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In Situ Detection of Hepatitis C Virus RNA in Salivary Glands

Chronic hepatitis C virus (HCV) infection has been associated with several extrahepatic manifestations, among these, to diseases with oral manifestations such as Sjögrens syndrome or sialadenitis. HCV-RNA has been detected in saliva and in salivary glands from patients with sialadenitis by polymerase chain reaction. However, morphological evidence of HCV replication in salivary gland cells is needed to support a role for HCV in causing sialadenitis or Sjögrens syndrome. We have used in situ hybridization and immunohistochemistry to analyze the presence of HCV-RNA of sense and antisense polarity and HCV core antigen, respectively, in salivary gland biopsies from 19 patients with chronic sialadenitis or Sjögrens syndrome (eight anti-HCV-positive; 11 anti-HCV-negative). HCV-RNA of both positive and negative polarity as well as HCV core antigen were detected in the epithelial cells of the salivary gland biopsies from all of the anti-HCV-positive patients but in none of the anti-HCV-negative cases. The percentage of HCV-infected cells ranged from 25 to 48.8% in the patients studied. In conclusion, we have shown that HCV infects and replicates in the epithelial cells from salivary glands of patients with Sjögrens syndrome or chronic sialadenitis. However, its implication in the pathogenesis of these diseases deserves future research.

Activation of NF-κB in Tubular Epithelial Cells of Rats With Intense Proteinuria Role of Angiotensin II and Endothelin-1

Abstract—The mechanisms by which persistent proteinuria induces interstitial inflammation and fibrosis are not well known, although nuclear factor-&kgr;B (NF-&kgr;B), which regulates the transcription of many genes involved in renal injury, could be implicated. In rats with intense proteinuria, we studied the renal activation of NF-&kgr;B as well as the potential involvement of the vasoactive hormones angiotensin II (Ang II) and endothelin-1 (ET-1). Uninephrectomized Wistar-Kyoto rats receiving 1 g/d of BSA had proteinuria but no renal morphological lesions at day 1. By contrast, tubular atrophy and/or dilation and mononuclear cell infiltration were observed after 8 or 28 days of BSA administration, coinciding with maximal proteinuria. In relation to control uninephrectomized rats, the renal cortex of nephritic rats showed an increment in the activation of NF-&kgr;B at all time periods studied. By in situ Southwestern histochemistry, NF-&kgr;B activity was mainly localized in proximal tubules, interstitial mononuclear cells, and, to a lesser extent, the glomeruli. The administration of the ACE inhibitor quinapril plus the ETA/ETB receptor antagonist bosentan during 28 days to BSA-overloaded animals diminished proteinuria, renal lesions, and NF-&kgr;B activity more markedly than single drugs. Cultured tubular epithelial cells exposed to BSA revealed an intense NF-&kgr;B activation in a time- and dose-dependent manner. Incubation of cells with receptor antagonists of Ang II (AT1: losartan and AT2: PD-123,319) or ET-1 (ETA: BQ123 and ETB: IRL1038) inhibited significantly the BSA-induced NF-&kgr;B activity (90%, 75%, 90%, and 60% of inhibition versus basal, respectively). Our results show that overload proteinuria causes NF-&kgr;B activation in tubular epithelial cells both in vivo and in vitro. The vasoactive peptides Ang II and ET-1 appear to be implicated in this effect. The results reveal a novel mechanism of perpetuation of renal damage induced by persistent proteinuria.

BPH and Inflammation: Pharmacological Effects of Permixon on Histological and Molecular Inflammatory Markers. Results of a Double Blind Pilot Clinical Assay

