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Pre-determined anatomical locations in the oral cavity were biopsied, and their histomorphology was characterised using haematoxylin and eosin staining (H&E). The most abundant cell type was of dendritic morphology. Lymphocyte foci were not evident in the palatoglossal folds or the gingiva. Immunohistochemical staining (IHC) for validated leukocyte markers followed, including CD3, CD20, CD79α, CD204, and Iba1. Consistent with H&E findings, CD204 immunoreactivity predominated amongst all niches. With the exception of the alveolar mucosa and palatoglossal folds, we also demonstrate a significant difference in the population of macrophages by region for only the Iba1 antigen (p

OBJECTIVES: To determine the role of keratin 17 (K17) as a predictive biomarker for response to chemotherapy by defining thresholds of K17 expression based on immunohistochemical tests that could be used to optimize therapeutic intervention for patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: We profiled K17 expression, a hallmark of the basal molecular subtype of PDAC, by immunohistochemistry in 2 cohorts of formalin-fixed, paraffin-embedded PDACs (n = 305). We determined a K17 threshold of expression to optimize prognostic stratification according to the lowest Akaike information criterion and explored the potential relationship between K17 and chemoresistance by multivariate predictive analyses. RESULTS: Patients with advanced-stage, low K17 PDACs treated using 5-fluorouracil (5-FU)-based chemotherapeutic regimens had 3-fold longer survival than corresponding cases treated with gemcitabine-based chemotherapy. By contrast, PDACs with high K17 did not respond to either regimen. The predictive value of K17 was independent of tumor mutation status and other clinicopathologic variables. CONCLUSIONS: The detection of K17 in 10% or greater of PDAC cells identified patients with shortest survival. Among patients with low K17 PDACs, 5-FU-based treatment was more likely than gemcitabine-based therapies to extend survival.

Ewing sarcoma (ES) is an uncommon mesenchymal neoplasm that typically develops as a bone mass, although up to 30% arise in extraskeletal sites. ES of the gastrointestinal (GI) and hepatobiliary tract is rare and may be misdiagnosed as other, more common neoplasms that occur in these sites. However, the correct classification of extraskeletal ES is important for timely clinical management and prognostication. We reviewed our experience of ES in the GI and hepatobiliary tract in order to further highlight the clinicopathologic features of these neoplasms and document the potential for misdiagnosis in this setting. The archives and consultation files of 6 academic institutions were retrospectively queried for cases of ES occurring in the GI and hepatobiliary tract. The histologic slides and ancillary studies were reviewed and clinical data were retrieved for each case through the electronic medical records, when available. Twenty-three patients with ES in the GI and/or hepatobiliary tract were identified from 2000 to 2022. Of these, 11 were women and 12 were men with a median age of 38 years (range, 2 to 64). Tumor locations included the pancreas (n=5), liver (n=2), stomach (n=3), colorectum (n=3), and small intestine (n=5), as well as tumors involving multiple organs, pelvis and retroperitoneum (n=5). Tumor size varied between 2 cm and 18 cm. Twenty were primary and 3 were metastases. Of the 23 cases, only 17% were initially diagnosed as ES. The most common misdiagnoses involved various forms of neuroendocrine neoplasia due to expression of synaptophysin and other neuroendocrine markers (22%). A wide variety of diagnoses including GI stromal tumor was considered due to aberrant CD117 expression (4%). The diagnosis of ES was ultimately confirmed by detection of the EWSR1 rearrangement in 22 cases. The remaining case was diagnosed using traditional immunohistochemistry. Follow-up information was available in 20 cases, with follow-up time varying between 2 and 256 months. Six patients with follow-up died of disease between 6 and 60 months following initial presentation. Our data indicate ES in the GI and hepatobiliary tract is commonly misdiagnosed leading to a delay in therapy. In light of the attendant therapeutic and prognostic implications, ES should be considered in the differential diagnosis of any GI or hepatobiliary tumor with epithelioid and/or small round cell morphology.

Mannheimia haemolytica-associated abomasitis has been clinically described as a cause of sudden death in lambs, but it is poorly characterized. We describe the pathological features of a severe fibrinonecrotizing abomasitis in 3 lambs that died suddenly. All 3 abomasums had a thickened submucosa due to edema and necrotic areas delimited by bands of degenerate neutrophils with slender nuclei (oat cells) and angiocentric distributions. The overlying mucosa was congested. Myriads of gram-negative coccobacilli were observed within the oat cell bands. M. haemolytica was isolated from the abomasum in all 3 animals and was serotyped as A2 in one of them. Pericarditis and pleuritis were observed in 2 of the lambs. Clostridium spp. were isolated in 1 lamb and detected by immunohistochemistry in the 3 animals, suggesting clostridial co-infection. M. haemolytica should be considered among the differential diagnoses of necrotizing abomasitis in lambs.

BackgroundS100A8 is a melanoma biomarker expressed in the melanoma-associated epidermal keratinocytes, but its diagnostic utility has not been compared with other biomarkers, including PRAME.ObjectivesTo compare the utility of S100A8 and PRAME immunohistochemistry (IHC) in the differential diagnosis of melanoma and naevi in a case-control study.MethodsA previously described cohort of 209 melanomas (case samples) and naevi (control samples) dual-immunostained for S100A8 and PRAME were included. For S100A8, previously reported scores indicating the proportion of tumour-associated epidermis stained (0 = indeterminate; 1 = 0-4%; 2 = 5-25%; 3 = 26-50%; 4 = 51-75%; 5 = > 75%) were utilized. PRAME IHC was reviewed by at least two reviewers and a consensus score assigned, with score indicating the proportion of tumour stained (0 = indeterminate; 1 = 0%; 2 = 1-50%; 3 = > 50%). A positive test was defined as > 50% staining.ResultsThe area under the receiver operating characteristic curves for S100A8 (0.833) and PRAME (0.874) were not significantly different from each other (P = 0.22). The diagnostic sensitivity and specificity were 42.4% [95% confidence interval (CI) 32.6-52.8%] and 98.2% (95% CI 93.6-99.8%) for S100A8, and 79.8% (95% CI 70.5-87.2%) and 87.3% (95% CI 79.6-92.9%) for PRAME, respectively. A combined test requiring both S100A8 and PRAME IHC positivity had a sensitivity of 39.4% (95% CI 29.7-49.7%) and specificity of 99.1% (95% CI 95.0-100.0%).ConclusionsS100A8 and PRAME have utility in the diagnostic workup of melanoma, with S100A8 being more specific and PRAME being more sensitive when using this threshold. Our findings suggest that these two immunohistochemical markers may favourably complement one another to improve the detection of melanoma.

Considering the lack of consensus related to the impact of selective IL-6 receptor inhibition on bone remodeling and the scarcity of reports, especially on large bone defects, this study proposed to evaluate the biological impact of the selective inhibitor of interleukin-6 receptor (tocilizumab) in an experimental model of critical calvarial defect in rats. In this preclinical and in vivo study, 24 male Wistar rats were randomly divided into two groups (n=12/group): defect treated with collagen sponge (CG) and defect treated with collagen sponge associated with 2 mg/kg tocilizumab (TCZ). The defect in the parietal bone was created using an 8-mm diameter trephine drill. After 90 days, the animals were euthanized, and tissue samples (skull caps) were evaluated through micro-CT, histological, immunohistochemistry, cytokines, and RT-qPCR analyses. Tocilizumab reduced mononuclear inflammatory infiltration (P0.05). The bone cells (osteoblasts, osteoclasts, and osteocytes) in the defect area were similar in both groups (P>0.05). Tocilizumab reduced inflammatory cytokines, decreased osteogenic protein, and increased proteases in a critical bone defect in rats. Ninety days after the local application of tocilizumab in the cranial defect, we did not find a significant formation of bone tissue compared with a collagen sponge.

