Immunochemical Changes Research Articles

Carisoprodol-Induced Histological and Immunohistochemical Changes in Spleen of Pregnant Rats

Background: Carisoprodol (Somadril®) is a centrally acting skeletal muscle relaxant that is commonly used to treat serious musculoskeletal disorders. Carisoprodol's mechanism of action is unknown and it is often attributed to its active metabolite, meprobamate. Objective: This study aimed to detect histopathological and immunohistochemical changes in the tissues of the splenic pregnant rats treated orally with Carisoprodol (Soma). Method: Thirty pregnant female albino rats were randomly categorized into three groups. The first group was a negative control and administered oral doses of distilled water. The other two groups were administered oral doses of carisoprodol in the distilled water equivalent to 10.8 mg and 21.6 mg/100g body weight/day respectively for 15 days from the 6th day to the 20th day of gestation. Results: Histological and immunohistochemical studies have been performed on the tissue of the spleen to detect histopathological and immunohistochemical changes. Compared to untreated (negative control) and pregnant rats treated with carisoprodol, the treated rats showed numerous histopathological and immunochemical changes. Conclusion: Treatment of pregnant rats with carisoprodol has induced many atrophic changes in the tissue of the spleen in pregnant rats, so the use of this drug during pregnancy should be under strict protection.

Correction of cytochemical changes in patients with occupational allergodermatosis

Professional allergodermatoses are accompanied by cytochemical and immunochemical changes in the body of patients. The use of ozone therapy techniques in this category of patients can accelerate the recovery period and normalize the above indicators.

Treating murine Kala-azar with a Mayan plant induces immunochemical changes.

Pentalinon andrieuxii Muell Arg is a Mexican-Central American plant anciently used by local people to treat cutaneous leishmaniasis. We evaluated a hexane extract of the root we called PAE for its chemical content and for its immunochemical and in vitro activity against Leishmania donovani and healing of experimental Kala-azar. Chemical analysis using gas chromatography coupled to mass spectrometry (GC-MS) identified hexadecanoic acid, hexadecanoic acid ethyl ester, 9, 12-octadecadienoic acid ethyl ester, octadecanoic acid ethyl ester, 9-octadecenoic acid ethyl ester and diethyl phthalate as the main compounds present in PAE. We also demonstrated PAE kills promastigotes and amastigotes in vitro and significantly reduces parasite loads in liver and spleen of infected Balb/c mice. PAE induces expression of NFkB/AP-1 transcription factors and production of IL-2 and IFN-γ by spleen cells of PAE treated but not in the untreated control mice. Furthermore, there were not IL-6, IL-10 nor TNF production in macrophages treated in vitro with PAE. We developed an affordable extract of P.andrieuxii effective to treat experimental Kala-azar in Balb/c mice.

Individual phases of contextual fear conditioning differentially modulate dorsal and ventral hippocampal GluA1-3, GluN1-containing receptor complexes and subunits

In contextual fear conditioning (CFC), the use of pharmacological and lesion approaches has helped to understand that there are differential roles for the dorsal hippocampus (DH) and the ventral hippocampus (VH) in the acquisition, consolidation and retrieval phases. Concomitant analysis of the DH and the VH in individual phases with respect to α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors and N-methyl-D-aspartate receptor subtype N1 (GluN1)-containing complexes (RCC) and subunits has not been reported so far. Herein, CFC was performed in mice that were euthanized at different time points. DH and VH samples were taken for the determination of RCC and subunit levels using BN- and SDS-PAGE, respectively, with subsequent Western blotting. Evaluation of spine densities, morphology, and immunohistochemistry of GluA1 and GluA2 was performed. In the acquisition phase levels of GluA1-RCC and subunits in VH were increased. In the consolidation phase GluA1- and GluA2-RCC levels were increased in DH and VH, while both receptor subunit levels were increased in the VH only. In the retrieval phase GluA1-RCC, subunits thereof and GluA2-RCC were increased in DH and VH, whereas GluA2 subunits were increased in the VH only. GluN1-RCC levels were increased in acquisition and consolidation phase, while subunit levels in the acquisition phase were increased only in the DH. The immunohistochemical studies in the individual phases in subareas of hippocampus supported immunochemical changes of GluA1 and GluA2 RCC's. Dendritic spine densities and the prevalence of thin spines in the acquisition phase of VH and mushroom spines in the retrieval phase of the VH and DH were increased. The findings from the current study suggest different receptor and receptor complex patterns in the individual phases in CFC and in DH and VH. The results propose that different RCCs are formed in the individual phases and that VH and DH may be involved in CFC.

