Periodic Acid-silver Methenamine Staining Research Articles

Trichoderma longibrachiatum-Associated Skin Inflammation and Atypical Hyperplasia in Mouse.

BackgroundThe relationship between infection and tumors has attracted increasing attention. Trichoderma spp. are often isolated from tumors. However, their potential role remains unclear. We recently reported the isolation of Trichoderma longibrachiatum from a patient with pulmonary spindle cell carcinoma that was confirmed as primary infection by application of laser capture microdissection and polymerase chain reaction. To explore whether the strain is pathogenic and whether it can cause atypical cell proliferation and infiltration of NK cells and T cells, we designed a mouse infection experiment.MethodsTwelve ICR mice were randomly separated into three groups. Cyclophosphamide was used to inhibit the immunity of mice. A mouse model of Trichoderma infection was successfully established by intracutaneous injection on the back skin with a suspension of strain PKUT180420015. The pathological manifestations of Trichoderma infection and the interaction between immune cells and fungi were observed by histopathology, immunohistochemistry and intensive fungal staining. Reisolation of the fungus was observed by infected tissue culture. The inoculated sites exhibited swelling 3 days after inoculation, and ulcers developed at approximately 14 days. Skin specimens were obtained and then cultured at 3, 7, and 14 days after inoculation. We selected mice 14 days after inoculation in Group 3, whose ulcers were the most typical, for histological analysis.ResultsInflammation, angioinvasion and necrosis were observed. Immunohistochemistry showed positive markers of Ki67, CD3, CD56, GZMB, and PRF. Periodic acid-Schiff staining, periodic acid-silver methenamine staining, and Calcofluor staining showed fungal spores in the vascular lumen, vascular walls and around the blood vessels.ConclusionsOur studies showed that a T. longibrachiatum strain (PKUT180420015) isolated from a biopsy specimen in a patient with pulmonary spindle cell carcinoma could induce atypical hyperplasia, with the expression of Ki67, CD3, CD56, GZMB, and PRF in mice. These data indicate that the fungus may be involved in inducing atypical hyperplasia or tumorigenesis.

Quality improvement Initiative to eliminate silver precipitate during Periodic Acid Silver Methenamine Stain (PAMS) automated protocol of renal biopsies at McGill University Health Centre

Abstract Introduction/Objective Annually, about 400 renal biopsies are processed at the McGill University Health Centre (MUHC) pathology laboratory located in Montreal, Canada. One of the stains used to visualize the glomerular basement membrane is Periodic Acid Silver Methenamine Stain (PAMS). In August 2020, a strong, granular precipitate of silver was noted during PAMS automated staining resulting in uninterpretable results and delay in the diagnosis. Based on a sample analysis, this problem affected 21 % of kidney biopsies. Methods/Case Report A cause-and-effect workflow was developed for systematic assessment of potential causes of the granular precipitate including pre-analytical and analytical factors. Some of the pre-analytical factors included length of time spent in transport before fixation and patient factors that predisposed precipitate formation. Analytical factors were categorized as fixation problems (temperature, pH, duration), embedding problems (parafilm temperature, cooling method, type of parafilm), slide preparation (temperature, water bath pH, dehydration and further processing steps), microtone parameters (microtone calibration, thickness, laboratory technologist expertise), automatic staining parameters (cartridge age, hematoxylin counterstain duration, wash-out period etc.) and coverslip placement (adhesive type, temperature, drying). Results (if a Case Study enter NA) Following our systematic approach, the cause of granular precipitate was identified as the timing of hematoxylin counterstain. A portion of renal biopsy tissue was taken from parafilm blocks of previouslly reported cases of patients with membranous glomerulonephritis to further test the hypothesis by introduction of various incubation times with the hematoxylin counterstain. Conclusion Best PAMS staining was attained when no hematoxylin counterstain was employed (instead, neutral red counterstain for 70 seconds was used). PAMS staining with hematoxylin counter stain for no more than 60 seconds was found to be acceptable for the interpretation of glomerular pathology.