Background: Distant metastases from lung cancer are commonly found in the brain, bone, and liver. Metastases to the breast from non-mammary malignancies are extremely rare, and their clinical presentations remain unclear. Case presentation: We herein report a case of bilateral breast metastases from anaplastic lymphoma kinase-positive advanced lung cancer in a 51-year-old Japanese male patient. During the course of systemic treatment for advanced lung cancer, computed tomography revealed bilateral breast enlargement without contrast enhancement, a finding consistent with gynecomastia. While other metastatic lesions responded to chemotherapy, both breast masses grew vertically like nodules. The breast masses were immunohistochemically diagnosed as metastases from lung cancer and were removed surgically. Simultaneous bilateral breast metastases from malignancies of other organs, like ones in this case, have rarely been described. Conclusions: It is important to keep in mind that breast metastases from nonmammary malignancies are a possible explanation for unusual breast findings in a patient with a history of malignancies. [ABSTRACT FROM AUTHOR]

Phosphatase and tensin homolog (PTEN) loss is associated with adverse outcomes in prostate cancer and can be measured via immunohistochemistry. The purpose of the study was to establish the clinical application of an in-house developed artificial intelligence (AI) image analysis workflow for automated detection of PTEN loss on digital images for identifying patients at risk of early recurrence and metastasis. Postsurgical tissue microarray sections from the Canary Foundation (n = 1264) stained with anti-PTEN antibody were evaluated independently by pathologist conventional visual scoring (cPTEN) and an automated AI-based image analysis pipeline (AI-PTEN). The relationship of PTEN evaluation methods with cancer recurrence and metastasis was analyzed using multivariable Cox proportional hazard and decision curve models. Both cPTEN scoring by the pathologist and quantification of PTEN loss by AI (high-risk AI-qPTEN) were significantly associated with shorter metastasis-free survival (MFS) in univariable analysis (cPTEN hazard ratio [HR], 1.54; CI, 1.07-2.21; P = .019; AI-qPTEN HR, 2.55; CI, 1.83-3.56; P < .001). In multivariable analyses, AI-qPTEN showed a statistically significant association with shorter MFS (HR, 2.17; CI, 1.49-3.17; P < .001) and recurrence-free survival (HR, 1.36; CI, 1.06-1.75; P = .016) when adjusting for relevant postsurgical clinical nomogram (Cancer of the Prostate Risk Assessment [CAPRA] postsurgical score [CAPRA-S]), whereas cPTEN does not show a statistically significant association (HR, 1.33; CI, 0.89-2; P = .2 and HR, 1.26; CI, 0.99-1.62; P = .063, respectively) when adjusting for CAPRA-S risk stratification. More importantly, AI-qPTEN was associated with shorter MFS in patients with favorable pathological stage and negative surgical margins (HR, 2.72; CI, 1.46-5.06; P = .002). Workflow also demonstrated enhanced clinical utility in decision curve analysis, more accurately identifying men who might benefit from adjuvant therapy postsurgery. This study demonstrates the clinical value of an affordable and fully automated AI-powered PTEN assessment for evaluating the risk of developing metastasis or disease recurrence after radical prostatectomy. Adding the AI-qPTEN assessment workflow to clinical variables may affect postoperative surveillance or management options, particularly in low-risk patients.

OBJECTIVE: Describe the clinical course and diagnostic and genetic findings in a cat with X-linked myotubular myopathy. CASE SUMMARY: A 7-month-old male Maine coon was evaluated for progressively worsening gait abnormalities and generalized weakness. Neurolocalization was to the neuromuscular system. Genetic testing for spinal muscular atrophy (LIX1) was negative. Given the progressive nature and suspected poor long-term prognosis, the owners elected euthanasia. Histopathology of skeletal muscle obtained post-mortem disclosed numerous rounded atrophic or hypotrophic fibers with internal nuclei or central basophilic staining. Using oxidative reactions mediated by cytochrome C oxidase and succinic dehydrogenase, scattered myofibers were observed to have central dark staining structures and a ring-like appearance. Given the cats age and clinical history, a congenital myopathy was considered most likely, with the central nuclei and ring-like changes consistent with either centronuclear or myotubular myopathy. Whole genome sequencing identified an underlying missense variant in myotubularin 1 (MTM1), a known candidate gene for X-linked myotubular myopathy. NEW OR UNIQUE INFORMATION PROVIDED: This case is the first report of X-linked myotubular myopathy in a cat with an MTM1 missense mutation. Maine coon cat breeders may consider screening for this variant to prevent production of affected cats and to eradicate the variant from the breeding population.

Background and aimsBarrett's esophagus (BE) is the precursor to esophageal adenocarcinoma. A major challenge is identifying the small group with BE who will progress to advanced disease from the many who will not. Assessment of p53 status has promise as a predictive biomarker, but analytic limitations and lack of validation have precluded its use. The aim of this study was to develop a robust criteria for grading abnormal immunohistochemical (IHC) expression of p53 and to test its utility as a biomarker for progression in BE.MethodsCriteria for abnormal IHC of p53 were developed in BE biopsies and validated with sequencing to assess TP53 mutations. The utility of p53 IHC as a biomarker for progression of BE was tested retrospectively in 561 patients with BE with or without known progression. The findings were prospectively validated in a clinical practice setting in 1487 patients with BE.ResultsAbnormal p53 IHC highly correlated with TP53 mutation status (90.6% agreement) and was strongly associated with neoplastic progression in the retrospective cohorts, regardless of histologic diagnosis (P < .001). In the retrospective cohort, abnormal p53 was associated with a hazard ratio of 5.03 (95% confidence interval, 3.88-6.5) and a hazard ratio of 5.27 (95% confidence interval, 3.93-7.07) for patients with exclusively nondysplastic disease before progression. In the prospective validation cohort, p53 IHC predicted progression among nondysplastic BE, indefinite for dysplasia, and low-grade dysplasia (P

AimsAdenomatoid tumours are mesothelial-derived benign neoplasms with a predilection for the genital tract. Extragenital sites are rare and can cause significant diagnostic challenges. Herein, we describe the clinicopathological features of a cohort of adenomatoid tumours involving the gastrointestinal tract and liver in order to more clearly characterise their histological findings and aid in diagnosis.Methods and resultsThe pathology databases at four institutions were searched for adenomatoid tumours involving the gastrointestinal tract or liver, yielding eight cases. Available clinicoradiological and follow-up data were collected from the medical records. Six tumours were incidentally discovered during imaging studies or at the time of surgical exploration for unrelated conditions; presenting symptoms were unknown in two patients. Histologically, the tumours were well-circumscribed, although focal ill-defined borders were present in four cases. No infiltration of adjacent structures was identified. Architectural heterogeneity was noted in five (63%) tumours; an adenoid pattern often predominated. The neoplastic cells were flattened to cuboidal with eosinophilic cytoplasm. Cytoplasmic vacuoles mimicking signet ring-like cells were present in five (63%) cases. Three (38%) cases showed involvement of the mesothelium with reactive mesothelial hyperplasia. Cytological atypia or increased mitotic activity was not identified. The surrounding stroma ranged from oedematous/myxoid to densely hyalinised. Immunohistochemistry confirmed mesothelial origin in all cases evaluated. No patients developed recurrence of disease.ConclusionsThe current study evaluates the clinicopathological findings in a collective series of gastrointestinal and hepatic adenomatoid tumours, correlating with those described in individually reported cases. We highlight common histological features and emphasise variable findings that could mimic a malignant neoplasm.

Chromophobe renal cell carcinoma (ChRCC) is a relatively rare subtype of RCC with a characteristic histologic appearance. Most ChRCCs are slow growing, but sarcomatoid differentiation and metastases can occur, indicative of aggressive behavior and poor prognosis. Herein, we characterize ten ChRCCs with aggressive components, defined as sarcomatoid change and/or metastasis. Immunohistochemistry (IHC) and next-generation sequencing were performed on available formalin-fixed paraffin-embedded tissue, with differential profiling of conventional and aggressive components. All ten cases showed a conventional component of renal tumor morphologically consistent with ChRCC: three had sarcomatoid change, four had metastases, and three had both sarcomatoid change and metastases. In the primary conventional components, a typical ChRCC IHC pattern (CK7+, CD117+, and CAIX-) was observed in 8 of 10 cases; 2 cases had rare CK7 staining. In the aggressive components, CD117 and/or CK7 was lost in 7 of 10 cases; 3 cases showed loss of both. Two of 10 cases showed significant CAIX staining in the aggressive component. All 7 cases that had molecular profiling performed showed characteristic chromosomal losses reported for ChRCC, with the aggressive components generally demonstrating more copy number complexity. Recurrent TP53 mutations (TP53m) were also seen; however, surprisingly, the conventional and aggressive components had no shared TP53m: a TP53m was private to aggressive components in 2 cases and to the conventional component in 1 case, and in 4 cases, components demonstrated different TP53m. Of the 21 pathogenic alterations identified in 7 tumors, only a PTEN splicing alteration was shared between both components in one case. In conclusion, ChRCC can have IHC staining patterns and molecular profile that differ between conventional and aggressive components. Interpretation of stains on metastases or small biopsies to determine histologic subtype can be misleading. The lack of shared pathogenic mutations between the two components supports a model in which aggressive ChRCC can have convergent subclones with different TP53m.