A histological study of the kidney structure of Van fish (Alburnus tarichi) acclimated to highly alkaline water and freshwater

Kidneys are important organs that play a role in ion and water regulation in different aquatic environments. The Van fish (Alburnus tarichi, Güldenstädt, 1814) has acclimated to the alkaline waters of the largest saline soda lake in the world. In this study, changes in kidney tissue were examined histologically in alkaline and freshwater as the fish entered freshwater for reproduction. In addition, immunological changes were examined for Na+, K+, ATPase (NKA), an important transmembrane protein in kidneys. A histological comparison of the kidneys of fish taken from both environments was made. The glomerular volume was larger in fish acclimated to freshwater, and the collecting tubules were larger in diameter and had thicker walls. The fish acclimated to alkaline lake water had reduced numbers of glomeruli of smaller size. NKA enzyme was present in tubules within kidneys in both environments. It was, however, observed more frequently in fish acclimated to freshwater. Although plasma osmolality and Cl− values decreased in the fish acclimated to freshwater, hematocrit values increased (p < 0.05). No changes were observed in the muscle water content between alkaline water- and freshwater-acclimated fish. We clearly demonstrate that histologic and immunochemical changes take place in the kidney of the Van fish acclimated to the different physicochemical characteristics of alkaline and freshwater environments.

Prenatal administration of lipopolysaccharide induces sex-dependent changes in glutamic acid decarboxylase and parvalbumin in the adult rat brain

RationaleRecent clinical studies suggest GABA-ergic system abnormalities as a neuropathological mechanism of schizophrenia. ObjectivesIn the present study, we examined the effect of chronic prenatal lipopolysaccharide (LPS) administration on immunohistochemical changes of glutamate decarboxylase (GAD67) and parvalbumin (PV)-expressing neurons in the medial prefrontal cortex and hippocampus of rats. ResultsThese data demonstrated that prenatal LPS administration during the final 2weeks of pregnancy induced schizophrenia-like behavioral symptoms, such as deficits in sensorimotor gating (prepulse inhibition) and impairments in social interactions and exploration, in adult offspring. Moreover, immunohistochemical analysis revealed that in our neurodevelopmental model of schizophrenia, decreases in the total number of PV- and GAD67-positive neurons in the medial prefrontal cortices of adult females prenatally exposed to LPS were observed, whereas these immunochemical changes were primarily detected in the hippocampus of males. Additionally, a decrease in PV-labeled axon terminals of GABA-ergic cells, likely reflecting the perisomatic inhibitory innervation of pyramidal neurons, was observed in the medial prefrontal cortices in both sexes. ConclusionThis study provided evidence of a key role for the GABA system in neurodevelopment associated with the etiopathogenesis of schizophrenia and showed that the observed changes are sex-dependent. Moreover, this study is the first to present a model of schizophrenia based on prenatal LPS administration, which not only produced behavioral abnormalities but also changed the cytoarchitecture of the GABA inhibitory system.

Effects of adrenergic agents on stress-induced brain microstructural and immunochemical changes in adult male Wistar rats

This preliminary study aims to explore how adrenergic agents modulate stress response and affect stress-induced behavioral and brain changes in rodents. A total of 40 adult male Wistar rats were subjected to chronic unpredictable mild stress (CUMS) and randomly divided into five groups. At 30 min before daily stress exposure, the rats were intraperitoneally injected with phentolamine (5 mg/kg), noradrenalin (1.0 mg/kg), propranolol (10 mg/kg), isoproterenol (0.05 mg/kg) or saline, respectively. Another 8 rats served as normal control and received daily saline injection without stress exposure. Open-field behaviors were tested at 1 day after the end of the 21 days of stress exposure. Blood samples were collected for serum corticosterone measurement. Brain sections containing hippocampus were stained with hematoxylin and eosin (H&E) as well as by immunohistochemistry for heat shock protein 70 (hsp70) and nitric oxide synthase type 2 (nos2) analyses. The experimental results demonstrated that repetitive dosing of noradrenalin, phentolamine, and propranolol during chronic stress might region-dependently attenuate stress-induced microstructural and biochemical changes in the hippocampus, although propranolol intensified stress-induced behavioral changes.

