Liver Macrophage Depletion Ameliorates The Effect of Mesenchymal Stem Cell Transplantation in a Murine Model of Injured Liver.

Mesenchymal stem cells (MSCs) therapy show different levels of effectiveness in the context of different types of liver damage, suggesting that the microenvironment of the injured liver is a key determinant for effective stem cell therapy. The objective was to assess the modulatory effect of hepatic stem cell niche components on the transplanted MSCs during liver injury induced by carbon tetrachloride (CCl4). Superparamagnetic iron oxide (SPIO)-labeled human MSCs were injected intravenously into mice treated with CCl4 and subjected to hepatic macrophage-depletion. Liver tissues were collected at different intervals post transplantation for subsequent histopathological, morphometric, immunohistochemical, gene expression and ultrastructural studies. The homing of the transplanted MSCs was evidenced by tracing them within the niche by iron staining and immunohistochemical studies. MSCs differentiated into hepatocyte-like cells and intimal smooth muscle cells as evidenced by their expression of human albumin and α-smooth muscle actin with a concomitant increase in the level of mouse hepatocyte growth factor. A post transplantation reduction in the liver fibro-inflammatory reaction was found and was promoted by liver macrophages depletion. Thus, it could be concluded from the present study that prior manipulation of the microenvironment is required to improve the outcome of the transplanted cells.

Expression of ERG Protein and TMRPSS2-ERG Fusion in Prostatic Carcinoma in Egyptian Patients.

AIM: Prostate cancer (PCa) is the second most common cancers in men worldwide. Its incidence can be influenced by several risk factors including genetic susceptibility. Therefore the search for the expression of a certain gene (ERG) and its rearrangement could give us clues for proper identification of PCa. And the study of ERG expression and its comparison to FISH in Egyptian patients can show whether ERG immunophenotype could be used instead of FISH, as it is cheaper. MATERIALS AND METHODS: This study was performed on 85 cases of PCa, showing 30 cases with HGPIN and 30 cases of prostatic hyperplasia. All were immunohistochemistry stained using ERG monoclonal rabbit antihuman antibody was used (clone: EP111). FISH analysis was performed in 38 biopsies of PCa cases to detect TMRPSS2-ERG rearrangement using the FISH ZytoLight TriCheck Probe (SPEC TMRPSS2-ERG). RESULTS: ERG expression was found in 26% of PCa cases and 20% of HGPIN cases. FISH analysis showed fusion of 21 cases of PCa (out of 22 cases showing ERG immunoexpression). CONCLUSION: Our findings emphasise that only malignant and pre-malignant cells and not benign cells from the prostate stain positive. ERG expression may offer a simpler, accurate and less costly alternative for evaluation of ERG fusion status in PCa.

Expression of FGFR3 Protein and Gene Amplification in Urinary Bladder Lesions in Relation to Schistosomiasis.

BACKGROUND: Bladder cancer represents the fifth most common malignancy worldwide and a major cause of cancer-related morbidity and death. Incidence and mortality rates have remained relatively constant over the past four decades. Urothelial bladder cancers have identified multiple risk factors. AIM: We aimed at evaluating the expression of the FGFR3 protein and gene amplification in the urothelial cells of neoplastic and non-neoplastic urothelial lesions of the urinary bladder, and correlation with tumour grade, stage and associated bilharziasis. MATERIAL AND METHODS: One hundred and five different urinary bladder lesions were studied, including 15 cystitis cases (9 bilharzial and 6 non-bilharzial cystitides), 75 urothelial carcinoma cases (18 bilharzial associated and 57 non-bilharzial associated) and 15 squamous cell carcinoma associated with bilharziasis, beside 5 control cases. Data concerning age, sex, tumour grade, stage, and associated bilharziasis were obtained. Each case was studied for FGFR3 expression, and FISH technique was applied on forty malignant cases that show high protein expression. RESULTS: The highest incidence of cystitis was in the fourth decade while of bladder cancer was in the seventh decade. Tumour grade was correlated significantly with tumour stage. FGFR3 correlates significantly with tumour grade, stage and with a bilharzial infestation. FGFR3 gene amplification was reported mainly in low grade and NNMBIC tumours. CONCLUSIONS: FGFR3 overexpression in malignant cases was significantly higher than in chronic cystitis. FGFR3 gene amplification was reported mainly in low grade and NNMBIC tumours. FGFR3 may be further studied as a subject for target therapy of bladder cancer.

