Detection of enteroviruses in the intestine of type 1 diabetic patients.

Enterovirus infections have been diagnosed more frequently in type 1 diabetic patients than in the healthy population, and enteroviruses have also been found in the pancreas of diabetic patients. Primary replication of the virus occurs in the gut, but there are no previous studies evaluating possible presence of virus in the intestine of diabetic patients. The purpose of this study was to investigate if enteroviruses can be found in small intestinal tissue of type 1 diabetic patients. Formalin-fixed, paraffin-embedded upper intestinal biopsy samples were analysed for the presence of enterovirus using in situ hybridization and immunohistochemistry. Enterovirus was detected by in situ hybridization in six (50%) of the type 1 diabetic patients (n = 12) but in none of the control subjects (n = 10, P = 0.015). Immunohistochemistry identified enterovirus in nine (75%) of the patients and one (10%) control subject (P = 0.004). The presence of the virus was confirmed by reverse transcription-polymerase chain reaction in one of the four patients from whom a frozen and unfixed sample was available. Intestinal morphology was normal in all study subjects. The results suggest that a substantial proportion of type 1 diabetic patients have an ongoing enterovirus infection in gut mucosa, possibly reflecting persistent enterovirus infection. This observation opens new avenues for further studies on the possible role of enteroviruses in human type 1 diabetes.

In vivo and in vitro degradation of a novel bioactive guided tissue regeneration membrane.

The aim of this study was to assess degradation of a novel bioactive guided tissue regeneration (GTR) membrane and to quantify the concurrent tissue responses. Pieces of membrane composed of poly-l-lactide, poly-d,l-lactide, trimethylenecarbonate and polyglycolide were dipped into an N-methyl-2-pyrroline (NMP) solution and implanted in the mandibles of 10 sheep. The animals were sacrificed at 6-104 weeks. Parallel in vitro degradation was analysed by measuring the inherent viscosity, water absorption and remaining mass. One of the 2 in vitro sets of membranes was prehandled with NMP. At 6-26 weeks in vivo, the gradually more degraded implants were surrounded by a fibrous network. At 52 and 104 weeks, the implants and fibrous networks were non-detectable. Foreign body granulomatous reactions were not observed. In vitro, the mass of the NMP-exposed membranes diminished linearly over the 2-year period down to 10%, while the non-NMP-exposed membrane maintained all their mass for the first 16 weeks. The membranes without NMP had absorbed significantly less water at weeks 4 and 8 than the other group. The inherent viscosity decreased relatively uniformly in the in vitro groups. In conclusion, the in vivo degradation was complete in 12 months with only mild histologic responses; the degradation in vitro may be slower. NMP accelerates the degradation.

Evidence for a structural abnormality of collagen VII in a patient with dystrophic epidermolysis bullosa inversa.

Recent studies indicate that in skin of patients with dystrophic epidermolysis bullosa (EB) inversa, anchoring fibrils have an abnormal ultrastructure, but the major protein of these fibrils, collagen VII, is expressed and detectable with antibodies at the dermo-epidermal junction. For molecular characterization of this rare EB phenotype, skin biopsies from a patient with dystrophic EB inversa were investigated with indirect immunofluorescence, immunoelectron microscopy, and immunoblotting. Ultrastructural analysis of clinically uninvolved skin showed sublamina densa splitting. In unblistered areas, focal groups of anchoring fibrils that appeared loosely polymerized and without a distinct crossbanding pattern were observed. Indirect immunofluorescence staining with antibodies to collagen VII exhibited a linear fluorescence at the dermo-epidermal junction and at the roof of a spontaneous blister. Immunoelectron microscopy demonstrated staining of the poorly assembled anchoring fibrils, but no significant reaction in areas where no fibrillar structures could be discerned. In contrast to normal control skin, immunoblotting showed immunoreactive collagen VII in both epidermal and dermal extracts. Moreover, the dermis-associated collagen VII appeared as a distinct doubleband that contained the tissue form of collagen VII (250-300 kD) and an additional band with a slightly smaller molecular weight. In epidermal extracts one band, of the size of the tissue form, was detected. The studies on the present patient suggest that a structural abnormality of collagen VII that prevents its aggregation to stable dimers or polymerization to distinct anchoring fibrils may contribute to the etiopathogenesis of dystrophic EB inversa.

