitivity of 78.4%% and specificity of 81.3%, the AUROC was 0.878.*Furthermore, in Reda et al.(16) the ROC curve for serum AFP in the differentiating HCC from liver cirrhotic patients, the cutoff value of 49.5 ng/ml yielded a sensitivity and a specificity of 43.3 % and 83.3%, respectively . When they increased the cutoff of AFP to 200 ng/ml, the sensitivity decreased to 23.3% and the specificity raised to 100%.In the present study, the ROC curve for GGT was significant (p. 0.002), and showed that at a cut off value of 69, diagnostic sensitivity for discriminating cirrhotic from HCC cases was with sensitivity of 64 %, specificity of 53.33%, positive predictive value of 69.6 % and negative predictive value 0f 47.1%.Thus, at these cut off points, serum Endoglin (CD 105) was found to have better diagnostic sensitivity, accuracy and larger AUC (0.912)than serum alpha fetoprtein (0.856) and GGT (0.709) as regards differentiating HCC cases from cirrhotic patients.As regards cutoff value of serum Endoglin, it was in this study 5.1 ng/ml in contrast to Reda et al. study,(16) which stated that it was 10.37 ng/ml and in Yagmur et al, study. (19) it was 6.9 (ng/ml). This variability may be because of different composition of patients groups in these studies. So in the present study as well

as, Reda et al. study, (16) and Yagmur et al, study, (19) serum Endoglin was found to have higher sensitivity, specificity and AUROC than AFP suggesting that serum endoglin may serve as a potential diagnostic marker for HCC. However, these studies differ in setting the optimal cut off for serum Endoglin for diagnosis of HCC. Hendy et al.,(20) who reported that as fibrosis progresses, serum Endoglin levels increase significantly and that circulating Endoglin levels correlated positively with the stage of hepatic fibrosis. Furthermore, Decai Yu et al. study (21) reported that Endoglin was not only present in neovessels in tumor tissues, but also more abundant in hepatic sinus endothelium in non-tumor tissues with cirrhosis. Therefore, CD105 may not be an appropriate targeting for ant angiogenesistherapy in HCC, especially with cirrhosis.The positive correlation between Endoglin expression and tumour growth and metastasis was mainly obtained by immunohistochemistry. Yang L et al. (22)

showed that there is a correlation of the intratumoural microvessel density (IMVD) with CD105 immunostaining, with the postoperative recurrence and metastasis of HCC. Interestingly, they could not find an association between liver cirrhosis and IMVD-CD105. Moreover, Yang L et al. (22)

noted that some of the observed patients with a relapse of the tumor had low IMVD. On the contrary, Ho et al. (13) postulated that IMVD by CD105 immunostaining in HCC did not correlate with the prognosis of patients. Interestingly, they conclude that the presence of well-diffuse patterns of CD105 expression in the adjacent non tumorousliver could predict early disease recurrence.Interestingly, there were no significant correlation between Serum Endoglin and AFP and GGT serum levels which is in contrast to what is reported by Reda et al,(16)

and Yagmur et al.(19) This difference maybe due to varied composition of patients groups in these studiesIn the current study, mean Endoglin (CD 105) was significantly correlated with Child Pugh score in both patients with cirrhosis without HCC andthose with HCC, but in Reda et al,.(16) there

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were positive correlation with Child Pugh score in liver cirrhosis group only. As regards tumor progression as graded by the BCLC, the current work showed that level of serum Endoglin (CD 105) in BCLC D than BCLC C than BCLC B than BCLC A.The present study showed no significant correlation between serum level of Endoglin (CD 105) and lymph node metastasis this may be due to limited number of cases as our study was conducted on just 50 cases of HCC.As regards number of lesions, our work showed that serum Endoglin (CD 105) levels were significantly higher in patients with multicentric HCC than in patient with single lesions. However, there were no statistically significant correlation between tumor size and serum Endoglin level. In this study we found that combined use of AFP and serum Endoglin have a sensitivity and specificity of 90% and 86.67% respectively in differentiating cirrhotic patient from those of HCC, and positive predictive value of 91.84% and negative predictive value of 83.87%.As regards combined use of GGT and serum Endoglin, it has a sensitivity and specificity of 88% and 76.67% respectively in differentiating cirrhotic patient from those of HCC, and positive predictive value of 86.27% and negative predictive value of 79.31%.As for the use of AFP,GGT and Endoglin we found that they have a sensitivity and specificity of 90% and 83.33% respectively in differentiatingcirrhotic patient from those of HCC, andpositive predictive value of 90% and negative predictive value of 83.33%.

ConclusionSerum endoglin might be used as a valuable serum marker of HCC development in cirrhotic HCV and HBV patients particularly in those with normal AFP level. Combined use of Serum Endoglin, AFP and GGT could represent a better diagnostic tool for HCC.

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infection. Clin Gastroenterol Hepatol 2010; 8(11): 924–33. 2. Brechot C, Gozuacik D, Murakami Y, Paterlini-Brechot P. Molecular bases for the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Semin Cancer Biol 2000;10:211–31.3. Estimated cancer incidence,mortality and prevalence worldwide in 2012.Available from: http://www.globocan.iarc.fr.[Accesed on: 14 feburary,2017].4. El-Zayadi AR, Badran HM, Barakat EM,AttiaMel-D, Shawky S, Mohamed MK, et al. Hepatocellular carcinoma in Egypt: a single center study over a decade. World J Gastroenterol 2005;11:51938.5. Lehman EM, Soliman AS, Ismail K, Hablas A, Seifeldin IA, Ramadan M, et al. Patterns of hepatocellular carcinoma incidence in Egypt from a population-based Cancer registry. Hepatol Res 2008;38:465–73.6. Egyptian Ministry of Health. Egyptian Ministry of Health Annual Report: 2007. Available online: http://www.mohp.gov.eg/main (accessed 2 May 2008).7. Yates SC, Hafez M, Beld M, Lukashov VV, Hassan Z, Carboni G, et al. Hepatocellular carcinoma in Egyptians with and without a history of hepatitis B virus infection: association with hepatitis C virus (HCV) infection but not with (HCV) RNA level. Am J Trop Med Hyg 1999;60:714–20.8. Kew M. Tumour markers of hepatocellular carcinoma. J Gastroenterol Hepatol 1989; 4:373–84.9. Gougos A, Letarte M. Primary structure of endoglin, an RGD-containing glycoprotein of human endothelial cells. J. Biol. Chem 1990; 265: 8361–4.10. Letarte M, Greaves A, Vera S. CD105 (endoglin) cluster report. In: Schlossman S.F, Boumsell L, Gilks W, Harlan J, Kishimoto T, Morimoto C, Ritz J, Shaw S, Silverstein R, Springer T, et al. Leukocyte Typing V: White Cell Differentiation Antigens. Oxford, UK:Oxford University Press 1995; 1756–9.11. Marrero JA, Feng Z, Wang Y, Nguyen MH, Befeler AS, Roberts LR, et al. Alpha-fetoprotein, des-gamma carboxyprothrombin, and lectin-bound alpha-fetoprotein in early hepatocellular carcinoma. Am J Gastroenterol 2016;22:8271–82.12. Yoshitomi H, Kobayashi S, Ohtsuka M, Kimura F, Shimizu H, Yoshidome H, et al. Specific expression of endoglin (CD105) in endothelial cells of intratumoral blood and