Fibrosis in Chronic Hepatitis C: Correlation between Immunohistochemically-Assessed Virus Load with Steatosis and Cellular Iron Content.

AIM: We aimed study impact of hepatocytic viral load, steatosis, and iron load on fibrosis in chronic hepatitis C and role of VEGF and VEGFR overexpression in cirrhotic cases in evolving HCC. MATERIAL AND METHODS: Total of 120 cases were included from TBRI and Beaujon Hospital as chronic hepatitis C (CHC), post-hepatitis C cirrhosis, and HCC. Cases of CHC were stained for Sirius red, Prussian blue and immunohistochemically (IHC) for HCV-NS3/NS4. HCC were stained IHC for VEGF and by FISH. RESULTS: Stage of fibrosis was significantly correlated with inflammation in CHC (P < 0.01). Noticed iron load did not correlate with fibrosis. Steatosis was associated with higher inflammation and fibrosis. The cellular viral load did not correlate with inflammation, steatosis or fibrosis. VEGF by IHC was significantly higher in cases of HCC when compared to cirrhotic group (P < 0.001). Amplification of VEGFR2 was confirmed in 40% of cases of HCC. Scoring of VEGF by IHC was the good indicator of VEGFR2 amplification by FISH (P < 0.005). CONCLUSION: Grade of inflammation is the factor affecting fibrosis in CHC. The degree of liver damage is not related to cellular viral load or iron load. Steatosis is associated with higher inflammation and fibrosis. VEGF by IHC is correlated with overexpression of VEGFR2 by FISH.

The clinical significance of HER2 protein amplification/expression in urinary bladder lesion.

OBJECTIVE: To evaluate HER2 oncoprotein expression by both immunohistochemical (IHC) staining and fluorescence in situ hybridisation (FISH) in different benign and malignant bladder lesions, and the effect of bilharzial infestation on this expression. PATIENTS AND METHODS: In a prospective controlled study, 72 patients were classified into a control group, and groups with cystitis, urothelial carcinoma, and squamous cell carcinoma (SCC). HER2 was detected using standard IHC staining and FISH in all groups. The correlation of HER2 expression with tumour type, stage and grade in relation to normal urothelium and cystitis was assessed. The effect of schistosomal infestation was evaluated. RESULTS: HER2 expression was statistically significantly higher in patients with malignant lesions than in the other groups, and in high-stage and -grade tumours than in low-stage and -grade tumours. The use of FISH increased the detection of HER2-positive tumours. Schistosomal infestation did not affect HER2 expression in patients with transitional cell carcinoma. CONCLUSION: High-stage and -grade bladder malignancies expressed HER2 much more than did benign lesions. FISH is more sensitive for detecting HER2 expression. The treatment of HER2-positive tumours might benefit from novel targeted-treatment protocols.

Superiority of fluorescent in situ hybridization over immunohistochemistry in detection of HER2 gene in carcinoma of the urinary bladder associated with and without schistosomiasis.