A case report of fungal infection associated acute fibrinous and organizing pneumonitis

BackgroundAcute fibrinous and organizing pneumonitis (AFOP) is an uncommon variant of acute lung injury, characterized by intra-alveolar fibrin and organizing pneumonia. Proposed etiologies include connective tissue diseases, infections, occupational exposure, drug reactions, and autoimmune disease. Here we present a rare case of fungal infection associated AFOP in patient with diabetes mellitus (DM) and review the relevant literature.Case presentationA 67-year-old man complained of cough, fever, dyspnea and hemoptysis. Patient experienced a rapidly progressive course exhibit diffuse predominant consolidation, ground glass opacities, and multifocal parenchymal abnormalities on chest computed tomography (CT). Antibacterial, antifungal, and antiviral treatments were ineffective. A CT-guided percutaneous lung biopsy was performed. Histologically, the predominant findings were as follows: alveolar spaces filled with fibrin and organizing loose connective tissues involving 70% of the observed region, pulmonary interstitial fibrosis, and small abscesses and epithelioid cell granuloma in the focal area. Result of periodic acid-silver methenamine stain was positive. The fungal pathogen from the sputum culture was identified as P. citrinum repeatedly over 3 times. Patient was diagnosed with DM during hospitalization. Corticosteroids combined with an antifungal therapy were effective. Follow-up for 4 months showed complete radiological resolution.ConclusionsAs this common “contaminant” can behave as a pathogen in the immunocompromised host, both clinicians and microbiologists should consider the presence of a serious and potentially fatal fungal infection on isolation of P. citrinum. Based on this case, it could be speculated that AFOP may be associated with fungal infection including P. citrinum.

Tacrolimus increases the expression level of the chemokine receptor CXCR2 to promote renal fibrosis progression.

Tacrolimus is one of the most used and effective immunosuppressive agents currently available in the clinic; however, its use is limited by nephrotoxicity, which is the main secondary effect of this drug. The mechanisms underlying tacrolimus-induced nephrotoxicity remain unknown. The present study aimed to investigate the mechanism underlying tacrolimus-induced nephrotoxicity and to identify novel potential targets. Masson staining, Sirius red staining and periodic acid-silver methenamine staining were used to observe kidney pathological changes. Immunohistochemical and immunofluorescent analyses were performed to examine the expression levels of vimentin, E-cadherin and α-smooth muscle actin (α-SMA). Transcriptomics and bioinformatics analyses were performed to investigate the nephrotoxicity mechanism induced by tacrolimus using RNA-sequencing, differentially expressed genes identification and annotation, and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. The present results demonstrated that compared with the normal control group, the tacrolimus nephrotoxicity group exhibited severe renal fibrosis (P<0.05), upregulated vimentin (P<0.01), downregulated E-cadherin (P<0.05) and upregulated α-SMA (P<0.01). Transcriptomics and bioinformatics analyses identified the pathway 'cytokine-cytokine receptor interaction' as the most significantly enriched (P<0.05). Moreover, KEGG pathway enrichment analysis identified that tacrolimus increased the expression levels of chemokine (C-X-C) motif ligand (CXCL)1, CXCL2 and CXCL3 and the chemokine receptor C-X-C chemokine receptor type 2 (CXCR2). Collectively, the present study suggested that tacrolimus increases the level of chemokine receptor CXCR2 to promote renal fibrosis progression, which is one of the potential mechanisms underlying tacrolimus-induced nephrotoxicity.

Allotransplantation using a diseased kidney: when a swallow makes a summer.