Immunochemical changes of calbindin, calretinin and SMI32 in ischemic retinas induced by increase of intraocular pressure and by middle cerebral artery occlusion

The reaction of neuroactive substances to ischemic conditions in the rat retina evoked by different methods was immunochemically evaluated in adult Sprague-Dawley rats. Ocular ischemic conditions were unilaterally produced by elevating intraocular pressure (EIOP) or by middle cerebral artery occlusion (MCAO). Two EF-hand calcium binding proteins, calbindin D28K (CB) and calretinin (CR), in the normal retina showed similar immunolocalization, such as the amacrine and displaced amacrine cells, the ganglion cells, and their processes, particularly CB in horizontal cells. CB immunoreactive neurons in the ganglion cell layer in both types of ischemic retinas were more reduced in number than CR neurons compared to those in a normal retina. The CB protein level in both ischemic retinas was reduced to 60-80% of normal. The CR protein level in MCAO retinas was reduced to about 80% of normal but increased gradually to the normal value, whereas that in the EIOP showed a gradual reduction and a slight recovery. SMI32 immunoreactivity, which detects a dephosphorylated epitope of neurofilaments-M and -H, appeared in the axon bundles of ganglion cells in the innermost nerve fiber layer of normal retinas. The reactivity in the nerve fiber bundles appeared to only increase slightly in EIOP retinas, whereas a moderate increase occurred in MCAO retinas. The SMI32 protein level in MCAO retinas showed a gradual increasing tendency, whereas that in the EIOP showed a slight fluctuation. Interestingly, the MCAO retinas showed additional SMI32 immunoreactivity in the cell soma of presumed ganglion cells, whereas that of EIOP appeared in the Müller proximal radial fibers. Glial fibrillary acidic protein (GFAP) immunoreactivity appeared in the astrocytes located in the nerve fiber layer of normal retinas. Additional GFAP immunoreactivity appeared in the Müller glial fibers deep in EIOP retinas and at the proximal end in MCAO retinas. These findings suggest that the neurons in the ganglion cell layer undergo degenerative changes in response to ischemia, although EIOP retinas represented a remarkable Müller glial reaction, whereas MCAO retinas had only a small-scaled axonal transport disturbance.

Murine Glut-1 transporter haploinsufficiency: Postnatal deceleration of brain weight and reactive astrocytosis

Glucose transporter type 1 (Glut-1) facilitates glucose flux across the blood–brain-barrier. In humans, Glut-1 deficiency causes acquired microcephaly, seizures and ataxia, which are recapitulated in our Glut-1 haploinsufficient mouse model. Postnatal brain weight deceleration and development of reactive astrogliosis were significant by P21 in Glut-1 +/− mice. The brain weight differences remained constant after P21 whereas the reactive astrocytosis continued to increase and peaked at P90. Brain immunoblots showed increased phospho-mTOR and decreased phospho-GSK3-β by P14. After fasting, the mature Glut-1 +/− females showed a trend towards elevated phospho-GSK3-β, a possible neuroprotective response. Lithium chloride treatment of human skin fibroblasts from control and Glut-1 DS patients produced a 45% increase in glucose uptake. Brain imaging of mature Glut-1 +/− mice revealed a significantly decreased hippocampal volume. These subtle immunochemical changes reflect chronic nutrient deficiency during brain development and represent the experimental correlates to the human neurological phenotype associated with Glut-1 DS.

Effect of QTY06 on lipopolysaccharide-induced chronic airway inflammation and MUC5ac secretion in rats

To investigate the effect of synthetic drug QTY06 on chronic airway inflammation and mucoprotein expression induced by intratracheal (i.t) instillation of lipopolysaccharide (LPS). Chronic airway inflammation was induced by i.t instillation of LPS in rats. Phospholipids content and the number of leucocytes in bronchoalveolar lavage fluid (BALF), pathological and immunochemical changes were examined 3 weeks after LPS instillation. The effect of QTY06 on chronic airway inflammation was observed. After treatment with QTY06, phospholipids in BALF was significantly increased, and the percentages of neutrophils and lymphocytes were decreased as well as the total number of leucocytes. Compared with the model group, pathological examination showed that tracheitis, bronchitis and pulmonary interstitial inflammation in QTY06 groups were significantly attenuated; epithelial damage was alleviated, infiltration of inflammatory cells reduced and the number of goblet cells decreased. QTY06 significantly decreased MUC5ac expression in trachea and bronchiole epithelium, and reduced the optical density and mucins area (%) as detected by image analysis in rats with chronic airway inflammation. QTY06 can reduce and inhibit the chronic airway inflammation induced by LPS in rats, and increase the content of phospholipids in pulmonary surfactant and inhibit the hypersecretion of airway mucins.

