CD31 Immunohistochemistry Research Articles

High Grade B-Cell Lymphoma with 11q Aberration Is Not a Rare Event in HIV-Associated Lymphoma

High grade B-cell lymphoma with 11q aberration/diffuse large cell lymphoma with 11q aberration (HGBL-11q; previously classified as Burkitt-like lymphoma with 11q aberration, a provisional category in WHO 4-revised) is a relatively new entity recognized by both the WHO-5 and International Consensus Classification (ICC). These lesions show high grade morphology which is often, but not always, similar to Burkitt lymphoma (BL), and a genetic composition that is similar to that of germinal center (GC) derived diffuse large cell lymphoma (DLBCL). While these lymphomas have been described in immunocompetent patients, where they occur usually in children/adolescents and adults under 60 years, they have been only been rarely described in HIV positive patients. In this study we examined the incidence of the 11q aberration in HIV positive lymphomas. Four tissue microarrays (TMA) containing 144 HIV-associated lymphomas (HIV-NHLs; 57 pre-ART; 16 females; median age 44 years) were examined for the 11q aberration by FISH using the ZytoLight SPEC 11q gain/loss Triple Color Probe (ZytoVision GmbH, Fischai1, Germany). The cases were classified morphologically, evaluated for cell of origin (Hans' criteria; immunohistochemistry for CD10, BCL6, MUM1), the presence of Kaposi sarcoma herpesvirus (KSHV; immunohistochemistry for LANA) and the Epstein Barr virus (EBV; in situ hybridization; EBER probe). Clinical data, when available, was obtained from the EMR. Of the 144 cases, 123 (85%) were evaluable by FISH, including 76 DLBCLs, 10 BL, 14 primary effusion lymphoma (PEL)/EC-PEL, 7 plasmablastic lymphomas, 1 marginal zone lymphomas and 15 polymorphic lymphoproliferative lesions. Four abnormal patterns were seen (Table 1) in 39 cases (32%): polysomy (≥ 3 copies of chromosome 11), 11q23q24 amplification (≥10 copies), variant or atypical 11q aberration (11q24 loss only) and classic or typical 11q aberration (11q23 gain and 11q24 loss). Although survival data was available in only a proportion of HIV+ patients with HGBL-11q, these patients showed in general better survival than patients with other chromosome 11 abnormalities (Table 1) and appear to trend to better survivals compared to HIV+ lymphoma patients without the 11q aberration (alive: 11-202 mo; median 73 mo; died 1 - 45 mo; median 6 mo). Our results demonstrate that chromosome 11 abnormalities are not an uncommon finding in HIV positive lymphoproliferative lesions. Furthermore, although previously only rarely described in HIV+ individuals, HIV-associated lymphomas with the classic or variant 11q aberration is not a rare event. As in immunocompetent patients, the HIV-associated high grade B cell lymphomas with 11q aberration are usually of germinal center origin and EBV negative and in at least some patients are associated with a relatively good prognosis. Thus, these findings indicate that HIV-associated high grade B cell lymphomas with 11q aberration exhibit similar biologic features as those arising in immunocompetent individuals.

T-Cell Lgll of the Γδ Subtype: Clinically Relevant Fact or Academically Interesting Finding?

Large granular lymphocyte leukemia (LGLL) is a rare group of mature T and NK cell lymphoproliferative disorders, with an annual incidence of 0.2 - 0.72 cases per 1 million individuals. Median age of onset is 65, with case prevalence roughly equivalent between sexes. T-Cell LGLL (T-LGLL) makes up 85-91% of cases. The majority of T-LGLL clones express Tαβ, with only 5% of cases expressing Tγδ. Outcomes and treatment data on this subtype is limited. Recent observational data suggest reduced overall survival (OS), increased incidence of cytopenias and associated autoimmune diseases, and favorable response to first line therapy with cyclosporine, in comparison to the Tαβ subtype. Here, we summarize disease biology and clinical presentation and provide review of recent literature describing clinical course and treatment options in the context of a patient case. A woman in her early 70's with osteoporosis, asthma and hypertension presented to hematology after she was found to have leukocytosis of 12.1* 103/µL during routine lab work. CBC w/differential showed lymphocytosis of 11.0* 103/µL with absolute neutrophil count of 496 *103/µL and thrombocytopenia (121* 103/µL). She was asymptomatic without history of malignancy or autoimmune disease, prior available labwork was normal and physical exam was unremarkable. Peripheral smear showed atypical lymphocytes, with flow cytometry demonstrating 50.45% clonality with CD2+, CD3+, CD4-, CD7+, CD8 +/- , CD16+, CD 56-, CD57+/-, Tγδ + phenotype. Bone marrow biopsy demonstrated hypercellular (70%) marrow with scattered lymphoid aggregates with clonal infiltration of 20% core cellularity by CD3 immunohistochemistry. Bone marrow flow demonstrated the T-Cell phenotype seen in peripheral blood. T-Cell clonality study demonstrated TCR gamma chain rearrangement. PET/CT showed mildly increased FDG uptake throughout bone marrow. Next-Generation Sequencing confirmed mutations of STAT3 (D661Y), ASXL1, and TET2. There is conflicting data on whether Tγδ positivity portends reduced OS compared to Tαβ. Observational studies show that Tγδ is more frequently symptomatic and conveys higher risk of cytopenias. Not all cases require treatment. Treatment is usually prompted by single-lineage cytopenias. First line single agent therapy with methotrexate (MTX), cyclosporine, and cyclophosphamide have shown similar overall response rates of 40-50%, though STAT3 mutated disease is associated with better response to MTX. STAT3 mutations are associated with more severe cytopenias, more frequent autoimmune/inflammatory disorders, and more frequently required therapy, however neither STAT mutations nor cytopenias convey decreased OS in Tγδ disease. STAT3 and STAT5b mutations occur in 40% of all LGLL cases, with STAT3 mutations associated with neutropenia and rheumatoid arthritis. Nearly two thirds of STAT3 genetic mutations are Y640F and D661Y. Our patient was initiated on prednisone, though due to poor tolerance was transitioned to MTX titrated to 12.5 mg weekly. She has remained on this dose with good tolerability for 18 months with resolution of lymphocytosis and improvement in neutropenia. T-LGLL has an OS of 9 years. Rarity of Tγδ disease has limited the ability to generate subtype specific survival data. The largest retrospective study to date included 137 Tγδ patients across 8 centers and provided a signal that Tγδ disease may predict reduced OS compared to the Tαβ subtype, however data was limited by disparate median follow-up time amongst cohorts due to high rates of censorship in the Tγδ cohort. Median OS was not reached at median follow up time of 48 months. This trend would be clinically expected given that Tγδ subtype has been consistently associated with severe cytopenias and autoimmune diseases, but should not be accepted until higher quality evidence is generated over longer median follow-up times. The same study found better responses to cyclosporine as first line therapy, an interesting observation as MTX has been generally accepted as first line therapy based on predominantly retrospective studies, though these studies did not stratify treatment responses by TCR phenotype. These findings underscore the need for prospective studies characterizing Tγδ specific disease course, survival analyses and treatment response.

