Immunohistochemical Staining Method Research Articles

ARNTL2 facilitates bladder cancer progression through potentiating ENO1-mediated glycolysis in a SLC31A1-independent and -dependent manner

BackgroundBasic helix-loop-helix ARNT like 2 (ARNTL2) is a transcription factor that controls the circadian rhythm. Amounts of studies have demonstrated the carcinogenic function of ARNTL2 in human malignant tumors albeit the underlying mechanisms remain poorly understood. We aimed to study the significance of ARNTL2 in bladder cancer (BLCA). MethodsImmunohistochemical staining, immunoblotting and the database from TCGA were used to analyze the clinical relevance of ARNTL2, enolase 1 (ENO1) and solute carrier family 31 member 1 (SLC31A1) in BLCA. The function of ARNTL2 was explored by cell proliferation assay, apoptosis, colony formation and xenografted tumorigenesis. The molecular mechanisms of ARNTL2-driving BLCA development were investigated by RT-qPCR, immunoblotting and luciferase assays. Glycolysis was checked by measuring glucose consumption and lactate production. ENO1 activity was assessed by using indicated assay kit. ResultsOverexpression of ARNTL2 facilitates the proliferation and tumorigenesis of BLCA cells through suppression of apoptosis and enhancement of glycolysis. Up-regulation of SLC31A1, ENO1, and enhancement of SLC31A1-mediated ENO1 activity were critical for ARNTL2-triggered glycolysis and malignant growth in BLCA cells. ARNTL2 was positively correlated with SLC31A1 and ENO1 in BLCA patients. High expression of ARNTL2, SLC31A1 or ENO1 predicted the poor prognosis of BLCA patients. Depletion of SLC31A1 and inhibition of glycolysis completely blunted the growth ability of BLCA cells. ConclusionIn summary, ARNTL2 facilitates the progression of BLCA via activating ENO1-mediated glycolysis in a SLC31A1-independent and -dependent manner. Inhibiting SLC31A1 and glycolysis may be an aspirational approach for the treatment of BLCA patients with overexpression of ARNTL2.

The value of immunohistochemical expression of SOX9 and CD34 in alopecia areata

ABSTRACT Background Alopecia areata (AA), an immune-mediated disorder, is marked by temporary, nonscarring hair loss. The bulge area is protected from immune attacks by immune privilege; however, recent studies demonstrated immune cells infiltrating the bulge area. Objective This study aims to investigate the immunohistochemical expression of the sex-determining region Y-box 9 (SOX9) and cluster of differentiation 34 (CD34) in AA patients as markers of hair follicle stem cells (HFSCs) and progenitor cells, respectively. Methods Immunohistochemical staining of SOX9 and CD34 was applied on skin samples of 20 AA patients and 20 healthy controls. Results SOX9 and CD34 were significantly lower in lesional samples of cases compared to perilesional and control skin biopsies. Furthermore, SOX9 level was negatively correlated with the severity of alopecia tool score (SALT score) among the studied AA patients. Moreover, lowered SOX9 expression was present in patients with recurrent attacks. Conclusions The significant reduction of stem cell markers (SOX9 and CD34) in our studied AA cases signifies the pathological affection of HFSCs and their progeny in AA. This is thought to cause a loss of competence in generating new hair in some AA cases, which needs to be validated in further research. Limitations of the study This study has a small sample size.

The differences between pure and mixed invasive micropapillary breast cancer: the epithelial–mesenchymal transition molecules and prognosis