BackgroundLymphocytic neoplasms with frequent reactive lymphocytes are uncommonly reported in dogs, and can pose a diagnostic challenge. Different diagnostic modalities such as cytology, flow cytometry, histopathology, immunohistochemistry, and clonality testing, are sometimes required for a diagnosis. This report illustrates the value of using a multi-modal diagnostic approach to decipher a complex lymphocytic tumor, and introduces immune repertoire sequencing as a diagnostic adjunct.Case presentationA 10-month-old Great Dane was referred for marked ascites. Cytologic analysis of abdominal fluid and hepatic aspirates revealed a mixed lymphocyte population including numerous large lymphocytes, yielding a diagnosis of lymphoma. Flow cytometrically, abdominal fluid lymphocytes were highly positive for CD4, CD5, CD18, CD45, and MHC II, consistent with T cell lymphoma. Due to a rapidly deteriorating clinical condition, the dog was euthanized. Post mortem histologic evaluation showed effacement of the liver by aggregates of B cells surrounded by T cells, suggestive of hepatic T cell-rich large B cell lymphoma. Immune repertoire sequencing confirmed the presence of clonal B cells in the liver but not the abdominal fluid, whereas reactive T cells with shared, polyclonal immune repertoires were found in both locations.ConclusionsT cell-rich large B cell lymphoma is a rare neoplasm in dogs that may be challenging to diagnose and classify due to mixed lymphocyte populations. In this case, the results of histopathology, immunohistochemistry and immune repertoire sequencing were most consistent with a hepatic B cell neoplasm and reactive T cells exfoliating into the abdominal fluid. Immune repertoire sequencing was helpful in delineating neoplastic from reactive lymphocytes and characterizing repertoire overlap in both compartments. The potential pitfalls of equating atypical cytomorphology and monotypic marker expression in neoplasia are highlighted.

Nucleolar stress has been implicated in the pathology and disease pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) from repeat expansions of GGGGCC in C9orf72 (C9-ALS/FTLD) but not in sporadic ALS (SALS). Previously we reported that antisense RNA transcripts are unique in C9-ALS because of their nucleolar localization in spinal motor neurons and correlation with TDP-43 mislocalization, the hallmark proteinopathy of ALS and FTLD. Here we report our further studies of 11 SALS, 11 C9-ALS and 11 control spinal cords. We find that nucleolar stress manifests specifically as shrinkage in nucleoli of C9-ALS spinal motor neurons. Nucleolar size reduction is greatest in similarly sized alpha motor neurons from C9-ALS cases and results are not skewed by the number of surviving neurons from each ALS spinal cord. Surprisingly, nucleolar shrinkage occurs before main pathological hallmarks-TDP-43 mislocalization or antisense RNA foci-appear and this suggest that nucleolar stress can precede pathology in C9-ALS, findings previously identified in C9-FTLD using sense RNA foci and dipeptide repeat proteins as pathological markers. Importantly, these observations are also seen in SALS motor neurons and thus nucleolar stress appears to be a significant and probably upstream problem in sporadic disease.

The term "cutaneous lymphadenoma" was coined in this journal for an unusual lymphoepithelial cutaneous adnexal neoplasm, possibly with immature pilosebaceous differentiation. Some authors further proposed that cutaneous lymphadenoma was an adamantinoid trichoblastoma. However, although a hair follicle differentiation is widely accepted, the fact that this is a lymphoepithelial tumor is not appropriately explained by the trichoblastoma hypothesis. Our goal was to further clarify the phenotypic and genotypic features of cutaneous lymphadenoma in a series of 11 cases. Histologically, a lobular architecture surrounded by a dense fibrous stroma was present in all cases. The lobules were composed of epithelial cells admixtured with small lymphocytes and isolated or clustered large Reed-Sternberg-like (RS-L) cells. The epithelial cells were diffusely positive for the hair follicle stem cell markers CK15, PHLDA1, and for androgen receptor. No immunostaining for markers of sebaceous differentiation was found. Intraepithelial lymphocytes were predominantly CD3+, CD4+, FoxP3+ T cells. RS-L cells showed both strong Jagged-1 and Notch1 cytoplasmic immunostaining. Androgen-regulated NKX3.1 nuclear immunostaining was present in a subset of large intralobular cells in all cases. Double immunostaining showed coexpression of NKX3.1 and CD30 in a subset of RS-L cells. No immunostaining for lymphocytic or epithelial markers was present in RS-L cells. EGFR, PIK3CA, and FGFR3 somatic mutations were found by next-generation sequencing in 56% of the cases. We consider that cutaneous lymphadenoma is a distinct benign lymphoepithelial tumor with androgen receptor and hair follicle bulge stem cell marker expression, RS-L cell-derived Notch1 ligand, and common EGFR gene mutations.

The Delta Smelt (Hypomesus transpacificus) is a small, semi-anadromous fish native to the San Francisco Bay-Delta Estuary and has been declared as critically endangered. Their olfactory biology, in particular, is poorly understood and a basic description of their sensory anatomy is needed to advance our understanding of the sensory ecology of species to inform conservation efforts to manage and protect them. We provide a description of the gross morphology, histological, immunohistochemical, and ultrastructural features of the olfactory rosette in this fish and discuss some of the functional implications in relation to olfactory ability. We show that Delta Smelt have a multilamellar olfactory rosette with allometric growth. Calretinin immunohistochemistry revealed a diffuse distribution of olfactory receptor neurons within the epithelium. Ciliated, microvillous and crypt neurons were clearly identified using morphological and immunohistochemical features. The olfactory neurons were supported by robust ciliated and secretory sustentacular cells. Although the sense of smell has been overlooked in Delta Smelt, we conclude that the olfactory epithelium has many characteristics of macrosmatic fish. With this study, we provide a foundation for future research into the sensory ecology of this imperiled fish.

Hereditary Diffuse Gastric Cancer (HDGC) syndrome is associated with CDH1 germline likely pathogenic/pathogenic variants. Carriers of CDH1 germline likely pathogenic/pathogenic variants are predisposed to diffuse gastric cancer and lobular breast cancer. This study aims to classify the CDH1 c.[715G>A] missense variant identified in a diffuse gastric cancer prone family by performing splicing studies. RT-PCR and subsequent cloning experiments were performed to investigate whether this variant completely disrupts normal splicing. This variant preferentially abolishes normal splicing through activation of a cryptic 3' acceptor splice site within exon 6 of CDH1, presumably leading to a premature protein truncation within first extracellular domain repeat of E-cadherin protein. Our results contributed to evidence necessary to resolve pathogenicity classification of this variant, indicating that this variant is to be classified as pathogenic.