Identifying coronary artery flow reduction and ischemia using quasistationary QT/RR-interval hysteresis measurements

Because myocardial ischemia induces QT/RR hysteresis, a correlation was hypothesized to exist between the extent of myocardial flow reduction and the magnitude of QT/RR hysteresis. Graded reductions in regional myocardial perfusion in the distribution of the left anterior descending coronary artery in open-chest pigs were used to model 1-vessel coronary artery disease. At each reduced level of left anterior descending coronary artery flow, the heart was electrically paced at progressively higher and lower rates between an initial control and maximum heart rate values. Digitized surface and intramyocardial electrograms and aortic pressure were used to measure QT/RR hysteresis, QT-interval adaptation, ST- and TQ-segment depression, and cardiac contractility. Intraexperimental blood samples were analyzed to assess inflammatory response (interleukin 6), oxidative stress (protein carbonyls), and myocyte injury (creatine kinase). Higher values of QT/RR hysteresis correlated with the severity of ischemia as assessed by TQ-segment depression in intramyocardial electrograms ( P = .002). Lower flow rates were strongly associated with higher values of QT/RR hysteresis and slower QT-interval adaptation ( P ≤ .004), and were less correlated with the magnitude of ST- and TQ-segment depression ( P ≥ .02). Significant increases in systemic measures of inflammation, oxidative stress, and cardiac myocyte injury and major decrease in cardiac contractility preceded the most severe stages of flow reduction (30% and 20% of normal flow). We determined QT/RR hysteresis index thresholds corresponding to these mechanical and immunochemical responses. QT/RR hysteresis is a strong indicator of reduced myocardial perfusion and may provide information for noninvasive assessment of mechanical and immunochemical changes associated with early stages of coronary artery disease.

Ultrastructural and Immunochemical Changes During the In Vitro Development of Plasmopara halstedii

ABSTRACT The asexual phase of the life cycle of Plasmopara halstedii, the causal agent of downy mildew of sunflower, plays a key role in the propagation of the disease. We investigated the morphological and ultrastructural changes that occur during the asexual development of the pathogen. Direct examination of infected cotyledons confirmed the presence of sporangiophores. In contact with water, important ultrastructural changes occurred, affecting the surface of zoosporangia, which became smoother, and their cytoplasm, which differentiated into flagellate zoospores. The subsequent encystment of zoospores was characterized by the synthesis of a cell wall and the loss of the flagella. In addition, two monoclonal antibodies (MAbs) specific for P. halstedii were used to analyze the immunochemical changes associated with these modifications. MAb 16A6, which bound to a 48-kDa glycoprotein, mainly labeled the surface of mobile or encysted zoospores and of mother cells of germ tubes. Conversely, MAb 2F9, which recognized highly glycosylated antigens, labeled the surface of zoosporangia and of flagellate zoospores, but not the encysted zoospores. These results provide new insights into the morphological and ultrastructural changes associated with the release and the encystment of zoospores which may be interesting targets for the development of new antimicrobial products.

Autosomal dominant dilated cardiomyopathy with atrioventricular block: a lamin A/C defect-related disease

ObjectivesWe investigated the prevalence of lamin A/C (LMNA) gene defects in familial and sporadic dilated cardiomyopathies (DCM) associated with atrioventricular block (AVB) or increased serum creatine-phosphokinase (sCPK), and the corresponding changes in myocardial and protein expression. BackgroundIt has been reported that familial DCM, associated with conduction disturbances or variable myopathies, is causally linked to LMNA gene defects. MethodsThe LMNA gene and myocardial ultrastructural and immunochemical changes were analyzed in 73 cases of DCM (49 pure, 15 with AVB [seven familial, eight sporadic], 9 with increased sCPK), four cases of familial AVB and 19 non-DCM heart diseases. The normal controls included eight heart donor biopsies for tissue studies and 107 subjects for LMNA gene studies. ResultsFive novel LMNA mutations (K97E, E111X, R190W, E317K, four base pair insertion at 1,713 cDNA) were identified in five cases of familial autosomal dominant DCM with AVB (5/15: 33%). The LMNA expression of the myocyte nuclei was reduced or absent. Western blot protein analyses of three hearts with different mutations showed an additional 30-kDa band, suggesting a degrading effect of mutated on wild-type protein. Focal disruptions, bleb formation and nuclear pore clustering were documented by electron microscopy of the myocyte nuclear membranes. None of these changes and no mutations were found in the nine patients with DCM and increased sCPK or in the disease and normal controls. ConclusionsThe LMNA gene mutations account for 33% of the DCMs with AVB, all familial autosomal dominant. Increased sCPK in patients with DCM without AVB is not a useful predictor of LMNA mutation.