PurposeInvasive micropapillary carcinoma (IMPC) of the breast is known for its high metastatic potential, but the definition of pure and mixed IMPC remains unclear.This retrospective cohort study aims to investigate the prognostic significance of the micropapillary component ratio and the expression of critical molecules of epithelial–mesenchymal transition (EMT), including E-cadherin (E-cad), N-cadherin (N-cad), CD44s, and β-catenin (β-cat), in distinguishing between pure and mixed IMPCs.MethodsWe analyzed 100 cases of locally advanced IMPC between 2000 and 2018 and excluded patients who received neoadjuvant chemotherapy. Pure IMPC was defined as having a micropapillary component of over 90%. A comprehensive recording of prognostic parameters was conducted. The IMPC areas were analyzed using the immunohistochemical (IHC) staining method on the microarray set for pure and mixed IMPC patients. Pearson's chi-square, Fisher’s exact tests, Kaplan–Meier analysis, and Cox proportional hazards analysis were employed.ResultsThe comparative survival analysis of the entire group, based on overall survival (OS) and disease-free survival (DFS), revealed no significant difference between the pure and mixed groups (P = 0.480, HR = 1.474 [0.502–4.325] and P = 0.390, HR = 1.587 [0.550–4.640], respectively). However, in the pure IMPC group, certain factors were found to be associated with a higher risk of short survival. These factors included skin involvement (P = 0.050), pT3&4 category (P = 0.006), a ratio of intraductal component (> 5%) (P = 0.032), and high-level expression of N-cad (P = 0.020). Notably, none of the risk factors identified for short OS in pure IMPC cases were observed as significant risks in mixed cases and vice versa. Furthermore, N-cad was identified as a poor prognostic marker for OS in pure IMPCs (P = 0.002).ConclusionThe selection of a 90% ratio for classifying pure IMPCs revealed significant differences in certain molecular and prognostic parameters between pure and mixed groups. Notably, the involvement of N-cadherin in the epithelial–mesenchymal transition (EMT) process provided crucial insights for predicting OS and DFS while also distinguishing between the two groups. These findings strongly support the notion that the pure IMPC subgroup represents a distinct entity characterized by unique molecular characteristics and behavioral patterns.

YY1 mediated DCUN1D5 transcriptional activation promotes triple-negative breast cancer progression by targeting FN1/PI3K/AKT pathway

Triple-negative breast cancer (TNBC) is more aggressive and has a higher metastasis rate compared with other subtypes of breast cancer. Due to the lack of drug-targetable receptors, chemotherapy is now the only available systemic treatment for TNBC. However, some patients might still develop drug resistance and have poor prognosis. Therefore, novel molecular biomarkers and new treatment targets are urgently needed for patients with TNBC. To provide molecular insights into TNBC progression, we investigated the function and the underlying mechanism of Defective in cullin neddylation 1 domain containing 5 (DCUN1D5) in the regulation of TNBC. By TCGA dataset and surgical specimens with immunohistochemical (IHC) staining method, DCUN1D5 was identified to be significantly upregulated in TNBC tumor tissues and negatively associated with prognosis. A series of in vitro and in vivo experiments were performed to confirm the oncogenic role of DCUN1D5 in TNBC. Overexpression of FN1 or PI3K/AKT activator IGF-1 could restore the proliferative and invasive ability induced by DCUN1D5 knockdown and DCUN1D5 could act as a novel transcriptional target of transcription factor Yin Yang 1 (YY1). In conclusion, YY1-enhanced DCUN1D5 expression could promote TNBC progression by FN1/PI3K/AKT pathway and DCUN1D5 might be a potential prognostic biomarker and therapeutic target for TNBC treatment.

Molecular mechanism of the influence of related genes expression in synovium tissue around shoulder joint of secondary frozen shoulder model rats on angiogenesis.