CD79 Expression Is Associated with Cell-of-Origin and Outcome in Diffuse Large B-Cell Lymphoma

Introduction: CD79B is a target of polatuzumab vedotin, an antibody-drug conjugate, which significantly improved the prognosis of both previously untreated and relapsed/refractory patients with diffuse large B-cell lymphoma (DLBCL). However, the biological and clinical significance of CD79B protein and gene expression have not been fully explored in DLBCL, thus we aimed at examining these relationships. Methods: We retrospectively analyzed de novo DLBCL patients, who were diagnosed and received rituximab-based immunochemotherapy from 2008 through 2018 in the Okayama Hematology Study Group from Japan. Immunohistochemistry (IHC) staining was performed using a CD79B antibody (AT107-2), and protein expression was assessed based on H-score as described in a previous study (Sehn LH et al. JCO 2020), integrating with publicly available representative bulk RNA sequencing DLBCL datasets (BCC cohort from Ennishi D et al. JCO 2019 and NCI cohort from Schmitz R et al. NEJM 2018). We also performed CD8 and MHC class-I IHC to evaluate the tumor microenvironment. Gene expression profile-based cell-of-origin (COO) classification was performed including double-hit signature (DHITsig), recently renamed the dark zone signature (DZsig), using the NanoString DLBCL90 assay. In addition, simultaneous epitope and transcriptome measurement in single cells from lymphoid tissues was conducted. Results: CD79B IHC was evaluable in 576 cases. DLBCL90 assay classified the entire cohort into 293 ABC (50.9 %), 189 GCB (32.8 %), 31 DZsig-positive (5.4 %) and 63 unclassified (10.9 %). Furthermore, we dichotomized the cohort into 288 CD79B high cases and 288 CD79B low cases according to the median CD79B H-score. A dynamic range of CD79B protein expression was observed across COO, where ABC-DLBCL showed the lowest values followed by GCB-DLBCL and DZsig-positive-DLBCL, in ascending order (Kruskal-Wallis test, P < .00001; Figure A). Indeed, CD79B low cases were significantly enriched in ABC-DLBCL (58 %) compared to GCB-DLBCL (26 %) and DZsig-positive-DLBCL (2 %), respectively (Chi-squared test, P < .001). Consistently, we revealed that CD79B expression was the lowest in ABC-DLBCL compared to GCB-DLBCL and DZsig-positive-DLBCL at the transcriptomic level (Kruskal-Wallis test, P = .011 for BCC cohort and P = .022 for NCI cohort). In addition, we identified different CD79B staining patterns, composed of 433 with cytoplasmic pattern (75 %), 86 with membranous pattern (14.9 %), and 52 cases being IHC negative. These patterns significantly varied across COO (Fisher's exact test, P = .015) and CD79B H-score was the highest in the membranous pattern followed by the cytoplasmic pattern (Kruskal-Wallis test, P < .0001). Of note, the composition of CD8 positive T-cells in CD79B low tumors was significantly higher than that of CD79B high tumors (Wilcoxon rank sum test, P < .0001). However, MHC class-I expression was decreased in CD79B low cases compared to CD79B high (Wilcoxon rank sum test, P = .0002), suggesting an immune escape mechanism with downregulation of MHC class-I in the presence of cytotoxic T-cells, which is often seen in solid cancers. Significant association of CD79B expression with COO further prompted us to evaluate CD79B expression in normal germinal center B cells. Notably, the single-cell simultaneous epitope and transcriptome analysis (CITEseq, n = 2) and single-cell RNAseq analysis (n = 6) of reactive lymph nodes revealed that both CD79B gene and protein expression were the lowest in cells exhibiting plasmablastic signatures, followed by light zone and dark zone B cells (Kruskal-Wallis test, P < .0001; Figure B), supporting the relation of CD79B expression to COO subtype in DLBCL. Regarding prognostic impact, CD79B low group had significantly poorer prognosis in the entire DLBCL cohort (Log-rank test, P = .0005 for overall survival (OS) and P = .008 for progression-free survival (PFS)) and in ABC-DLBCL (Log-rank test, P = .003 for OS and P = .031 for PFS). Moreover, CD79B protein expression was significantly associated with OS after adjusting for International Prognostic Index in the entire DLBCL cohort (Cox regression model; P = .035). Conclusion: Our study identifies distinct CD79B expression patterns across COO subtypes, with CD79B low cases enriched in ABC-DLBCL and demonstrating poorer prognosis, suggesting its potential as a prognostic marker and for targeted therapies.