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lymphatic vessels in pancreatic cancer. Pancreas 2008; 37: 275–81.13. Ho JW, Poon RT, Sun CK, Xue WC, Fan ST. Clinicopathological and prognostic implications of endoglin (CD105) expression in hepatocellular carcinoma and its adjacent non-tumorous liver. World Journal of Gastroenterology 2005; 11: 176–81. 14. Fonsatti E, ALtomonte M, Nicotra MR, Natali PG, Maio M. Endoglin (CD105): a powerful therapeutic target on tumor-associated angiogenetic blood vessels. Oncogene 2003; 22: 6557–63. 15. Yu D, Zhuang L, Sun X, Chen J, Yao Y, Meng K, Ding Y. Particular distribution and expression pattern of endoglin (CD105) in theliver of patients with hepatocellular carcinoma. BMC Cancer 2007; 7: 122. 16. Redaa M.A., Khalifa M.O., Elhosary Y., Kandil D. and Emara A. . Endoglin (CD105) as a novel serum marker for hepatocellular carcinoma in Egyptian patients with chronic liver disease. Egyptian Liver Journal 2013;3:92–96.17. Debruyne EN, Delanghe JR. Diagnosing and monitoring hepatocellular carcinoma with alpha-fetoprotein: new aspects and applications. Clin Chim Acta2008; 395:19-26.18. Chang TS, Wu YC, Tung SY, Wei KL, Hsieh YY, Huang HC, et al. Alpha Fetoprotein Measurement Benefits Hepatocellular Carcinoma Surveillance in Patients with Cirrhosis. Am J Gastroenterol 2015; 110: 836-44

19. Yagmur E, Mohamed R, Stanzel S, Hellerbrand C, Lammert F, Trautwein C, et al. Elevation of endoglin (CD105) concentrations in serum of patients with liver cirrhosis and carcinoma. Eur J Gastroenterol Hepatol 2007; 19:755–61.20. Hendy OM, El Haddad OK, El Lattef HA, Ehsan NA, El Sharnouby JA. The diagnostic and prognostic significance of circulating and intrahepatic endoglin in liver fibrosis associated HCV infection. Afro-Arab Liver J 2008;7:41–7.21. Decai Yu, Linyuan Zhuang, Xitai Sun, Jun Chen, Yongzhong Yao,Kui Meng, et al. Particular distribution and expression pattern of endoglin (CD105) in the liver of patients with hepatocellular carcinoma. BMC Cancer 2007; 7:122.22. Yang L, Lu W, Huang G,Wang W. Correlation between CD105 expression and postoperative recurrence and metastasis of hepatocellular carcinoma. BMC Cancer 2006; 6:110.

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Original Article

Serum Thioredoxin in Cirrhotic Hepatitis C Virus Patients with and without Hepatocellular Carcinoma

Ayman El-Shayeb1, Soraya Hamouda1, Akram Deghady2, Doaa El-Wazzan1, Rafik Mehanna1;1TropicalMedicine Department, 2ClinicalPathology Department, Faculty of Medicine, Alexandria University.

ABSTRACTHepatocellular carcinoma (HCC) is one of the most serious complications of liver cirrhosis. In most cases, HCC develops within an established background of chronic liver disease (70–90% of all patients). The incidence of HCC in individuals with hepatitis C virus (HCV) cirrhosis is 3–5% per year. Therefore, evaluation of biomarkers that predict early occurrence of HCC in patients with HCV induced liver cirrhosis is of great clinical value from the diagnostic and prognostic points of view. Aim of the work: The aim of this study was to assess the level of thioredoxin as a potential marker for HCC in cirrhotic HCV patients.Patients and methods: The study was conducted on sixty patients classified into two groups: group 1 consisted of 30 patients of HCV with liver cirrhosis; group 2 included 30 patients with HCC on top of HCV induced liver cirrhosis. Moreover 20 healthy subjects were taken as control group. Serum thioredoxin was measured by ELISA. Results: Serum thioredoxin was significantly higher in HCC (group 2) than liver cirrhosis patients (group 1) (<0.001). Moreover, significant positive correlation was found between serum thioredoxin level and Barcelona clinic liver cancer (BCLC), tumor size, infiltration of liver and portal vein thrombosis in cases of HCC. (Kruskal Wallis test (H)=18.778,Spearman coefficient (r)=0.639,Mann Whitney test(MW)= 0.000, MW=19.000 respectively) (P<0.001, P<0.001, P<0.001,P<0.001 respectively).Diagnosis of HCC among patients with HCV infection and cirrhosis could be suggested when serum thioredoxin is assessed at a cutoff value of>50ng/ml. Conclusion: serum thioredoxin might be used as a valuable surrogate serum marker of HCC development in cirrhotic HCV patients.

IntroductionChronic HCV is the most common cause of chronic liver disease. (1)The infection is often asymptomatic, but chronic infection can lead to scarring of the liver and ultimately to cirrhosis.Liver cirrhosis is the end-stage condition of a multistep process, initiating from a primary hepatotoxic effect and resulting in insufficiency of liver function due to overwhelming hepatocyte damage.(2)HCC is the 4thmost common cancer and 3rd leading cause of cancer death worldwide.(3) HCV is the most important risk factor for HCC in western European and North American countries, since epidemiological studies have shown up to 70% of patients with HCC have anti-HCV antibody in their serum.(4)Alpha-fetoprotein (AFP) is a 70-kD glycoprotein consisting of 591 amino acids and 4% carbohydrate residues, encoded by a gene on chromosome 4q11-q13, normally produced during gestation by the fetal liver and yolk sac, AFP has been utilized as a marker for HCC, despite its low sensitivity and positive predictive value.(5) Moreover, it has been

estimated that up to 30% of HCC patients have normal AFP levels.(6) Therefore, novel biomarkers are needed to be used for early detection of cases with HCC.(7)Thioredoxinsare proteins that act as antioxidants by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange. It plays a role in many important biological processes, including redox signaling.(8) It basically comprises the small redox protein thioredoxin, nicotinamide adenine dinucleotide phosphate, in its reduced form (NADPH), and thioredoxin reductase(Thioredoxin R), a large homodimericselenzo enzyme controlling the redox state of thioredoxin.(9)

Aim of the workThe aim of this work was to assess the clinical significance of serum thioredoxin asmarker of HCC occurrence in cirrhotic HCV patients.

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Patients and MethodsAll patients were selected from Tropical Medicine Department, Faculty of Medicine,Alexandria University in the duration between January 2016 till January 2017.This study was carried out on 60patients classified into two groups. Group 1 consisted of 30patients with HCV induced liver cirrhosis.Group 2 included 30 patients of HCV induced liver cirrhosis with HCC. Moreover 20 healthy subjects were taken as control (group 3).Exclusion Criteria: Patients with concurrent co-infection with hepatitis B,diabetes mellitus, coronary heart disease, pulmonary fibrosis or active pulmonary disease, renal disease, alcoholics and smokers, those suffering from fever, patients with autoimmune disease and any malignancy other than HCC. Ethical approval: Theethical committee of Faculty of Medicine, Alexandria University,approved this study. Written informed consent was obtained from all patients.Methods: All patients were subjected to thorough history taking and proper clinical examination. Laboratory investigationsincluding: complete blood count (CBC);Completeurine and stool analysis; liver function tests; renal function tests, viralhepatitis markers, serum antinuclear antibody (ANA), Anti - smooth muscle antibody (ASMA) and AFP. Serum thioredoxin was measured by ELISA kits format number (201-12-1453). Abdominal imaging including: Ultrasound examination of the abdomen and triphasic C.T scan (was done for patients with U/Spositive hepatic focal lesions).