Growth and differentiation factor 15 (GDF-15), also known as macrophage inhibitory cytokine 1 (MIC-1), may act as both a tumor suppressor and promotor and, by regulating NF-κB and macrophage signaling, promote early prostate carcinogenesis. To determine whether expression of these two inflammation-related proteins affect prostate cancer susceptibility, dual immunostaining of benign prostate biopsies for GDF-15 and NF-κB was done in a study of 503 case-control pairs matched on date, age, and race, nested within a historical cohort of 10,478 men. GDF-15 and NF-κB expression levels were positively correlated (r = 0.39; p

BACKGROUND: The potential for low-grade (grade group 1 [GG1]) prostate cancer (PCa) to progress to high-grade disease remains unclear. OBJECTIVE: To interrogate the molecular and biological features of low-grade PCa serially over time. DESIGN, SETTING, AND PARTICIPANTS: Nested longitudinal cohort study in an academic active surveillance (AS) program. Men were on AS for GG1 PCa from 2012 to 2017. INTERVENTION: Electronic tracking and resampling of PCa using magnetic resonance imaging/ultrasound fusion biopsy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: ERG immunohistochemistry (IHC) and targeted DNA/RNA next-generation sequencing were performed on initial and repeat biopsies. Tumor clonality was assessed. Molecular data were compared between men who upgraded and those who did not upgrade to GG ≥ 2 cancer. RESULTS AND LIMITATIONS: Sixty-six men with median age 64 yr (interquartile range [IQR], 59-69) and prostate-specific antigen 4.9 ng/mL (IQR, 3.3-6.4) underwent repeat sampling of a tracked tumor focus (median interval, 11 mo; IQR, 6-13). IHC-based ERG fusion status was concordant at initial and repeat biopsies in 63 men (95% vs expected 50%, p

Spix's cavy is a potentially good experimental model for research on reproductive biology and sexual development. The aim of the present study was to evaluate the ontogeny of the steroidogenic enzymes involved in testicular androgen synthesis during prenatal development. Testes were investigated on Days 25, 30, 40 and >50 of gestation. Immunohistochemistry and immunoblotting were used to establish the site and relative amount of androgenic enzymes, including 5α-reductase, cytosolic 17β-hydroxysteroid dehydrogenase (17β-HSDI) and mitochondrial microsomal 3β-hydroxysteroid dehydrogenase (3β-HSDII), throughout prenatal development. The testicular parenchyma began to organise on Day 25 of gestation, with the development of recognisable testicular cords. The mesonephros was established after Day 25 of gestation and the ducts differentiated to form the epididymis, as testicular cords were beginning to proliferate and the interstitium to organise by Day 30 of gestation, continuing thereafter. The androgen-synthesising enzymes 5α-reductase, 17β-HSDI and 3β-HSDII were evident in Leydig cells as they differentiated at all subsequent gestational ages studied. In addition, immunoblotting showed an increase in immunoreactivity for the enzymes at Days 30 and 40 of gestation (P

BackgroundWe previously reported the specificity of a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum for immunostaining of cholinergic neuronal cell bodies and fibers in the human colon. In this study, we investigate 3D architecture of intrinsic cholinergic innervation in the human sigmoid colon and the relationship with nitrergic neurons in the enteric plexus.MethodsWe developed a modified CLARITY tissue technique applicable for clearing human sigmoid colon specimens and immunostaining with hpChAT antiserum and co-labeling with neuronal nitric oxide synthase (nNOS) antibody. The Z-stack confocal images were processed for 3D reconstruction/segmentation/digital tracing and computational quantitation by Imaris 9.2 and 9.5.Key resultsIn the mucosa, a local micro-neuronal network formed of hpChAT-ir fibers and a few neuronal cell bodies were digitally assembled. Three layers of submucosal plexuses were displayed in 3D structure that were interconnected by hpChAT-ir fiber bundles and hpChAT-ir neurons were rarely co-labeled by nNOS. In the myenteric plexus, 30.1% of hpChAT-ir somas including Dogiel type I and II were co-labeled by nNOS and 3 classes of hpChAT-ir nerve fiber strands were visualized in 3D images and videos. The density and intensity values of hpChAT-ir fibers in 3D structure were significantly higher in the circular than in the longitudinal layer.Conclusions and inferencesThe intrinsic cholinergic innervation in the human sigmoid colon was demonstrated layer by layer for the first time in 3D microstructures. This may open a new venue to assess the structure-function relationships and pathological alterations in colonic diseases.

Follicular lymphoma (FL) is an indolent B-cell neoplasm of germinal center origin. Standard treatment regimens consist of anti-CD20 therapy with or without chemotherapy. While high response rates to initial therapy are common, patients ultimately relapse or have progressive disease. Clinical risk factors such as the Follicular Lymphoma International Prognostic Index (FLIPI) have been identified, but there is a need for prognostic and predictive biomarkers. We studied markers of lymphoma cells and tumor microenvironment by immunohistochemistry in tissue samples from patients enrolled in 1 of 4 phase 2 trials of anti-CD20-based biological therapy for previously untreated grades 1 to 2 or 3A FL. Results were correlated with progression-free survival (PFS) and PFS status at 24 months. The 4 trials included 238 patients (51.1% male, median age: 55 y) with stage III, IV, or bulky stage II disease. By FLIPI, 24.6% had low-risk, 56.8% had intermediate-risk, and 18.6% had high-risk disease. The outcome differed significantly for patients treated with lenalidomide and rituximab (CALGB 50803) compared with the other 3 trials (median: PFS not reached vs. 3.0 y, hazard ratio=3.47, 95% confidence interval: 2.11-5.72); therefore, data were stratified by clinical trial (CALGB 50803 vs. all others) and adjusted for FLIPI risk group. Among 154 patients with available tissue, interfollicular BCL6 positivity, interfollicular CD10 positivity, and elevated Ki67 proliferation index ≥30% within neoplastic follicles were each associated with inferior PFS and a high risk of the early event by PFS status at 24 months. We identify promising biomarkers for FL risk stratification that warrant further validation in phase 3 trials.

BackgroundArginases (ARG isoforms, ARG-1/ARG-2) are key regulatory enzymes of inflammation and tissue repair; however, their role after neonatal brain hypoxia (H) and hypoxia-ischemia (HI) remains unknown.MethodsC57BL/6 mice subjected to the Vannucci procedure on postnatal day (P9) were sacrificed at different timepoints. The degree of brain damage was assessed histologically. ARG spatiotemporal localization was determined via immunohistochemistry. ARG expression was measured by Western blot and activity spectrophotometrically.ResultsARG isoform expression increased during neurodevelopment (P9-P17) in the cortex and hippocampus. This was suppressed with H and HI only in the hippocampus. In the cortex, both isoforms increased with H alone and only ARG-2 increased with HI at 3 days. ARG activity during neurodevelopment remained unchanged, but increased at 1 day with H and not HI. ARG-1 localized with microglia at the injury site as early as 4 h after injury, while ARG-2 localized with neurons.ConclusionsARG isoform expression increases with age from P9 to P17, but is suppressed by injury specifically in the hippocampus and not in the cortex. Both levels and activity of ARG isoforms increase with H, while ARG-1 immunolabelling is upregulated in the HI cortex. Evidently, ARG isoforms in the brain differ in spatiotemporal localization, expression, and activity during neurodevelopment and after injury.ImpactArginase isoforms change during neurodevelopment and after neonatal brain HI. This is the first study investigating the key enzymes of inflammation and tissue repair called arginases following murine neonatal brain HI. The highly region- and cell-specific expression suggests the possibility of specific functions of arginases. ARG-1 in microglia at the injury site may regulate neuroinflammation, while ARG-2 in neurons of developmental structures may impact neurodevelopment. While further studies are needed to describe the exact role of ARGs after neonatal brain HI, our study adds valuable data on anatomical localization and expression of ARGs in brain during development and after stroke.

Clear cell meningioma represents an uncommon variant of meningioma that typically affects children and young adults. Although an enrichment of loss-of-function mutations in the SMARCE1 gene has been reported for this subtype, comprehensive molecular investigations are lacking. Here we describe a molecularly distinct subset of tumors (n = 31), initially identified through genome-wide DNA methylation screening among a cohort of 3093 meningiomas, of which most were diagnosed histologically as clear cell meningioma. This cohort was further supplemented by an additional 11 histologically diagnosed clear cell meningiomas for analysis (n = 42). Targeted DNA sequencing revealed SMARCE1 mutations in 33/34 analyzed samples, accompanied by a nuclear loss of expression determined via immunohistochemistry and a decreased SMARCE1 transcript expression in the tumor cells. Analysis of time to progression or recurrence of patients within the clear cell meningioma group (n = 14) in comparison to those with meningioma WHO grade 2 (n = 220) revealed a similar outcome and support the assignment of WHO grade 2 to these tumors. Our findings indicate the existence of a highly distinct epigenetic signature of clear cell meningiomas, separate from all other variants of meningiomas, with recurrent mutations in the SMARCE1 gene. This suggests that these tumors may arise from a different precursor cell population than the broad spectrum of the other meningioma subtypes.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with CCR5- donor cells is the only treatment known to cure HIV-1 in patients with underlying malignancy. This is likely due to a donor cell-mediated graft-versus-host effect targeting HIV reservoirs. Allo-HSCT would not be an acceptable therapy for most people living with HIV due to the transplant-related side effects. Chimeric antigen receptor (CAR) immunotherapies specifically traffic to malignant lymphoid tissues (lymphomas) and, in some settings, are able to replace allo-HSCT. Here, we quantified the engraftment of HSC-derived, virus-directed CAR T cells within HIV reservoirs in a macaque model of HIV infection, using potentially novel IHC assays. HSC-derived CAR cells trafficked to and displayed multilineage engraftment within tissue-associated viral reservoirs, persisting for nearly 2 years in lymphoid germinal centers, the brain, and the gastrointestinal tract. Our findings demonstrate that HSC-derived CAR+ cells reside long-term and proliferate in numerous tissues relevant for HIV infection and cancer.