CD34+ Megakaryocytes Associate with Myelodysplastic Syndromes and Related Cytogenetic Abnormalities, but Not Other Hematological Disorders

Background: CD34 is a transmembrane phosphoglycoprotein that is a marker of hematopoietic stem cells and is used clinically to aid in enumeration of blast cells for identification of advanced myeloid neoplasms. Megakaryocytes are differentiated cells from the myeloid lineage that reside in the bone marrow and generally stain negative for CD34 (CD34-). However, CD34 positive (CD34+) megakaryocytes have been observed in hematological disorders such as myelodysplastic syndrome, myeloproliferative neoplasms, and acute leukemias. CD34+ megakaryocytes are proposed to be relatively specific for malignant hematologic conditions and have been reported to associate with a worse prognosis. However, the general clinical, biochemical, and cytogenic characteristics for bone marrow cases of CD34+ megakaryocytes versus CD34- megakaryocytes remains unclear. Objective: We sought to determine the clinical, biochemical, and cytogenetic characteristics associated with finding CD34+ megakaryocytes on bone marrow biopsies. Methods: We identified 281 independent, index bone marrow biopsy cases between 2011-01-01 and 2017-12-31 from Vancouver General Hospital, Canada that underwent CD34 immunohistochemistry at clinical request. The clinical, biochemical, and cytogenetic characteristics of patients were compared based on megakaryocytes being CD34+ vs CD34- on immunohistochemistry. Cases of CD34+ megakaryocytes were defined as having >5% of megakaryocytes staining CD34+, but sensitivity analyses were performed using different percentage cut-offs of 10, 30, and 50%. Results: Out of 281 cases, 134 had CD34+ megakaryocytes (58.2% male sex; mean age = 64.1 years [SD = 15.2 years]) and 147 had CD34- megakaryocytes (57.1% male sex; mean age = 60.0 years [SD = 16.6 years]). There was a significant difference in the distribution of diagnostic categories observed between cases of CD34+ versus CD34- megakaryocytes: myelodysplastic syndromes 35.1% (n=47) versus 21.8% (n=32), acute myeloid leukemias 20.9% (n=28) versus 21.1% (n=31), non-diagnostic etiology 17.9% (n=24) versus 14.3% (n=21), and myeloproliferative neoplasms 10.4% (n=14) versus 19.7% (n=29) (Figure A; Chi-square p-value=0.0004). Sensitivity analyses using different percentage cut-offs to define cases of CD34+ from CD34- megakaryocytes yielded similar results. The differences in biochemical characteristics between cases of CD34+ versus CD34- megakaryocytes were generally unremarkable. The median (IQR) for biochemical parameters between cases of CD34+ and CD34- megakaryocytes were hemoglobin 99 (28) versus 100 (34) g/L, mean corpuscular volume 94 (14) versus 93 (10) fL, neutrophil count 2.20 (3.68) versus 2.46 (3.63) x10 9/L, platelet count 98 (158) versus 112 (194) x10 9/L, and bone marrow blasts 7 (4) versus 3 (14)%. Lastly, there were notable trends in cytogenetic results associated with 90 cases with CD34+ versus 84 cases with CD34- megakaryocytes including: complex karyotypes for 15.6% (n=14) versus 6.0% (n=5), isolated deletion 5q for 7.8% (n=7) versus 1.2% (n=1), and/or isolated trisomy 8 for 5.6% (n=5) versus 2.4% (n=2). Similar cytogenetic results were observed when cases were restricted to only cases of CD34+ versus CD34- megakaryocytes with a diagnosis of myelodysplastic syndrome (Figure B). Conclusion: The presence of CD34+ megakaryocytes is more common in cases of myelodysplastic syndrome relative to other neoplastic hematologic disorders, but does not appear to help with discriminating between neoplastic and non-neoplastic hematological disorders ( e.g. non-diagnostic etiology). Cases with CD34+ megakaryocytes tend to associate with complex karyotype and deletion 5q abnormalities relative to cases with CD34- megakaryocytes.