Immunoelectron microscopic localization of a progesterone-inducible protein (avidin) in the chick oviduct mucosa.

Avidin, a specific progesterone inducible protein, was localized in the oviduct magnum mucosa of chicks treated with diethylstilbestrol (DES) or DES plus progesterone, using ultrastructural immunoperoxidase techniques. Diffusion technique and the double or triple layer peroxidase staining method were applied. The results obtained by immunoelectron microscopic peroxidase techniques with high resolution power indicated that progesterone stimulation in the DES-treated chicks resulted in avidin production in the goblet cells of the oviduct epithelium. The sensitive-antiperoxidase staining method revealed a slight avidin production in many goblet cells of chicks treated with only DES. This method also showed some avidin-positive ciliated epithelial cells in chicks treated with progesterone. This results suggest that some ciliated epithelial cells may have functional or metabolic properties characteristic of secretory goblet cells.

Characterization of the phenotype of smooth muscle cells in human fetal aorta on the basis of ultrastructure, immunofluorescence, and the composition of cytoskeletal and cytocontractile proteins.

The phenotype of smooth muscle cells (SMCs) in the aortic media of 7 human fetuses (14-20 weeks of gestation) was examined with transmission electron microscopy, immunofluorescence microscopy, and gel electrophoresis of the cytoskeletal and cytocontractile proteins. Ultrastructurally, virtually all medial cells were identified as SMCs having a poorly differentiated phenotype with a cytoplasm rich in rough endoplasmic reticulum and organelles, and with only a few myofilaments. All medial cells stained intensely with antibodies to vimentin, but only in a 20-week-old fetus could we find a few SMCs staining with antibodies to desmin. Nor was desmin detectable with SDS gel electrophoresis followed by immunoblotting, while clear bands corresponding to vimentin, myosin, and actin were present. In isoelectric focusing and two-dimensional gel electrophoresis beta-actin was the most prominent of the 3 actin isoforms in all cases. The present results show that SMCs in the media of fetal human aorta have a poorly differentiated phenotype, which morphologically and biochemically resembles that previously described in the aorta of fetal and newborn rat, in the arterial intima after endothelial injury, in atherosclerotic lesions, and after spontaneous modulation of medial SMCs in culture.

Immunoelectron microscopic localization of immunoglobulins and complement in human renal glomeruli.

A study was undertaken to evaluate some processing variables affecting the immunoelectron microscopic demonstration of immunoglobulins and complement (C3) in human glomeruli. Percutaneous biopsies were performed on 28 patients with various types of glomerulonephritis. Light microscopic, electron microscopic, and immunofluorescence examinations were performed by routine methods. For immunoelectron microscopy, fixation in paraformaldehyde (PA) or periodate-lysine-paraformaldehyde (PLP) was used. With the diffusion technique, using tissue chopper or cryostat sections, human immunoglobulin (Ig)G, IgA, IgM, and C3 were localized in glomeruli with peroxidase-labeled antisera. Using PLP and the tissue chopper sections, good ultrastructure was achieved. The antigens could be demonstrated in intramembranous, subepithelial, subendothelial, or mesangial immune deposits. Penetration of antibodies and quality of peroxidase reaction in the cryostat sections did not differ from that of the tissue chopper sections. Freezing and thawing, however, resulted in inferior morphology. If PA was used, the antigens could not be reliably demonstrated. The results of light microscopy, electron microscopy, and immunofluorescence microscopy were in good agreement with those from the immunoperoxidase procedure. The present study shows that PLP preserves well the antigenicity of human immunoglobulins and C3, resulting in good ultrastructure. PA fixation, on the contrary, caused a loss of antigenicity before an adequate ultrastructure could be achieved.

Expression of HSP-65 in jejunal epithelial cells in patients clinically suspected of coeliac disease.