INTRODUCTION The role of infiltrating cells (I.C.), commonly observed in the adenoma interstitial tissue, is unknown. We tested the hypothesis that I.C. are related with BPH progression by: phenotypically characterising these cells; quantifying the expression of lymphokines and growth factors; investigating the response to Permixon (P) in a clinical study. Permixon is a lipido sterolic extract of Serenoa repens possessing pharmacological activities and widely used in the treatment of men with BPH. MATERIAL AND METHODS A multicenter open pilot study of two parallel groups on BPH patients was carried out. They were randomized to receive either oral Permixon (P) 160 mg bid for three months or to be followed for 3 weeks without any treatment before surgery (control group C). Strict inclusion and exclusion criteria were applied to conform homogeneous groups, avoiding interferences of inflammatory drugs or others. Baseline clinical profile was almost identical in both groups in terms of age (65.7+/-5.1 vs. 67.1+/-5.8 years), IPSS (19.8+/-6.1 vs. 19.0+/-5.8), prostate volume (64.8+/-18.9 vs. 71.5+/-29.3cc), Q(max) (9.6+/-3.2 vs. 10.6+/-2.6 ml/s), and Q(L) (4.0+/-1.1 vs. 3.5+/-0.7). Surgery was ultimately performed on 29 patients (17C, 12P) by TURP or retropubic adenomectomy. Adenoma samples were routinely stained with HE and later prepared for immunohistochemical studies using CD3, CD20 and CD68 antibodies. Counting of positives cells, lymphoid aggregates and foci were done using EnVision technique and the Tech Mate processor. Cytokines, growth factors and eicosanoids were determined by Elisa kits following the manufactured recommendation. RESULTS HISTOLOGICAL: A difference was observed in the number of lymphocytes B between C (91.4+/-44.1) and P treated (58.2+/-53.7) groups (p=0.097). BIOLOGICAL MARKERS: TNFalpha and IL-1beta were dramatically lower in the Permixon treated group. Other parameters did not show significant changes. CLINICAL: IPSS in the Permixon treated group was significantly reduced (p<0.006) from 20.0+5.9 to 14.9+3.8 after three months of treatment. COMMENTS The BPH inflammatory hypothesis was tested in humans. Our pilot study shows a significant reduction of some inflammatory parameters in prostatic tissues of patients treated with Permixon. These biological findings justify a pharmacological effect of this drug on the inflammatory status of the adenoma. A correlation with clinical improvement was observed.

Increased prevalence of p53 overexpression from typical endometriosis to atypical endometriosis and ovarian cancer associated with endometriosis

OBJECTIVE To evaluate the expression of p53, c-erb-B-2, MIB1 and Bcl-2 in normal endometrium, endometriosis, atypical endometriosis and ovarian cancer associated with endometriosis, looking for immunohistochemical markers that may help determine endometriosis with premalignant potential. STUDY DESIGN Between 1948 and 1999, 410 epithelial ovarian cancers and 521 cases of endometriosis were surgically treated at Fundación Jiménez Díaz. Pathology reports and slides were reviewed. Four groups were defined: (1) endometriosis/cancer (n=17); (2) atypical endometriosis (n=6); (3) endometriosis (n=17); (4) endometrium (n=7). Tumors and controls were immunostained and evaluated for expression of p53, c-erb-B-2, MIB1 and Bcl-2. Statistical analysis was performed using Chi-square for linear trends, Fisher exact and Kruskal-Wallis tests. RESULTS Of the 410 cancers, 17 (4.1%) had associated endometriosis and of the 521 endometriosis, 6 (1.2 %) had atypical changes. Fourteen of 17 (82.4%) cancers associated with endometriosis and all atypical endometriosis had p53 overexpression. Only 2 of 17 (11.8%) endometriosis and none of the endometriums had mutant p53 (P<0.01). We found a trend towards increased expression of MIB1 (0.073) in the cancer and atypical endometriosis groups, and no differences in expression of Bcl-2 or c-erb-B-2. The sensitivity and specificity of p53 as a marker for the diagnosis of atypical endometriosis and cancer associated with endometriosis were 87%; CI 95% (73.2-100%) and 92% (80.6-100%), respectively. When comparing all groups, the mean positive p53 and MIB1 cell count was statistically significant (P=0.01). CONCLUSIONS Overexpression of p53 in atypical endometriosis and cancer associated with endometriosis is a common finding and may be used to identify endometriosis with premalignant potential.

The impact of medical therapy on surgery for benign prostatic hyperplasia: a study comparing changes in a decade (1992–2002)

In a study from Madrid, patients from one centre were retrospectively reviewed, comparing those who had surgery for BPH in the first half of 1992 to that of 2002. The authors concluded that surgery was currently less common in older patients, and in patients who had received medical treatment for longer. They also found that open surgery was more common in those who required surgery, perhaps because the increase in prostate size was not suppressed by the medical therapy predominantly prescribed in the decade in question.

Histological damage in chronic hepatitis C is not related to the extent of infection in the liver.

It has not been completely elucidated whether the liver injury induced by the hepatitis C virus (HCV) is due to direct cytopathic damage or to an immune-mediated response against HCV-infected hepatocytes. In this work, we have determined the percentage of HCV-infected hepatocytes, the histological activity index, and the viremia levels in chronically HCV-infected patients with different grades of liver injury to investigate any possible correlation between them. For that purpose, liver biopsies from 27 patients with HCV chronic hepatitis were analyzed by in situ hybridization. This technique revealed that the percentage of infected hepatocytes ranged from 0.04% to 83.6%. Regarding the viremia levels, HCV RNA concentration ranged from 1.8 x 10(3) to 1.4 x 10(6) genome copies/ml. A significant correlation (r = 0.54; P = 0.003) between the percentage of infected hepatocytes and the viremia levels was found. In contrast, no correlation was observed between the percentage of HCV-infected hepatocytes or the viremia levels and the histological activity index. In conclusion, we have shown that the HCV viremia reflects the extent of the infection in the liver and that the liver injury in chronic HCV infection is not directly related to either the number of infected hepatocytes or the serum HCV RNA concentration.