Decellularized and de-epithelialized placenta membranes have widely been used as scaffolds and grafts in tissue engineering and regenerative medicine. Exceptional pro-angiogenic and biomechanical properties and low immunogenicity have made the amniochorionic membrane a unique substrate which provides an enriched niche for cellular growth. Herein, an optimized combination of enzymatic solutions (based on streptokinase) with mechanical scrapping is used to remove the amniotic epithelium and chorion trophoblastic layer, which resulted in exposing the basement membranes of both sides without their separation and subsequent damages to the in-between spongy layer. Biomechanical and biodegradability properties, endothelial proliferation capacity, and in vivo pro-angiogenic capabilities of the substrate were also evaluated. Histological staining, immunohistochemistry (IHC) staining for collagen IV, and scanning electron microscope demonstrated that the underlying amniotic and chorionic basement membranes remained intact while the epithelial and trophoblastic layers were entirely removed without considerable damage to basement membranes. The biomechanical evaluation showed that the scaffold is suturable. Proliferation assay, real-time polymerase chain reaction for endothelial adhesion molecules, and IHC demonstrated that both side basement membranes could support the growth of endothelial cells without altering endothelial characteristics. The dorsal skinfold chamber animal model indicated that both side basement membranes could promote angiogenesis. This bi-sided substrate with two exposed surfaces for cultivating various cells would have potential applications in the skin, cardiac, vascularized composite allografts, and microvascular tissue engineering.

Diabetic kidney disease (DKD) is a key microvascular complication of diabetes, with few therapies for targeting renal disease pathogenesis and progression. We performed transcriptional and protein studies on 103 unique blood and kidney tissue samples from patients with and without diabetes to understand the pathophysiology of DKD injury and its progression. The study was based on the use of 3 unique patient cohorts: peripheral blood mononuclear cell (PBMC) transcriptional studies were conducted on 30 patients with DKD with advancing kidney injury; Gene Expression Omnibus (GEO) data was downloaded, containing transcriptional measures from 51 microdissected glomerulous from patients with DKD. Additionally, 12 independent kidney tissue sections from patients with or without DKD were used for validation of target genes in diabetic kidney injury by kidney tissue immunohistochemistry and immunofluorescence. PBMC DKD transcriptional analysis, identified 853 genes (p < 0.05) with increasing expression with progression of albuminuria and kidney injury in patients with diabetes. GEO data was downloaded, normalized, and analyzed for significantly changed genes. Of the 325 significantly up regulated genes in DKD glomerulous (p < 0.05), 28 overlapped in PBMC and diabetic kidney, with perturbed FcER1 signaling as a significantly enriched canonical pathway. FcER1 was validated to be significantly increased in advanced DKD, where it was also seen to be specifically co-expressed in the kidney biopsy with tissue mast cells. In conclusion, we demonstrate how leveraging public and private human transcriptional datasets can discover and validate innate immunity and inflammation as key mechanistic pathways in DKD progression, and uncover FcER1 as a putative new DKD target for rational drug design.

Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin. Current approaches have poor precision, require large amounts of tissue, and are difficult to standardize. This paper presents an immuno-mass spectrometry imaging method using gadolinium (Gd)-labeled anti-dystrophin antibodies and laser ablation-inductively coupled plasma-mass spectrometry to simultaneously quantify and localize dystrophin in muscle sections. Gd is quantified as a proxy for the relative expression of dystrophin and was validated in murine and human skeletal muscle sections following k-means clustering segmentation, before application to DMD patients with different gene mutations where dystrophin expression was measured up to 100 µg kg-1 Gd. These results demonstrate that immuno-mass spectrometry imaging is a viable approach for pre-clinical to clinical research in DMD. It rapidly quantified relative dystrophin in single tissue sections, efficiently used valuable patient resources, and may provide information on drug efficacy for clinical translation.

BackgroundMolecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein aggregation. A lead MT, called CLR01, has been demonstrated to inhibit the aggregation and toxicity of multiple amyloidogenic proteins in vitro and in vivo. Previously, we evaluated the effect of CLR01 in the 3 × Tg mouse model of Alzheimer's disease, which overexpresses mutant human presenilin 1, amyloid β-protein precursor, and tau and found that subcutaneous administration of the compound for 1 month led to a robust reduction of amyloid plaques, neurofibrillary tangles, and microgliosis. CLR01 also has been demonstrated to inhibit tau aggregation in vitro and tau seeding in cell culture, yet because in Alzheimer's disease (AD) and in the 3 × Tg model, tau hyperphosphorylation and aggregation are thought to be downstream of Aβ insults, the study in this model left open the question whether CLR01 affected tau in vivo directly or indirectly.MethodsTo determine if CLR01 could ameliorate tau pathology directly in vivo, we tested the compound similarly using the P301S-tau (line PS19) mouse model. Mice were administered 0.3 or 1.0 mg/kg per day CLR01 and tested for muscle strength and behavioral deficits, including anxiety- and disinhibition-like behavior. Their brains then were analyzed by immunohistochemical and biochemical assays for pathological forms of tau, neurodegeneration, and glial pathology.ResultsCLR01 treatment ameliorated muscle-strength deterioration, anxiety-, and disinhibition-like behavior. Improved phenotype was associated with decreased levels of pathologic tau forms, suggesting that CLR01 exerts a direct effect on tau in vivo. Limitations of the study included a relatively short treatment period of the mice at an age in which full pathology is not yet developed. In addition, high variability in this model lowered the statistical significance of the findings of some outcome measures.ConclusionsThe findings suggest that CLR01 is a particularly attractive candidate for the treatment of AD because it targets simultaneously the two major pathogenic proteins instigating and propagating the disease, amyloid β-protein (Aβ), and tau, respectively. In addition, our study suggests that CLR01 can be used for the treatment of other tauopathies in the absence of amyloid pathology.

Cancer is the second leading cause of death in the United States. Although screening facilitates prevention and early detection and is one of the most effective approaches to reducing cancer mortality, participation is low-particularly among underserved populations. In a large, preregistered field experiment (n = 7711), we tested whether deadlines-both with and without monetary incentives tied to them-increase colorectal cancer (CRC) screening. We found that all screening invitations with an imposed deadline increased completion, ranging from 2.5% to 7.3% relative to control (ps

BACKGROUND:Renal cell carcinoma (RCC) is a highly vascular tumor and patients with low risk metastatic RCC of clear-cell histological sub-type (mccRCC) are treated with tyrosine-kinase inhibitors (TKIs), sunitinib, as the first-line of treatment. Unfortunately, TKI resistance eventually develops, and the underlying molecular mechanism is not well understood. METHODS:RCC cell-line with metastatic clear-cell histology (Caki-1), and patient samples were analysed to identify the role of Y-box binding protein 1 (YB-1) and ATP-binding cassette sub-family B member 1 (ABCB-1) in acquired sunitinib-resistance development. Caki-1 was conditioned with increasing sunitinib doses to recapitulate acquired resistance development in clinics. Sunitinib-conditioned and wild-type Caki-1 were subjected to cell viability assay, scratch assay, chicken embryo chorioallantoic membrane engraftment and proteomics analysis. Classical biochemical assays like flow cytometry, immunofluorescent staining, immunohistochemical staining, optical coherence tomography imaging, Western Blot and RT-PCR assays were applied to determine the possible mechanism of sunitinib-resistance development and the effect of drug treatments. Publicly available data was also used to determine the role of YB-1 upregulation in ccRCC and the patients' overall survival. RESULTS:We demonstrate that YB-1 and ABCB-1 are upregulated in sunitinib-resistant in vitro, ex vivo, in vivo and patient samples compared to the sensitive samples. This provides evidence to a mechanism of acquired sunitinib-resistance development in mccRCC. Furthermore, our results establish that inhibiting ABCB-1 with elacridar, in addition to sunitinib, has a positive impact on reverting sunitinib-resistance development in in vitro, ex vivo and in vivo models. CONCLUSION:This work proposes a targeted therapy (elacridar and sunitinib) to re-sensitize sunitinib-resistant mccRCC and, possibly, slow disease progression.