BACKGROUND: Coeliac disease (CD) can be classified both clinically and biologically an autoimmune disease. A close relationship obtains between heat shock proteins (HSPs) and numerous autoimmune diseases. HSPs are overexpressed when protecting the host against environmental insult. We sought here to establish whether dietary gluten is such a stress stimulus in patients clinically suspected of CD, and whether the expression of HSP-65 associates with densities of intraepithelial gammadelta+ T cells and/or with expression of mucosal HLA-DR. METHODS: Seventy-eight children with clinical suspicion of CD underwent a jejunal biopsy. Monoclonal antibodies were used to stain jejunal epithelial HSP-65, intraepithelial lymphocytes and mucosal HLA-DR. Serum IgA-class endomysial autoantibodies (EMA) were measured by an indirect immunofluorescence method. CD susceptibility HLA DQA1*0501 and DQB1*0201 alleles (HLA DQ2) were determined. RESULTS: Enhanced expression of epithelial cell mitochondrial HSP-65 was found in 80% (16/20) of coeliacs and in 24% (14/58) of children excluded for the disease, but in only 7% (2/28) of control subjects (p < 0.001, p = 0.049, respectively). Children with enhanced expression of HSP-65 had significantly higher gammadelta+ T cell densities than those with normal HSP-65 expression. A clear association between HSP-65 and serum IgA-class EMA were also ascertained in patients with normal jejunal mucosal morphology. HLA DQ2 positivity did not correlate with the HSP-65 expression. CONCLUSIONS: Gluten might be an environmental insult not only in CD patients but also in some patients excluded for the disease on biopsy. Enhanced expression of epithelial cell stress proteins might be an indicator of such an insult.

Enterochromaffin cell density in the gastric mucosa of patients with chronic renal failure.

Thirty patients with chronic renal failure (CRF) and 30 age- and sex-matched controls were assessed for gastrointestinal diseases by gastroscopy, serum gastrin determination, and routine clinical and laboratory evaluation. Biopsy specimens from their gastric oxyntic mucosa were immunohistochemically stained with monoclonal antibodies against serotonin (5-hydroxytryptamine) and chromogranin A, the latter staining all gastric endocrine cells, the former disclosing serotonin-containing enterochromaffin (EC) cells only. The average EC cell density (cells/mm2) in the CRF patients was significantly lower than in the controls: 2.6 vs 12.9 (p = 0.0005). The EC cell counts also correlated negatively with serum gastrin values (p = 0.0031). The densities of the chromogranin-positive cells did not differ between CRF patients (74 cells/mm2) and controls (76 cells/mm2) (p = 0.7559). We conclude that, in addition to the previously known findings of hypoacidity, persistent hypergastrinaemia, and G and parietal cell hyperplasia, CRF also reduces the number of oxyntic EC cells. The negative correlation between EC cell density and serum gastrin levels reflects the complex interplay between different endocrinological activities in the gastrointestinal tract.

Depletion of Langerhans cells in cervical HPV infection is associated with replication of the virus.

We studied the quantity of Langerhans cells in 36 patients with cervical human papillomavirus (HPV) infection. Significantly fewer Langerhans cells (p < 0.05) were found in patients with compared to those without DNA tetraploidy. Similarly, patients positive for HPV 16/18 DNA by in situ hybridization or antipeptide IgA antibodies to HPV 18 tended to have fewer Langerhans cells than those negative for HPV 16/18 DNA or IgA antibodies. Our results suggest that depletion of Langerhans cells is associated with productive HPV 16/18 infection in the cervical epithelium.

Immunofluorescence microscopy of paraffin-embedded human kidney specimens obtained by fine-needle aspiration biopsy.