The study aimed to explore the pathogenesis of secondary frozen shoulder and its influence on synovium tissue and angiogenesis by constructing a rat secondary frozen shoulder model along with transforming growth factor. 40 healthy male rats aged 8 weeks were divided into Sham group (n=10, no modeling treatment), Control group (n=10, modeling treatment), Low group (n=10, modeling treatment, and 10 mL/d transforming growth factor), and High group (n=10, modeling treatment, and 20 mL/d transforming growth factor). Hematoxylin and Eosin (HE) method was used for histological detection, and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and immunohistochemical staining method were adopted to detect the expression of Matrix metalloproteinase-14 (MMP-14), mitogen-activated protein kinase (p38MAPK), and Vascular endothelial growth factor (VEGF). Compared with Sham group, the range of abduction and external rotation of rat glenohumeral joint in Control group, Low group, and High group was significantly reduced, and High group had the smallest range. Compared with the Sham group, the synovium in the Control group, the Low group, and the High group had obvious hyperplasia, and the blood vessels were significantly increased. Immunohistochemical staining and RT-PCR results showed that compared with Sham group, MMP-14, p38 MAPK, and VEGF in Control group, Low group, and High group all increased significantly, among which High group increased most. The secondary frozen shoulder is mainly manifested as synovial hyperplasia and increased blood vessels, which are related to the induction of MMP-14, p38 MAPK, and VEGF by transforming growth factor, which reveals the pathogenesis of secondary frozen shoulder to a certain extent, and lays a foundation for subsequent clinical treatment of secondary frozen shoulder.

Current state of in situ gene expression studiesin animal tissues

Morphological studies of farm animals are most often conducted using simple preparation and staining techniques. The study of the processes of embryogenesis, postembryonic features of the development of organs and tissues, as well as the effect of various substances remains to be elucidated not only using histochemical and immunohistochemical staining methods, but also using RNA in situ hybridization and transcriptome in situ sequencing. Aspects of many cellular and tissue processes for cattle, pigs and chickens in the context of comparative physiology have not yet been studied. The high productivity of farm animals is associated with the intensive functioning of all organs and systems of the body. The influence of the agricultural management of farm animals and its consequences on the development of an organism in ontogeny deserve a separate area of research from the point of in situ gene expression. Despite rapid development of transcriptome sequencing technologies, which result in the discovery of new candidate genes for many processes, RNA in situ hybridization remains the gold standard for their validation. This review briefly presents modern techniques and their modifications for studying in situ gene expression. Transcriptome studies that have been implemented in cattle, pigs and chickens as model organisms include: in situ RNA hybridization using ZZ-probes, tyramide signal amplification, hybridization chain reaction, digoxigenin-labeled probes, RT-PCR, single cell transcriptome sequencing, in situ transciptome sequencing. This paper is a review of the results of studies on cattle, pigs and chickens. The results of research in this area are relevant for understanding the features of adaptation mechanisms at the transcriptomic level in highly productive animals under industrial conditions in order to search for new markers of valuable agricultural traits. It should be noted that in literature there are very few studies using RNA in situ hybridization, despite the availability and simplicity of the method.

Immunohistochemical Analysis of Sperm Aquaporin-3 Expression in Men with Varicocele

Objective: The primary objective of this study is to detect potential alterations in sperm cells in cases of varicocele using immunohistochemical methods. Materials and Methods: In this study, semen samples obtained with consent from infertile males aged between 25-40 years were analyzed for the presence of varicocele and other determined factors, and were divided into two groups: a normozoospermic control group and a group diagnosed with varicocele. Direct swim-up techniques were utilized to separate spermatozoa with high motility. Immunohistochemical staining methods were employed to visualize protein expression, and the results were photographed at high magnification for documentation. Results: This research was conducted on sperm samples collected from males diagnosed with varicocele and from a healthy control group. The findings suggest that the presence of aquaporin-3 protein is associated with low or negative expression in sperm cells in the presence of varicocele. Immunohistochemical analyses have revealed a reduced or absent expression of aquaporin 3 in patients with varicocele compared to healthy individuals. Conclusion: The results of this study clearly demonstrate the adverse effects of varicocele on the structural and functional integrity of spermatozoa. Given that aquaporin-3 protein is critical for the motility of spermatozoa, the reduced expression or absence of this protein sheds light on the molecular mechanisms of male infertility. Keywords: Sperm DNA fragmentation, immunohistochemistry, Varicocele, protein expression, male infertility, aquaporin-3.

Enhancing Colorectal Cancer Tumor Bud Detection Using Deep Learning from Routine H&E-Stained Slides.