Paragangliomas are neuroendocrine tumors of the autonomic nervous system that are variably clinically functional and have a potential for metastasis. Up to 40% occur in the setting of a hereditary syndrome, most commonly due to germline mutations in succinate dehydrogenase (SDHx) genes. Immunohistochemically, paragangliomas are characteristically GATA3-positive and cytokeratin-negative, with loss of SDHB expression in most hereditary cases. In contrast, the rare paragangliomas arising in the cauda equina (CEP) or filum terminale region have been shown to be hormonally silent, clinically indolent, and have variable keratin expression, suggesting these tumors may represent a separate pathologic entity. We retrospectively evaluated 17 CEPs from 11 male and 6 female patients with a median age of 38 years (range 21-82), none with a family history of neuroendocrine neoplasia. Six of the 17 tumors demonstrated prominent gangliocytic or ganglioneuromatous differentiation. By immunohistochemistry, none of the CEPs showed GATA3 positivity or loss of SDHB staining; all 17 CEPs were cytokeratin positive. Genome-wide DNA methylation profiling was performed on 12 of the tumors and compared with publicly available genome-wide DNA methylation data. Clustering analysis showed that CEPs form a distinct epigenetic group, separate from paragangliomas of extraspinal sites, pheochromocytomas, and other neuroendocrine neoplasms. Copy number analysis revealed diploid genomes in the vast majority of CEPs, whereas extraspinal paragangliomas were mostly aneuploid with recurrent trisomy 1q and monosomies of 1p, 3, and 11, none of which were present in the cohort of CEP. Together, these findings indicate that CEPs likely represent a distinct entity. Future genomic studies are needed to further elucidate the molecular pathogenesis of these tumors.

Galactoceles are benign lesions formed at the expense of breast glandular tissue. Its occurrence in boys is exceptional, with isolated reports in the scientific literature. We present the case of a 2-year-old boy who debuted with a unilateral breast enlargement of 18 months of evolution, with no other associated symptoms. Initial hormonal study showed no alterations. Ultrasonography showed the presence of a homogeneous 5-cm retroareolar cyst. Surgical excision was indicated. During the procedure, abundant milk drained from the lesion. The histopathological study, supported by GATA-3 immunohistochemical staining, confirmed the diagnosis of galactocele. The range of possible differential diagnoses for unilateral or bilateral breast enlargement in boys is wide and includes neoplastic, vascular, and hormonal etiologies. Galactocele, which is an exceptional entity in this group, should be considered. Immunohistochemical techniques such as GATA-3, characteristic of the breast epithelium, can contribute to the diagnosis. [ABSTRACT FROM AUTHOR]

BackgroundMelanocytic tumors are often challenging and constitute almost one in four skin biopsies. Immunohistochemical (IHC) studies may assist diagnosis; however, indications for their use are not standardized.MethodsA test set of 240 skin biopsies of melanocytic tumors was examined by 187 pathologists from 10 US states, interpreting 48 cases in Phase I and either 36 or 48 cases in Phase II. Participant and diagnosis characteristics were compared between those who reported they would have ordered, or who would have not ordered IHC on individual cases. Intraobserver analysis examined consistency in the intent to order when pathologists interpreted the same cases on two occasions.ResultsOf 187 participants interpreting 48 cases each, 21 (11%) did not request IHC tests for any case, 85 (45%) requested testing for 1 to 6 cases, and 81 (43%) requested testing for ≥6 cases. Of 240 cases, 229 had at least one participant requesting testing. Only 2 out of 240 cases had more than 50% of participants requesting testing. Increased utilization of testing was associated with younger age of pathologist, board-certification in dermatopathology, low confidence in diagnosis, and lesions in intermediate MPATH-Dx classes 2 to 4. The median intraobserver concordance for requesting tests among 72 participants interpreting the same 48 cases in Phases I and II was 81% (IQR 73%-90%) and the median Kappa statistic was 0.20 (IQR 0.00, 0.39).ConclusionSubstantial variability exists among pathologists in utilizing IHC.

Cerebral microbleeds (CMB) are a common MRI finding, representing underlying cerebral microhemorrhages (CMH). The etiology of CMB and microhemorrhages is obscure. We conducted a pathological investigation of CMH, combining standard and immunohistological analyses of postmortem human brains. We analyzed 5 brain regions (middle frontal gyrus, occipital pole, rostral cingulate cortex, caudal cingulate cortex, and basal ganglia) of 76 brain bank subjects (mean age ± SE 90 ± 1.4 years). Prussian blue positivity, used as an index of CMH, was subjected to quantitative analysis for all 5 brain regions. Brains from the top and bottom quartiles (n = 19 each) were compared for quantitative immunohistological findings of smooth muscle actin, claudin-5, and fibrinogen, and for Sclerosis Index (SI) (a measure of arteriolar remodeling). Brains in the top quartile (i.e. with most extensive CMH) had significantly higher SI in the 5 brain regions combined (0.379 ± 0.007 vs 0.355 ± 0.008; p

The ability to rapidly change gene expression patterns is essential for differentiation, development, and functioning of the brain. Throughout development, or in response to environmental stimuli, gene expression patterns are tightly regulated by the dynamic interplay between transcription activators and repressors. Nuclear receptor corepressor 1 (NCoR1) and silencing mediator for retinoid or thyroid-hormone receptors (SMRT) are the best characterized transcriptional co-repressors from a molecular point of view. They mediate epigenetic silencing of gene expression in a wide range of developmental and homeostatic processes in many tissues, including the brain. For instance, NCoR1 and SMRT regulate neuronal stem cell proliferation and differentiation during brain development and they have been implicated in learning and memory. However, we still have a limited understanding of their regional and cell type-specific expression in the brain. In this study, we used fluorescent immunohistochemistry to map their expression patterns throughout the adult mouse brain. Our findings reveal that NCoR1 and SMRT share an overall neuroanatomical distribution, and are detected in both excitatory and inhibitory neurons. However, we observed striking differences in their cell type-specific expression in glial cells. Specifically, all oligodendrocytes express NCoR1, but only a subset express SMRT. In addition, NCoR1, but not SMRT, was detected in a subset of astrocytes and in the microglia. These novel observations are corroborated by single cell transcriptomics and emphasize how NCoR1 and SMRT may contribute to distinct biological functions, suggesting an exclusive role of NCoR1 in innate immune responses in the brain.

Human papillomavirus (HPV) is a principal driver for most oropharyngeal squamous cell carcinomas (OPSCCs), where it is strongly associated with improved survival. HPV is much less frequently detected in squamous cell carcinomas arising in nonoropharyngeal sites (non-OPSCCs), and its pathogenic role and prognostic value in these tumors is unclear. We evaluated the clinicopathologic features of 52 non-OPSCCs considered HPV-positive based upon p16 immunohistochemistry and direct HPV detection using RNA in situ hybridization (ISH), DNA ISH, or real-time DNA polymerase chain reaction. The HPV-positive non-OPSCCs were from the larynx (n=27), oral cavity (n=21), and hypopharynx (n=4). While most cases (n=34, 65%) showed classic histologic features of HPV-positive OPSCC, including endophytic growth, minimal keratinization, and hyperchromatic nuclei without koilocytic changes, a subset (n=13, 25%) were characterized by exophytic growth, exuberant surface hyperkeratosis and parakeratosis, marked nuclear pleomorphism, and prominent koilocytic atypia. These antithetical features were highly reminiscent of the warty variant of HPV-positive squamous cell carcinoma described in anogenital sites. Compared with tumors without warty features, the warty tumors presented at lower stage and were not associated with lymph node metastasis, local recurrence, or distant spread (4 y disease-free survival of 100% vs. 66%, P=0.069). The presence of transcriptionally active HPV as detected by RNA ISH suggests a pathogenic role for HPV in these nonoropharyngeal sites. While most HPV-positive non-OPSCCs are morphologically similar to their tonsillar counterparts, this study highlights a previously unrecognized warty variant that may be associated with a highly favorable clinical outcome.