In this issue of Nephrology, Dialysis, Transplantation, Mirza et al. [1] present a very interesting but also puzzling case of membranous nephropathy (MN) transplanted in a patient with a history of long-term diabetes mellitus and severe atherosclerosis. Simultaneous heart and kidney transplantation were carried out at a time when the recipient’s estimated glomerular filtration rate was 28 mL/min and proteinuria 1+. His native kidney histology was unknown and although the aetiology of his nephropathy was multifactorial, it can be safely ascertained that the grafted kidney benefitted, in a broad sense, from a ‘non-immunologic’ environment. This case thus provides a unique opportunity to observe the outcome of immune deposits after cessation of the immunological aggression. The donor was a young woman who had died from a hypertensive subarachnoid haemorrhage. Prior to her death, she was not proteinuric. A pre-transplant biopsy of the donor’s kidney led to the discovery of lesions of Stages II, III and IV MN, a puzzling finding in the absence of proteinuria. The authors hypothesize that the preservation of the glomerular filtration barrier to proteins might be explained by the mild podocyte injury that was observed on the four serial biopsies that were performed up to Day 812. Each yielded a substantial number of glomeruli and showed by light microscopy, electron microscopy and immunofluorescence, a striking improvement of the initially severe membranous lesions. As in the donor, and despite persistence of Stages II and III glomerular basement membrane (GBM) lesions and immunoglobulin and complement staining, proteinuria was never significant and the kidney recipient maintained good function. This case is exceptional, although not the first one describing progressive wash-out of membranous immune deposits in kidneys transplanted into a non-immunologic environment. Parker et al. [2] reported the case of a patient with end-stage renal disease (ESRD) secondary to Type II diabetes mellitus who received a cadaveric renal transplant from a 37-year-old woman who died of cerebral infarction. A biopsy of the donor’s kidney performed at the time of transplantation showed MN by electron microscopy. There was immediate graft function and the recipient continued to have good kidney function 3 years post-transplantation. Nakazawa et al. [3] presented the case of a 41-year-old man grafted with a cadaver kidney. An allograft biopsy obtained during the operation showed spike formation on periodic acid-silver methenamine staining and deposition of IgG along the glomerular capillary loop on immunoperoxidase staining. Immunofluorescence staining for IgG persisted in the specimens obtained on Day 11 and after 4 weeks, but markedly decreased in the specimen obtained 7 weeks after transplantation. Electron-dense deposits also decreased in amount, but irregular thickening of the glomerular basement membrane with spikes, electron-lucent lesions and small amounts of electron-dense deposits remained 20 months after the transplantation. Akioka et al. [4] transplanted a kidney from a father, diagnosed with MN and mild proteinuria, to his son. At 39 months after transplantation, allograft biopsy showed a wash-out of electron-dense deposits and electron-lucent lesions. These reports are interesting but were not analyzed as meticulously as Mirza et al. did in their case. Their recipient’s kidney was studied by a pre-transplant histology followed by four biopsies up to 27 months post-transplantation. Along with light and electron microscopy, they used a wide panel of antibodies for immunofluorescence including staining for type-M phospholipase A2 receptor (PLA2R) that was negative. This is suggestive of a secondary aetiology of membranous glomerulonephritis (GN) but does not exclude a PLA2R-unrelated primary form [5]. PLA2R is a major target antigen in autoimmune idiopathic membranous GN. PLA2R is revealed in normal human glomeruli, and in idiopathic MN, PLA2R and IgG4 are shown to co-localize within subepithelial deposits. Early recurrence of MN in kidneys transplanted to recipients with circulating anti-PLA2R antibodies lends credit to a pathogenic role of these antibodies [6–8]. Their decrease IN F O C U S

Effects of vitamin E on mercuric chloride-induced renal interstitial fibrosis in rats and the antioxidative mechanism

