Immunoperoxidase Staining Research Articles

Multi-Tissue Controls and Multiplex Immunocytochemistry in Pulmonary Cytology

Introduction: The World Health Organization 2021 lung cancer classification highlights the central role of immunohistochemistry (IHC) in diagnostic pathology. Despite traditional IHC being essential, its limitation to one marker per tissue section brings challenges, particularly when facing cytological limitedly sized samples. To overcome these challenges, multiplex immunocytochemistry (mICC) techniques offer the simultaneous detection of multiple markers from a single section. These advances complement the highly complex imaging techniques that enable additional analyses of cellular interactions. Methods: The present study outlines a comprehensive mICC methodology of an automated multiplex immunoperoxidase staining method and multiple tissue hybrid controls for ICC/mICC. Protocols are presented in detail and demonstrate a careful approach to optimizing various markers for diagnostic workup including immunotherapy. Conclusion: Multiplex IHC/ICC emerges as a transformative force in biomedical diagnostics and research. Beyond simultaneous marker detection, it unravels complexities within tissues – unveiling co-localization nuances, deciphering expression patterns, and enhancing understanding of cellular populations. As personalized treatments gain prominence, the study emphasizes the heightened importance of diagnostic tools and sample adequacy. The present methodological study, encapsulating an automated multiplex immunoperoxidase staining method, symbolizes a stride towards precision in pulmonary carcinoma diagnosis. Multi-tissue controls represent a key element in quality assurance in pathology laboratories.

Histiocytic Sarcoma

Histiocytic sarcoma is a rare and frequently missed diagnosis. With unusual and varied presentations, it typically indicates a rapid patient decline and poor outcomes. The diagnosis requires a high degree of clinical suspicion. In this case, we explore the progression of illness in a 56-year-old white male who initially presented with a 2-week history of soft-tissue nodules scattered throughout his head, neck, torso, and limbs, as well as shortness of breath and knee pain. After a thorough workup including computed tomography imaging of the neck, chest, and lower extremity, as well as a biopsy of a nodule with immunoperoxidase staining, a diagnosis of histiocytic sarcoma was established. Due to the severity of his malignancy, his hospital course was complicated by a deep vein thrombosis. Clinicians should be mindful of the risk of acute decompensation in such cases and can employ the various tenets of osteopathic theory to improve patient quality of life. Depending on the severity of illness, physicians may proceed to facilitate end-of-life measures with grace and dignity.

An up-to-date myopathologic characterisation of facioscapulohumeral muscular dystrophy type 1 muscle biopsies shows sarcolemmal complement membrane attack complex deposits and increased skeletal muscle regeneration

The aim of this study was to identify key routinely used myopathologic biomarkers of FSHD1. Needle muscle biopsies were taken in 34 affected muscles (m. quadriceps femoris (QF), n = 20, m. tibialis anterior (TA), n = 13, m. biceps brachii, n = 1) from 22 patients (age, 53.5 (10) years; M = 12, F = 10). Eleven patients had more than one biopsy (2xQF, n = 1; QF+TA, n = 9; 2xQF+TA, n = 1). Histochemistry, immunoperoxidase, and immunofluorescence stainings were performed and compared to age and muscle type matched muscle specimens of 11 healthy controls. Myopathologic features observed in our FSHD1 cohort were internalized nuclei, type 1 fibre hypertrophy and NADH central clearances/cores. We observed a prominent inflammatory response with MAC deposits, MHC I expression, and muscle regeneration that correlated with the inflammatory score. Our up-to-date characterization of FSHD1 points towards MHC I, MAC, and embryonic Myosin Heavy Chain/muscle regeneration as useful myopathologic readouts of FSHD1.

Antiviral activity of bovine type III interferon against bovine viral diarrhea virus is greatly reduced in bovine turbinate cells due to limited expression of IFN lambda receptor 1 (IL-28Rα).