Detection of TT Virus DNA in Liver Biopsies by in Situ Hybridization

A novel hepatitis-associated virus named TT virus (TTV) has been isolated. However, its hepatotropism has not been proven. We have retrospectively analyzed the presence of TTV-DNA by polymerase chain reaction (PCR) and in situ hybridization in liver biopsies from 30 patients with liver disease (15 TTV-DNA-positive and 15 TTV-DNA-negative in serum), and prospectively in serum and liver from eight patients with normal liver histology. TTV-DNA was detected by PCR in the liver from the 15 patients with serum TTV-DNA and in serum and liver of two of the eight patients without liver disease. TTV-DNA titers in liver were 10 times higher than in serum, although no correlation between TTV-DNA titers in serum and liver were observed. In situ hybridization shows positive signals in the hepatocytes of the 17 patients infected by TTV but in none of the TTV-DNA-negative patients by PCR. No morphological changes were observed in the hepatocytes showing hybridization signals. The percentage of positive hepatocytes ranged from 2.1% to 30% and correlated with the TTV-DNA titers in liver (r = 0.54; P = 0.037). In conclusion, our results show that TTV is able to infect liver cells although they do not support a role for TTV in causing liver disease.

Acute renal failure associated to paroxysmal nocturnal haemoglobinuria leads to intratubular haemosiderin accumulation and CD163 expression

Decreased renal function has been observed in diseases with intravascular haemolysis, including paroxysmal nocturnal haemoglobinuria (PNH). However, the mechanisms via which haemoglobin enhances renal damage in this pathology are not fully known. We report a case of acute renal failure associated to PNH and extensive haemosiderin deposits in tubular cells. Renal biopsy also revealed a strong immunostaining of CD163 (a haemoglobin scavenger receptor expressed in macrophages) and oxidative stress markers (NADPH-p22 phox and haeme oxigenase-1) in areas with deposits of iron. This fact provides evidence for a pathogenic role for free haemoglobin in tubulointerstitial renal injury in human PNH disease.

Sebaceous carcinoma of the vulva.

Extraocular sebaceous carcinoma is an uncommon neoplasm usually localized on the head and neck. Sebaceous glands are abundant on the vulva, but vulvar sebaceous carcinoma is an uncommon neoplasm. To our knowledge, there are only five previously reported cases of sebaceous carcinoma on this location. We report an additional case of vulvar sebaceous carcinoma associated with Bowens disease in the overlying epidermis. The patient also had bowenoid papulosis involving the skin of labia majora. We analyzed by immunohistochemistry, Southern blot hybridization, and polymerase chain reaction (PCR) techniques for the presence of DNA of human papilloma viruses (HPVs) in the specimen of sebaceous carcinoma and in lesions of bowenoid papulosis. Immunohistochemistry, Southern blot hybridization, and PCR studies in specimens of bowenoid papulosis lesions and sebaceous carcinoma did not detect DNA of HPVs. A significant increase in intranuclear p53 staining was demonstrated in several areas of neoplastic aggregations of sebaceous carcinoma.

Inhibition of JAK2 protects renal endothelial and epithelial cells from oxidative stress and cyclosporin A toxicity.

Cyclosporin A is an immunosuppressant drug widely used in solid organ transplantation, but it has nephrotoxic properties that promote oxidative stress. The JAK2/STAT pathway has been implicated in both cell protection and cell injury; therefore, we determined a role of JAK2 in oxidative stress-mediated renal cell injury using pathophysiologically relevant oxidative challenges. The AG490 JAK2 inhibitor and overexpression of a dominant negative JAK2 protein protected endothelial and renal epithelial cells in culture against peroxide, superoxide anion and cyclosporin A induced cell death while reducing intracellular oxidation in cells challenged with peroxide and cyclosporin A. The decrease in Bcl2 expression and caspase 3 activation, induced by oxidative stress, was prevented by AG490. In mouse models of ischemia/reperfusion and cyclosporin A nephrotoxicity, AG490 decreased peritubular capillary and tubular cell injury. Our study shows that JAK2 inhibition is a promising renoprotective strategy defending endothelial and tubular cells from cyclosporin A- and oxidative stress-induced death.