To observe the effects of vitamin E (Vit E) on mercuric chloride (HgCl2)-induced renal interstitial fibrosis (RIF) in rats and discuss its antioxidative mechanism. A total of 32 Sprague-Dawley rats were randomly assigned to three groups: normal group, model group and Vit E group. RIF was induced by oral administration of HgCl(2) at a dose of 8 mg/kg body weight once a day for 9 weeks. Rats in Vit E group were administered with Vit E capsule at 100 mg/kg body weight, and rats in normal and model groups were treated with normal saline. At the end of the 9th week, rats were sacrificed and renal hydroxyproline (Hyp)'s trichrome and periodic acid-silver methenamine (PASM) staining. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and contents of glutathione (GSH) and malondialdehyde (MDA) in kidney tissue were tested with commercial kits. The expressions of nuclear factor-κB (NF-κB), inhibitor-κB (IκB), phospho-IκB (p-IκB) and tumor necrosis factor-α (TNF-α) were determined by Western blot. The expression of α-smooth muscle actin (α-SMA) was assayed by Western blot and immunofluorescent staining. Renal Hyp content, HE, Masson's trichrome and PASM staining results and α-SMA expression confirmed development of HgCl2-induced RIF in rats. Oxidative stress markers GSH, GSH-Px and MDA confirmed oxidative stress in RIF rats. Compared with model rats, rats in Vit E group had lower kidney Hyp content (P<0.01). GSH and MDA contents decreased significantly in Vit E group compared with model group (P<0.01). The expressions of NF-κB and IκB had no significant difference among all groups (P>0.05). In Vit E group, the expressions of p-IκB and TNF-α decreased significantly compared with model group (P<0.01). The expression of α-SMA in Vit E group was also decreased significantly compared with model group (P<0.01). Vit E has a protective effect on experimental RIF induced by HgCl(2) in rats and it is related to inhibition of lipid peroxidation, which involves blocking of NF-κB signaling pathway and the activation of cells producing extracellular matrix.

Genetic Podocyte Lineage Reveals Progressive Podocytopenia with Parietal Cell Hyperplasia in a Murine Model of Cellular/Collapsing Focal Segmental Glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a progressive renal disease, and the glomerular visceral cell hyperplasia typically observed in cellular/collapsing FSGS is an important pathological factor in disease progression. However, the cellular features that promote FSGS currently remain obscure. To determine both the origin and phenotypic alterations in hyperplastic cells in cellular/collapsing FSGS, the present study used a previously described FSGS model in p21-deficient mice with visceral cell hyperplasia and identified the podocyte lineage by genetic tagging. The p21-deficient mice with nephropathy showed significantly higher urinary protein levels, extracapillary hyperplastic indices on day 5, and glomerular sclerosis indices on day 14 than wild-type controls. X-gal staining and immunohistochemistry for podocyte and parietal epithelial cell (PEC) markers revealed progressive podocytopenia with capillary collapse accompanied by PEC hyperplasia leading to FSGS. In our investigation, non-tagged cells expressed neither WT1 nor nestin. Ki-67, a proliferation marker, was rarely associated with podocytes but was expressed at high levels in PECs. Both terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy failed to show evidence of significant podocyte apoptosis on days 5 and 14. These findings suggest that extensive podocyte loss and simultaneous PEC hyperplasia is an actual pathology that may contribute to the progression of cellular/collapsing FSGS in this mouse model. Additionally, this is the first study to demonstrate the regulatory role of p21 in the PEC cell cycle.

Ultrastructural and Immunohistochemical Analysis of Fibrous Long–Spacing Collagen Fibrils in Malignant Mesothelioma

Three cases of biphasic mesothelioma and 2 cases of sarcomatoid mesothelioma were investigated using light and electron microscopy. In 2 of the 3 cases of biphasic mesotheliomas, fibrous long-spacing (FLS) collagen fibrils were discovered with a symmetrical cross-striation of 130 nm in periodicity. However, no connection between the FLS fibrils and usual collagen fibrils were observed. Periodic acid silver methenamine stain revealed unstained bands with periods of 130 nm in FLS fibrils, whereas the usual collagen fibrils showed continuous positive staining. All 3 cases of biphasic mesotheliomas showed deposits of hyaluronic acid, whereas both cases of sarcomatoid mesotheliomas showed little hyaluronic acid. As a high concentration of hyaluronic acid induces the formation of FLS collagen fibrils in vitro, the authors propose that FLS fibrils from mesothelioma may be special structures that occur as the tropocollagens are assembled into new collagen fibrils in the presence of hyaluronic acid.