The antiviral activity of recombinant bovine interferon lambda 3 (bovIFN-λ3) against bovine viral diarrhea virus (BVDV) has been demonstrated in vitro in Madin-Darby bovine kidney cells (MDBK) and in vivo in cattle. However, anti-BVDV activity of bovIFN-λ3 has not been studied in bovine respiratory tract epithelial cells, supposedly a primary target of BVDV infection when entering the host by the oronasal route. Here we investigated the anti-BVDV activity of bovIFN-λ3 in bovine turbinate-derived primary epithelial cells (BTu) using BVDV infection and immunoperoxidase staining, TCID50, RT-qPCR, DNA and transcriptome sequencing, and transfection with plasmids containing the two subunits, IL-28Rα and IL-10Rβ that constitute the bovIFN-λ3 receptor. Our immunoperoxidase staining, RT-qPCR, and TCID50 results show that while BVDV was successfully cleared in MDBK cells treated with bovIFN-λ3 and bovIFN-α, only the latter, bovIFN-α, cleared BVDV in BTu cells. Preincubation of MDBK cells with bovIFN-λ3 before BVDV infection was needed to induce optimal antiviral state. Both cell types displayed intact type I and III IFN signaling pathways and expressed similar levels of IL-10Rβ subunit of the type III IFN receptor. Sequencing of PCR amplicon of the IL-28Rα subunit revealed intact transmembrane domain and lack of single nucleotide polymorphisms (SNPs) in BTu cells. However, RT-qPCR and transcriptomic analyses showed a lower expression of IL-28Rα transcripts in BTu cells as compared to MDBK cells. Interestingly, transfection of BTu cells with a plasmid encoding IL-28Rα subunit, but not IL-10Rβ subunit, established the bovIFN-λ3 sensitivity showing similar anti-BVDV activity to the response in MDBK cells. Our results demonstrate that the sensitivity of cells to bovIFN-λ3 depends not only on the quality but also of the quantity of the IL-28Rα subunit of the heterodimeric receptor. A reduction in IL-28Rα transcript expression was detected in BTu as compared to MDBK cells, despite the absence of spliced variants or SNPs. The establishment of bovIFN-λ3 induced anti-BVDV activity in BTu cells transfected with an IL-28Rα plasmid suggests that the level of expression of this receptor subunit is crucial for the specific antiviral activity of type III IFN in these cells.

Establishment of a superinfection exclusion method for pestivirus titration using a recombinant reporter pestiviruses.

Pestiviruses are classified into two biotypes based on their cytopathogenicity. As the majority of pestivirus field isolates are noncytopathogenic, their titration requires alternative methods rather than direct observation of cytopathogenic effects, such as immunostaining using specific antibodies or interference with cytopathogenic strains. However, these methods require microscopic observation to assess virus growth, which is time- and labor-intensive, especially when handling several samples. In this study, we developed a novel luciferase-based pestivirus titration method using the superinfection exclusion phenomenon with recombinant reporter pestiviruses that possessed an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). In this method, swine kidney cells were inoculated with classical swine fever virus (CSFV) and superinfected with the reporter CSFV vGPE-/HiBiT 5 days postinoculation. Virus titer was determined based on virus growth measured in luminescence using the culture fluid 3 days after superinfection; the resultant virus titer was comparable to that obtained by immunoperoxidase staining. Furthermore, this method has proven to be applicable for the titration of border disease virus (BDV) by superinfection with both the homologous reporter BDV and heterologous reporter CSFV, suggesting that this novel virus titration method is a simple technique for automated virus detection based on the luciferase system.

Individual Keratinocyte Necroses in the Epidermis Are Apoptotic Keratinocytes in the Skin.

The patient was a 44-year-old woman with Stevens-Johnson syndrome due to receiving Baktar® (sulfamethoxazole trimethoprim) medication at our outpatient dermatology clinic. The epidermis, dermis, and subcutaneous adipose tissue samples showed numerous necrotic keratinocytes in the epidermis. Apoptotic nuclei were visualized as diaminobenzidine brown deposits with immunoperoxidase staining for cleaved caspase-3. The cleaved caspase-3-positive findings were consistent with eosinophilic material that appeared to be necrotic cells within the epidermis. Therefore, these eosinophilic materials may be apoptotic bodies. Generally speaking, eosinophilic cells are considered necrotic keratinocytes, especially in Japan. To the best of our knowledge, no studies have used apoptotic immunohistochemical markers to examine whether these structures are apoptotic in a human specimen.

Favipiravir Protects Enterocytes From Cell Death After Inflammatory Storm.