Ileocecal involvement in intestinal Behçet's disease and Crohn's disease: comparison of clinicopathological and immunophenotypic features.

Intestinal Behçet's disease (BD) predominantly affects the ileocecal region and is currently diagnosed based on endoscopic features and clinical manifestations. It is difficult to distinguish between intestinal BD and Crohn's disease (CD) due to similar patient populations, gastrointestinal involvement, extraintestinal manifestations, and long-term recurrent course. In this study we aimed to compare the clinicopathological and immunophenotypic features of intestinal BD to CD. The medical and pathological records of 29 cases of intestinal BD and 120 cases of CD diagnosed at Sir Run Run Shaw Hospital were retrospectively analyzed. Immunohistochemistry for CD3, CD20, FOXP3, myeloperoxidase, and quantitative analysis of the infiltrating inflammatory cells was conducted. Intestinal BD with ileocecal ulcer had a higher incidence of abdominal pain and a higher erythrocyte sedimentation rate than CD, while chronic diarrhea was more common in CD. Excessive neutrophils in the mucosal lamina propria, neutrophilic exudate on the ulcer surface, and prominent lymphocytic infiltration in ulcer tissues were statistically more frequent in intestinal BD than in CD. The numbers of FOXP3+ T cells, CD3+ T cells, and CD20+ B cells in biopsy tissue from intestinal BD were significantly higher than CD, but the ratio of FOXP3+ T cells to CD3+ T cells was not statistically different. Besides the typical clinical and endoscopic findings, diagnostic biopsies from the ileocecal region in intestinal BD show some histological and immunophenotypic features that are different from CD, which may be useful in distinguishing these two entities.

IFN-γ Triggered IFITM2 Expression to Induce Malignant Phenotype in Elderly GBM.

Advanced age is an important risk factor for the worse clinical presentation of gliomas, especially glioblastoma (GBM). The tumor microenvironment (TME) in elderly GBM (eGBM) patients is considerably different from that in young ones, which causes the inferior clinical outcome. Based on the data from the Chinese Glioma Genome Atlas RNA sequence (CGGA RNA-seq), the Cancer Genome Atlas RNA array (TCGA RNA-array), and gene set enrichment (GSE) 16011 array sets, the differential genes and function between eGBM (≥ 60years old) and young GBM (yGBM, 20-60years old) groups were explored. Immunohistochemistry (IHC) was utilized to depict the abundance of CD8+cells (the main resource of IFN-γ) and IFITM2 protein expression in GBM samples. Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting (WB) were performed to verify the link between IFN-γ and IFITM2. Moreover, the small-interfering RNA (siRNA) of IFITM2 was used to explore the function of IFITM2 in GBM. Characterized by inflammatory TME and higher IFITM2 expression, eGBM harbored a shorter survival time. Chemotaxis and inflammatory cytokine-related genes were enriched in the eGBM group, with more infiltrative CD8+T cells. The IHC of CD8 and IFITM2-staining could demonstrate these results. In addition, the IFN-γ response pathway was activated in eGBM and resulted in a dismal outcome. Next, it was found that IFITM2 triggered by IFN-γ played a key role in IFN-γ-induced malignant phenotype in eGBM.

Evaluation of immune infiltrate according to the HER2 status in colorectal cancer

Background and aimsIn colorectal cancer (CRC), HER2 targeting is a promising treatment and immune infiltrate is an important area of research and strategy. Data regarding HER2 status and immune infiltrate are lacking. The aim of this study was to compare the immune infiltrate between HER2 amplified and non-amplified categories in proficient MisMatchRepair (pMMR)/microsatellite stable (MSS) CRC. MethodsHER2 immunohistochemistry (IHC) and fluorescence in situ hybridization were performed in a retrospective series of 654 CRC. Lymphocyte infiltrate was analysed by anti-CD3, CD8 and CD4 IHC and evaluated digitally using QuPath software. ResultsAmong the 654 CRC, we first observed a decreased CD3+ and CD8+ infiltrate between HER2 amplified (all IHC 3+ except one 2+) and non-amplified HER2 2+ IHC CRC (p = 0.059 and 0.072 respectively). A supplementary analysis of 258 pMMR/MSS CRC from the previous cohort, displaying all the IHC scores (0, 1+, 2+, 3+), showed a lower CD3+ infiltrate between HER2 amplified versus HER2 0 (p = 0.002), 1+ (p = 0.088) and non-amplified 2+ (p = 0.081) IHC cases. ConclusionsOur original findings suggest that in pMMR/MSS CRC, the immune infiltrate is reduced in HER2 amplified versus other HER2 categories. These data might be useful for future strategies combining anti-HER2 treatments and immune checkpoint inhibitors and need to be confirmed in larger CRC cohorts.

Effect of a portable light emitting diode device on wound healing in a rat model.