A case report of glomerulopathy-associated podocytic infolding in a patient with tumor lysis syndrome

A 59-year-old man underwent total gastrectomy and splenectomy for gastric cancer 14 months before admission. The pathological diagnosis was neuroendocrine carcinoma of the stomach. Eight months after the operation, systemic chemotherapy with irinotecan and cisplatin was started because of multiple metastases to lymph nodes. After two courses of chemotherapy, renal function continued to decline. Renal biopsy showed acute tubular necrosis with cast formation, where needle crystallization was found. These clinicopathological findings suggested that tumor lysis syndrome was the cause of acute renal insufficiency. Moreover, diffuse, global bubbling and focal segmental spike formation were revealed by periodic acid-silver methenamine stain in the glomerular basement membrane. Electron microscopy showed an infolding of the cytoplasm of podocytes into the basal basement membrane and spotty electron-lucent areas. These ultrastructural findings, but not epimembranous deposits, corresponded with the bubbling on PAM staining. The present case was a rare case of glomerulopathy associated with podocytic infolding, which was not associated with collagen disease but with tumor lysis syndrome.

The kidney expression of matrix metalloproteinase-9 in the diabetic nephropathy of Kkay mice

Background Disorder of the extracellular matrix has been suggested to play a role in the development of diabetic nephropathy. The aim of the present study was to examine the expression of matrix metalloproteinase-9 (MMP-9) in the kidney of Kkay mice, a type 2 diabetic model. Methods Eight Kkay control mice (KA) and eight Kkay diabetic mice (KB) were fed with the same diets for 4 months and sacrificed after glucose levels and weight were measured. The renal pathological changes were examined by periodic acid–Schiff and periodic acid–silver methenamine staining, and the glomerulosclerosis indexes (GIs) were calculated. The levels of MMP-9 protein in the kidney tissue were assessed by immunohistochemistry. The gene expression of MMP-9 was measured by reverse transcription polymerase chain reaction. Results The diabetic group had morphological findings of diabetic nephropathy, and their GI (240.0±17.3) was much higher than that of the control group (118.4±15.1) ( P<.01). The mean positive area of MMP-9 protein in the glomeruli of Kkay mice in the KB group was higher than that in the KA group. The expression of MMP-9 messenger RNA level in the diabetic KB group was up-regulated when compared with the KA group. Conclusion The expression of MMP-9 in the kidney of mice with diabetic nephropathy was enhanced, and MMP-9 might be involved in the development of diabetic nephropathy.

A simple method for the specific detection of Ren-1 renin

Ren-1 and Ren-2 renin are expressed in the kidneys of all mice and in the submandibular gland of several mouse strains. The present study determined the usefulness of modified periodic acid silver-methenamine (PAM) staining for the specific detection of Ren-1 renin. Conventional paraffin sections were prepared from kidneys of ICR, BALB/cA, C57BL/6Cr, C3H/HeN, DBA/2Cr, angiotensin II type 1a receptor gene knockout (AT1aKO) mice, Wistar rats and a human, and submandibular glands of C57BL/6Cr and DBA/2Cr mice. Sections were analyzed for the presence of renin using PAM and immunohistochemistry. PAM reactions were terminated at generally or weakly intense (weak PAM staining; W-PAM). In addition, kidneys of DBA/2Cr mice were fixed using various fixatives (formalin, PFA, PLP, Zamboni's, Bouin's, or Carnoy's) and treated using identical procedures. Although PAM-positive reactions were observed in juxtaglomerular (JG) cells, W-PAM reactions were particularly specific for these cells. These findings were observed in all mouse strains. Immunohistochemistry using mirror sections suggested that a W-PAM-positive reaction detected renin. This hypothesis was confirmed by the results from AT1aKO mice. Briefly, W-PAM detected an expansion of renin-positive areas in AT1aKO mice. Rat and human kidneys and mouse submandibular glands were negative for W-PAM. Levels of JG cell detection by W-PAM were similar in samples fixed in formalin, PFA, PLP, or Zamboni's. The present findings show that W-PAM can identify Ren-1 renin, but not Ren-2, rat or human renin. The W-PAM method is useful for the specific detection of Ren-1 renin.

Silver staining of collagen fibrils in cartilage

Direct visualization of individual collagen fibrils by light microscopy in human cartilage was achieved by applying a periodic acid-silver methenamine stain on plastic sections. Collagen fibrils, 100 nm in diameter or thicker, were delineated individually by light microscopy and were easily traced for a length beyond 100 microns. Thinner fibrils not readily visible optically were identified if arranged in compact bundles as occurring in the superficial zone of articular cartilage.