Recently, we have introduced an atraumatic fine-needle aspiration biopsy method to obtain human glomeruli for morphologic investigation. In the present study, immunofluorescence microscopy of paraffin-embedded, fine-needle specimens is described. The specimens were obtained by aspiration with a 10-mL syringe fitted to the fine-needle prepared from a lumbar puncture needle (Jintan Terumo). Embedding of the specimens into conventional paraffin blocks was carried out after pelleting them by centrifugation between processing steps in conical centrifuge tubes. Sections from the blocks were collected on small pieces of GelBond film (FMC Corporation) instead of objective slides, which prevented the detachment of small sections during enzyme treatment. Localization then was performed on deparaffinized trypsin-digested sections using fluorescein-labeled antibodies. The choice of fixative and digestive enzymes was found to have a marked effect on the localization; periodate-lysine-paraformaldehyde fixative and trypsin digestion gave the most reliable results.

Human papillomavirus types 16 and 18 in adenocarcinoma of the uterine cervix.

Many reports have shown a link between human papillomavirus (HPV) and cervical squamous neoplasia. However, the association of HPV with cervical adenocarcinoma has been studied less extensively. The authors evaluated the presence of HPV-DNA in 106 patients with adenocarcinoma of the uterine cervix by in situ hybridization, using 35S-labeled probes for HPV 16 DNA and HPV 18 DNA. The overall prevalence of HPV-DNA was 18% (19 of 106). HPV 16 was present in 2 (2%) cases, HPV 18 was observed in 15 (14%) cases, and both HPV 16 and HPV 18 were found in 2 (2%) cases. There was a correlation between HPV-DNA positivity and tumor stage (P less than 0.01) and tumor size (P less than 0.05), but there was no relationship between HPV-DNA positivity and tumor differentiation, proliferation (S-phase fraction), ploidy, lymph node metastases, or five-year survival rate. These results suggest that HPV 18 DNA is associated with cervical adenocarcinoma but the presence of HPV 18 has no influence on overall survival.

IgA-IgM nephropathy. A subgroup of primary mesangial glomerulonephritis.

The renal biopsy material of Tampere University Central Hospital comprises 1992 renal biopsy specimens, accessioned during the years 1978-1989. Among these, there were three cases of mesangial glomerulonephritis with a peculiar type of immunofluorescent reactivity. Striking mesangial deposits of both IgA and IgM were found in glomeruli, whereas C3 deposits were absent or present in slight amounts. The light microscopic findings ranged from mild mesangial glomerulonephritis to more advanced forms of sclerosing glomerulopathy. Electron microscopic examination disclosed an increase of mesangial matrix, together with mesangial and paramesangial electron-dense deposits. Two of the patients had microscopic hematuria associated with proteinuria, and one had isolated proteinuria. The authors propose that this group of cases may represent a new subgroup of primary mesangial glomerulonephritis that has not been described previously. They differ immunohistologically from both IgA nephropathy and IgM nephropathy, and therefore could be designated as IgA-IgM nephropathy.

Intratumor variation in cell proliferation in breast carcinoma as determined by antiproliferating cell nuclear antigen monoclonal antibody and automated image analysis.

Immunohistochemical detection of the proliferating cell nuclear antigen (PCNA) represents a potentially useful tool for the study of tumor proliferative activity. To study the intratumor heterogeneity of tumor growth, 88 breast carcinomas were immunostained with the anti-PCNA antibody 19F4 and analyzed with the CAS 200 image analysis system (Cell Analysis System, Inc., Lombard, IL). For each sample, 12 fields from both the central and the peripheral areas of the tumor were measured. The proportion of PCNA-positive nuclear area in the whole tumor (PCNAt score) varied from 0.7% to 45.2% (median, 14.4%). There was considerable intratumor heterogeneity in the staining for PCNA. In 79% of the specimens, the PCNA score was higher in peripheral areas than in the center of the tumor, the average difference being +3.4% (range, -9.2- +15.1%; P < 0.0001, Student's t-test). The S-phase fraction, determined by DNA flow cytometry of the same tumors, varied from 2.0% to 32.6% (median, 10.0%). The PCNA score showed a significant correlation with the S-phase fraction (r = 0.469, P < 0.001). Most divergent results were those with high PCNA scores and low S-phase fraction; possible explanations for this are discussed. The PCNA score also was related to the histologic grade of the tumors (P = 0.03, analysis of variance). In conclusion, proliferation indices obtained from different areas of a tumor can differ significantly because of intratumor heterogeneity in growth fractions. The PCNA immunostaining correlates with well-known prognostic factors (S-phase fraction and histologic tumor grade) in breast carcinoma.