HER2 is an oncogene encoding a type 1 tyrosine kinase growth factor receptor and the role of HER2 in the development of numerous types of human cancer is still understood and correlates with clinical outcome, poor prognosis, it is a predictor factor for poor response to chemotherapy. HER2 overexpression is associated with reduced disease free and overall survival. Patients who have HER2 negative expression have a poor prognosis. The aim of the present study is to explore the accuracy of detection of expression of HER2 protein by two different techniques of immunohistochemistry (IHC) and gene amplification by fluorescent in situ hybridization (FISH). The two techniques were applied to sixty two patients that included different cell types of carcinoma of the bladder, benign bilharzial lesions and control. Characteristics of the 62 patients are: 10 chronic cystitis, 19 squamous cell carcinoma (SCC) with schistosomiasis, 33 urothelial carcinoma (UC) schistosomal and non-schistosomal, ten healthy individuals without schistosomiasis served as controls. Gene amplification of HER2 was done using FISH and protein expression of HER2 by IHC. The study was applied on archival data of formalin-fixed paraffin embedded tissues and patient clinical data and follow up for 5 years. Overexpression of HER2 protein was found in 30/52 (57.7%). Fourteen cases had score of 2+, and sixteen cases had score of 3+. Using FISH technique it showed more accurate detection of HER2 gene as those fourteen cases who had score of 2+ had been found to be 5 out of 14 were positive for gene over expression, the other sixteen who had score of 3+ all were positive for gene amplification. HER2 protein and gene was found to be significantly overexpressed in carcinoma of the bladder in both cell types SCC and UC with or without schistosomiasis compared to the benign lesions and control groups (P <0.01) by both techniques. There is significant increase in expression of HER2 protein and gene in SCC compared to UC (P< 0.01). In UC overexpression of HER2 protein and gene was evident in all stages Ta, T1, T2-4. HER2 protein and gene overexpressed in different grades of UC. In SCC HER2 protein and gene had overexpression in different stages and grades.

Evaluation of the diagnostic value of serum and tissue apoptotic cytokeratin-18 in patients with chronic hepatitis C.

BACKGROUND AND STUDY AIMS: Hepatitis C virus (HCV) is considered the most common aetiology of chronic liver disease (CLD) in Egypt. The disease severity ranges from mild illness to cirrhosis and hepatocellular carcinoma. A role for apoptosis in liver damage caused by HCV chronic infection has been suggested. Cytokeratin 18 (CK-18) is the major intermediate filament protein in the liver and is a known caspase substrate in hepatocyte apoptosis. Therefore, we analysed the serum and tissue levels of CK-18 in patients with chronic HCV infection to evaluate its role in hepatocyte apoptosis. We also correlated CK-18 expression with the severity of hepatic pathology. PATIENTS AND METHODS: This study examined 80 Egyptian patients with liver disease. There were 69 patients with chronic hepatitis C and 11 patients with hepatitis C-induced cirrhotic changes. Fifteen healthy controls were also included in the study. The levels of CK-18 fragment were quantified in paired serum and liver biopsy samples. RESULTS: The serum and tissue CK-18 levels were reduced in chronic HCV patients compared to early cirrhosis patients. This result indicates that serum levels of CK-18 and the hepatic expression of CK-18 might play an important role in disease progression. The serum and tissue levels of CK-18 were significantly increased and directly correlated with inflammation severity, stage of fibrosis, and ALT levels in the chronic HCV group and the cirrhotic liver group. There was no significant difference in viral load between patient cohorts. CONCLUSION: The serum level and the hepatic expression of CK-18 are related to disease activity and are directly correlated with METAVIR scoring. This result suggests that serum CK-18 levels may be useful for monitoring disease activity in chronic HCV and liver cirrhosis patients.

Insulin resistance: a major factor associated with significant liver fibrosis in Egyptian patients with genotype 4 chronic hepatitis C.