Over the past years, inflammatory bowel disease (IBD) treatment has become more targeted, anticipating the use of immune-modifying therapies at an earlier stage. During the treatment process prevention and management of viral infections hold significant importance. The protective role of favipiravir on enterocytes which are affected by inflammation is still unknown. We aim to analyze the effects of favipiravir on enterocytes after an inflammatory condition. We conducted a 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay to assess the cytotoxicity of favipiravir on intestinal epithelioid cells (IEC-6). To mimic the inflammation model in cell culture conditions, we exposed IEC-6 cells to tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). The cells were categorized into four groups: control, inflammation model, application of favipiravir before inflammation (prophylactic), and application of favipiravir after inflammation (treatment). We assessed the presence and distribution of caspase 1, caspase 3, interleukin6 (IL6), interleukin 8 (IL8), mixed lineage kinase domain-like protein (MLKL), receptor-interacting protein kinase 1 (RIPK1), and TNF-α using indirect immunoperoxidase staining. TNF-α and IL8 levels were analyzed with enzyme-linked immunosorbent assay (ELISA) in a culture medium. Caspase 1 was observed to be strong (+++) in the treatment group and weak (+) in the prophylactic group compared to the inflammation group. Caspase 3 was weak (+) in the inflammation group, and it was strong (+++) in the prophylactic and treatment group, the increase in the treatment group was significant. Therefore administering favipiravir before inducing inflammation appears to control the inflammatory caspase pathway in intestinal enterocytes, protecting them from inflammatory responses, while the caspase 3-dependent apoptotic pathway may not be active in enterocytes during inflammation. IL6 and IL8 were negative (-) in control, IL6 was weak (+) in inflammation and favipiravir treated groups; IL8 increased significantly in favipiravir groups compared to control and inflammation groups. Consequently, favipiravir may trigger IL6 release, initiating the inflammatory pathway and potentially enhancing IL8 interactions with other cytokines. TNF-α immunoreactivity was strong (+++) in the inflammation group, while it was moderate (++) in favipiravir-administered groups. MLKL immunoreactivity was strong (+++) in all groups, RIPK1 was weak (+) in control, strong (+++) in the inflammation and treatment group, moderate (++) in the prophylactic group, and the increase in inflammation and treatment group was significant compared to control. Our findings suggest that in the treatment group, necroptosis was triggered by increased MLKL and RIPK1, key players in inflammation and cell death. After immunocytochemical evaluation, our findings suggest that, after the onset of inflammation, favipiravir may play a role in cell death by increasing necroptosis rather than apoptosis.

FRI214 Adrenal Adenoma With Biochemical Findings Suggestive Of Cushing's Disease

Abstract Disclosure: A. Calimag: None. U. Tarabichi: None. M. Ahmad: None. M. Kazi: None. Introduction: We are presenting a case of a young female with prior history of multiple endocrinopathies presenting with a left adrenal adenoma with biochemical findings suggestive of Cushing's disease. Case Presentation: A 22-year-old female with a history of Type 1 Diabetes Mellitus was found to have a left adrenal adenoma 2.8 cm on CT. Absolute washout of 85 % and a relative washout of 63%, having 12 Hounsfield units (HU) on the non-contrast phase, findings suggestive of a benign adenoma. Further work up including 1 mg Dexamethasone suppression test (DST) showed unsuppressed plasma ACTH 17 pg/ml and total cortisol 9.4 mcg/dL. 8 mg DST showed ACTH 12 pg/ml, AM cortisol 8.8 mcg/dL, and Dexamethasone 991 ng/dl. Late night salivary cortisol 0.07 ug/dl, and 0.09 ug/dl, 24 hr urine cortisol 40.7. Other labs showed Aldosterone level was normal at 6.7, Renin level was also normal at 1.1. 24 hr urine catecholamines were Norepinephrine 35 mcg/24 hr, Epinephrine 3.9 mcg/24 hr, while urine Metanephrine level was <0.24, and normetanephrine level at 0.34. TSH 1.2mcU/ml. One day prior to surgery ACTH level was suppressed at 2.1 pg/ml drawn from a different lab. MRI of the Pituitary showed a slightly enlarged pituitary gland with uniform contrast enhancement and no definite lesion. The patient underwent a laparoscopic left adrenalectomy. Pathology showed an adrenal cortical neoplasm consistent with adrenal cortical adenoma, with no significant cytologic atypia, mitotic activity, or necrosis. Immunoperoxidase stain, the tumor is positive for inhibin and negative for PAX8. Post-op she developed adrenal insufficiency which was treated with hydrocortisone. The follow-up cortisol level was 11.3 mcg/dL when the patient was off hydrocortisone. Discussion: In this case, the biochemical exam showed elevated cortisol with ACTH not fully suppressed at both 1 mg and 8 mg DST which was done twice, and normal 24-hour urine cortisol. Determining whether this is adrenal vs pituitary mediated was one of the clinical dilemmas of this case. Initially, the patient’s ACTH was unsuppressed however the day before surgery ACTH was suppressed and the patient’s cortisol level normalized after the left adrenalectomy. Immunoassays for ACTH levels may occasionally be susceptible to analytical errors. (1-2) There are several case series and studies mentioning assay interference when using Immulite Assay versus Cobas ACTH assay. (1-2) Therefore, it is important to analyze clinical findings and biochemical testing and if lab tests are discordant, recheck using a different analytical platform in a different lab or an alternate ACTH assay to confirm ACTH levels. (1-2)