Melanocyte-Associated T Cell Epitopes Can Function as Autoantigens for Transfer of Alopecia Areata to Human Scalp Explants on Prkdcscid Mice

Alopecia areata is a tissue restricted autoimmune condition affecting the hair follicle, resulting in hair loss. The goal of this study was to test the hypothesis that the autoantigen of alopecia areata is melanocyte associated. Potential autoantigens were tested in the human scalp explant/Prkd(scid) CB-17 mouse transfer system. Scalp T cells from lesional (bald) alopecia areata scalp were cultured with antigen-presenting cells, and antigen, along with interleukin-2. The T cells were then injected into autologous lesional scalp grafts on SCID mice, and hair regrowth was measured. Hair follicle homogenate was used as an autoantigen control. T cells cultured with melanoma homogenate induced statistically significant reduction in hair growth (p <0.01 by ANOVA). HLA-A2-restricted melanocyte peptide epitopes were then tested with lesional scalp T cells from HLA-A2-positive alopecia areata patients. Melanocyte-peptide-activated T cells significantly reduced the number of hairs regrowing in two experiments with six patients (p <0.001 by ANOVA). Injected scalp grafts showed histologic and immunochemical changes of alopecia areata. The most consistent peptide autoantigens were the Gp100-derived G9-209 and G9-280 peptides, as well as MART-1 (27-35). Melanocyte peptide epitopes can function as autoantigens for alopecia areata. Multiple peptides were recognized, suggesting epitope spreading.

Alopecia areata is a T-lymphocyte mediated autoimmune disease: lesional human T-lymphocytes transfer alopecia areata to human skin grafts on SCID mice.

Much evidence suggests that alopecia areata is a tissue restricted autoimmune disease. Alopecia areata responds to immunosuppressive agents, and is associated with other tissue restricted autoimmune diseases, including autoimmune thyroiditis and vitiligo. Furthermore, hair regrows when involved scalp is transplanted to nude mice. This study was undertaken to determine whether alopecia areata is mediated by T lymphocytes. Involved scalp from alopecia areata patients was grafted onto SCID mice. Additional biopsies from lesional scalp of the same patients were used to isolate T lymphocytes. These T lymphocytes were cultured with hair follicle homogenate, as well as autologous antigen presenting cells. The T lymphocytes were then injected into autologous scalp grafts on the SCID mice, which had regrown hair. Injection of scalp T lymphocytes resulted in hair loss. Hair loss was associated with the histologic and immunochemical changes of alopecia areata, including perifollicular infiltrates of T cells, along with HLA-DR and ICAM-1 expression by the follicular epithelium. Scalp T lymphocytes that had not been cultured with hair follicle homogenate did not have this effect. Preliminary data suggests hair loss requires a collaboration between CD8+ and CD4+T cells. These studies have demonstrated that alopecia areata can be induced by the transfer of T cells that recognize a hair follicle autoantigen.

Neuronal abnormalities in microtubule-associated protein 1B mutant mice.

Microtubules play an important role in establishing cellular architecture. Neuronal microtubules are considered to have a role in dendrite and axon formation. Different portions of the developing and adult brain microtubules are associated with different microtubule-associated proteins (MAPs). The roles of each of the different MAPs are not well understood. One of these proteins, MAP1B, is expressed in different portions of the brain and has been postulated to have a role in neuronal plasticity and brain development. To ascertain the role of MAP1B, we generated mice which carry an insertion in the gene by gene-targeting methods. Mice which are homozygous for the modification die during embryogenesis. The heterozygotes exhibit a spectrum of phenotypes including slower growth rates, lack of visual acuity in one or both eyes, and motor system abnormalities. Histochemical analysis of the severely affected mice revealed that their Purkinje cell dendritic processes are abnormal, do not react with MAP1B antibodies, and show reduced staining with MAP1A antibodies. Similar histological and immunochemical changes were observed in the olfactory bulb, hippocampus, and retina, providing a basis for the observed phenotypes.

