Effect of the antihypertensive drug enalapril on oxidative stress markers and antioxidant enzymes in kidney of spontaneously hypertensive rat.

Oxidative stress has been suggested to play a role in hypertension and hypertension induced organ damage. This study examined the effect of enalapril, an antihypertensive drug, on oxidative stress markers and antioxidant enzymes in kidney of spontaneously hypertensive rat (SHR) and Nω -nitro-L-arginine methyl ester (L-NAME) administered SHR. Male rats were divided into four groups (SHR, SHR+enalapril, SHR+L-NAME, and SHR+enalapril+L-NAME). Enalapril (30 mg kg(-1) day(-1)) was administered from week 4 to week 28 and L-NAME (25 mg kg(-1) day(-1)) was administered from week 16 to week 28 in drinking water. Systolic blood pressure (SBP) was measured during the experimental period. At the end of experimental periods, rats were sacrificed; urine, blood, and kidneys were collected for the assessment of creatinine clearance, total protein, total antioxidant status (TAS), thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), and catalase (CAT), as well as histopathological examination. Enalapril treatment significantly enhanced the renal TAS level (P < 0.001) and SOD activity (P < 0.001), reduced the TBARS levels (P < 0.001), and also prevented the renal dysfunction and histopathological changes. The results indicate that, besides its hypotensive and renoprotective effects, enalapril treatment also diminishes oxidative stress in the kidneys of both the SHR and SHR+L-NAME groups.

Nitric oxide (no), citrulline - no cycle enzymes, glutamine synthetase and oxidative stress in anoxia (hypobaric hypoxia) and reperfusion in rat brain.

Nitric oxide is postulated to be involved in the pathophysiology of neurological disorders due to hypoxia/ anoxia in brain due to increased release of glutamate and activation of N-methyl-D-aspartate receptors. Reactive oxygen species have been implicated in pathophysiology of many neurological disorders and in brain function. To understand their role in anoxia (hypobaric hypoxia) and reperfusion (reoxygenation), the nitric oxide synthase, argininosuccinate synthetase, argininosuccinate lyase, glutamine synthetase and arginase activities along with the concentration of nitrate /nitrite, thiobarbituric acid reactive substances and total antioxidant status were estimated in cerebral cortex, cerebellum and brain stem of rats subjected to anoxia and reperfusion. The results of this study clearly demonstrated the increased production of nitric oxide by increased activity of nitric oxide synthase. The increased activities of argininosuccinate synthetase and argininosuccinate lyase suggest the increased and effective recycling of citrulline to arginine in anoxia, making nitric oxide production more effective and contributing to its toxic effects. The decreased activity of glutamine synthetase may favor the prolonged availability of glutamic acid causing excitotoxicity leading to neuronal damage in anoxia. The increased formation of thiobarbituric acid reactive substances and decreased total antioxidant status indicate the presence of oxidative stress in anoxia and reperfusion. The increased arginase and sustained decrease of GS activity in reperfusion group likely to be protective.

Synovial membrane cytokine profiles in reactive arthritis secondary to intravesical bacillus Calmette-Guérin therapy.

We describe the cellular infiltrate and cytokine profile in sequential synovial membrane biopsies from a patient with acute followed by chronic synovitis after intravesical bacillus Calmette-Guérin (BCG) therapy for an in situ transitional cell carcinoma of the bladder. Histological and immunohistochemical analysis of 3 synovial biopsies were done sequentially over a 9 month period. The patient was HLA-B27 positive, but HLA-DR4 negative, and did not have the "shared epitope." Unlike other cases, this patient's arthritis did not respond initially to nonsteroidal antiinflammatory drugs and was exacerbated by corticosteroid therapy. The synovitis took a neutrophilic form, with marked synovial membrane content of interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha). It subsequently developed into chronic lymphoplasmacytoid synovitis, similar to rheumatoid arthritis (RA), with decreased IL-8 but continuing IL-1 and TNF-alpha production in the synovial membrane. The synovitis resolved to a fibrotic synovium with residual thickening of the synovial lining layer and continued production of TNF-alpha. Thus, during the evolution of this arthritis, the synovial layer and continued production of TNF-alpha. Thus, during the evolution of this arthritis, the synovial membrane yielded a cellular infiltrate and cytokine content that had marked similarities with that seen in RA; however, the arthritis eventually remitted spontaneously.