BI11 Cutaneous presentation of Kaposi sarcoma following allogeneic haematopoietic stem cell transplantation for myeloma

Abstract Iatrogenic Kaposi sarcoma (KS) is a relatively frequent occurrence following solid-organ transplants (0–5%), in part due to prolonged immunosuppression. However, its occurrence after haematopoietic stem cell transplantation (HSCT) is rare, with only 15 cases described in the literature. We report a case of KS presenting in the skin of a patient who had received a matched unrelated donor allogeneic HSCT for IgG kappa myeloma. A 55-year-old HIV-negative white man with myeloma underwent an allogeneic HSCT from a matched, unrelated donor following conditioning chemotherapy with fludarabine–melphalan and antithymocyte globulin. Post-transplant, he was maintained on ciclosporin as graft-versus-host disease (GVHD) prophylaxis. On day 14 post-transplant, he developed a widespread eczematous eruption that rapidly evolved into an erythroderma; a skin biopsy was consistent with acute GVHD. He was managed with superpotent topical steroids and a 5-day course of oral prednisolone. He began extracorporeal photopheresis (ECP) on day 33 post-transplant, having failed to respond adequately to systemic steroids. He also completed four cycles of rituximab. Ciclosporin was stopped by day 130 post-transplant. He had completed 14 cycles of ECP by day 139 post-transplant, with resolution of cutaneous GVHD; however, he had developed a rapidly growing solitary dusky-red nodule on his right neck that was excised due to diagnostic uncertainty. Histology revealed a dermal haemorrhagic exophytic vascular tumour composed of lobules and sheets of epithelioid cells with an eosinophilic cytoplasm and pleomorphic nuclei. Immunoperoxidase staining was negative for S100, MNF-116 and AE1/AE3. The same cells were strongly and diffusely positive for the vascular markers CD31, CD34 and ERG. The lesional cells were positive for human herpesvirus (HHV)-8 confirming a diagnosis of Kaposi sarcoma. He subsequently developed a second lesion on his left forearm, despite being off all immunosuppression. A repeat bone biopsy confirmed that he remains in morphological remission from his myeloma. Positron emission tomography–computed tomography has not identified extracutaneous disease. KS following HSCT is rare, but it can be fatal. Skin lesions are the dominant clinical presentation. HHV-8 has an important role in the pathogenesis, and transplant-related KS may occur secondary to reactivation of the virus as a result of significant immunosuppression following HSCT. However, it could also occur as primary infection or be transmitted with donor cells. The outcome of KS after HSCT is variable. A watch and wait strategy can be applied if immune recovery is expected. In our patient, it developed 5 months after HSCT; shortly after immunosuppression had been tapered, he remains well and will be closely monitored for further suspicious lesions.