Light-emitting diode (LED) lights produce a variety of wavelengths that have demonstrable efficacy in therapeutic and aesthetic fields. However, a repetitive treatment regimen is required to produce treatment outcomes, which has created a need for portable LED devices. In this study, we aimed to develop a portable therapeutic LED device and investigate its healing effect on excisional wounds in a rat model. The 35 × 35 mm-sized LED device was used on a total of 30 rats with full-thickness wounds that were divided into two groups depending on radiation intensity (11.1 and 22.2 mW/cm2 group). LED irradiation was performed every 24 h for 30 min, over 14 days, in direct contact with the wound. Percentage wound closure was measured by photographic quantification and was assessed histologically using haematoxylin and eosin (H&E) and Masson's Trichrome staining, and immunohistochemistry for Vascular endothelial growth factor (VEGF) and CD31. Percentage wound closure was significantly higher in 22.2 mW/cm2 irradiated wounds than that in the control wounds on days 7 and 10. The area of collagen deposition was remarkably larger in 22.2 mW/cm2 irradiated wounds than that in the control, with more horizontally organized fibres. CD31 immunostaining confirmed a significant increase in the number of microvessels in 22.2 mW/cm2 irradiated wounds than that in the control wounds, although there was no difference in VEGF immunostaining. Our novel portable LED device accelerates wound healing in a rat model, raising the possibility that portable LED devices can combine convenience with accessibility to play an innovative role in wound dressing.

Hepatic T‐cell lymphoma in a blue‐fronted Amazon parrot (Amazona aestiva) and subcutaneous T‐cell lymphoma in a grey parrot (Psittacus erithacus)

AbstractT‐cell lymphoma was diagnosed in a mature blue‐fronted Amazon parrot (Amazona aestiva) and a mature grey parrot (Psittacus erithacus). The Amazon parrot was diagnosed with soft tissue sarcoma in the neck and T‐cell hepatic lymphoma. The sarcoma was surgically removed, but chemotherapy for the lymphoma was declined by the owner. This parrot survived 24 months without treatment until it was euthanased for dyspnoea, the cause of which was not investigated. The grey parrot was diagnosed with subcutaneous T‐cell lymphoma on the left side of the neck that likely originated from the thymus. The mass was surgically excised without complications. The bird remained healthy 24 months after diagnosis, at which point follow‐up was lost. This case series documents the clinical presentation and diagnostic workup of T‐cell lymphoma in two Psittacidae patients. In both cases, T‐cell lymphomas were confirmed by histological examination and CD3 immunohistochemistry.

The effect of growth hormone on ovarian function recovery in a mouse model of ovarian insufficiency.

To evaluate the effects and mechanisms of action of growth hormone (GH) in the recovery of ovarian function in ovarian insufficiency induced by cyclophosphamide (CP) in a mouse model. After inducing ovarian insufficiency by administering 400 mg/kg of CP intraperitoneally to 6-week-old ICR mice, the mice were divided into four groups (control, CP, 1 mg/kg GH, and 2 mg/kg GH) with 10 mice in each group. GH was administered a week later for 7 days. Five mice from each group were sacrificed the next day, and their ovaries were collected for histological examination. The remaining mice were superovulated for in vitro fertilization (IVF). The terminal deoxynucleotidyl transferase dUTP-nick end labeling assay was performed to detect apoptosis. Masson's trichrome staining was used to analyze the degree of fibrosis. To quantify angiogenesis, CD31 immunohistochemistry was performed. Angiogenesis-related gene expression profiles were assessed using quantitative reverse transcription polymerase chain reaction. CP induced the loss of non-growing (primordial and primary) follicles while GH significantly protected primordial follicles and increased follicular quality. The CP group showed a decrease in fertilization and blastocyst formation rates in IVF. In contrast, the GH treatment group showed dose-dependent enhanced IVF outcomes. Furthermore, GH treatment decreased apoptosis and stromal fibrosis and increased angiogenesis. Many genes involved in angiogenesis, especially Leptin (Lep), platelet endothelial cell adhesion molecule 1 (Pecam-1), and angiogenin (Ang) were up-regulated in the GH treatment groups. GH treatment may promote the recovery of ovarian function in ovarian insufficiency induced by the administration of CP via decreasing apoptosis and stromal fibrosis and upregulating Lep, Pecam-1, and Ang genes.

Saponins of Panax japonicus ameliorates cardiac aging phenotype in aging rats by enhancing basal autophagy through AMPK/mTOR/ULK1 pathway

Heart disease is a significant health concern for elderly individuals, with heart aging being the primary cause. Recent studies have shown that autophagy can play a protective role in preventing cardiac aging. Our previous research confirmed that Chikusetsu saponin IVa, a fundamental component of Saponins of Panax japonics (SPJ), can enhance basic autophagy levels in cardiomyocyte of isoproterenol induced cardiac fibrosis mice. However, it remains unclear whether SPJ possesses a protective effect on cardiac dysfunction during the natural aging process. Rats were randomly divided into four groups: adult control group (6 months old), aging group (24 months old), aging group treated with 10 mg/kg SPJ, and aging group treated with 30 mg/kg SPJ. The heart function, blood pressure, and heart mass index (HMI) were measured. Hematoxylin and eosin staining (H&E) and Wheat Germ Agglutinin (WGA) staining were used to observe the changes in morphology, while Masson staining was used to examine collagen deposition in the rat hearts and CD45 immunohistochemistry was conducted to examine the macrophage infiltration in heart tissues. TUNEL kit was used to detect apoptosis level of cardiomyocyte, and western blot was used to evaluate autophagy-related proteins as well as AMPK/mTOR/ULK1 pathway-related markers. SPJ treatment improved the cardiac function, reduced HMI, attenuated myocardial fiber disorder, inhibited inflammatory cell infiltration, and decreased collagen deposition and cardiomyocyte apoptosis in aging rats. Additionally, SPJ treatment decreased the expression of aging-related proteins and restored the expression of autophagy-related markers. SPJ activated autophagy through the activation of AMPK, which in turn increased the phosphorylation of ULK1(Ser555), while inhibited the phosphorylation of mTOR and ULK1(Ser757). Our study demonstrates that SPJ improves the cardiac function of aging rats by enhancing basal autophagy through the AMPK/mTOR/ULK1 pathway. These results offer a theoretical foundation and empirical evidence to support the clinical advancement of SPJ in enhancing age-related cardiac dysfunction.