Electron-Microscopic and Inimunohistochemical Study of Beta-2-Microglobulin-Related Amyloidosis

beta 2-Microglobulin (beta 2-MG)-related amyloidosis has been reported as a complication in long-term hemodialysis patients. We observed beta 2-MG amyloid deposits in synovial sheaths, bone cysts and gastric mucosa. They showed unique ultrastructural features, that is bundles or nodules consisting of curved or linear amyloid fibrils, associated with various cell reactions. The electron-microscopic histochemical study showed that they strongly stained with periodic acid-silver methenamine stain. A similar phenomenon was noticed in the spicules or bundles of amyloid fibrils in primary and secondary renal amyloidosis. With the cationic reagent toluidine blue 0, proteoglycan-like structures were observed around amyloid bundles and nodules, but not on each fibrils. Based on these results, we postulate that there is a close relationship between ultrastructural features and histochemical characteristics in beta 2-MG amyloid fibrils.

Microcyst formation in the plasmodial slime moldDidymium iridis

Myxamoebae ofDidymium iridis were removed from the bacterial food source and induced to encyst by transfer to 10 mM phosphate buffer. After 24 hours of induction approximately 90% of the myxamoebae had differentiated into microcysts. The kinetics of encystment were not significantly affected by pH or osmolarity of the encystment medium. Early stages of encystment were distinguished by the appearance of autophagic vacuoles and an extracellular “slime-like” sheath. The outer wall layer, consisting of dense fibrils, was unevenly deposited after 4 hours. An electron-lucent, second wall layer appeared between 5–10 hours followed by a densely packed, third wall layer adjacent to the plasma membrane. Wall formation appeared to involve smooth-membraned vesicles of possible Golgi origin. The vesicle contents and outer wall layer reacted with the periodic acid-silver methenamine stain for polysaccharide. The density of intramembrane particles of the protoplasmic fracture face increased during encystment with a gradual formation of aggregates of particles.

An Electron Microscopic Study of the Location of κ-Casein in Casein Micelles by Periodic Acid-Silver Methenamine Staining

The periodic acid-silver methenamine staining method for carbohydrate was applied to ultrathin sections of fixed casein micelles to determine the location of κ-casein in these particles. Staining for 60min resulted in the deposition of silver in uniformly distributed regions throughout all sizes of micelles. A shorter staining procedure (10min) produced evenly distributed silver grains in small micelles (apparent diameter less than 100nm) while in large micelles (apparent diameter more than 220nm) the metal grains were deposited in the outer portions with few in the center regions. This is interpreted as indicating that while κ-casein as a whole is distributed throughout the casein micelle, the more highly glycosylated molecules of the protein are located in the outer portions of the particle.

Electron histochemistry and ultrastructural localization of carbohydrate-containing substances in the sheath of Volvox.

The location and characteristics of carbohydrate-containing structures within the intact sheath of Volvox were studied by 3,3'-diaminobenzidine tetrahydrochloride-osmium, colloidal iron, colloidal thorium, ruthenium red and periodic acid-silver methenamine staining. The sheath consists of external and internal fibrillar layers separated by a tripartite structure. The external layer reacts positively with 3,3'-diaminobenzidine tetrahydrochloride, colloidal iron, colloidal thorium and ruthenium red, indicating that it contains acid mucosaccharides. Staining in the external layer is abolished by Ba(OH)2 treatment. The tripartite structure and internal fibrillar layer contain periodic acid reactive groups which do not occur in the external layer. Under certain conditions, reactions between the cationic dyes and the internal material were also observed. It is postulated that the internal matrix of the sheath contains glycoproteins or a mixture of acid mucosaccharides and glycoproteins. Possible functions of the sheath material are discussed.

Electron Microscopic Studies of Kidney with Periodic Acid Silver Methenamine Staining

Electron Microscopic Studies of Kidney with Periodic Acid Silver Methenamine Staining