In Vivo Prevention of Corticosteroid-Induced Skin Atrophy by Tretinoin in the Hairless Mouse Is Accompanied by Modulation of Collagen, Glycosaminoglycans, and Fibronectin

In an earlier study we showed that tretinoin could prevent corticosteroid-induced skin atrophy in hairless mice. In this study, we examined the histochemical, biochemical, and immunochemical changes that accompanied the atrophy and its prevention. Mice were treated dorsally for 3 weeks in the morning and afternoon (AM:PM) as follows: 1) vehicle:vehicle, 2) steroid:vehicle, 3) steroid:tretinoin. Tretinoin concentration was 0.05% in an ethanol:propylene glycol vehicle. The steroid was clobetasol propionate (0.05%). The normally sparse dermal glycosaminoglycans were further reduced by steroid:vehicle treatment and increased to greater than vehicle:vehicle amounts by steroid:retinoid. Mast cells were similarly affected. Biochemical quantification of glycosaminoglycans confirmed the histochemical findings. Collagen, non-collagenous protein, and total protein content were reduced by the steroid. The latter two were returned to more normal levels by tretinoin whereas with collagen there was only a trend toward normal levels. Fibronectin, which was increased by the steroid:vehicle treatment, was reduced to more normal levels by steroid:tretinoin. We conclude that tretinoin has the ability to prevent the major steroid-induced biomechanical changes in hairless mouse dermal connective tissue that contribute to atrophy.

Evaluation of immunochemical changes in ovalbumin due to reaction with oxidizing methyl linoleate by an enzyme linked immunosorbent assay.

1. An enzyme linked immunosorbent assay (ELISA) was used to study the immunochemical properties of ovalbumin altered by the reaction with oxidizing methyl linoleate under various conditions. 2. The results indicated that the values of ELISA decreased with an increase of the degree of ovalbumin following reaction with oxidizing methyl linoleate. 3. The antigenicity of ovalbumin, when examined by competitive inhibition assay, reduced about 20-fold as compared with native ovalbumin when reacted with oxidizing methyl linoleate at 50 degrees C and water activity (0.68) for 30 days.

Characterization of two anti-DNA antibodies bearing distinct idiotypes. Correlation with clinical manifestations

Spontaneous idiotype shift of anti-DNA antibodies in systemic lupus erythematosus was shown to be associated with changes in the clinical manifestations of the disease. Characterization of two anti-DNA antibody preparations from the same lupus patient showed that lupus nephritis was associated with antibodies showing higher specific antigen-binding activity and avidity to DNA and presenting more cationic quality upon isoelectric focusing and Western blot analysis than the ones found in patients without renal involvement. Lupus cerebritis was associated with anti-DNA antibodies with different idiotypic characteristics not shared by anti-DNA antibodies present in lupus nephritis. Lupus cerebritis anti-DNA antibodies had less specific antigen-binding activity and avidity to DNA and did not show cationic quality. Idiotypic and immunochemical changes of anti-DNA antibodies in lupus may be associated with various clinical manifestations.

Renal distribution of collagen types III, IV and V in various glomerular diseases

The purpose of this study is to examine the immunochemical changes of the glomerular basement membrane (GBM) and the mesangium, in pretreated paraffin-embedded sections with trypsin by utilizing monoclonal antibodies to type III (anti-III), type IV (anti-IV) and type V (anti-V) collagens. We observed 6 normal kidneys and 44 kidneys with various renal diseases. In normal human kidney the staining with anti-IV demonstrated GBM, mesangium, Bowman's BM, tubular BM and capillary BM. Anti-V was also seen in the interstitium. On the other hand, anti-III stained only interstitium. Thickened GBM in membranoproliferative glomerulonephritis (MPGN) and diabetic nephropathy, and irregular GBM in Membranous Nephropathy and Alport's syndrome were also evident in anti-IV stain, while widened mesangial area was seen in anti-V rather than anti-IV stain. In severely proliferative GN, anti-III as well as anti-IV and anti-V was detected in the mesangium in spite of existence of neither adhesion nor Bowman's gap. In MPGN type II, anti-III was observed along the GBM. In obsolescent glomeruli, anti-IV was not always detected although anti-V was constantly seen. On the other hand, anti-III was markedly positive in the crescents and obsolescent glomeruli. These results suggest that it is possible for mesangial, endothelial and epithelial cell to produce several types of collagens and type III collagen is closely related to the process of the glomerular obsolescence.