Sodium orthovanadate exhibits anti-angiogenic, antiapoptotic and blood glucose-lowering effect on colon cancer associated with diabetes.

The presence of type 2 diabetes mellitus increases the risk of developing the colon cancer. The main objective of this study was to determine the role of sodium orthovanadate (SOV) in colon cancer associated with diabetes mellitus by targeting the competitive inhibition of PTP1B. For in vivo study, high fat diet with low dose streptozotocin model was used for inducing the diabetes mellitus. Colon cancer was induced by injecting 1,2-dimethylhydrazine (25mg/kg, sc) twice a week. TNM staging and immunohistochemistry (IHC) was carried out for colon cancer tissues. In vitro studies like MTT assay, clonogenic assay, rhodamine-123 dye assay and annexin V-FITC assay using flow cytometry were performed on HCT-116 cell line. CAM assay was performed to examine the anti-angiogenic effect of the drug. Sodium orthovanadate reduces the blood glucose level and tumor parameters in the animals. In vitro studies revealed that SOV decreased cell proliferation dose dependently. In addition, SOV induced apoptosis as depicted from rhodamine-123 dye assay and annexin V-FITC assay using flow cytometry as well as p53 IHC staining. SOV showed reduced angiogenesis effect on eggs which was depicted from CAM assay and also from CD34 and E-cadherin IHC staining. Our data suggest that SOV exhibits protective role in colon cancer associated with diabetes mellitus. SOV exhibits anti-proliferative, anti-angiogenic and apoptotic inducing effects hence can be considered for therapeutic switching in diabetic colon cancer.

Immunohistochemical and ultrastructural identification of telocytes in the lamina propria of human vaginal mucosa

Since their relatively recent discovery, telocytes (TCs) have been described as peculiar cells strategically positioned in the stromal tissue component of multiple organ systems of the mammalian body including female reproductive organs (i.e., ovary, uterine tube, and uterus). Nevertheless, current knowledge of TCs in the vagina is very limited. The present study was therefore undertaken to investigate the existence and characteristics of TCs in the stromal tissue of human vaginal mucosa by means of immunohistochemistry, immunofluorescence confocal microscopy, and transmission electron microscopy. In the vaginal lamina propria, TCs were first identified by CD34 immunohistochemistry that revealed the presence of CD34+ stromal cells arranged in networks, especially around blood vessels. Double immunofluorescence confocal microscopy allowed to precisely distinguish the perivascular networks of CD34+ stromal cells lacking CD31 immunoreactivity from adjacent CD31+ microvessels. All the perivascular networks of TCs/CD34+ stromal cells situated in the vaginal lamina propria coexpressed platelet-derived growth factor receptor α, which strengthened their identification as TCs. Instead, vaginal mucosal TCs were immunophenotypically negative for c-kit/CD117. The ultrastructural examination confirmed the presence of TCs, namely stromal cells with characteristic cytoplasmic processes (i.e., telopodes) forming labyrinthine networks around blood vessels and releasing extracellular vesicles. Together, our morphological findings provide the first comprehensive demonstration that TCs reside in the human vaginal lamina propria, thus paving the way for further investigation of their putative functions in vaginal mucosal homeostasis and pathophysiology.

Single-cell RNA sequencing of neurofibromas reveals a tumor microenvironment favorable for neural regeneration and immune suppression in a neurofibromatosis type 1 porcine model.

Neurofibromatosis Type 1 (NF1) is one of the most common genetically inherited disorders that affects 1 in 3000 children annually. Clinical manifestations vary widely but nearly always include the development of cutaneous, plexiform and diffuse neurofibromas that are managed over many years. Recent single-cell transcriptomics profiling efforts of neurofibromas have begun to reveal cell signaling processes. However, the cell signaling networks in mature, non-cutaneous neurofibromas remain unexplored. Here, we present insights into the cellular composition and signaling within mature neurofibromas, contrasting with normal adjacent tissue, in a porcine model of NF1 using single-cell RNA sequencing (scRNA-seq) analysis and histopathological characterization. These neurofibromas exhibited classic diffuse-type histologic morphology and expected patterns of S100, SOX10, GFAP, and CD34 immunohistochemistry. The porcine mature neurofibromas closely resemble human neurofibromas histologically and contain all known cellular components of their human counterparts. The scRNA-seq confirmed the presence of all expected cell types within these neurofibromas and identified novel populations of fibroblasts and immune cells, which may contribute to the tumor microenvironment by suppressing inflammation, promoting M2 macrophage polarization, increasing fibrosis, and driving the proliferation of Schwann cells. Notably, we identified tumor-associated IDO1 +/CD274+ (PD-L1) + dendritic cells, which represent the first such observation in any NF1 animal model and suggest the role of the upregulation of immune checkpoints in mature neurofibromas. Finally, we observed that cell types in the tumor microenvironment are poised to promote immune evasion, extracellular matrix reconstruction, and nerve regeneration.