De novo expression of P-glycoprotein associated with poor prognosis in high grade non-Hodgkin's lymphoma.

Chemotherapy has proven to be effective in the management of high grade non-Hodgkin's lymphoma (NHL). However drug resistance remains a major clinical obstacle to effective disease control. One such well documented system involves multidrug resistance (MDR), characterized by the overexpression of a 170 kDa membrane protein termed P-glycoprotein (Pgp). Analysis of P-glycoprotein was done on lymph node biopsies obtained from 109 patients with high grade NHL. Patients were followed up for time periods ranging from 7 to 26 months after chemotherapy or until recurrent/residual disease was diagnosed. Analysis of Pgp in tissue samples was carried out by immunocytochemistry and Western blot. Of the 109 patients, 36 had either residual or recurrent disease. All these patients had a second lymph node biopsy done which was also analyzed for Pgp. P-glycoprotein was detected by immunocytochemistry in only 5 of the 73 patients who remained disease-free, while it was expressed in 26 of the 36 patients with residual/recurrent disease. All the 36 tissue samples of the latter group and 42 of the 73 biopsies of the disease-free group were re-analyzed by Western blot. Only one of the 42 samples from the disease-free group showed a positive Western blot reaction while 30 of the 36 samples from patients with recurrent/residual disease gave a positive reaction. Thirty-four of the 36 repeat biopsy samples from patients with recurrent/residual disease were positive for Pgp. Correlation analysis, thus showed significant relationship between prognosis and detection of Pgp by immunocytochemistry (r=0.85, p<0.001) and Western blot (r=0.90, p<0.001). Moreover, the odds ratio of a tumor positive for Pgp not responding to chemotherapy was 35.36 (CI 11.03, 113.37). Thus evaluation for Pgp may prove to be a useful prognostic marker for high grade NHL, and may also prove useful in designing or altering chemotherapy protocols in such patients.

The abnormalities of nailfold capillaries in scleroderma as assessed by video image analysis and photomicroscopy.

Scleroderma is a systemic connective tissue disease in which the diagnosis in supported by morphological changes in nailfold capillary size and density. These changes are open to observer bias. In this paper we describe 2 objective methods that allow quantitative definition of capillary changes, video image analysis (VIA) and photomicroscopy. VIA was used to assess 15 healthy control subjects and 22 patients with scleroderma. Scleroderma patients had a significantly larger capillary diameter (43 microns versus 20 microns, p = 0.0001) and capillary density was reduced by a mean factor of 0.5. Image stored on computer will facilitate serial assessments of nailfold capillary changes and possibly provide information on disease progression.

Rheumatic manifestations of the myelodysplastic syndromes: a comparative study.

BACKGROUND: The myelodysplastic syndromes (MDS) are a group of common haematological disorders that increase in incidence with age. Case reports have suggested an arthritis associated with myelodysplasia. This comparative study reviews patients with myelodysplasia and patients with a myeloproliferative disorder (MPD) as the control group. AIM: To document the rheumatological manifestations in patients with MDS and to determine if there is an association between MDS and an inflammatory arthritis/vasculitis. METHODS: Between July 1990 and July 1995 all patients with a known diagnosis of MDS and MPD attending the Haematology clinics of two teaching hospitals were reviewed. There were 87 MDS patients and 86 MPD patients identified. Twenty-six of the MDS patients and 28 of the MPD patients attended a clinical review by a single examiner. A history of joint symptoms, skin rashes, family and drug history was obtained. Physical examination and serology were routinely performed. The case notes of the remaining patients were reviewed by a single observer. Approval was obtained from the Ethics Committee at both hospitals. RESULTS: The two patient groups were matched for sex and age. There were equal numbers of patients with osteoarthritis, rheumatoid arthritis and crystal arthritis in the two groups. The significant finding was the presence of a seronegative inflammatory arthritis in five patients in the MDS group. One patient had both a seronegative arthritis and a cutaneous leukocytoclastic vasculitis, and another a cutaneous leukocytoclastic vasculitis only. These rheumatic manifestations were not seen in the MPD group. Five of six patients were treated with prednisolone and responded impressively. The rheumatological symptoms preceded the diagnosis of MDS in two of the six cases. CONCLUSIONS: A seronegative arthritis is an associated finding in MDS. The arthritis can precede the development of the bone marrow disorder, and can be a guide to the diagnosis of this haematological disorder in elderly patients presenting with an inflammatory arthritis and cytopenias.