ResultsBy Child Pugh classification, Group 1 contained 6 patients Child A, 10 patients Child B and 14 patients Child C. While group 2 contained 5 patients with Child score A, 11 patients with Child score B and 19 patients with Child score C. (Table 1).Among studied HCC patients, 5 patients were BCLC A, 5 patients were BCLC B, 6 patients were BCLC C, and 14 patients were BCLC D.14 patients (46.7.3%) had single HCC lesions, 2 patients (6.7%) had 2 HCC lesions, 7 patients had 3 HCC lesions and 7 patients had more than 3 HCC lesions. Thetumor size ranged from 1.5 cm to 15 cm with a mean of 6.0 ± 4.09 cm. (table 1). Among studied HCC patients, 5 patients (16.7%) had infiltrative HCC lesions, 5 patients (16.7%) had LNS metastases and 10 patients (33.3%) had portal vein thrombosis. (Table 1). As regard to AFP, in the control group it ranged between 2.40ng/ml – 8.40ng/ml with a meanof 5.67 ± 1.65ng/ml while in group (1) it ranged between 1.50ng/ml – 86.30ng/mlwith a mean of 8.28 ± 15.32ng/ml and in group (2) it ranged between 10ng/ml –153000ng/ml with a mean of5288.26 ± 27906.99ng/ml (the median was 14.50).There was statistically significant differencesbetween the three groups (P<0.001). (Table 1). As regard to serum thioredoxin, in the control group it ranged between 3.0ng/ml –15.0 ng/ml with a mean of 9.35 ± 3.03ng/mlwhile in group (1) it ranged between 20.0ng/ml – 50.0ng/ml with a mean of 37.43± 8.83ng/ml and in group (2) it ranged between 43.0 ng/ml – 230.0ng/ml with amean of 123.70 ± 56.52 ng/ml. There was statistically significant differences between the three groups (P<0.001). (Table 1).

Table 1: Baseline characteristics of the HCC, liver cirrhosis and control casescontrol Liver cirrhosis HCC

Child Pugh A= 6, B= 10, C= 14 A= 5, B= 11, C= 19BCLC A= 5, B= 5, C= 6, D= 14

Tumor number 1=14, 2 = 2, 3 = 2, >3= 7Tumor size (cm) 6.0 (1.5 – 15)

Infiltrative 5LNS metastases 5

Portal vein thrombosis 10AFP (ng/ml) 5.67 (2.40 – 8.40) 8.28 (1.50 – 86.30) 5288.26 (10.0 – 153000.0)

serum thioredoxin (ng/ml) 9.35 (3.0 – 15.0) 37.43(20.0 – 50.0) 123.70(43.0 – 230.0)

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In HCC group, there was no significant correlation between AFP and serumthioredoxin in which P value equal 0.260.In cirrhosis group, serum thioredoxin levels were found to be the lowest in Child A followed by patients with Child B where they ranged from 31ng/ml to 39ng/ml with amean value of 35.30 ± 2.54ng/ml, followed by Child C patients where they ranged from 40 ng/ml to 50ng/ml with a mean value of 44.93 ± 3.41ng/ml. There was significant difference between mean values of serum thioredoxin in the three groups (p <0.001).

(Figure 1). In HCC group, serum thioredoxin levels were found to be the lowest in Child A patients where they ranged from 43ng/ml to 85ng/ml with a mean of 66.60 ± 15.98ng/ml,followed by patients with Child B where they ranged from 48 ng/ml to 220ng/ml with a mean value of 103.91 ± 51.88ng/ml,followed by Child C patients where they ranged from 100 ng/ml to 230ng/ml with a mean value of 159.64 ± 44.74ng/ml. There was significant difference between mean values of serum thioredoxin in the three groups (p <0.001). (Figure 1)

Figure (1): Relation between child Pugh score and serum thioredoxin in Cirrhosis and HCC groups (n = 30)

In HCC group, serum thioredoxin levels were found to be the lowest in BCLC Apatients where they ranged from 43ng/ml to 70 ng/ml with a mean of 60.60±10.45ng/ml,followed by patients with BCLC B where they ranged from 48ng/ml to 150ng/ml with a mean value of 84.60±38.55ng/ml, followed by BCLC C patients where they ranged from

85ng/ml to 220ng/ml with a mean value of 125.0±54.04ng/ml, followed by patients with BCLC D where they ranged from 100ng/mlto 230ng/ml with a mean value of 159.64±44.74ng/ml. There was significant difference between mean values of serum thioredoxin in the three groups (p <0.001).(Figure 2)

Figure (2): Relation between BCLC and serum thioredoxin in HCC group (n = 30)

In HCC group, 14 patients had a single lesion; their serum thioredoxin levels ranged from 65ng/ml to 230ng/ml with a mean value

of 148.93±62.73ng/ml. 2 patients had two lesions, their serum thioredoxin levels ranged from 43ng/ml to 60ng/ml with a

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mean value of 51.50±12.02ng/ml. 7 patients had three lesions, their serum thioredoxin levels ranged from 70ng/ml to 170ng/mlwith a mean value of 112.86±44.71ng/ml. 7patients had more than three lesions, their serum thioredoxin levels ranged from 48ng/ml to 160ng/ml with a mean value of 104.71±33.35ng/ml. There was no significant difference between mean values of serum thioredoxin in the four groups(p=0.059).There was significant positive correlation between tumor size and serum thioredoxin (P < 0.001).In HCC group, 25 patients had no infiltrative HCC, their serum thioredoxin levels ranged from 43ng/ml to 180ng/ml with a mean value of 105.64 ±42.04ng/ml, while 5 patients had infiltrative HCC and their serum thioredoxin levels ranged from 190ng/ml to 230ng/ml with a mean value of 214.0 ± 18.17ng/ml. Mean level of serum thioredoxin was statistically significantly higher in patients with infiltrative HCC than those without (p <0.001). In HCC group , 25 patients had no LNS metastases, their serum thioredoxin levels ranged from 43ng/ml to 230ng/mlwith a mean value of 119.44 ± 53.40ng/ml,while 5 patients had LNS metastases and their serum thioredoxin levels ranged from 85ng/ml to 230ng/ml with a mean value of 145.0 ± 73.31ng/ml. Mean level of serum thioredoxin was not statistically significantly higher in patients with LNS metastases than those without (p =0.419).In HCC group, 20 patients had no portal vein thrombosis, their serum thioredoxin levels ranged from