Presence of Activated (Phosphorylated) STAT3 in Radiation Necrosis Following Stereotactic Radiosurgery for Brain Metastases.

Brain radiation necrosis (RN) is a subacute or late adverse event following radiotherapy, involving an exacerbated inflammatory response of the brain tissue. The risk of symptomatic RN associated with stereotactic radiosurgery (SRS) as part of the treatment of brain metastases (BMs) has been a subject of recent investigation. The activation of the signal transducer and activator of transcription 3 (STAT3) was shown in reactive astrocytes (RA) associated with BMs. Given that the pathophysiological mechanisms behind RN are not fully understood, we sought to investigate the role of STAT3 among other inflammatory markers in RN development. A mouse model of RN using clinical LINAC-based SRS was designed to induce brain necrosis with the administration of 50 Gy in a single fraction to the left hemisphere using a circular collimator of 5 mm diameter. Immunohistochemistry and multiplex staining for CD4, CD8, CD68, GFAP, and STAT3 were performed. For validation, eleven patients with BMs treated with SRS who developed symptomatic RN and required surgery were identified to perform staining for CD68, GFAP, and STAT3. In the mouse model, the RN and perinecrotic areas showed significantly higher staining for F4/80+ and GFAP+ cells, with a high infiltration of CD4 and CD8 T-lymphocytes, when compared to the non-irradiated cerebral hemisphere. A high number of GFAP+pSTAT3+ and F4/80+pSTAT3+ cells was found in the RN areas and the rest of the irradiated hemisphere. The analysis of human brain specimens showed that astrocytes and microglia were actively phosphorylating STAT3 in the areas of RN and gliosis. Phosphorylated STAT3 is highly expressed in the microglia and RA pertaining to the areas of brain RN. Targeting STAT3 via inhibition represents a promising strategy to ameliorate symptomatic RN in BM patients undergoing SRS.

High Tumor-Infiltrating Lymphocyte Count Is Associated with Distinct Gene Expression Profile and Longer Patient Survival in Advanced Ovarian Cancer.

Cancer-related immunity plays a significant role in the outcome of ovarian cancer, but the exact mechanisms are not fully explored. A retrospective, real-life observational study was conducted including 57 advanced ovarian cancer patients. Immunohistochemistry for CD4+, CD8+, and CD45+ was used for assessing tumor-infiltrating immune cells. Furthermore, an immune-related gene expression assay was performed on 12-10 samples from patients with less than and more than 1-year overall survival (OS), respectively. A higher number of CD4+ (p = 0.0028) and CD45+ (p = 0.0221) immune cells within the tumor microenvironment were associated with longer OS of patients. In a multivariate setting, higher CD4+ T cell infiltration predicted longer OS (p = 0.0392). Twenty-three differentially expressed genes-involved in antigen presentation, costimulatory signaling, matrix remodeling, metastasis formation, and myeloid cell activity-were found when comparing the prognostic groups. It was found that tumor-infiltrating immune cell counts are associated with peculiar gene expression patterns and bear prognostic information in ovarian cancer. SOX11 expression emerged and was validated as a predictive marker for OS.

Evaluation of a new technique using artificial intelligence for quantification of plasma cells on CD138 immunohistochemistry.

The diagnosis of plasma cell neoplasms depends on the accurate quantification of plasma cells, traditionally done by immunohistochemical CD138 staining of bone marrow biopsies. Currently, there is no fully satisfactory reference method for this quantification. In our previous study, we compared the commonly used overview estimation method (method A) with a novel method for counting plasma cells in three representative areas (method B). Results showed comparable concordance parameters between the two methods. In this follow-up study, we compared the previously evaluated methods with a digital analysis method (method C) that uses artificial intelligence in open-source software, QuPath. Archived CD138 immunohistochemically stained trephine sections of bone marrow samples used in our previous study were used (n = 33). Reviewers selected three representative areas on each sample by taking images with a light microscope and camera. Digital analysis was performed using the positive cell detection function in QuPath. The entire process was repeated by each reviewer to test intraobserver concordance (concordance correlation coefficient [CCC]) in addition to interobserver concordance (intraclass correlation coefficient [ICC]). Intraobserver concordance of method C showed strong agreement for all reviewers with the lowest CCC = 0.854. Interobserver concordance for method C using ICC was 0.909 and 0.949. This showed high interobserver agreement with significant differences between method C and previously assessed method A (ICC = 0.793 and 0.713) and method B (ICC = 0.657 and 0.658). We were able to successfully count CD138-positive plasma cells in bone marrow biopsies using artificial intelligence. This method is superior to both manual counting and overview estimation, regardless of tumour load.