Incedental Cancer of Prostate Among Patienst Undergoimg Transurethral Resection of Prostate aged more than 50 Years

Objective: To determine the incidental cancer of prostate among patienst undergoimg transurethral resection of prostate aged more than 60 years at tertiary care Hospital Subjects and Methods: This cross-sectional asutyd was done at pathology daerpatemnt of LUMHS from January 2019 to December 2020. Satudy was done after atking ethical approval fron ethical review commetee of LUMHS Jamshoro. TURP specimens underwent overnight processing after being fixed ino 10% neutral buffered formalin. Each block had a single section taken from it that was stained with hematoxylin and eosin and inspected histologically. On the glass slides, all foci were marked, and the tumour volume was calculated as a visual representation of the tumor's percentage of the specimen's total surface area. According on the 2005 International Association of Urological Pathol (ISUP) consensus recommendations, Gleason scoring was performed. With immunoperoxidase staining, small tumour foci were confirmed. pT1a disease was defined as incidental tumor in 5% of TURP specimens. pT1b disease was defined as incidental tumor in > 5% of TURP specimens. All the data was collected via suyd profoema. SPSS version 26 was used for the data analysis. Results: A toat of 800 cases were studied the incidence of prostate carcinoma was estimated 39.25%. mean age of the patients was 66.95+9.37 years. Most of the cases had, tumor grade II to V as 18.3%, 18.3%, 17.0% and 36.9% respectively. As per estimated tumor invasion, most of the cases had tumor invasion was >25% as 22.8% ahd tumor invesion 26-50%, 27.6% and 51-75% and 30.1% had 76-100%. Perineural invasion was observed in 42.3%of the cases and lymphovascular invasion was only in 3.2% of the acses. Conclusion: The incidence of prostate carcinoma was observed to the highly frrequenct as estimated 39.25% and most of the cases had, tumor grade II to V. Keywords: Prostate, incidental cancer, stages

Detection and verification of neurodegeneration after traumatic brain injury in the mouse: Immunohistochemical staining for amyloid precursor protein.

Previous studies of human traumatic brain injury (TBI) have shown diffuse axonal injury as varicosities or spheroids in white matter (WM) bundles when using immunoperoxidase-ABC staining with 22C11, a mouse monoclonal antibody against amyloid precursor protein (APP). These findings have been interpreted as TBI-induced axonal pathology. In a mouse model of TBI however, when we used immunofluorescent staining with 22C11, as opposed to immunoperoxidase staining, we did not observe varicosities or spheroids. To explore this discrepancy, we performed immunofluorescent staining with Y188, an APP knockout-validated rabbit monoclonal that shows baseline immunoreactivity in neurons and oligodendrocytes of non-injured mice, with some arranged-like varicosities. In gray matter after injury, Y188 intensely stained axonal blebs. In WM, we encountered large patches of heavily stained puncta, heterogeneous in size. Scattered axonal blebs were also identified among these Y188-stained puncta. To assess the neuronal origin of Y188 staining after TBI we made use of transgenic mice with fluorescently labeled neurons and axons. A close correlation was observed between Y188-stained axonal blebs and fluorescently labeled neuronal cell bodies/axons. By contrast, no correlation was observed between Y188-stained puncta and fluorescent axons in WM, suggesting that these puncta in WM did not originate from axons, and casting further doubt on the nature of previous reports with 22C11. As such, we strongly recommend Y188 as a biomarker for detecting damaged neurons and axons after TBI. With Y188, stained axonal blebs likely represent acute axonal truncations that may lead to death of the parent neurons. Y188-stained puncta in WM may indicate damaged oligodendrocytes, whose death and clearance can result in secondary demyelination and Wallerian degeneration of axons. We also provide evidence suggesting that 22C11-stained varicosities or spheroids previously reported in TBI patients might be showing damaged oligodendrocytes, due to a cross-reaction between the ABC kit and upregulated endogenous biotin.