Expression of p53 in leukoplakia and squamous cell carcinoma of the oral mucosa: correlation with expression of Ki67.

Aim-To study p53 expression in relation to proliferative status in normal and nondysplastic, dysplastic and malignant lesions of the oral mucosa.Method-The standard avidin-biotin complex (ABC) immunohistochemical staining method was used to study the expression of p53 and Ki67 on frozen sections of oral leukoplakias and carcinomas.Results-Of the leukoplakia and carcinoma samples, 70% expressed p53 in over 5% of cells. In normal mucosa less than 5% of cells expressed p53. The proliferation index, as assessed by expression of Ki67, was highest in the malignant lesions (43%) and lowest in normal mucosa (11%). Statistical analysis revealed that expression of both p53 and Ki67 was correlated significantly with the histopathological stage of the tumour. However, expression of p53 was not correlated with that of Ki67. In leukoplakia lesions with proliferative features p53 immunostaining was less intense than in non-proliferative lesions; this difference was statistically significant.Conclusions-These results emphasise the potential of Ki67 and p53 as biomarkers of carcinogenesis in oral cancer and may also serve as intermediate points for cancer prevention programmes, such as the oral chemopreventive trials. Factors other than p53 may have a more important role in the deregulation of proliferation in pre-malignant oral lesions.

Potential biological markers for the staging of tumor progression in oral mucosa: a multivariate analysis.

A recent trend in cancer control programmes is the development of early detection strategies and chemoprevention of premalignant lesions. The present study evaluates the potential of selected markers in the biological staging of tumor progression in oral mucosa for better management of the disease. The expression patterns of various cytokeratin protein types such as 10/11, 13 & 16, 19, 18, 14 and pancytokeratin, involucrin, ras p21, epidermal growth factor (EGF) and its receptor (EGFR) were assessed immunohistochemically in various stages of tumor progression in oral mucosa. Statistical analyses such as the Kruskal-Wallis one-way ANOVA, Spearman's rank correlation and multiple regression analysis were carried out to see which proteins have a significant association with tumor progression in oral mucosa. Statistical analysis showed that the expression patterns of cytokeratin types 10/11, 14 and 19, involucrin and epidermal growth factor were significantly correlated with tumor progression in oral mucosa in both univariate and multivariate analysis. Thus the biological stage of a lesion can be calculated from the multiple regression equation derived for these proteins, which could be more useful in assessing the stage of tumor progression in oral mucosa than histopathological grading.

Ultrastructural analysis of the adjacent epithelium of oral squamous cell carcinoma.

Fifteen biopsies of the immediate adjacent epithelium of oral squamous cell carcinoma were examined under light and electron microscopy. Light microscopic examination of one micron thick sections revealed that the majority of lesions (67%) had hyperplastic or mildly dysplastic epithelium while the remaining (33%) had moderate to severe dysplasia. Ultrastructural observations showed that all these lesions had subcellular alterations similar to those seen in frank malignant oral tissue, particularly in the lower half of the epithelium. Important ultrastructural changes observed included bizarre nuclei of basal and lower spinal cells, enlarged and multiple nucleoli, presence of interchromatin and perichromatin granules, loss of desmosomes and marked spongiosis as well as disturbed cellular maturation sequences in the keratinocytes evidenced by abnormal and irregular distribution of maturation markers such as keratohyalin granules and tonofilaments. The present study thus shows the value of electron microscopy in the detection of malignant changes in the adjacent epithelium of oral squamous cell carcinoma.

A study of scleroderma in South Australia: prevalence, subset characteristics and nailfold capillaroscopy.