Granulomatous mural folliculitis in 16 domestic goats: Infection with malignant catarrhal fever viruses and colocalization with ovine herpesvirus-2 using in situ hybridization.

Granulomatous mural folliculitis (GMF) is an uncommon reaction pattern occasionally observed in nonadapted ruminant hosts infected with malignant catarrhal fever viruses. This report characterizes GMF and concurrent cutaneous lesions in 16 goats with crusting dermatitis using histochemistry including hematoxylin and eosin, periodic acid-Schiff, and Grocott's methenamine silver, and immunohistochemistry for CD3, CD20, ionized calcium binding adaptor molecule 1, and cytokeratin AE1/3. Infiltrates in all 16 GMF cases consisted of macrophages and fewer T lymphocytes, and variably included eosinophils, multinucleated histiocytic giant cells, and/or neutrophils. Formalin-fixed paraffin-embedded skin and fresh skin samples from caprine GMF cases were tested using pan-herpesvirus nested conventional polymerase chain reaction (PCR) and partial sequencing, ovine herpesvirus-2 (OvHV-2) real-time PCR, and OvHV-2 colorimetric in situ hybridization (ISH). Five of 16 goats with GMF (31%) were PCR positive for malignant catarrhal fever viruses, including caprine herpesvirus 3 in 1 goat and OvHV-2 in 4 goats. Three goats also had positive intranuclear OvHV-2 hybridization signal in follicular keratinocytes, among other cell types, localized to areas of GMF. Herpesviruses were not detected in the formalin-fixed paraffin-embedded skin of 9 goats without GMF. This case series describes relatively frequent detections of malignant catarrhal fever viruses in the skin of goats with GMF, including the first report of caprine herpesvirus 3, and localizes OvHV-2 infected follicular keratinocytes within areas of GMF.

Reassessing Breast Cancer-Associated Fibroblasts (CAFs) Interactions with Other Stromal Components and Clinico-Pathologic Parameters by Using Immunohistochemistry and Digital Image Analysis (DIA).

Breast cancer (BC) stroma has CD34- and αSMA-positive cancer-associated fibroblasts (CAFs) differently distributed. During malignant transformation, CD34-positive fibroblasts decrease while αSMA-positive CAFs increase. The prevalence of αSMA-positive CAFs in BC stroma makes microscopic examination difficult without digital image analysis processing (DIA). DIA was used to compare CD34- and αSMA-positive CAFs among breast cancer molecular subgroups. DIA-derived data were linked to age, survival, tumor stroma vessels, tertiary lymphoid structures (TLS), invasion, and recurrence. Double immunostaining for CD34 and αSMA showed different CAF distribution patterns in normal and BC tissues. Single CD34 immunohistochemistry on supplemental slides quantified tumor stroma CD34_CAFs. Digital image analysis (DIA) data on CAF density, intensity, stromal score, and H-score were correlated with clinico-pathologic factors. CD34/αSMA CAF proportion was significantly related to age in Luminal A (LA), Luminal B (LB), and HER2 subtypes. CD34_CAF influence on survival, invasion, and recurrence of LA, LB-HER2, and TNBC subtypes was found to be significant. The CD34/αSMA-expressing CAFs exhibited a heterogeneous impact on stromal vasculature and TLS. BC stromal CD34_CAFs/αSMA_CAFs have an impact on survival, invasion, and recurrence differently between BC molecular subtypes. The tumor stroma DIA assessment may have predictive potential to prognosis and long-term follow-up of patients with breast cancer.

Effects of different obesogenic diets on joint integrity, inflammation and intermediate monocyte levels in a rat groove model of osteoarthritis.

Introduction: Obesogenic diets aggravate osteoarthritis (OA) by inducing low-grade systemic inflammation, and diet composition may affect OA severity. Here, we investigated the effect of diet on joint damage and inflammation in an OA rat model. Methods: Wistar-Han rats (n = 24) were fed a chow, a high-fat (HF) diet, or a high-fat/high-sucrose (HFS) for 24weeks. OA was induced unilaterally 12weeks after the diet onset by groove surgery, and compared to sham surgery or no surgical intervention (contralateral limb). Knee OA severity was determined by OARSI histopathology scoring system. At several timepoints monocyte populations were measured using flow cytometry, and joint macrophage response was determined via CD68 immunohistochemistry staining. Results: Groove surgery combined with HF or HFS diet resulted in higher OARSI scores, and both HF and HFS diet showed increased circulating intermediate monocytes compared to chow fed rats. Additionally, in the HFS group, minimal damage by sham surgery resulted in an increased OARSI score. HFS diet resulted in the largest metabolic dysregulation, synovial inflammation and increased CD68 staining in tibia epiphysis bone marrow. Conclusion: Obesogenic diets resulted in aggravated OA development, even with very minimal joint damage when combined with the sucrose/fat-rich diet. We hypothesize that diet-induced low-grade inflammation primes monocytes and macrophages in the blood, bone marrow, and synovium, resulting in joint damage when triggered by groove OA inducing surgery. When the metabolic dysregulation is larger, as observed here for the HFS diet, the surgical trigger required to induce joint damage may be smaller, or even redundant.