Tumor budding refers to a cluster of one to four tumor cells located at the tumor-invasive front. While tumor budding is a prognostic factor for colorectal cancer, counting and grading tumor budding are time consuming and not highly reproducible. There could be high inter- and intra-reader disagreement on H&E evaluation. This leads to the noisy training (imperfect ground truth) of deep learning algorithms, resulting in high variability and losing their ability to generalize on unseen datasets. Pan-cytokeratin staining is one of the potential solutions to enhance the agreement, but it is not routinely used to identify tumor buds and can lead to false positives. Therefore, we aim to develop a weakly-supervised deep learning method for tumor bud detection from routine H&E-stained images that does not require strict tissue-level annotations. We also propose Bayesian Multiple Instance Learning (BMIL) that combines multiple annotated regions during the training process to further enhance the generalizability and stability in tumor bud detection. Our dataset consists of 29 colorectal cancer H&E-stained images that contain 115 tumor buds per slide on average. In six-fold cross-validation, our method demonstrated an average precision and recall of 0.94, and 0.86 respectively. These results provide preliminary evidence of the feasibility of our approach in improving the generalizability in tumor budding detection using H&E images while avoiding the need for non-routine immunohistochemical staining methods.

N-myc downstream–regulated gene 1 can promote vasculogenic mimicry and angiogenesis in urothelial carcinoma

Urothelial carcinoma (UC) of the bladder is a common cause of cancer-related death worldwide. Vasculogenic mimicry (VM) is a process by which the malignant cells can generate vascular-like structures formed of periodic acid–Schiff (PAS) positive/CD31 negative extracellular matrix independent of angiogenesis and thus promotes tumor progression. N-myc downstream–regulated gene 1 (NDRG1) is a protein that can modulate tumor angiogenesis; however, its role in regulating tumor angiogenesis and VM formation has not been previously investigated in UC. This study aims to evaluate the role of intra-tumor microvessel density (MVD) (as a surrogate measure of angiogenesis), VM, and NDRG1 in UC and their correlation with different clinicopathologic features, then assess the correlation between them in UC. Sixty specimens of UC of the bladder were included. PAS-CD31 immunohistochemical double staining method was used to evaluate the intra-tumor MVD and VM. Immunohistochemical expression of NDRG1 was also examined. VM and NDRG1 expression were detected in 41.7% and 83.3% of UC specimens respectively. The mean of intra-tumor MVD, VM area, and NDRG1 was significantly higher in tumors with higher grade, lymphovascular invasion, and higher T stage. NDRG1 expression was positively correlated with MVD and VM. We can suggest that MVD, VM, and NDRG1 may serve as poor prognostic markers for UC. The positive correlation between NDRG1 and both MVD and VM may provide the first evidence that NDRG1 can induce tumor angiogenesis and VM in UC which may offer a novel pathway for further therapeutic strategies.

Exploring the Mechanism of Acupuncture's Influence on the Protease Activity and Free Radical Damage in Synovial Fluid of Rheumatoid Arthritis Rats Induced by Type II Collagen from the Release of Active Oxygen.

Acupuncture was studied to investigate the mechanism of its effect on protease vitality and free radical damage in Type I CIA rats induced by type II collagen. The study divided rats into a control group (injected with physiological saline, n = 10), a model group (injected with type II collagen, n = 10), and an intervention group (injected with type II collagen + acupuncture ST36 and GB39, 3 times a week, for a total of 4weeks, n = 10) based on the different injected drugs. Then, various indicators of the mice were experimentally tested using joint index scoring, H&E histological staining, protein blotting, and immunohistochemistry staining methods. Acupuncture ST36 and GB39 can reduce arthritis scores, histological staining scores, and increase MVD in CIA rats. And reduce protease levels, alleviate inflammation, synovial hyperplasia, and angiogenesis. In addition, the intervention group TNF-α, IL-1β and IL-6 mRNA were reduced, and the clearance rates of hydrogen peroxide free radicals and nitric oxide free radicals were increased. The expression levels of ROS and MDA decrease, while the expression levels of SOD increase It has been proved that acupuncture at ST36 and GB39 can inhibit the release of ROS, reduce protease activity, inflammation, synovial hyperplasia, angiogenesis and free radical damage, thus reducing the severity of CIA (Collagen-Induced Arthritis) in rats.