Reduced E-cadherin expression is associated with invasiveness and unfavorable prognosis in breast cancer.

E-cadherin (E-cad) is a calcium-dependent, epithelial cell adhesion molecule whose reduced or lost expression has been associated with tumor dedifferentiation and increased metastatic potential in human carcinomas. The authors studied immunohistochemically E-cad expression in frozen sections of 362 breast carcinomas using a monoclonal antibody (HECD-1). The immunohistochemical detection of reduced E-cad expression was confirmed by mRNA in situ hybridization with two different oligonucleotide probes. THe proportion of tumors with reduced or lost E-cad expression increased significantly from pure intraductal carcinomas (20%, 4 of 20) through invasive ductal (IDCs; 52%, 124 of 239) to recurrent carcinomas (64%, 18 of 28; chi square test for trend, P = .004). Invasive lobular carcinomas (ILCs) and IDCs differed from each other in their E-cad expression. None of the ILCs (n=55) retained normal E-cad expression in contrast to 48% (115 of 239) of the IDCs. In 259 primary IDCs, reduced E-cad expression was associated with high histologic grade (chi square test for trend, P < .001), negative estrogen receptor status (ER; Fisher's exact test; P = .042), and marginally with axillary node involvement (Fisher's exact test, P = .063). In a subset of 109 primary IDC patients whose clinical follow-up was available (median follow-up 51 months), reduced E-cad expression was associated with shortened disease-free survival (DFS; Mantel-Cox test, P = .027). In Cox's multivariate regression analysis, progesterone receptor status (P = .018) and E-cad expression (P = .072) were selected as independent predictors of DFS. Our findings provide clinical evidence that loss of normal E-cad expression is an indicator of increased invasiveness and dedifferentiation in breast carcinoma. E-cad is a potentially important prognostic factor in primary IDCs.

Immunofluorescence of immunoglobulins and complement in kidneys taken at necropsy.

The immunofluorescence of immunoglobulins and complement components in kidney specimens taken at necropsy was investigated to determine the persistence of antigenicity of immune reactants. Of 74 consecutive necropsies, 12 cases had positive glomerular fluorescence. The pattern and intensity were followed up for up to 15 days. Along with necropsy specimens, tissue samples of normal looking kidney from 14 nephrectomies were also studied. Two of these specimens turned out to be positive in the immediate immunofluorescence study. To rule out possible false positive staining after death immunofluorescence findings in all nephrectomy specimens were followed up for up to 19 days. The presence of immunoglobulins and complement could be shown for between 12 and 15 days after death; no changes in immunofluorescence findings occurred during this period. It is concluded that immunofluorescence provides valuable information when immunologically mediated reactions need to be clarified in necropsy kidneys.

Immunoglobulin and complement deposition in glomeruli of 756 subjects who had committed suicide or met with a violent death.

AIMS: To study immune deposits in renal glomeruli. METHODS: Tissue was obtained from 756 necropsy cases from people who had committed suicide or met with a violent death. Glomerular immune deposits were examined by immunofluorescence microscopy and a light microscopy. The clinical histories of all the decreased were studied to ascertain reasons for the deposits. RESULTS: Immune deposits were found in glomeruli in 91 (12%) cases. In 52 (6.8%) cases mesangial IgA was observed as a solitary finding in 34 (4.5%), and was accompanied by other immunoglobulins in 18 (2.4%). Mesangial IgM was present in 19 (2.5%) and IgG in 11 cases (1.5%). Two cases had capillary IgG (0.3%). Light microscopic examination showed mesangial enlargement in eight of the cases with mesangial IgA. These included one with IgA glomerulonephritis diagnosed before death. Two cases with normal glomerular morphology and mesangial IgA deposits had clinical laboratory evidence of renal disease. In two subjects with normal glomerular morphology, mesangial IgM and microscopic haematuria were present. In one case with capillary IgG membranous glomerulonephritis was detected. CONCLUSIONS: Ten cases had mesangial IgA together with morphological or clinical laboratory findings suggestive of renal disease. If all these are regarded as IgA glomerulonephritis, then its prevalence can be estimated at 1.3%. For IgM glomerulonephritis, a prevalence of 0.3% was deduced.