43ng/ml to 190ng/ml with a mean value of 95.80 ± 39.95ng/ml, while 10 patients had portal vein thrombosis and their serum thioredoxin levels ranged from 95ng/ml to 230ng/ml with a mean value of 179.50 ± 41.66ng/ml. Mean level of serum thioredoxin was statistically significantly higher in patients with portal vein thrombosis than those without (p <0.001).Receiver operatingcharacteristic curves (ROC curves) were done to estimate the cutoff points discrminating cirrhosis from HCC of serum thioredoxin as well as AFP.1- Serum thioredoxin:The ROC curve for serum thioredoxin was significant (<0.001)(AUC=0.986)and showed that the cutoff point discriminating cirrhotic cases from HCC cases was 50ng/ml, with sensitivity of 93.33%, specificity of 100%, positive predictive value of 100% and negative predictive value of 93.7% ( figure3 , table 2).2- Alpha-fetoprotein: The ROC curve for serum AFP was significant (<0.001) (AUC=0.919) and showed that the cutoff point discriminating cirrhotic cases from HCC cases was 11ng/ml, with sensitivity of 73.33%, specificity of 86.67%, positive predictive value of 77.3% and negative predictive value of 76.5% (figure 3 , table 2).Thus, at these cut off points, serum thioredoxin was found to have better diagnostic sensitivity, accuracy and larger AUC than AFP as regards differentiating cirrhosis from HCC.

Figure (3): ROC curve for serum AFP and serum thioredoxin to predict HCC cases vs cirrhosis

Serum AFP Serum ThioredoxinAUC 0.919* 0.986*

P <0.001* <0.001*

95%CI(LL - UL) 0.838 – 1.0 0.96 – 1.0

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Table (2): Agreement (sensitivity, specificity) of serum AFP and serum thioredoxinto predict HCC cases vs cirrhosis

AUC Cut off Sensitivity Specificity PPV NPVSerum AFP (ng/ml) 0.919 ≤4.9 56.67 75.0 77.3 53.6Serum thioredoxin (ng/ml) 0.986 >15 100.0 100.0 100.0 100.0

DiscussionThe diagnosis of HCC particularly in the early stage of the disease is a challenging issue particularly with the use of AFP only as a predictor of HCC development whichhas a low sensitivity. (10) One candidate marker for the diagnosis, progression and prognosis of multiple malignant tumors is serum thioredoxin which is the major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state, which is reduced by electrons from NADPH via thioredoxinreductase. (11)

Thioredoxin was found to be highly expressed in many malignancies and the expression level of thioredoxin has been positively correlated with tumor grade, tumor stage and early recurrence in many cancer types. (12) Normal AFP levels are present in as many as 30% of patients at time of diagnosis and usually remain low, even with advanced HCC (10). With values of the magnitude from 400ng/ml to 500 ng/ml, the specificity of AFP is close to 100% but at a cost to the sensitivity which falls below 45%.(13) In a study using 20 ng/ml as the cut-off point, the sensitivity rose to 78.9%, although the specificity declined to 78.1%.(14)

The positive predictive value (PPV) of AFP is low, ranging from 9% to 32%.(15)In this study, serum AFP was found to be significantly higher in the HCC group in comparison to the cirrhotic and control groups, this is in agreement with Jun Li et al study. (16) Moreover, the cut off for serum AFP to diagnosis HCC was 11ng/ml, with sensitivity of 73.33% and specificity of 86.67%. The AUROC was 0.919* with p<0.001. In the Jun Li et al study, (16) the cut off for serum AFP to diagnose HCC was 18.5ng/ml, with sensitivity of 78.4%% and specificity of 81.3%, the AUROC was 0.878*.In the current study, serum thioredoxin was found to be significantly

higher in the HCC group in comparison to the cirrhotic and control groups this is in agreement with Jun Li et al study (16) andMiyazaki et al study. (17) Moreover the cut off for serum thioredoxin to diagnosis HCC was 50ng/ml, with sensitivity of 93.33% and specificity of 100%. The AUROC was 0.986* with p<0.001. In the Jun Li et al study, (16) the cut off for serum thioredoxin to diagnosis HCC was 20.5ng/ml, with sensitivity of 84.3% and specificity of 91.8%. The AUROC was 0.946*.So our study and Jun Li et al study, (16) agreed that serum thioredoxin has higher sensitivity, specificity and AUROC than AFP but they were different in setting the optimal cut off for serum thioredoxin for diagnosis of HCCsuggesting that serumthioredoxin may serve as a potential diagnostic marker for HCC. In our study, thioredoxin level was found to be significantly higher in cirrhotic patients than in the healthy control group suggesting that serum thioredoxin may serve as a potential diagnostic marker for liver cirrhosis. Using the best cut-off value (20ng/ml), the sensitivity and specificity were 100% and 100% respectively. (AUROC was 1.000*with p <0.001). Thioredoxin stimulates the growth of a variety of tumor cell lines(18)by reducing equivalents for DNA synthesis,(19) reducinghydrogen peroxide(20) and by acting as an electron donor to human plasma glutathione peroxidase ,(21) activation of transcription factors that regulate cell growth,(22) and an increase in the sensitivity of cells to other cytokines and growth factors.(23)It is important to discuss whether thioredoxin in HCC patients have pathological roles or just was as indicator of oxidative stress or inflammation. Serum thioredoxin levels have been reported to decline significantly after surgical removal of HCC, and may be produced and secreted by HCC cells. (24)It was previously reported that serum levels of

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thioredoxin increase relative to the degree of hepatic fibrosis, and that high serum concentrations of thioredoxin may indicate advanced hepatic fibrosis.(25)

Correspondingly, Sumida et al.(24) suggested that thioredoxin may therefore be responsible for the pathological mechanism of HCV-related hepatic fibrogenesis, and Tamai et al.(26) reported that serum thioredoxin levels were potential clinical biomarkers that predict patient prognosis in HCV-related HCC. Thus, thioredoxin might play animportant role in the process of HCC rather than just was a diagnosis marker.AS regards number of lesions, the current work showed that there was no significant correlation between serum thioredoxin levels and the number of tumor lesions which was against the study held by Jun Li et al., (16) that detected a positive correlation between the serum thioredoxin levels and the tumor number. The current study revealed significant correlation between Serum thioredoxin and tumor size, serum thioredoxin was significantly increased in relation to the tumor size with P value < 0.001which was in agreement with the study held by Jun Li et al. (16)In the present study, thioredoxin level was found to be significantly higher in patients with infiltrative HCC than in patient without infiltration, but due to limited number of cases, more studies are needed before using serum thioredoxin as a marker for diagnosis of infiltration.AS regards LNS metastases, our work showed that there was no significant correlation between serum thioredoxin levels and the LNS metastases which was against the study held by Jun Li et al., (16) that detected a positive correlation between the serum thioredoxin levels and LNS metastases but again only 5 patients had LNS metastases and so more studies are still needed. The current study showed significant correlation between serum level ofthioredoxin and malignant portal vein thrombosis, which was in agreement with the study held by Jun Li et al. (16) but our current study didn't include cases of benign portal vein thrombosis so we don't know if the benign portal vein thrombosis have an effect

on serumthioredoxin or not. In the present research, mean serum thioredoxin was significant as regard child score in both patients with cirrhosis without HCC and those with HCC. Serum thioredoxin tended to increase as liver disease progressed from Child-Pugh class A to C which was in agreement with the study held by Jun Li et al. (16) As regards tumor progression asgraded by the BCLC, the current workshowed that level of serum thioredoxintended to increase as tumor progressed from BCLC class A to D which was in agreement with the study held by Jun Li et al. (16)

ConclusionSerum thioredoxin might be used as a valuable surrogate serum marker of HCC development in cirrhotic HCV patients.