Experimental study on the preservation of amputated limb by low-temperature HTK preservation solution arterial perfusion

Objective: To evaluate the effect of HTK (Histidine-Tryptophan-Ketoglutarate solution) infusion on the protection and replantation of amputated limb. Methods: In experiment 1, the amputated limbs of male New Zealand white rabbits were preserved by different preservation methods, including low-temperature HTK solution perfusion group, low-temperature normal saline perfusion group, low-temperature preservation group and non-perfusion group at normal temperature. The homogenates of muscle tissue at different time points were collected for biochemical detection. H&E (hematoxylin and eosin) staining was performed on muscle tissue. Bax, Bcl-2 protein immunohistochemical staining and Tunel method were used to detect apoptosis in muscle, nerve, and vascular tissues. The ultrastructure of cells was observed by transmission electron microscope. In experiment 2, the amputated limbs of rabbits were treated with HTK perfusion and cryopreservation without perfusion, respectively. The survival of rabbits and their limbs were observed, and detected by H&E, TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) and scanning electron microscopy. Statistical analysis was performed using SPSS 22 (SPSS Inc., Chicago, IL, USA). χ2 test, student’s t-test, and ANOVA (Analysis of Variance) were used to compare the categorical and continuous variables. Results: Experiment 1: With the extension of disconnection time, there were statistical differences between the biochemical indexes of the HTK liquid perfusion group and those of the non-low temperature non-perfusion group, while some indexes of the other groups were different. Immunohistochemical staining of tissues (muscle, nerve, and blood vessel) in each group showed that the number of cells stained by anti-apoptotic protein BCL-2 in each group significantly increased with the extension of time, while the number of cells stained by pro-apoptotic protein BAX2 in each group significantly decreased. Tunel test showed that compared with other treatment groups, the apoptosis rate of the HTK liquid group was significantly decreased. Experiment 2: The survival rate of the experimental group was 60%, and that of the control group was only 30%. H&E staining, the TUNEL method and scanning electron microscopy suggested that HTK infusion could reduce ischemia-reperfusion injury in muscle tissue. Conclusion: 1. Perfusion preservation of severed limbs at 4 ℃ can effectively reduce tissue damage caused by ischemia and hypoxia, and the effect is stronger than direct preservation. 2. HTK solution and normal saline were used for one-time perfusion of the severed limbs of rabbits, and the preservation effect of HTK solution was the best. 3. HTK solution was used for one-time perfusion of severed hind limbs of rabbits, which was refrigerated at 4 ℃ and then replanted (ischemia time was 3–5 h). Compared with the replanted limbs after cold storage alone, the survival rate was higher, and the postoperative ischemia and hypoxia injury of surviving limbs were less.

Evidence for chronic headaches induced by pathological changes of myodural bridge complex

Clinical studies have shown that there may be a certain relationship between pathological changes of the myodural bridge complex (MDBC) and chronic headaches of unknown cause. But there is still a lack of experimental evidence to explain the possible mechanism. This study aims to further confirm this relationship between MDBC and chronic headaches and explore its potential occurrence mechanism in rats. Bleomycin (BLM) or phosphate-buffered saline (PBS) was injected into the myodural bridge fibers of rats to establish the hyperplastic model of MDBC. After 4 weeks, the occurrence of headaches in rats was evaluated through behavioral scores. The immunohistochemistry staining method was applied to observe the expression levels of headache-related neurotransmitters in the brain. Masson trichrome staining results showed that the number of collagen fibers of MDBC was increased in the BLM group compared to those of the other two groups. It revealed hyperplastic changes of MDBC. The behavioral scores of the BLM group were significantly higher than those of the PBS group and the blank control group. Meanwhile, expression levels of CGRP and 5-HT in the headache-related nuclei of the brain were increased in the BLM group. The current study further confirms the view that there is a relationship between pathological changes of MDBC and chronic headaches of unknown cause. This study may provide anatomical and physiological explanations for the pathogenesis of some chronic headaches of unknown cause.