Low apoptotic activity in primary prostate carcinomas without response to hormonal therapy.

Proliferative and apoptotic activities, as well as p53 protein expression, of ten untreated primary prostate carcinomas that showed extremely poor response to hormonal therapy (primary androgen independent prostate carcinomas) were compared with the stage- and grade-matched primary tumor specimens with favorable response to hormonal therapy (androgen dependent prostate carcinomas). The mean proliferative activity measured by Ki-67 immunohistochemistry was slightly higher in the primary androgen independent prostate carcinomas (8.70+/-5.24) than in the androgen dependent prostate carcinomas (7.09+/-2.68; p=0.27). The mean apoptotic activity by in situ end-labeling technique in the primary androgen independent prostate carcinomas (0.96+/-1.03) was less than half of that in the androgen dependent prostate carcinomas (2.75+/-0.98; p=0.0001). Ten percent of the androgen dependent prostate tumors showed p53 protein expression, whereas 30% of the primary androgen independent prostate tumors were immunopositive for p53 (p=0.30). In summary, we have shown that apoptotic activity in the primary androgen independent prostate carcinomas is significantly lower than in the matched androgen dependent prostate carcinomas while the proliferative activity remains unaffected. These results suggest that primary androgen independent prostate carcinomas may have genetic properties, such as inactivation of the p53 gene, that enable them to escape apoptosis caused by androgen ablation.

Clinical course and treatment of IgA nephropathy.

Impairment of renal function, severe proteinuria and arterial hypertension are the strongest clinical predictors of an unfavorable outcome in IgA nephropathy (IgAN). Glomerulosclerosis and interstitial fibrosis are the most reliable histologic prognostic markers. Metabolic syndrome and insulin resistance probably affect the clinical course of the disease. Among the known gene polymorphism it seems that there is a link between the ACE gene D allele and the progression of IgAN. Elevated blood pressure should be actively treated. The target blood pressure is 130/80 mmHg or less and the goal should also be to reduce proteinuria. Several large-scale trials are currently testing corticosteroids and other drugs in the treatment of IgAN.

The nephrotic syndrome in IgA glomerulonephritis: response to corticosteroid therapy.

The nephrotic syndrome was observed in eight out of 170 patients with IgA glomerulonephritis (5%). Three patients had mild glomerular alterations, all of them were normotensive, had normal renal function and responded to treatment with corticosteroids. In five patients moderate to marked mesangial changes associated with segmental sclerosing or proliferative lesions were seen. These patients were hypertensive and four of them had renal insufficiency. Three were treated with corticosteroids without response.

Glomerular IgA deposits in patients with celiac disease.

Glomerular immunopathology was studied in 25 patients with newly diagnosed celiac disease. None had clinical signs of renal disease. Glomeruli were obtained by fine-needle aspiration biopsy. The specimens were processed and studied by indirect immunofluorescence for immunoglobulins and complement. Mesangial IgA was found in 8 of the patients. It occurred occasionally together with slight IgG or IgM, but C3 was not seen in these patients. IgA-class circulating immune complexes (CIC), antireticulin antibodies (ARA), antigliadin antibodies (AGA), and rheumatoid factor (RF) occurred significantly more often in the patients with mesangial IgA than in the 17 patients having no mesangial IgA. The patients with mesangial IgA also had significantly higher mean levels of serum IgA, IgA-ARA and IgA-AGA than those without. The results suggest that glomerular mesangial deposits of IgA occur frequently in untreated celiac disease and that they are in some way associated with circulating IgA-class antibodies and immune complexes. In this situation IgA seems to be deposited without being able to induce clinically overt glomerulonephritis, a circumstance that may be related to the lack of complement in the deposits.