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function. Molecular and cellular biology 2004; 24:9414-23.9. Bindoli A, Rigobello MP, Scutari G, Gabbiani C.; Casini A.; Messori L. Thioredoxinreductase: a target for gold compounds acting as potential anticancer drugs. Coordination Chemistry Reviews 2009; 253:1692–707.10. Colombo M. Screening for cancer in viral hepatitis. Clin Liver Dis 2001; 5:109–22. 11. Holmgren, A. Thioredoxin. Annu. Rev. Biochem 1985; 54: 237-7112. Kakolyris S, Giatromanolaki A, Koukourakis M,Powis G, Souglakos J,Sivridis Eet al. Thioredoxin expression is associated with lymph node status and prognosis in early operable nonsmall cell lung cancer. Clin Cancer Res 2001; 7:3087–9113. Gupta S, Bent S, Kohlwes J. Test characteristics of alpha-fetoprotein for detecting hepatocellular carcinoma in patients with hepatitis C. A systematic review and critical analysis. Ann Intern Med 2003; 139:46–50.14. Taketa K. Alpha-fetoprotein. J Med Technol 1989; 33:1380.15. Befeler AS, Di BisceglieAM. Hepatocellular carcinoma: diagnosis and treatment.Gastroenterology2002; 122:1609–19.16. Jun Li, Zhang-Jun Cheng, Yang Liu, Zhen-Lin Yan, Kui Wang, Dong Wu, et al. Serum thioredoxin is a diagnostic marker for hepatocellular carcinoma.Oncotarget 2015; 6: 9551–63.17. Miyazaki K, Noda N, Okada S, Hagiwara Y, Miyata M, Sakurabayashi I, et al EIevated serum level of Thioredoxin in patients with Hepatocellular Carcinoma. Biotherapy 1998; 11:277–88.18. Nakamura H, Masutani H, Tagaya Y, Yamauchi A, Inamoto T, Nanbu Y, et al.Expression and growth-promoting effect of adult t-cell leukemia-derived factor a human thioredoxin homologue in hepatocellular carcinoma. Cancer1992; 69:2091–7.

19. Jordan A, Reichard P. Ribonucleotidereductases. Annual review of biochemistry1998; 67:71–98.20. Spector A, Yan GZ, Huang RR, McDermott MJ, Gascoyne PR, Pigiet V. The effect of H2O2 upon thioredoxin- enriched lens epithelial cells.Journal of Biological Chemistry 1988;263:4984–90.21. Björnstedt M, Xue J, Huang W, Akesson B, Holmgren A. The thioredoxin and glutaredoxin systems are efficient electron donors to human plasma glutathione peroxidase. Journal of Biological Chemistry1994; 269:29382–4.22. Powis G, Mustacich D, Coon A. The role of the redox protein thioredoxin in cell growth and cancer. Free Radical Biology and Medicine 2000;29:312–22.23. Iwata S, Hori T, Sato N, Hirota K, Sasada T, Mitsui A, et al. Adult T cell leukemia (ATL)-derived factor/human thioredoxin prevents apoptosis of lymphoid cells induced by L-cystine and glutathione depletion: possible involvement of thiol-mediated redox regulation in apoptosis caused by pro-oxidant state. The Journal of Immunology 1997; 158:3108–17.24. Sumida Y, Nakashima T, Yoh T, Nakajima Y, Ishikawa H, Mitsuyoshi H, et al. Serum thioredoxin levels as an indicator of oxidative stress in patients with hepatitis C virus infection. J Hepatol 2000;33:616–2225. Sumida Y, Nakashima T, Yoh T, Furutani M, Hirohama A, Kakisaka Y, et al. Serum thioredoxin levels as a predictor of steatohepatitis in patients with nonalcoholic fatty liver disease. JHepatol 2003; 38:32–8.26. Tamai T, Uto H, Takami Y, Oda K, Saishoji A, Hashiguchi M, et al. Serum manganese superoxide dismutase and thioredoxin are potential prognostic markers for hepatitis C virus-related hepatocellular carcinoma. World J Gastroenterol 2011; 17:4890–8.

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Original Article

The Effects of Common House Fly (Musca Domestica) Larvae-Derived Substances on Wound Healing in Mice Model

Doaa Elsaid Said Ahmed1, Hoda Mahmoud Khalifa2, Radwa GalalDiab1, Lamiaa Moustafa Abdel Samad3, Marwa Ahmed Meheissen4, Hala Elsayed Diab Basiony1

1Department of Medical Parasitology, Faculty of Medicine, University of Alexandria, Egypt,2Department of Histology and Cell Biology, Faculty of Medicine, University of Alexandria, Egypt,3Department of Zoology, Faculty of Science, University of Alexandria, Egypt, 4Department of Medical Microbiology, Faculty of Medicine, University of Alexandria, Egypt.

ABSTRACTAmong different maggot extracts, chitosan, is considered one of the most important extracts. The use of high molecular weight chitosan (2,000,000 MW) with high DD (85%) was decided to make the ultimate use of the quantitatively high solubility of N-acetyl glucosamine molecules process of healing. Aim of The work:Chitosan and gut extracts of M. domestica larvae were used in the assessment of wound healing. Material and methods Experimental animals: Males of albino Swiss mice (n= 30/group),6-8 weeks old and 20-30 g were raised at the animal house of the Medical Parasitology Department. All mice were treated and sacrificed in accordance with the Alexandria University Ethics committee according to the guidelines of animal experimentation in immunocompetent and immunocompromised mice. Results revealed early and good remodeling of gut extracts treated wounds in both immunocompetent and immunocompromised mice. While, chitosan treated mice showed good result only in immunocompetent and to lesser extent in immunocompromised. Conclusions: The present study showed high quality wound healing process in terms of normal cytoarchitecture achievement in the side of gut-extracts from M. domestica maggots.

IntroductionInsects and insect derivatives have been widely used in folk medicine across the world since ancient times(1). The use of insects is particularly common in China, and in many other countries including Brazil, Mexico, India, Africa, and South Korea (2).At present, there are approximately 300 medicinal insects distributed in 70 genera, 63 families, and 14 orders. An estimated 1700 traditional Chinese medicine prescriptions include medicinal insects or insect-derived crude drugs (3). The housefly (Musca domestica) belongs to the order of Diptera. The housefly larvae have been used clinically to cure malnutritional stagnation, decubital necrosis, osteomyelitis, ecthyma, and lip boil which was described by Li Shizhen (1518–1593 AD) in the pharmaceutical text of Compendium of Materia Medica (4). Recently, effects of antioxidant(5), antibacterial, and in vitro antitumor properties of the protein extracts of housefly larvae have been reported(6,7).Additionally, the oil of housefly larva could be used as a natural ointment to heal burn

wound (8).The aim of the present work was to study the effect of the house fly (M. domestica)-derived larval extract (chitosan and alimentary secretions) on the healing process of experimental surgical wounds in immune-competentand immunosuppressed mice.