BACKGROUND AND AIMS: The role of insulin resistance (IR) in chronic hepatitis C genotype 4 (CHC-4) patients is still under assessment. The aims of this study are to assess the prevalence and predictors of IR and its influence along with clinical, metabolic, virological, and histological factors on the severity of liver fibrosis in 100 Egyptian patients with CHC-4. PATIENTS AND METHODS: In 100 untreated patients with CHC-4, IR was assessed using the Homeostasis Model Assessment and defined greater than 3. By logistic regression (LR), independent factors associated with IR and significant fibrosis (SF=fibrosis, Metavir score≥F2) were assessed in nondiabetic and noncirrhotic patients. RESULTS: One hundred patients were included; 54% were men and 46% were women. The mean age of the patients was 40.46±9.41 years. Of the total patients, 55% were overweight and 28% were obese. Metabolic syndrome was observed in 26% of patients; five of them were known to be diabetic. All patients were genotype 4. Most of our patients had mild viremia (<2 00 000 IU/ml), whereas only 16% had higher viral load (>2 00 000 IU/ml). There was no correlation between IR and hepatitis C virus viremia (r=-0.069; P=0.492). Necroinflammation was moderate-severe (A2-A3) in 25% of patients. SF (F2-F4) was found in 46% of patients and 11% had cirrhosis (F4). Most of our patients, 54%, had moderate steatosis and 21% had severe steatosis. IR was present in 46% of patients; 39 (42.9%) were nondiabetic, which is correlated significantly with BMI (r=0.395; P<0.01). IR was found to increase significantly with the fibrosis stage (P=0.001), insignificant fibrosis, 18.5%, SF (F2-F4), 71.4%, and cirrhosis (F4), 100%. By LR, IR was independently and significantly associated with age more than 40 years, obesity (BMI>30 kg/m), SF, and severe steatosis (>30%). IR was also significantly associated with metabolic syndrome. SF was present in 46 patients (46%). It was associated with IR, moderate-severe necroinflammation, and severe steatosis. By LR, in noncirrhotic patients, SF was associated with age more than 40 years, obesity (BMI>30 kg/m), moderate/severe liver inflammation, and severe steatosis. CONCLUSION: In CHC-4 patients, IR is highly prevalent and independently associated with age, obesity, SF, and severe steatosis. Management of IR might significantly improve the prognosis of CHC-4 patients.

Subpleural versus deep lung biopsies obtained during pleuroscopy for histological examination: an experimental animal study.

BACKGROUND: Lung biopsies obtained during medical pleuroscopy using coagulating forceps could represent a cost-effective alternative to surgical lung biopsies in patients with diffuse parenchymal lung diseases (DPLD). However, it is not clear whether these patients should undergo deeplung biopsies rather than more superficial subpleural lung biopsies. OBJECTIVES: The aim of this experimental animal study was to compare gross and microscopic features of deep and subpleural pleuroscopic lung biopsy samples. METHODS: Six male sheep (median weight 40 kg) underwent lung biopsies via pleuroscopy under general anesthesia. The following parameters were studied: weight, size, quality of the parenchyma and visceral pleura, parenchymal vessels and bronchial tissue. RESULTS: The mean number of biopsies taken per animal was 4.5 ± 1.22 and 4.83 ± 1.33 (p = 0.36) for deep and subpleural biopsies, respectively. The mean size of deep and subpleural biopsies was 1.758 ± 0.478 and 1.283 ± 0.851 cm(2), respectively (p = 0.0006). The mean weight of deep biopsies and subpleural biopsies was 0.156 ± 0.092 and 0.145 ± 0.047 mg, respectively (p = 0.83). No statistically significant difference was found between subpleural and deep biopsies regarding the mean quality scores of parenchyma-pleura (p = 0.36), vessels (p = 0.36), or bronchial tissue (p = 0.20). CONCLUSION: Both subpleural biopsies obtained during pleuroscopy and deep lung biopsy specimens obtained by electrocautery in animal subjects provided satisfactory material for histologic examination. Therefore, in DPLD, where the subpleural layers are involved, subpleural biopsies obtained during pleuroscopy might be sufficient for establishing an accurate diagnosis.

Prevalence of microscopic colitis in patients with chronic diarrhea in Egypt: a single-center study.

BACKGROUND/AIM: Microscopic colitis (MC) is diagnosed when a patient with chronic watery non-bloody diarrhea (CWND) has an endoscopically normal colon, but colonic biopsies show unique inflammatory changes characteristic of lymphocytic or collagenous colitis. MC is a disorder of unknown etiology. Studies comparing the prevalence of the disease in developing countries as compared to developed countries may shed more light on the possibility of a post-infectious etiology. Most data on the incidence and prevalence of MC are from developed countries where it accounts for 4-13% of cases of CWND. There are only a few reports from developing countries. Two studies from Peru and Tunis, with high prevalence of infectious gastroenteritis, revealed MC in 40% and 29.3% of cases of CWND, respectively. The aim of this study was to investigate the prevalence of MC in patients presenting with CWND in Egypt. MATERIALS AND METHODS: A total of 44 patients with CWND of unexplained etiology who had undergone full colonoscopy with no macroscopic abnormalities between January 2000 and January 2010 were assessed retrospectively. RESULTS: The histological appearance of MC was identified in 22 (50%) patients. Twelve (55%) patients were male and 10 (45%) female. Mean age was 40 years (range: 20-65 years). Twenty (91%) of MC cases had lymphocytic colitis and 2 (9%) had collagenous colitis. CONCLUSIONS: The prevalence of MC in Egyptian patients with CWND is high when compared to that in developed countries. MC mainly affects young and middle-aged patients and it is more commonly of the lymphocytic type.