Identifying Hmga2 preserving visual function by promoting a shift of Müller glia cell fate in mice with acute retinal injury

BackgroundUnlike in lower vertebrates, Müller glia (MG) in adult mammalian retinas lack the ability to reprogram into neurons after retinal injury or degeneration and exhibit reactive gliosis instead. Whether a transition in MG cell fate from gliosis to reprogramming would help preserve photoreceptors is still under exploration.MethodsA mouse model of retinitis pigmentosa (RP) was established using MG cell lineage tracing mice by intraperitoneal injection of sodium iodate (SI). The critical time point for the fate determination of MG gliosis was determined through immunohistochemical staining methods. Then, bulk-RNA and single-cell RNA seq techniques were used to elucidate the changes in RNA transcription of the retina and MG at that time point, and new genes that may determine the fate transition of MG were screened. Finally, the selected gene was specifically overexpressed in MG cells through adeno-associated viruses (AAV) in the mouse RP model. Bulk-RNA seq technique, immunohistochemical staining methods, and visual function testing were used to elucidate and validate the mechanism of new genes function on MG cell fate transition and retinal function.ResultsHere, we found the critical time point for MG gliosis fate determination was 3 days post SI injection. Hmga2 was screened out as a candidate regulator for the cell fate transition of MG. After retinal injury caused by SI, the Hmga2 protein is temporarily and lowly expressed in MG cells. Overexpression of Hmga2 in MG down-regulated glial cell related genes and up-regulated photoreceptor related genes. Besides, overexpressing Hmga2 exclusively to MG reduced MG gliosis, made MG obtain cone’s marker, and retained visual function in mice with acute retinal injury.ConclusionOur results suggested the unique reprogramming properties of Hmga2 in regulating the fate transition of MG and neuroprotective effects on the retina with acute injury. This work uncovers the reprogramming ability of epigenetic factors in MG.

Cortisol as a cerebral cortex neurons apoptosis regulator in acute phase of ischemic stroke (clinical and pathological study)

In response to ischemic stroke (IS) a natural activation of the stress-realizing system occurs. The features of this activation influence the outcome of the acute period and the prognosis of recovery and can be adjusted. At the same time, the role of the stress-realizing system in the pathogenesis of IS is still unexplored.Objective: to investigate the effect of peripheral blood cortisol concentration on the regulation of apoptosis of neurons of the cerebral cortex in the acute phase of IS.Material and methods. A prospective clinical and pathological study was performed. It included 9 patients with IS in the left middle cerebral artery territory who were admitted to hospital and died in the hyperacute phase of IS and had no infectious complications, allergic reactions or oncological diseases and who did not undergo thrombolysis. The cerebral cortex was examined. Neuron-specific enolase (NSE), protein 53 (p53), caspase 3, caspase 8, Fas receptor (CD95), and Fas apoptotic inhibitory molecule 2 (FAIM2) were determined on the slices using an indirect immunoperoxidase immunohistochemical staining method. A total of 567 microscopic fields were analysed for the group of patients with IS and 63 fields for the control group (three people). Before death, the blood concentrations of sFas, sFasL, cortisol, adrenocorticotropic hormone, adrenaline and norepinephrine were determined by enzyme immunoassay (the control group consisted of 28 people).Results. Significant correlation was found between the proportion of casp3-positive neurons and the concentration of cortisol in peripheral blood in zones 2 (r=0.263; p<0.01) and 3 (r=0.383; p<0.01). In the 2nd zone, significant negative correlation was found with the concentrations of sFas (r=-0.177; p<0.05) and sFasL (r=-0.164; p<0.05); in the 3rd zone, significant positive correlation was found with the ratio of the concentrations of sFasL and sFas (r=0.240; p<0.01). The proportion of Fas-positive neurons in the cerebral cortex correlated significantly with the concentration of the soluble form of this molecule (for the 1st zone – r=0.222, for the 2nd zone – r=0.438, for the 3rd zone – r=0.289; p<0.01) and the ratio of the concentrations of sFasL and sFas (respectively: r=0.231, r=0.266 and r=0.281; p<0.01) in the peripheral blood.Conclusion. Peripheral blood cortisol concentration is a factor that determines the regulation of apoptosis of neurons in the cerebral cortex in the acute phase of IS.