Patients and MethodsI. Experimental animals: Males of albino Swiss mice (n= 30/group),6-8 weeks old and 20-30 g were raised at the animal house of the Medical Parasitology Department, Faculty of Medicine, Alexandria University. Each animal was maintained in an individual cage, under controlled environmental conditions (12-h light/dark cycle, temperature 23±2◦C and humidity (55±10%) with water and commercial food. All mice were treated and sacrificed in accordance with the Alexandria University Ethics committee according to the guidelines of animal experimentation. II. Rearing of M.domestica larvae: (9) The larvae were reared from the adult M. domestica after being identified morphologically in Department of

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zoology, Faculty of Science, University of Alexandria. Adult M. domestica flies were collected and bred in cages closed by wire mesh. Adults were maintained on carbohydrate diet and water. Eggs were laidon beef liver pieces. Part of these eggs wasleft to hatch and release the larvae which pupate and mature into adults to maintain the breeding cycle.III. Chitosan extraction and characterization: (10, 11)One part of the eggs was left to hatch and the third instar larvae was collected, homogenized to extract chitin thatwill be converted into chitosan by chemical treatment.IV. Gut extraction: (12) Eggs were collected after eight hours of oviposition from the liver by using fine brush. The obtained egg suspension was transferred aseptically to a test tube containing Dakin’ssolution for one minute to ensure safe cleaning of the hatched larvae. Eggs were transferred to sterile blood agar petri dishes sealed by polyvinyl chloride plastic film. The petri dish agars were then incubated under suitable laboratory conditions until hatching. (13) Third instar larvae were dissected according to the method of Linville and Wells. (14, 15) The Collected gut extracts were pooled in aliquots then stored at -20 ̊C till used. V. Immunosuppression induction:Immunosuppression was induced by cyclophosphamide (Endoxan) in subgroups Ib, IIb1 and IIb2, given as two intra-peritoneal doses (70 mg/kg each) one week apart. (16) Wounding was done 48 hours after the second dose. Two extra doses were given at the 2nd and 3rd week after the initial dose to maintain the immune suppression state till the end of the experiment. (17) VI.Experimental design and protocol: Animals were divided into six groups (n= 30/group) and were anesthetized for the surgical procedure using 2% xilazine chloridrate (10 mg/kg) and 10% ketamine chloridrate (115 mg/kg) in subcutaneous injections. (18)Astandard circular wound (one cm of diameter) was performed on the dorsal thoracic region using circular tempelate andsurgical scissors. Groups were divided asfollows: (Ia): competent non-treated, (Ib): suppressed non-treated, (IIa1): competent chitosan treated, (IIa2): competent gut

extracts treated, (IIb1): suppressed chitosan treated, (IIb2): suppressed gut extracts treated. The clinical characteristics of the experimental wounds were observed considering the following aspects: edema, hyperemia, secretion, scab, granulation, epithelialization and scar tissues. Wound sizewas measured using a sterile ruler at each day of biopsy to calculate the percent of wound contraction using the following formula: [Initial wound size/mm−specific day wound size/mm/Initial wound size/mm×100]. (19) At each time of biopsy, on the 3rd, 7th and 14th days after surgery, 6 animals were drawn from the experimental groups, subjected to subcutaneous anesthesia and euthanized by cervical disruption. After this procedure, the skin around the area of the wound was removed, extending one centimeter beyond each dorsal and ventral edge, and internally until the muscular layer. Immediately after the withdrawal of skin, the samples were transferred to formaldehyde (10%, v/v) for a maximum period of 48 h for the processing of histological sections. VII.Measurement of epidermal thickness: (20)

Histopathological images of H&E-stained sections on the 14th days post-wounding were taken with a Zeiss microscope then transferred to a computer with a Panasonic GP-KR222 camera and converted to BMP files. A Full epidermal thickness was measured. For each biopsy specimen, six different areas representing the lesion were measured and the average was calculated.VIII. Collagen quantification: (21, 22) Three Masson’s trichrome-stained histological images were captured from each group at 100x magnification. By using the threshold colour tool in the ImageJ software (NIH), the areas stained in green were selected and in that way we could indirectly calculate the mean collagen fraction in each group. The ratio of green pixels above the threshold to total pixels in the image was used to calculate the percentage of collagen in each sample. IX. Immunohistochemical staining:(23, 24)Immunohistochemistery was done using the streptavidin-biotin-peroxidase complex(Thermo scientific. Egypt) method against Cytokeratin Cocktail AE1/AE3:

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mouse monoclonal antibodies (Bio SB)(New Test Comp. Egypt) to evaluate the state of differentiation and proliferation of keratinocytes by measuring their keratin content. X. Microbiological assessment of wound contamination:(25) Specimens from wounds were collected by sterile swabs, which were rolled over the wound area (1×1 cm) using quantitative culture technique at different time interval (3rd, 7th, 14th). Swabs were aseptically placed in a tube containing 5ml of saline. Using a 100-fold dilution, finally 0.1 ml were pipetted and inoculated on nutrient agar plate and grown aerobically at 37°C for 24 h. Bacterial colonies were counted at x104 bacterial organism/ ml.

Results1) Criteria of the prepared chitosan: The prepared chitosan had 85% DD (high DD) and 2,000,000 MW (high MW).2) Clinical

observation of mice: a) Naked eye appearance of wounds: An observable thick dry scab was apparent on the 3rd day post wounding in all groups. On the 7th day post-wounding, the scab was still present with the absence of any macroscopic signs of inflammation or infection in all studied groups. While, on the 14th day post wounding, non-treated immunocompetent (Ia) and immunosuppressed (Ib) subgroupsand subgroup IIb1 (immunosuppressed chitosan treated) showed delay in the reappearance of hair. Group IIa1 (immunocompetent chitosan treated) and IIb2 (immunosuppressed gut treated) showed partial progress in the reappearance of hair. However, in group IIa2 (immunocompetent gut treated), the skin was pink, smooth and was completely covered with hair.b) Measurement of wound surface area: (tableI)

Table (I): Measurement of wound surface area on the 3rd and 7th days post wounding in both immunocompetent and immunosuppressed groups:

SubgroupWoundSizein cm

Group Ia(n = 6)

Group Ib(n = 6)

Group IIa1(n = 6)

Group IIa2(n = 6)

Group IIb1(n = 6)

Group IIb2(n = 6) F p

Day 3Mean 1.20 1.22 0.80 1.02 0.92 1.07 4.366* 0.004*

±SD. 0.26 0.26 0.14 0.18 0.08 0.15Median 1.15 1.15 0.80 1.0 0.90 1.0

Sig. bet. Grps p1=0.011*, p2=0.558,p3=0.096,p4=0.744p5=1.000, p6=0.891, p7=0.997, p8=0.377, p9=0.744

Day 7Mean 0.87 0.78 0.38 0.78 0.73 0.78 8.822* <0.001*

±SD. 0.24 0.12 0.15 0.08 0.12 0.08Median 0.85 0.75 0.45 0.80 0.75 0.80

Sig. bet. Grps p1<0.001*, p2=0.907, p3=0.989, p4=1.000p5=0.907, p6=0.002*,p7=1.000, p8<0.001*, p9=0.989

By measuring wound size in the studied groups we found that significant reduction in wound size in group IIa1 in comparison to group Ia. Regarding the immunosuppressed groups, there was no significant reduction in wound size in group IIb1 in comparison to group Ib. and similarly group IIb2.Initial wound expansion was observed in all groups other than chitosan treated ones, with no reduction in original size until the 7th day post wounding. Wound contraction occurred at

the same rate following the 7th day postwounding which was mostly indifferent till the point of complete healing. Finally, eventual healing occurred at the same time on day 14 post wounding with exception of group IIa1, where wound closure occurred on the 10th day post wounding (Fig. 1).However, the results show no difference in term of duration of wound closure except in group IIa1.