Cardiomyocyte (CM) proliferative potential varies considerably across species. While lower vertebrates and neonatal mammals retain robust capacities for CM proliferation, adult mammalian CMs lose proliferative potential due to cell-cycle withdrawal and polyploidization, failing to mount a proliferative response to regenerate lost CMs after cardiac injury. The decline of murine CM proliferative potential occurs in the neonatal period when the endocrine system undergoes drastic changes for adaptation to extrauterine life. We recently demonstrated that thyroid hormone (TH) signaling functions as a primary factor driving CM proliferative potential loss in vertebrates. Whether other hormonal pathways govern this process remains largely unexplored. Here we showed that agonists of glucocorticoid receptor (GR) and vitamin D receptor (VDR) suppressed neonatal CM proliferation. We next examined CM nucleation and proliferation in neonatal mutant mice lacking GR or VDR specifically in CMs, but we observed no difference between mutant and control littermates at postnatal day 14. Additionally, we generated compound mutant mice that lack GR or VDR and express dominant-negative TH receptor alpha in their CMs, and similarly observed no increase in CM proliferative potential compared to dominant-negative TH receptor alpha mice alone. Thus, although GR and VDR activation is sufficient to inhibit CM proliferation, they seem to be dispensable for neonatal CM cell-cycle exit and polyploidization in vivo. In addition, given the recent report that VDR activation in zebrafish promotes CM proliferation and tissue regeneration, our results suggest distinct roles of VDR in zebrafish and rodent CM cell-cycle regulation.

The handling-induced dark neuron is a histological artifact observed in brain samples handled before fixation with aldehydes. To explore associations between dark neurons and immunohistochemical alterations in mouse brains, we examined protein products encoded by Cav3 (neuronal perikarya/neurites), Rbbp4 (neuronal nuclei), Gfap (astroglia), and Aif1 (microglia) genes in adjacent tissue sections. Here, dark neurons were incidental findings from our prior project, studying the effects of age and high-fat diet on metabolic homeostasis in male C57BL/6N mice. Available were brains from 4 study groups: middle-aged/control diet, middle-aged/high-fat diet, old/control diet, and old/high-fat diet. Young/control diet mice were used as baseline. The hemibrains were immersion-fixed with paraformaldehyde and paraffin-embedded. In the hippocampal formation, we found negative correlations between dark neuron hyperbasophilia and immunoreactivity for CAV3, RBBP4, and glial fibrillary acidic protein (GFAP) using quantitative image analysis. There was no significant difference in dark neuron hyperbasophilia or immunoreactivity for any protein examined among all groups. In contrast, in the hippocampal fimbria, old age seemed to be associated with higher immunoreactivity for GFAP and allograft inflammatory factor-1. Our findings suggest that loss of immunohistochemical reactivity for CAV3, RBBP4, and GFAP in the hippocampal formation is an artifact associated with the occurrence of dark neurons. The unawareness of dark neurons may lead to misinterpretation of immunohistochemical reactivity alterations.

The immune mechanisms that cause tissue injury in lupus nephritis have been challenging to define. The advent of high-dimensional cellular analyses, such as single-cell RNA sequencing, has enabled detailed characterization of the cell populations present in small biopsy samples of kidney tissue. In parallel, the development of methods that cryopreserve kidney biopsy specimens in a manner that preserves intact, viable cells, has enabled the uniform analysis of tissue samples collected at multiple sites and across many geographic areas and demographic cohorts with high-dimensional platforms. The application of these methods to kidney biopsy samples from patients with lupus nephritis has begun to define the phenotypes of both infiltrating and resident immune cells, as well as parenchymal cells, present in nephritic kidneys. The detection of similar immune cell populations in urine suggests that it might be possible to non-invasively monitor immune activation in kidneys. Once applied to large patient cohorts, these high-dimensional studies might enable patient stratification according to patterns of immune cell activation in the kidney or identify disease features that can be used as surrogate measures of efficacy in clinical trials. Applied broadly across multiple inflammatory kidney diseases, these studies promise to enormously expand our understanding of renal inflammation in the next decade.

Behavioral and clinical studies suggest a critical role of substance P (SP)/neurokinin-1 receptor (NK-1R) signaling in alcohol dependence. Here, we examined regulation of GABA transmission in the medial subdivision of the central amygdala (CeM) by the SP/NK-1R system, and its neuroadaptation following chronic alcohol exposure. In naïve rats, SP increased action potential-dependent GABA release, and the selective NK-1R antagonist L822429 decreased it, demonstrating SP regulation of CeM activity under basal conditions. SP induced a larger GABA release in alcohol-dependent rats accompanied by decreased NK-1R expression compared to naïve controls, suggesting NK-1R hypersensitivity which persisted during protracted alcohol withdrawal. The NK-1R antagonist blocked acute alcohol-induced GABA release in alcohol-dependent and withdrawn but not in naïve rats, indicating that dependence engages the SP/NK-1R system to mediate acute effects of alcohol. Collectively, we report long-lasting CeA NK-1R hypersensitivity corroborating that NK-1Rs are promising targets for the treatment of alcohol use disorder.

BACKGROUND: Colorectal cancer (CRC) screening remains underused, especially in safety-net systems. The objective of this study was to determine the effectiveness, costs, and cost-effectiveness of organized outreach using fecal immunochemical tests (FITs) compared with usual care. METHODS: Patients age 50-75 years eligible for CRC screening from eight participating primary care safety-net clinics were randomly assigned to outreach intervention with usual care vs usual care alone. The intervention included a mailed postcard and call, followed by a mailed FIT kit, and a reminder phone call if the FIT kit was not returned. The primary outcome was screening participation at 1 year and a microcosting analysis of the outreach activities with embedded long-term cost-effectiveness of outreach. All statistical tests were two-sided. RESULTS: A total of 5386 patients were randomly assigned to the intervention group and 5434 to usual care. FIT screening was statistically significantly higher in the intervention group than in the control group (57.9% vs 37.4%, P

BackgroundColorectal cancer (CRC) screening remains underused, especially in safety-net systems. The objective of this study was to determine the effectiveness, costs, and cost-effectiveness of organized outreach using fecal immunochemical tests (FITs) compared with usual care.MethodsPatients age 50-75 years eligible for CRC screening from eight participating primary care safety-net clinics were randomly assigned to outreach intervention with usual care vs usual care alone. The intervention included a mailed postcard and call, followed by a mailed FIT kit, and a reminder phone call if the FIT kit was not returned. The primary outcome was screening participation at 1 year and a microcosting analysis of the outreach activities with embedded long-term cost-effectiveness of outreach. All statistical tests were two-sided.ResultsA total of 5386 patients were randomly assigned to the intervention group and 5434 to usual care. FIT screening was statistically significantly higher in the intervention group than in the control group (57.9% vs 37.4%, P

PurposeAlthough intracranial meningiomas are the most common primary brain tumor in adults, treatment options are few and have traditionally been limited to surgical resection and radiotherapy. Additional targeted therapies and biomarkers are needed, especially as complete surgical resection is frequently not feasible in many patients.MethodsNon-pathologic brain tissue from 3 patients undergoing routine autopsies and tumor specimens from 16 patients requiring surgical resection for meningioma were collected. EMP2 protein expression was evaluated by immunohistochemistry and western blot analysis. EMP2 mRNA expression was also investigated using surgical specimens and validated by analysis of several independent NCBI GEO databases.ResultsEMP2 mRNA expression levels were found to be higher in meningioma relative to non-pathologic meninges (P = 0.0013) and brain (P = 0.0011). Concordantly, strong EMP2 protein expression was demonstrated in 100% of meningioma specimens from all 16 patients, with no observable protein expression in normal brain tissue samples from 3 subjects (P