BACKGROUND: Scleroderma is a systemic rheumatic disorder seen in a wide range of clinical specialties. AIMS: To establish the prevalence and mortality rates of scleroderma in South Australia (SA), to determine the relative frequency and characteristics of the three principal subsets (diffuse, limited and overlap), and to examine the role of nailfold capillaroscopy in subset identification and implied prognosis. METHODS: Outpatient and discharge diagnostic indexes from five major teaching hospitals in SA were reviewed between February 1987 and November 1993. A total of 215 patients with scleroderma were identified. Case notes of 115 of these patients were reviewed in order to validate scleroderma diagnosis, and subset characteristics such as sex, mean age at diagnosis, extent of skin involvement, internal organ involvement and serology were analysed. Fifty-two of these patients were then examined prospectively to confirm positive discharge diagnosis, and nailfold capillaroscopy was performed on these patients. RESULTS: The point prevalence of scleroderma in SA for 1993 was estimated to be 208/10 This figure is a conservative estimate and is higher than most other reported series. The female to male ratio was 4:1. The majority of patients had limited disease with a ratio of 6:1:1.6 limited vs diffuse vs overlap. Systemic involvement excluding the oesophageal components limited disease was found predominantly in the diffuse group. Autoimmune serology was positive in 90% of patients, with Scl-70 being more common in diffuse scleroderma, anti-centromere antibody (ACA) in the limited form and anti-ribonucleoprotein (RNP) in the overlap form. Nailfold capillaroscopy was useful in predicting disease-subtype as capillary dilatation was observed predominantly in limited disease, capillary dropout in diffuse disease. CONCLUSIONS: Scleroderma is more common in SA than previously recognised. Limited disease is more common than diffuse or overlap disease, carries a better prognosis and in associated with ACA. Nailfold capillaroscopy is a useful tool in disease assessment and may provide useful diagnostic and prognostic information.

Cirrhosis in patients with rheumatoid arthritis receiving low dose methotrexate.

We describe three patients with RA who developed cirrhosis while taking low dose methotrexate (MTX). This report includes a review of the risk factors for cirrhosis occurring in association with MTX. Two of these patients were part of a prospective study to quantify changes in pericellular and total collagen in 76 patients with RA receiving low dose pulse MTX, giving a point prevalence of cirrhosis of 2.63%.

Expression of galactoside-specific endogenous lectins and their ligands in human oral squamous cell carcinoma.

Human endogenous lectins have a wide spectrum of biological functions. The present study analyses the expression of beta-galactoside specific and N-acetyl-D-galactosamine specific endogenous lectins in oral squamous cell carcinomas using biotinylated neoglycoproteins. The expression pattern of beta-galactosyl-containing glycoconjugates or ligands of beta-galactoside specific lectins in these tissues was also studied using an endogenous biotinylated lectin, the human 14-kDa lectin. For comparison a galactoside specific plant lectin from mistletoe, Viscum album was also employed. The results demonstrate that oral squamous cell carcinomas mainly express accessible binding sites for lactosylated neoglycoprotein (90%) while few carcinomas expressed mild amount of N-acetyl-D-galactosamine specific binding sites (40%). There was no difference in the binding patterns of these probes between well and less differentiated carcinomas. Expression of these neoglycoprotein binding sites were mostly concentrated in immature basaloid cells, indicating a possible association with cell proliferation. The binding pattern of D-galactosyl specific lectins (human 14-kDa and mistletoe lectins) showed conspicuous differences. This feature emphasizes the caution that needs to be exercised in interpreting the biological significance of results attained using plant lectins on human tissue.

Alterations in expression of basement membrane proteins during tumour progression in oral mucosa.

Expression of three basement membrane proteins--collagen IV, laminin and fibronectin--was studied in normal, hyperplastic, dysplastic and neoplastic conditions of the oral mucosa using immunohistochemistry. Collagen IV and laminin exhibited similar staining patterns, while fibronectin showed a different pattern of expression. The expression of collagen IV and laminin also demonstrated an inverse correlation between staining intensity, thickness and basement membrane continuity in various stages of tumour progression. In contrast to the continuous and intense staining of basement membrane in normal oral mucosa with collagen IV and laminin antibodies, severe dysplasia and carcinoma exhibited discontinuous, thin and weakly stained basement membrane. The expression of fibronectin showed a direct correlation with extent of tumour progression. In normal mucosa, expression of fibronectin was almost absent, whereas in carcinoma intense expression of fibronectin was evident in the basement membrane and basal cells. These results emphasize the value of basement membrane proteins as biological markers for assessing oral carcinogenesis.

Co-expression of ras p21 and epidermal growth factor receptor during various stages of tumour progression in oral mucosa.