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Fig.1: Progress of wound healing in mice starting from the day of wounding till complete wound closure in both immunocompetent and immunosuppressed groups.

a) Histopathological results: i) Hematoxylin and Eosin (H&E) staining: Histological examination of H&E stained sections of group (Ia) showed Complete re-epithelialization and complete closure of the wound with no hair follicles and sebaceous glands was observed 14th day post-wounding (Plate I. Fig. a). The epidermal thickness was decreased (191.55±39.61) (table II).Normalization of the epidermis was evident 14 days post wounding in group IIa1 with limited number of newly formed hair follicles and sebaceous glands penetrating into the underlying dermis (Plate I. Fig. b). Furthermore, the epidermal thickness showed significant reduction (99.37±40.09) when compared to group Ia (table II). While, in group IIa2 the dermal bed was rich in hair follicles and sebaceous glands with normalization of the epidermis at day 14 post-wounding (Plate I. Fig. c). Statistically significant decrease in the epidermal thickness

of group (IIa2) (57.80±14.26) as compared to group Ia. Regarding the immunosuppressed group, complete closure of the wound was noticed 14 days post wounding of group Ib with thickened slightly vacuolated epidermis (263.64±89.55) with absence of hair follicles and sebaceous glands (Plate I. Fig. d).Normalization of the epidermis was evident 14 days post wounding with decreased epidermal thickness and less vacuolation, but this was statistically not significant (229.07±63.31) when compared to group (Ib), with no associated hair follicles or sebaceous glands (Plate I. Fig. e). on the other hand, group IIb2 showed remodeled epidermis 14 days post wounding with moderate number of associated hair follicles and sebaceous glands (Plate I. Fig. f). The epidermal thickness of IIb2 showed significant decrease (130.07±30.14) when compared to group Ib.

Plate I:H&E stained skin section of different studied group 14th days post wounding:

Figure a:Skin section of group Ia 14 days post wounding (d ×100)

Figure b:Skin section of group IIa1 14th days post wounding (×40)

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Figure c:Skin section of group IIa214 days post wounding (×40)

Figure d:Skin section of group Ib 14 days post wounding (d×100)

Figure e:Skin section of group IIb1 14 days post wounding (×40)

Table (II): Epidermal thickness of the different studied groupsat 7th and 14th days post-wounding(distance in pixel)

SubgroupEpidermalthickness

GroupIa(n = 6)

Group Ib(n = 6)

Group IIa1(n = 6)

Group IIa2(n = 6)

Group IIb1(n = 6)

Group IIb2(n = 6) F P

Day 14Mean 191.55 263.64 99.37 57.80 229.07 130.07 13.903* <0.001*

±SD. 39.61 89.55 40.09 14.26 63.31 30.14Median 188.45 259.66 94.32 58.01 232.63 116.28

Sig. bet. grps p1=0.048*,p2=0.001*,p3=0.857,p4=0.001*

p5=0.190, p6=0.002*,p7=0.188, p8=0.738, p9=0.028*

ii) Trichrome stain: Examination of samples of group Ia for collagen fibers on day 14 post wounding depicted deposition of homogenous amorphous intercellular substance and few collagen fibers aligned horizontally in one direction that were more densely stained (Plate II, fig. a). Group IIa1 revealed denser mature collagen fiber staining of the dermis 14 days post wounding (Plate II, fig. b). Stained sections of group IIa2 14 days post wounding showed increased amount of lightly stained new collagen alternating with darker mature ones on the dermis underneath the normal looking epidermis (Plate II, fig. c).The immunosuppressed group Ib immature lightly stained irregularly oriented collagen fibers 14 days post wounding (Plate II, fig.

d).Denser reaction of immature collagen was evident 14 days post wounding of groupIIb1with evident highly cellular less fibrous sub-epidermal area(Plate II, fig. e).While, group IIb2 marked increase of both lightly stained new collagen fibers intermingled with darkly stained mature collagen fibers were evident in the dermis 14 days post wounding(Plate II, fig. f).In immunocompetent mice, there were significant increase in collagen in group IIa2 with a mean collagen fraction of (226.99±8.55) in comparison with group Ia that showed a mean collagen fraction of (184.53±12.96), while in group IIa1 there was an increase in collagen(212.28±12.88) compared to group Ia but this was statistically insignificant. Regarding the immunosuppressed mice, there was

41

significant decrease in collagen in group Ib (118.97±1.57) in comparison with group Ia (184.53±12.96). While in the experimental immunosuppressed groups IIb1, IIb2 there

was significant increase in collagen (188.42±16.29), (196.99±10.28) respectively in comparison with group Ib (118.97±1.57).

Plate II: Trichrome stained skin section of different studied groups:

Figure a: Skin section of group Ia14 days post wounding (×100)

Figure b: skin section of group IIa1 14 days post wounding (×100)

Figure c: Skin section of group IIa2 14 days post wounding (×100)

Figure d: Skin section of group Ib 14 days post wounding (×100)

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Figure e: Skin section of group IIb1 14 days post wounding (×100)

Figure f: Skin section of group IIb2 14 days post wounding (×100)

Table (III): Mean collagen fraction of the different studied groups at 14th days post-woundingSubgroup

MeanCollagenfraction

Group Ia(n = 3)

Group Ib(n = 3)

Group IIa1(n = 3)

Group IIa2(n = 3)

Group IIb1(n = 3)

Group IIb2(n = 3) F p

Day 14Mean 184.53 118.97 212.28 226.99 188.42 196.99 32.147* <0.001*

±SD. 12.96 1.57 12.88 8.55 16.29 10.28Median 183.20 118.98 210.40 225.97 181.37 195.91

Sig. bet. grps p1=0.093, p2=0.007*,p3<0.001*, p4<0.001*

p5<0.001*, p6=0.181, p7=0.063, p8=0.625, p9=0.934

iii) Immunohistochemical results: The re-epithelialized skin of group Ia showed very weak reaction (scant brown color) as compared by the rest of un-wounded skin (plate III, Fig. a). Intense positive reaction in all epidermal layers and in the newly formed hair follicles was encountered 14 days post wounding (deep brown color) in group IIa1(plate III, Fig. b). While, in group IIa2 the reaction was intense after 14 days in both neo-epidermis and newly formed hair follicles (plate III, Fig. c). In group Ib, the

results showed limited reaction in the re-epithelialized tissue 14 days post wounding (scant brown color) (plate III, Fig. d). Group IIb1showed mild positive reaction that was more intense only in stratum corneum after 14 days (brown color) (plate III, Fig. e). On the other hand, group IIb2 intense reaction (deep brown color) in both epidermal layers as well as newly formed hair follicles was found 14 days post wounding (plate III, Fig. f).