Neutrophil extracellular traps (NETs) and Th-2 dominant immune responses in chronic granulomatous chromobalstomycosis.

Chromoblastomycosis (CBM), a chronic, granulomatous, suppurative mycosis of the skin and subcutaneous tissue, is caused by several dematiaceous fungi. The formation of granulomas, tissue proliferation, and fibrosis in response to these pathogenic fungi is believed to be intricately linked to host immunity. To understand this complex interaction, we conducted a comprehensive analysis of immune cell infiltrates, neutrophil extracellular traps (NETs) formation, and the fibrosis mechanism in 20 CBM lesion biopsies using immunohistochemical and immunofluorescence staining methods. The results revealed a significant infiltration of mixed inflammatory cells in CBM granulomas, prominently featuring a substantial presence of Th2 cells and M2 macrophages. These cells appeared to contribute to the production of collagen I and III in the late fibrosis mechanism, as well as NETs formation. The abundance of Th2 cytokines may act as a factor promoting the bias of macrophage differentiation toward M2, which hinders efficient fungal clearance while accelerates the proliferation of fibrous tissue. Furthermore, the expression of IL-17 was noted to recruit neutrophils, facilitating subsequent NETs formation within CBM granulomas to impede the spread of sclerotic cells. Understanding of these immune mechanisms holds promise for identifying therapeutic targets for managing chronic granulomatous CBM.

Multiplex Cyclic Fluorescent Immunohistochemistry.

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

Synovial Hemangioma of the Stifle Joint in a Kangal Dog

A 10-year-old intact male Kangal dog presented with a mass located in the left stifle joint. The mass was 1.2x0.8x1 cm in size. Hematoxylin-eosin and immunohistochemical staining methods were used for histopathological evaluation. Histopathological examination revealed a significant proliferation in synoviocytes. Also, vascular channels lined with well-differentiated endothelial cells were detected. Immunohistochemically, mild CD31 expression was observed in the endothelial cells of the vascular channels. Actin expression was detected in smooth muscle cells of the vascular channels. In addition, vimentin expression was observed in the stromal connective tissue around blood vessels. Based on these findings, the mass was diagnosed as synovial hemangioma. In this case report, synovial hemangioma is rarely observed in animals has been reported.

Expression of transferrin receptor 1 and β1-integrins correlates with estrogen receptor status and immune infiltration in breast cancer

To study of transferrin receptor 1 (CD71) expression by primary breast cancer cells in correlation with tumor cell phenotype. Determination of BC phenotype: immunohistochemical staining method (immunofluorescence). Antibodies to ER (estrogen receptors), KL-1 (pancytokeratin), CD71 (transferrin receptor), CD29 (β1-integrins). CD45, CD3, CD4, CD8, CD20 infiltration was also evaluated. ZEISS microscope (AXIOSKOP; Germany), method of G.J. Hammerling et al. Statistical processing: IBM-SPSS Statistics v.21. 63% of BC cases had CD71+ phenotype. CD71-mosaic tumors were observed in 14.4%. β1-integrin expression was monomorphic in 51.6% of cases and mosaic in 38.7%. 85% of ER-positive tumors were CD71-positive with a monomorphic type of reaction; p=0.014. Among ER-negative tumors, CD71-negative reactions were 2-fold more frequent and the monomorphic type was less frequent. ER-positive tumors were CD29-positive in 73%; p=0.031. 45.5% of ER+ tumors were CD29-monomorphic. Among ER-negative tumors, the frequency of CD29-monomorphic tumors was 55%. Significant infiltration by CD3+ cells was predominant in CD71-positive tumors; p=0.016. In the CD29-monomorphic phenotype, CD45+ infiltration was 31.3%, and in the mosaic phenotype, 67.1%. BC aberrantly expresses transferrin receptors, β1-integrins. CD71 expression is associated with ER expression. ER-positive tumors are often monomorphic for CD71. Prominent CD3+ infiltration was present in CD71+ tumors. Expression of β1-integrins correlated with ER+ status and weak immune infiltration.