Ras p21 and epidermal growth factor receptor (EGFR) have commonly been implicated in a variety of malignancies. The present study immunohistochemically analysed the relationship between the expression of ras p21 and EGFR in normal, premalignant and malignant lesions of the oral mucosa. Except for normal mucosa, all other lesions showed a more or less similar expression pattern of both ras p21 and EGFR. It was also observed that the expression of both proteins was mostly confined to the basal or basaloid cells of the leucoplakia and carcinoma lesions, respectively. Normal keratinizing type mucosa showed expression of ras p21 and EGFR in the basal and lower spinal cells. However, in non-keratinizing mucosa the expression of ras p21 was totally negative while EGFR was restricted to the basal cells. Statistical analysis revealed a good correlation between the expression of ras p21 and EGFR with the various histological stages of tumour progression. The presence of these proteins in immature dividing cells of premalignant and malignant lesions thus emphasizes their relationship with cell proliferation.

Differential expression of cytokeratin proteins during tumour progression in oral mucosa.

The expression pattern of cytokeratin filaments in epithelia has been shown to be dependent on their type and grade of differentiation. The type of expression of cytokeratin in a cell may also be altered during carcinogenesis or other pathological conditions. The present study examined the alterations in expression of various cytokeratin types during different stages of tumour progression in the oral mucosa. Six monoclonal anti-cytokeratin antibodies were used for the study. Conspicuous staining differences with these antibodies were evident between normal keratinizing and non-keratinizing mucosa. These differences can be correlated to the differentiation pattern of the normal mucosa types. Antibodies specific to cytokeratin types 10/11, 19 and 14 showed significant correlation with stage of tumour progression. In addition cytokeratin type 18 also showed prominent differences in expression between different stages of tumour progression. These alterations in cytokeratin expression suggest that the terminal differentiation pathway of keratinocytes is disturbed during oral carcinogenesis. The results of the present study also emphasize the potential of using cytokeratin filaments as markers in the biological staging of oral premalignant and malignant lesions.

Alterations in expression of terminal differentiation markers of keratinocytes during oral carcinogenesis.

Expression of cytoskeletal proteins has been shown to be dependent on the differentiation status of the tissue. In the present study, expression of two cytoskeletal proteins normally present in terminally differentiated keratinocytes, namely cytokeratin types 10/11 and involucrin, were studied in different stages of tumour progression in the oral mucosa. Results showed that cytokeratins 10/11 and involucrin strongly correlated with the differentiation status of cells. High expression was observed in non-dysplastic hyperplastic epithelium as compared to normal, dysplastic and neoplastic epithelium. In addition, various grades of dysplasia showed an inverse correlation with expression of these proteins. Statistical analysis of the results also showed a negative correlation between the differentiation of upper spinal cells and the stage of tumour progression. It therefore appears that the proteins studied may be useful as markers for epithelial carcinogenesis.

Ultrastructure of oral squamous cell carcinoma: a comparative analysis of different histological types.

Twenty-five oral carcinomas and five normal oral epithelial specimens were studied using light and electron microscopy. All histological types (well differentiated squamous cell carcinoma, moderately differentiated squamous cell carcinoma, poorly differentiated squamous cell carcinoma, verrucous carcinoma and spindle cell carcinoma) were seen in the study sample. In addition, 1 case of carcinoma in situ was also present. The normal oral epithelium consisted of three keratinising types (gingiva) and two non-keratinising types (buccal mucosa). The ultrastructural features of oral carcinomas showed good correlation with the features seen in light microscopy. The differentiation status of the lesions showed a relationship with cell and nuclear size, tonofilament and keratin content as well as few other cellular abnormalities. It was also observed that the fine details revealed by electron microscopy were often a means of explaining the characteristic histopathological features of oral carcinoma.

Immunohistochemical localization of epidermal growth-factor and its receptor in normal, premalignant and malignant oral-mucosa.

An important growth factor involved in epithelial carcinogenesis is the epidermal growth factor (EGF). The present study analyze the expression pattern of EGF and its receptor (EGFR) in different stages of tumour progression in oral mucosa (normal epithelium, non-dysplastic and dysplastic leukoplakias and carcinomas). Alterations in expression pattern of EGFR was not significant in the various tissues from normal mucosa to malignancy. In all these stages EGFR positivity was confined to the immature basal or basaloid cells. EGF however showed marked alterations in expression in different stages of tumour progression in oral mucosa. Most of the malignant cells were positive for EGF antibodies while, only a few lower layers of cells were stained in normal mucosa and in dysplastic leukoplakia lesions, more number of cells expressed EGF than normal tissue. The present study thus shows the autocrine role of the EGF and EGFR and the possibility of using EGF as a marker for tumour progression in oral mucosa.