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Plate III: Immuno-keratin stain of skin sections of different studied groups:

Figure a: Skin section of group Ia 14 days post wounding (×400)

Figure b: Skin section of group IIa1 14 days post wounding (×400)

Figure c: Skin section of group IIa2 14 days post wounding (×400)

Figure d: Skin section of group Ib 14 days post wounding (×400)

Figure e: Skin section of group IIb1 14 days post wounding (×400)

Figure f:Skin section of group IIb2 14 days post wounding (×400)

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Microbiological results: Staphylococci were identified by their colonial morphology on blood agar. They appeared as round, convex, 1-4 mm in diameter and with a sharp border. They were surrounded with clear zone of beta-hemolysis and had only weak yellow pigmentation. On the 3rd and 7th day post-wounding, immunocompetent mice showed no significant colonial reduction in the treated groups IIa1 and IIa2 versus the corresponding immunocompetent control group Ia (table IV). Regarding the immunosuppressed groups, there was significant decrease in the number of

colonies in IIb1 with a mean number of colonies of [(0.83±1.33), (1.42±2.01) respectively] in comparison to Ib (20.77±23.07) on the 3rd days post-wounding. The same was observed on the 7th

days post-wounding, where there was significant colonial reduction in IIb1 [(0.88±1.11), (0.18±0.33) respectively] when compared to the group Ib (18.63±24.37) (table IV).Besides, we observed significant colonial reduction in the subgroup (Ia) (1.0±1.67), when compared by itscorresponding immunosuppressed group (Ib) (18.36±24.37) on the 7th day post wounding.

Table (IV):The number of staphylococcal colonies on the 3rd and 7th day post wounding from swabs in all studied groups

SubgroupNumberOf colonies (×104)

Group Ia(n = 6)

Group Ib(n = 6)

Group IIa1(n = 6)

Group IIa2(n = 6)

Group IIb1(n = 6)

Group IIb2(n = 6) F p

Day 3Mean 5.50 20.77 4.94 0.33 0.83 1.42 3.506* 0.013*

±SD. 7.45 23.07 4.36 0.52 1.33 2.01Median 4.0 11.0 4.50 0.0 0.0 0.25

Sig. bet. grps p1=1.000, p2=0.947,p3=0.021*,p4=0.026*

p5=0.124, p6=0.980, p7=1.000, p8=0.967, p9=1.000Day 7Mean 1.0 18.63 0.85 0.47 0.88 0.18 3.231* 0.019*

±SD. 1.67 24.37 1.0 0.73 1.11 0.33Median 0.0 5.0 0.45 0.0 0.40 0.0

Sig. bet. grps p1=1.000, p2=1.000, p3=0.046*,p4=0.035*

p5=0.049*, p6=1.000, p7=1.000, p8=1.000, p9=1.000

DiscussionAmong different maggot extracts, chitosan, is considered one of the most important extracts. The use of high molecular weight chitosan (2,000,000 MW) with high DD (85%) was decided to make the ultimate use of the quantitatively high solubility of N-acetyl glucosamine molecules in the process of healing. This goes with Choi et al, 2001(26)

where N-acetyl glucosamine moieties in chitosan were reported to be the key factor in wound healing. Lemos and Terra, 1991(27)

showed that M. domestical arvalmidgut display in cells and luminal contentsaproteolytic activity with properties similar to cathepsin D. Actually, wound healing in immunosuppressed patients has been recently considered a major global issue that increases the risk of complication of surgical

wound healing. Anderson and Hamm, 2014(28) stated that chemotherapeutic agents cause delayed inflammatory phase of healing, decreased fibrin deposition and collagen synthesis, and delayed wound contraction. Inspection of wound showed a dry thick scab formed on the 3rd day post wounding in all groups. A dry scab can maintain a sterile wound exudate beneath it, thus preventing dehydration and contamination of the wound, to optimize conditions for healing as explained by Dai et al, 2011.(29) Wound contraction is an important parameter for wound closure in mice.(30) The results showed non-difference in duration of wound healing among different studied groups in the present study. Our only exception was observed in immunocompetent chitosan treated mice

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(IIa1); in which complete wound closure happened on the 10th day post-wounding even without initial wound expansion. This could be explained by a potential stimulatory effect of chitosan on myofibroblasts. In case of immunosuppression, chitosan treatment resulted in significantly higher collagen fraction in immunosuppressed chitosan treated group (IIb1) versus the corresponding immunosuppressed non-treated group (Ib).This could be explained by the activation of fibroblasts which are the main source of collagen. Further more, Kas, 1997 showed that chitosan could be used to prevent fibroplasia in wound healing and to stimulate tissue growth and differentiation in culture. In our study, we further assessed the wound healing by immunohistochemistry to interpret the effect of extracted materials on skin keratinocytes. Anggraeni et al, 2012(31)

considered keratinocyte migration to the edge of the wound through wound gaps is the earliest stage of wound recovery. Thus, keratinocyte migration is considered the basis of wound healing, followed by fibroblast proliferation and granulation tissue formation. (32) Owing to their characteristic distribution in different epidermal layers, researchers considered keratin proteinsspecific markers of epithelial differentiation.(33, 34) One of the most important intracellular markers of wound healing is cytokeratin. Cytokeratin AE1/AE3 used in this study isa mix of two different clones of cytokeratin monoclonal antibodies.The present study showed high quality wound healing process in terms of normal cytoarchitecture achievement in the side of gut-extracts from M. domestica maggots. The role of M. domestica larval gut contents on wound healing was assessed in the present work. It is worth meaning to reveal that to our knowledge, we are the first to assess M. domestica larval extract on wound healing. We depended on the results of Lemos and Terra, 1991(27) who showed that M. domestical arval midgut acid proteinasedisplayaproteolytic activity with properties similar to cathepsin D (CD)proteinase. CD enzymatic activity is known to be involved in the processes of epithelial

differentiation, epidermal barrier function and wound healing.(35) This could be related to keratinocytes activation that plays a vital role in wound healing through epidermal differentiation and remodeling. Epidermal remodeling was assessed by measuring the epidermal thickness in H&E stained skin sections taken from gut extracts-treatedwounds in the present study. The results strongly pointed to the activation of keratinocytes by gut extracts. Larval gut extracts showed significantly higher collagen fraction in both immunocompetent (IIa2) and immunosuppressed (IIb2) subgroups. This could be explained by enhancement of the action of several enzymes in the gut of M.domestica maggots that have a dramatic role in the process of efficient wound healing and the building of well-formed cutaneous cytoarchitecture. Our results placed M.domestica larvae as a relevant and reliable substitute to the famous L. sericata larvae in the field of MDT. Thus, now then, M.domestica could be seen in a new scope being a beneficial insect rather than a filthy fly, mainly concerned with mechanical transmission of pathogens.

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