Comparative phytochemical studies on the roots of Polygala azizsancarii and P. peshmenii and neuroprotective activities of the two xanthones

Six known sucrose mono-, di- and triesters and five xanthone derivatives were isolated from the roots of Polygala peshmenii Eren, Parolly, Raus & Kürschner which is a narrow species endemic to Türkiye. Among the xanthones, 1,7-dihydroxy-2,3-methylenedioxy-5,6-dimethoxy-xanthone is an undescribed compound isolated for the first time from a natural source. The studies on the roots of P. azizsancarii Dönmez have resulted in the isolation of four known compounds including sucrose mono-, di- and triesters. The structures of the sucrose esters and xanthones isolated from P. azizsancarii and P. peshmenii were established by spectroscopic methods, including 1D-NMR (1H NMR, 13C NMR, DEPT-135), 2D-NMR (COSY, NOESY, HSQC, HMBC). Neuroprotective activities of two xanthones, 1,3,6-trihydroxy-2,5,7-trimethoxyxanthone and 3-O-β-D-glucopyranosyloxy-1,6-dihydroxy-2,5,7-trimethoxyxanthone isolated from the roots of P. azizsancarii were evaluated in vitro using in a cellular model of Alzheimer's disease. SKNAS human neuroblastoma cells were used in the study and treated with different consecrations of Aβ₂₅₋₃₅ oligomer for up to 48 h. Cell viability was evaluated using MTT assay. The distribution of β-amyloid, α-synuclein, tau, JAK2, STAT3, caspase 3 and BMP-2 were investigated using indirect immunoperoxidase staining. Our results suggested that both xanthones control tau aggregation with no effect on β-amyloid plaque formation. In addition, for neuronal pathophysiology in AD cell model, decreased distributions of JAK/STAT3 and BMP2 signaling pathways were demonstrated, therefore they play a role in the protective effect on neurons in neurodegenerative disease. A significant decrease in caspase 3 immunoreactivity was detected after the administration of both compounds in AD cells. Therefore, both compounds control neuronal pathophysiology and rescue cell death in AD disease.

Early Syphilis Presenting as Thickened Penile Plaques With an Embedded Recurrent Ulcer (Chancre Redux): A Case Report

Introduction: The manifestations of syphilis are varied, and serology can establish the diagnosis early, especially in rare cases. We report a case of chancre redux (a rare recurrence of primary syphilis), which was embedded within a secondary syphilitic penile skin plaque. There were missed opportunities at earlier diagnosis as serology for syphilis was not ordered. Case presentation: A 56-year-old man presented with thickened penile plaques for five months. He reported a small penile ulcer approximately one month prior. There was no history of other skin lesions or rash. However, clinically there was an asymptomatic, indurated ulcer embedded within a plaque which was swab-positive for syphilis by PCR. A punch biopsy of a plaque was spirochaete-positive using an immunoperoxidase stain. The patient’s lesions resolved three weeks after treatment with intramuscular benzathine penicillin. Discussion: Uncommonly reported even in the preantibiotic era, chancre redux is now rare. The ulcer usually recurs at or near the site of the original chancre, and has similar morphological features. The skin lesions of secondary syphilis can exhibit remarkable morphological variety. The most common skin manifestation, a generalized macular rash, was not present at any time in this case. Rather, there were only a couple of nonspecific penile plaques. If biopsy is performed, histologic findings are variable, though typically the inflammatory infiltrate includes plasma cells. A special immunoperoxidase stain can highlight spirochaetes in biopsy sections. Conclusion: This case highlights the importance of considering syphilis in the differential diagnosis of persistent, atypical penile lesions and underscores the need for appropriate serological testing in such instances.

Late Onset Pseudolymphomatous Reaction to Blue Tattoo Pigment Precipitated by Covid Vaccination

Tattoos can lead to a variety of cutaneous conditions such as allergic or contact dermatitis, lichenoid dermatitis, pseudolymphomatous reactions and granulomatous responses such as foreign body granulomas that may have a sarcoidal histology simulating sarcoidosis.[1] Pseudolymphomatous reactions are less common and may clinically and histologically resemble cutaneous lymphomas. Pseudolymphomatous reactions may develop in response to injections, Borrelia infections and arthropod bites. Diagnosis of cutaneous pseudolymphoma is usually based on clinicopathologic correlation coupled with skin biopsy and ancillary tests such as immunoperoxidase stains and genetic testing when necessary. Treatment is generally successful with application of topical corticosteroid preparation or injection of intralesional corticosteroids and in some cases, surgical excision. Here, we describe the case of a 61-year-old male who presented with 3-4 mm papules contained within the blue region of a multicolored tattoo on his upper right arm that had been present for several years. The reaction developed shortly following a Covid vaccination.