Homing of transplanted bone marrow cells in livers of Schistosoma mansoni-infected mice.

The efficiency of differentiation of bone marrow cells (BMCs) into hepatocytes in vivo and its importance in physiopathological processes is still debated. Murine schistosomiasis was used as a liver injury model and unfractionated male mice BMCs were transplanted through intrahepatic injection into non-irradiated Schistosoma mansoni-infected female mice on their 16th week post-infection. Two weeks after bone marrow transplantation, mice were sacrificed on a weekly basis until 10 weeks. Tracing of male donor-derived cells in female recipient mice livers was carried out by the detection of Y chromosome expression by fluorescent in situ hybridization (FISH) and also of chromodomain Y-linked (CDYL) protein by indirect immunofluorescence (IF). Their transformation into hepatocytes was studied by double labelling indirect IF using antibodies directed against CDYL and mouse albumin. Histopathological and electron microscopic examinations revealed the presence of small hepatocyte-like cells in the periportal tracts and in between the hepatocytes facing the sinusoids. Donor-derived cells showing Y chromosome by FISH and expressing CDYL protein by IF were recovered in the infected transplanted livers. The initial number of these cells increased with increased post-transplantation time. Cells were mainly localized in the periphery of schistosoma granuloma. Few donor-derived cells appeared within the hepatic parenchymal tissue and showed positivity for albumin secretion by double labelling with IF. We suggest that transplanted bone marrow stem cells can repopulate the Schistosoma-infected liver of immunocompetent mice. Their differentiation is a complex event controlled by many factors and needs to be further characterized extensively. The extent and type of liver injury and the number of transplanted cells are important variables in the process of stem cell engraftment and differentiation into functioning hepatic cells that still need to be defined.

Value of α-smooth muscle actin and glial fibrillary acidic protein in predicting early hepatic fibrosis in chronic hepatitis C virus infection.

INTRODUCTION: α-Smooth muscle actin (α-SMA)-positive hepatic stellate cells (HSCs) are pericytes responsible for fibrosis in chronic liver injury. The glial fibrillary acidic protein (GFAP), commonly expressed by astrocytes in the central nervous system, is expressed in vivo in the liver in a subpopulation of quiescent stellate cells. The reports concerning GFAP expression in human liver are still conflicting. The aim of the study is investigation the utility of GFAP compared to α-SMA as an indicator of early activated HSCs, in predicting fibrosis in chronic hepatitis C (CHC) patients. MATERIAL AND METHODS: With immunohistochemistry and a semi-quantitative scoring system, the expressions of α-SMA and GFAP on HSCs in liver biopsies from patients with pure CHC (n = 34), hepatitis C virus-induced cirrhosis (n = 24), mixed CHC/schistosomiasis (n = 11) and normal controls (n = 10) were analysed. RESULTS: The immunoreactivity of α-SMA and GFAP in perisinusoidal, periportal and pericentral areas was assessed. α-Smooth muscle actin and GFAP-positive HSCs were significantly increased in all diseased groups compared with normal controls. In pure CHC with or without cirrhosis, perisinusoidal α-SMA-positive HSCs were predominant in relation to GFAP-positive cells. On the other hand, GFAP-positive cells were predominant in the group of schistosomiasis as compared with the other diseased groups. It was noticed that expression of GFAP on perisinusoidal HSCs in CHC patients sequentially decreased with the progression of fibrosis. CONCLUSIONS: Glial fibrillary acidic protein could represent a more useful marker than α-SMA of early activation of HSCs in CHC patients and seems to be an early indicator of hepatic fibrogenesis.

SEN virus infection in Egyptian patients with chronic hepatitis C and patients undergoing hemodialysis.