Severe loss of excitatory synapses in key brain regions is thought to be one of the major mechanisms underlying stress-induced cognitive impairment. To date, however, the identity of the affected circuits remains elusive. Here we examined the effect of exposure to repeated multiple concurrent stressors (RMS) on the connectivity of the posterior parietal cortex (PPC) in adolescent male mice. We found that RMS led to layer-specific elimination of excitatory synapses with the most pronounced loss observed in deeper cortical layers. Quantitative analysis of cortical projections to the PPC revealed a significant loss of sensory and retrosplenial inputs to the PPC while contralateral and frontal projections were preserved. These results were confirmed by decreased synaptic strength from sensory, but not from contralateral, projections in stress-exposed animals. Functionally, RMS disrupted visuospatial working memory performance, implicating disrupted higher-order visual processing. These effects were not observed in mice subjected to restraint-only stress for an identical period of time. The PPC is considered to be a cortical hub for multisensory integration, working memory, and perceptual decision-making. Our data suggest that sensory information streams targeting the PPC may be impacted by recurring stress, likely contributing to stress-induced cognitive impairment.SIGNIFICANCE STATEMENT Repeated exposure to stress profoundly impairs cognitive functions like memory, attention, or decision-making. There is emerging evidence that stress not only impacts high-order regions of the brain, but may affect earlier stages of cognitive processing. Our work focuses on the posterior parietal cortex, a brain region supporting short-term memory, multisensory integration, and decision-making. We show evidence that repeated stress specifically damages sensory inputs to this region. This disruption of synaptic connectivity is linked to working memory impairment and is specific to repeated exposure to multiple stressors. Altogether, our data provide a potential alternative explanation to ailments previously attributed to downstream, cognitive brain structures.

Brain microbleeds are increased in chronic kidney disease (CKD) and their presence increases risk of cognitive decline and stroke. We examined the interaction between CKD and brain microhemorrhages (the neuropathological substrate of microbleeds) in mouse and cell culture models and studied progression of microbleed burden on serial brain imaging from humans. Mouse studies: Two CKD models were investigated: adenine-induced tubulointerstitial nephritis and surgical 5/6 nephrectomy. Cell culture studies: bEnd.3 mouse brain endothelial cells were grown to confluence, and monolayer integrity was measured after exposure to 5-15% human uremic serum or increasing concentrations of urea. Human studies: Progression of brain microbleeds was evaluated on serial MRI from control, pre-dialysis CKD, and dialysis patients. Microhemorrhages were increased 2-2.5-fold in mice with CKD independent of higher blood pressure in the 5/6 nephrectomy model. IgG staining was increased in CKD animals, consistent with increased blood-brain barrier permeability. Incubation of bEnd.3 cells with uremic serum or elevated urea produced a dose-dependent drop in trans-endothelial electrical resistance. Elevated urea induced actin cytoskeleton derangements and decreased claudin-5 expression. In human subjects, prevalence of microbleeds was 50% in both CKD cohorts compared with 10% in age-matched controls. More patients in the dialysis cohort had increased microbleeds on follow-up MRI after 1.5 years. CKD disrupts the blood-brain barrier and increases brain microhemorrhages in mice and microbleeds in humans. Elevated urea alters the actin cytoskeleton and tight junction proteins in cultured endothelial cells, suggesting that these mechanisms explain (at least in part) the microhemorrhages and microbleeds observed in the animal and human studies.

PurposeTo investigate the molecular and cellular mechanisms of cataract induced by cold temperatures in young lenses of wild-type C57BL/6J (B6), wild-type 129SvJae (129), and filensin knockout (KO) mice. To determine how lens intermediate filament proteins, filensin (BFSP1) and CP49 (BFSP2), are involved in the formation of cold cataract.MethodsThe formation of cold cataract was examined in enucleated lenses at different temperatures and was imaged under a dissecting microscope. Lens vibratome sections were prepared, immunostained with different antibodies and fluorescent probes, and then imaged with a laser confocal microscope to evaluate the protein distribution and the membrane and cytoskeleton structures in the lens fibers.ResultsPostnatal day 14 (P14) wild-type B6 lenses showed cataracts dependent on cold temperatures in interior fibers about 420-875 µm (zone III) and 245-875 µm (zone II and zone III) from the lens surface, under 25 °C and 4 °C, respectively. In contrast, wild-type 129 (with CP49 gene deletion) and filensin KO (on the B6 background) lenses did not have cold cataracts at 25 °C but displayed a reduced cold cataract, especially in zone III, at 4 °C. Immunofluorescent staining data revealed that CP49 and filensin proteins were uniformly distributed in fiber cell cytosols without cold cataracts but accumulated or aggregated in the cell boundaries of the fibers where cold cataracts appeared.ConclusionsCP49 and filensin are important components for the formation of cold cataract in young B6 mouse lenses. Accumulated or aggregated CP49 and filensin beaded intermediate filaments in fiber cell boundaries might directly or indirectly contribute to the light scattering of cold cataract. Cold cataract in zone II is independent of beaded intermediate filaments. CP49 and filensin intermediate filaments and other lens proteins probably form distinct high molecular organizations to regulate lens transparency in interior fibers.

Insulin resistance is a major factor in obesity-linked type 2 diabetes. PPARγ is a master regulator of adipogenesis, and small molecule agonists, termed thiazolidinediones, are potent therapeutic insulin sensitizers. Here, we studied the role of transcriptional co-activator with PDZ-binding motif (TAZ) as a transcriptional co-repressor of PPARγ. We found that adipocyte-specific TAZ knockout (TAZ AKO) mice demonstrate a constitutively active PPARγ state. Obese TAZ AKO mice show improved glucose tolerance and insulin sensitivity compared to littermate controls. PPARγ response genes are upregulated in adipose tissue from TAZ AKO mice and adipose tissue inflammation was also decreased. In vitro and in vivo mechanistic studies revealed that the TAZ-PPARγ interaction is partially dependent on ERK-mediated Ser112 PPARγ phosphorylation. As adipocyte PPARγ Ser112 phosphorylation is increased in obesity, repression of PPARγ activity by TAZ could contribute to insulin resistance. These results identify TAZ as a new factor in the development of obesity-induced insulin resistance.

Aims/hypothesisThe conserved hypoxia inducible factor 1 α (HIF1α) injury-response pro-survival pathway has recently been implicated in early beta cell dysfunction but slow beta cell loss in type 2 diabetes. We hypothesised that the unexplained prolonged prediabetes phase in type 1 diabetes may also be, in part, due to activation of the HIF1α signalling pathway.MethodsRNA sequencing (RNA-Seq) data from human islets with type 1 diabetes or after cytokine exposure in vitro was evaluated for activation of HIF1α targets. This was corroborated by immunostaining human pancreases from individuals with type 1 diabetes for 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), the key effector of HIF1α-mediated metabolic remodelling, and by western blotting of islets and INS-1 832/13 cells exposed to cytokines implicated in type 1 diabetes.ResultsHIF1α signalling is activated (p = 4.5 × 10-9) in islets from individuals with type 1 diabetes, and in human islets exposed in vitro to cytokines implicated in type 1 diabetes (p = 1.1 × 10-14). Expression of PFKFB3 is increased fivefold (p

The current study was designed to evaluate the pathogenesis, pathology and immune response of female genital tract infection with Chlamydia trachomatis L2c, the most recently discovered lymphogranuloma venereum strain, using a porcine model of sexually transmitted infections. Pigs were mock infected, infected once or infected and re-infected intravaginally, and samples were obtained for chlamydial culture, gross and microscopic pathology, and humoral and cell-mediated immunity. Intravaginal inoculation of pigs with this bacterium resulted in an infection that was confined to the urogenital tract, where inflammation and pathology were caused that resembled what is seen in human infection. Re-infection resulted in more severe gross pathology than primary infection, and chlamydial colonization of the urogenital tract was similar for primary infected and re-infected pigs. This indicates that primary infection failed to induce protective immune responses against re-infection. Indeed, the proliferative responses of mononuclear cells from blood and lymphoid tissues to C. trachomatis strain L2c were never statistically different among groups, suggesting that C. trachomatis-specific lymphocytes were not generated following infection or re-infection. Nevertheless, anti-chlamydial antibodies were elicited in sera and vaginal secretions after primary infection and re-infection, clearly resulting in a secondary systemic and mucosal antibody response. While primary infection did not protect against reinfection, the porcine model is relevant for evaluating immune and pathogenic responses for emerging and known C. trachomatis strains to advance drug and/or vaccine development in humans.