ИЗУЧЕНИЕ РЕПРОДУКЦИИ ВИРУСА МЕШОТЧАТОГО РАСПЛОДА ПЧЕЛ ИММУНОЦИТОХИМИЧЕСКИМ МЕТОДОМ IN VITRO В КУЛЬТУРАХ КЛЕТОК

The article presents the results of studying the reproduction of the sacbrood virus (SBV) in cells of heterologous origin using the method of immunocytochemical analysis. To identify the localization of the SBV antigen of bees during its reproduction in sensitive cell cultures of heterologous lines, the method of immunoperoxidase staining of infected cells (IPS) was used, as a result of which sites of specific antibody/antigen interaction were observed on the cell membrane and in the form of inclusions in the cytoplasm.

A rare case of nephrotic syndrome and Tangier disease.

Rarely, disorders of lipid metabolism cause nephrotic syndrome with progressive kidney disease. Tangier disease is a rare condition belonging to this family of lipid disorders; however, it is not associated with kidney disease. We report a patient presenting with nephrotic syndrome, leading to the unmasking of Tangier disease. A 34-year-old man presented with ankle oedema, nephrotic-range proteinuria and hypoalbuminaemia. Kidney biopsy demonstrated membranous nephropathy with features of immunoperoxidase staining, suggesting a secondary aetiology. Acute serology was negative. Imaging showed lymphadenopathy with splenomegaly suggestive of lymphoproliferative disorder. Bone marrow biopsy revealed foamy macrophages with widespread lipid deposition. Genomic sequencing revealed a pathological homozygous variant for ATP-binding cassette subfamily A member 1 (ABCA1) c.1510-1G > A, consistent with Tangier disease. Review of the ultrastructural kidney biopsy features demonstrated, in addition to membranous subepithelial and intramembranous usual-type electron-dense deposits, intramembranous osmiophilic lipid deposits similar to those in LCAT deficiency. The patient's renal function gradually declined (serum creatinine 133µmol/L); therefore, he was started on rituximab. Metabolic disorders causing nephrotic syndrome are rare and even more so their association with membranous nephropathy. These should be considered in cases with unexplained persistent nephrotic syndrome with progressive kidney disease and lipid deposits on renal biopsy.

AMACR Expression is a Potential Diagnostic Marker in Apocrine Lesions of Breast, and is Associated with High Histologic Grade and Lymph Node Metastases in Some Invasive Apocrine Breast Cancers

BackgroundCarcinoma with apocrine differentiation (AC) is a subtype of breast carcinoma with apocrine features in >90% of the tumor. Molecular studies demonstrate AC has high expression of androgen receptor (AR) mRNA. Pure AC lack estrogen receptor (ER), progesterone receptor (PR), and express AR, with variable human epidermal growth factor 2 (HER2) status. Currently, in triple negative AC, no targetable therapies or specific diagnostic markers exist. Materials and Methodsα-Methylacyl CoA racemase (AMACR) expression was investigated as a marker of apocrine differentiation using a single-plex immunoperoxidase stain, and a novel AMACR/p63 dual stain in a subset of cases, across 1) benign apocrine lesions (apocrine metaplasia, adenosis) 2) apocrine DCIS (ADCIS), 3) AC/ invasive ductal carcinoma (IDC) with apocrine features, 4) non-apocrine triple negative breast cancer (TNBC) and 5) IDC, no special type. A sub-set of cases were evaluated by tissue microarray. ResultsAMACR expression was increased in both AC and ADCIS, with minimal expression in benign breast tissue, TNBC and IDC, NST cases. In invasive cases, those with positive AMACR (>5% positivity) were significantly associated with higher histologic grade (P = .006), initial N stage (chi squared 0.044), and lack of ER or PR expression (both P < .001), with no correlation with overall survival. Analysis of TCGA breast cancer datasets revealed AMACR expression was significantly higher in molecularly defined apocrine carcinomas relative to basal and luminal subtypes. Moreover, high AMACR expression predicted worse relapse-free and distant-metastasis free survival, among both ER-/PR-/Her2- and ER-/PR-/Her2+ breast cancer cohorts (log-rank P = .081 and .00011, respectively). ConclusionAMACR represents a promising diagnostic and prognostic marker in apocrine breast lesions. Further study is needed to determine the biologic and clinical significance of this protein in AC.