The SEN virus has been tentatively linked to transfusion-associated non-A to E hepatitis. The aim of the present study was to 1) determine the prevalence of SEN virus among Egyptian patients with hepatitis C virus (HCV)-related chronic liver disease and patients undergoing hemodialysis and 2) demonstrate the clinical effect of SEN virus infection on coexistent hepatitis C in terms of severity and probability of developing hepatocellular carcinoma. Polymerase chain reaction was used to detect SEN virus-D and SEN virus-H DNA in serum samples of 74 patients with HCV-related chronic liver disease, 45 uremic patients undergoing maintenance hemodialysis, and 28 healthy controls. SEN virus-D/H DNA was detected in 13.5% of patients with chronic liver disease, 11.1% of patients undergoing hemodialysis, and 7.1% of healthy controls, with no significant differences between patients and the control group. Clinical and biochemical measures did not significantly differ between SEN virus-infected and noninfected patients in the chronic liver disease group or the hemodialysis group. The rate of SEN virus infection was significantly higher in patients with chronic liver disease and hepatocellular carcinoma (33.3%) than in those with chronic liver disease only (8.5%) (P < .05). In conclusion, SEN virus does not seem to be a common infection in Egyptian patients. It has no apparent influence on the severity of coexistent HCV-related chronic liver disease but could be a risk factor for hepatocellular carcinoma in such patients. Further studies are needed to define the etiopathogenic role of SEN virus infection in the development of hepatocellular carcinoma.

Gastrointestinal stromal tumors (GISTs): clinical presentation, diagnosis, surgical treatment and its outcome.

This study included 13 selected patients treated by surgical excision for lesions that proved postoperatively to be gastrointestinal stromal tumors (GISTs) by histopathological and immunohistochemistry studies. The demographic, clinical and operative reports data were collected. Eight cases were gastric GISTs, four cases were small bowel GISTs (jejunum 1 & ileum, 3) and GIST of the sigmoid colon was in one patient. Eight cases presented at the emergency department due to hematemesis (3), gastrointestinal obstruction (3), bowel perforation (1) and severe bleeding per rectum (1). Three cases presented with a feeling of abdominal fullness and ill-defined palpable abdominal mass. Two cases were discovered incidentally during GIT endoscopy for dyspepsia. Diagnosis of GISTs was presumed on clinical basis and operative findings from gross morphological features. Complete resection (R0) was achieved for 12 tumors (92.3%). The immunohistochemistry profile was positive for C-kit for all cases. One operative death was due to massive pulmonary embolism. Postoperative complications occurred in three (23%) as upper GIT bleeding (1), biliary gastritis (1) and wound infection (1), and one (7.69%) of ileum tumor recurrence.

Epidemiologic approach for early detection and control of renal and urinary tract diseases in rural populations.

The dipstick testing, microscopic examination of urine and urine cytology were performed for inhabitants from two rural villages (El Shobak El Sharki, V.1 & El Katta, V.2) in Giza G. The proliferating cell nuclear antigen (PCNA) and Schistosoma haematobium antigen were done by immuno-histochemical stain to confirm diagnosis. Also, they were subjected to medical questionnaire, clinical examination, ultra-sonography of kidneys and urinary tract. The results showed that V.2 had higher percentage of haematuria, proteinuria, glucosuria and lower urinary tract infection than V.1. Crystaluria was higher in V.1. Sensitivity of dipstick testing compared to microscopic examination was 26.6%, & specificity was 78.7%. Lower urinary tract infection cytologically detected was 44.2% sensitivity & 62.5% specificity compared to pyuria detected by microscopic examination of urine. Among those suffering variable urinary abnormalities, schistosome antigen was not detected in any fixed urine samples in comparison to corresponding confirmed positive controls. Urine cytology detected urinary tract infection, crystaluria, dysplasia and atypia, squamous metaplasia and transitional cell carcinoma (TCC). PCNA positivity was found in TCC (100%), dysplasia (50%) and squamous metaplasia (28.6%). So, microscopic examination of urine proved valuable for tract abnormalities as pyuria, haematuria and crystaluria. Also, urine cytology is a must for malignancy of urinary tract especially in adult males.