Comparative Study of Some Staining Methods for the Demonstration of Helicobacter pylori in Gastric Biopsies in Gombe State, Nigeria

Background: Currently there are different staining methods available for the demonstration of Helicobacter pylori in gastric biopsies sections. Most of these methods are technically demanding, slow and expensive with varying sensitivity and specificity. Aims: This study was to compare some of these techniques for the detection of H. pylori in routine gastric biopsy tissue sections in order to ascertain their reliability and effectiveness. Methodology: Sixty-five positive and seven negative cases of H. pylori were selected based on the results of rapid urease test, serology test and histopathological examination of the tissue sections. Histopathological examinations of sections stained by Hematoxylin and Eosin staining method (H&E), Giemsa staining method, modified Giemsa staining method, modified McMullen staining method and immunohistochemical staining were performed. Results: The sections were evaluated by two independent observers. The sensitivity and specificity for the techniques were as follow: immunohistochemistry, 100% and 100%; modified Giemsa stain, 96.9% and 85.7%; Giemsa stain, 92.3% and 81.4%; modified McMullen stain, 89.2% and 52.9%; H&E, 90.8% and 77.1%. The interobserver agreement was analyzed by Kappa (k) statistics using SPSS version 23 software. The agreement between the two observers was k: 0.859 (98.4%) for immunohistochemistry; k: 0.777 (96.9%) for modified Giemsa stain; k: 0.533 (95.2%) for Giemsa stain; k: 0.267 (92.1%) for modified McMullen stain; and k: 0.421 (93.7%) for H&E stain. Conclusion: The best results were obtained by the immunohistochemical staining and the Giemsa stainings. Although the immunohistochemistry staining method gives the best result and is very reliable, but it is fairly time consuming and expensive. Therefore, considering the cost, applicability and the reliability of the modified Giemsa or Giemsa stain, we recommend both for the detection of H. pylori in gastric biopsies sections especially in resource constrained laboratories.

Kırıkkale İlinde Spermophilus xanthoprymnus ve Meriones tristrami’de Glutatyon S-Transferaz-Alfa ve Glutatyon S-Transferaz-Pi Ekspresyon Düzeylerinin Yaşam Koşulları ve Doğal Habitat Farklılıkları Açısından İncelenmesi

Glutathione S-transferase (GST) is a multifunctional enzyme that provides homeostasis by catalyzing the first step in the formation of the end product mercapturic acid in the detoxification metabolic pathway. Being found in mammals, insects, fish, birds, annelids, molluscs, and many microorganisms, GST takes part the elimination of toxic substances taken into body by consuming food, and their transport by binding non-substrate ligands (e.g. heme and bilirubin) with GSH. In addition, it can prevent reactive electrophilic compounds from harming the body by covalent bonding similar compounds to each other. These xenobiotic acceptors affected by GST include nitrogen halogen compounds, organophosphates, and polycyclic aromatic hydrocarbons. Xenobiotics are oxygenated by this enzyme system, the next mechanism of oxygenated products is more oxygenation, and these products become more easily soluble in water. In this study, Glutathione S-Transferase was detected in the liver tissue of Spermophilus xanthoprymnus and Meriones tristrami and its characteristic features were determined. For this purpose, the animals were anesthetized with sodium pentobarbital and their liver tissues were harvested. After necessary preparations were completed, the samples were analyzed by using immunohistochemical staining method and the expressions of GST isozymes were determined. As a result, glutathione s-transferase-alpha and glutathione s-transferase-pi expression levels were found to differ in Spermophilus xanthoprymnus and Meriones tristrami samples obtained from different localities of Kırıkkale province. Differences in GST enzyme expression in these species indicate that both species differ in their detoxification capacity and response to xenobiotics.