Microglia in Human Postmortem Brain Samples: Quantitative Ultrastructural Analysis of Scanning Electron Microscopy Images.

In this protocol, we describe the specific steps required to prepare human postmortem brain samples for ultrastructural microglial analysis. A detailed procedure is provided to improve the ultrastructural quality of the samples, using aldehyde fixatives followed by immunoperoxidase staining of allograft inflammatory factor 1 (AIF1, also known as IBA1), a marker of myeloid cells, and cluster of differentiation 68 (CD68), a marker of phagolysosomal activity. Additionally, we describe an osmium-thiocarbohydrazide-osmium (OTO) post-fixation method that preserves and increases the contrast of cellular membranes in human postmortem brain samples, as well as the steps necessary to acquire scanning electron microscopy (SEM) images of microglial cell bodies. In the last section, we cover the quantitative analysis of various microglial cytoplasmic organelles and their interactions with other parenchymal elements.

Oogenesis and expression of PCNA and vitellogenin in Geophagus native to the Amazon basin in pre- and post-hydroelectric dam periods

Oogenesis in fish is a process that involves cell proliferation, growth and maturation. In rivers blocked by hydroelectric dams, these factors can be altered and affect the reproduction mechanism of the species. Regarding this scenario, the present study aimed to describe oogenesis in two cichlids native to the Amazon basin, Geophagus argyrostictus and G. altifrons, during pre- and post-dam periods of the Xingu River. Females of both Geophagus species were captured and biometric measurements were taken. Afterwards, the fish were euthanized, and the gonads were removed and subjected to histological processing for light microscopy, scanning electron microscopy and immunoperoxidase staining. Oocyte diameter and zona radiata thickness were also measured. The ovaries appeared in pairs, where according to their shape, size, vascularization and cell organization, five stages were described for both species. In the post-dam period, there was a reduction in the mean diameter of the oocytes, especially type IV, and a decrease in the thickness of the zona radiata, mainly, in G. argyrostictus. In both species, there was a greater presence of oocyte atresia in the post-dam period. PCNA immunoreactivity was more intense in type I and II oocytes, while vitellogenin immunoreactivity occurred in the cytoplasm and follicular cells of oocytes III and IV. These data suggest that because of changes in the level/flow of the river, the gonads of these two species adjust to the new environment, with a decrease in mean diameter and zona radiata thickness of the oocytes, which can interfere with the reproduction of the animals.

A Rare Case of Paratesticular Cystadenoma with Ovarian Stroma Found by Scrotal Ultrasound on a Cirrhotic Patient with Elevated Alpha-fetoprotein

Abstract Introduction/Objective Paratesticular cystadenoma with ovarian stroma is a rare benign cyst-forming neoplasm with less than 10 cases reported in the literature. Typically, it presents in men aged 11 to 68 years (average 30) and is asymptomatic. The tumor is hypothesized to represent the efferent ductulus undergoing metaplastic change, the vestigial remnants of Mullerian duct, or mesothelium of the tunica vaginalis. Most tumors are considered benign, but a few cases of malignant transformation have been reported. Methods/Case Report We present a case of a 54-year-old male with a history of cirrhosis and elevated alpha-fetoprotein (17.85 ng/ML) who was found to have a paratesticular mass by scrotal ultrasound. Clinical history was obtained from review of electronic medical record. CD10 (Novocastra Clone 56C6) and progesterone receptor (PR) (Leica Clone 16) Immunoperoxidase stains were performed following the vendor recommendations. Surgical excision revealed a 0.6-cm polypoid nodule on tunica albuginea that was entirely submitted. Microscopically, the tumor was comprised of cystic spaces lined by cuboidal epithelium identical to that of the Fallopian tubes. There was a sparse spindle-shaped stroma underlying the epithelium similar to ovarian stroma. No nuclear atypia or mitosis was found. Immunoperoxidase stains showed the epithelium was positive for PR, and the stroma was positive for CD10. No evidence of germ cell tumor was identified. Results (if a Case Study enter NA) NA. Conclusion Characteristic histologic and immunophenotypic findings can aid in the diagnosis of paratesticular cystadenoma with ovarian stroma. Surgical resection for histological confirmation and long-term monitoring for the potential recurrence and metastatic spread are required.