Immunohistochemical Detection Research Articles

Thoracic Endometriosis Detected During Medical Thoracoscopy in a Tertiary Care Setting - A Retrospective Observational Study

Background: Thoracic endometriosis is a very rare clinical situation mostly presenting with pleural diseases, that is often misdiagnosed or missed due to the lack of specific clinical features. This report aims to explore the clinical characteristics, thoracoscopic findings, histopathology, treatment, and prognosis of women with thoracic endometriosis, providing a reference for the diagnosis and management of thoracic endometriosis. Design/Methods: Clinical data of 3 patients diagnosed with pleural endometriosis from among 50 consecutive thoracoscopic procedures conducted in a tertiary care hospital in North Kerala were analysed from electronic hospital records. The clinical features, thoracoscopic findings, histopathological features, treatment process, and prognosis were summarized. Results: Among the 3 female patients with pleural endometriosis, there were 2 cases of catamenial pneumothorax and one case of catamenial haemothorax. The age ranged from 43 years to 44 years. All the 3 cases were diagnosed by thoracoscopic pleural biopsy. Conclusions: Thoracic endometriosis syndrome is extremely rare, and clinical diagnosis is always missed or delayed due to nonspecific symptoms. Diagnosis mainly relies on pathological findings and immunohistochemical detection. Most cases were managed conservatively with hormonal therapy or by inducing surgical menopause.

Cardiac SARS-CoV-2 Infection, Involvement of Cytokines in Postmortem Immunohistochemical Study

In the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, significant attention was given to pulmonary manifestations. However, cardiac involvement is increasingly recognized as a critical factor influencing the prognosis, leading to myocardial damage, heart failure, acute coronary syndromes, potentially lethal arrhythmic events, and sudden cardiac death. Despite these findings, there is a lack of studies detailing the necroscopic, macroscopic, and microscopic cardiac changes associated with SARS-CoV-2. This study aimed to investigate the presence of SARS-CoV-2 viral proteins in cardiac tissue using immunohistochemical techniques to assess viral tropism. The analysis of cardiac tissue samples from deceased subjects, in different stages of conservation, confirmed to be positive for SARS-CoV-2 via reverse transcriptase-polymerase chain reaction (RT-PCR), showed immunopositivity for the SARS-CoV-2-NP viral antigen in 33% of cases. Notably, the presence of leukocyte infiltrates sufficient for diagnosing lymphocytic myocarditis was not observed. The central proinflammatory cytokines involved in the pathogenetic mechanism of coronavirus disease 19 (COVID-19) were researched using the immunohistochemical method. A significant increase in cytokine expression was detected, indicating myocardial involvement and dysfunction during SARS-CoV-2 infection. These findings suggest that the immunohistochemical detection of SARS-CoV-2 viral antigens and inflammatory cytokine expression in cardiac tissue could be crucial for a proper forensic assessment of the cause of death, even in sudden cardiac death.

Pudilan Anti-inflammatory Oral Liquid and Organic Acid Component from Taraxaci Herba Attenuate Allergic Asthma in Young Mice through Toll-like Receptor 2/Toll-like Receptor 4 Signaling Pathway

Abstract Objective: Allergic asthma (AA) is a chronic airway inflammatory disease characterized by airway hyper-responsiveness (AHR). Pudilan anti-inflammatory oral liquid (PDL) along with its main medicinal material, Taraxaci Herba (Taraxacum mongolicum Hand.-Mazz, TH) has been widely used to treat upper respiratory tract infections. Research has shown that the major ingredient of TH, the organic acid component (OAC), possesses favorable AA activity. However, the attenuated effects of PDL and OAC from TH (TH-OAC) on AA and their possible mechanisms remain poorly understood. This study analyzed the attenuating effects of PDL and TH-OAC on AA and the underlying mechanisms. Methods: Young BALB/c mice were sensitized and stimulated to develop asthma using ovalbumin. Histological examinations were performed by hematoxylin and eosin staining. Western blotting, immunohistochemistry, and protein expression detection of toll-like receptor 2 (TLR2), TLR4, and orosomucoid 1-like protein 3 (ORMDL3) were performed to detect the presence of inflammatory components in the lung tissue. The messenger RNA (mRNA) expression levels were determined using quantitative real-time polymerase chain reaction. Results: Results showed that PDL and TH-OAC alleviated augmented AHR and typical asthmatic pathological changes, including inflammatory infiltration and thickening of the alveolar wall. They also significantly reduced the levels of the immunoglobulin E, IL-4, IL-5, IL-6, tumor necrosis factor-α, and Nitric oxide (NO) in lung tissues of mice. Protein and mRNA expression levels of TLR2, TLR4, and ORMDL3 were downregulated following treatment with PDL and TH-OAC. Conclusions: PDL and TH-OAC can reduce asthma-induced inflammatory damage to the bronchi. These results provide a theoretical basis for the treatment of asthma in clinical settings.

Role of P63 in Benign and Malignant Lesions of Breast

Introduction: Breast carcinoma is leading cause of cancer death in women. Breast lesions constitute heterogeneous group of diseases with wide variety of etiologies ranging from inflammatory-benign- malignant lesions. There are several reported markers for immunohistochemical detection of myoepithelial cells. Smooth muscle specific proteins, such as smooth muscle actin, smooth muscle myosin heavy chain, calponin and h-caldesmon are used to highlight myoepithelium. p63 antibody is myoepithelial cell marker that selectively stains nuclei. It is negative in stromal, myofibroblastic and adipocytic cells.Aims and Objectives: The aim of this study was to establish role of p63 expression in distinguishing benign breast lesions, premalignant lesions and malignant tumors of breast.Materials and Methods: 30 cases were selected from core biopsy, lumpectomy and mastectomy specimens ofbreast received at department of Pathology, Chalmeda Anand Rao Institute of Medical Sciences during the period of January 2021 to December 2021 and were studied prospectively. All specimens were processed according to CAP protocol and reported . Immunohistochemistry was performed to determine p63 expression in those specimens. p63 expression was evaluated as continuous positive/discontinuous positive/negative.Ethical Approval: This study was reviewed and approved by institute ethics committee, CAIMS, Karimnagar.Results: Among total 30 cases, 18 cases (60%) were benign lesions and all were positive for p63 expression. 3 cases (9.99%) were premalignant and were least positive for p63 expression. All malignant cases 9 cases (29.99%) were negative for p63 expression.Conclusion: The Positive correlation was seen between histomorphological features and p63 scoring in all the lesions, So p63 is good Immunohistochemical marker for evaluating breast lesions.

Abstract LB422: Specific activation of the Epstein-Barr Virus BGLF4 locus via lowered DNA methylation in EBV-associated CNS lymphoproliferative disease

Abstract Introduction: Epstein-Barr virus positive (EBV+) central nervous system lymphoproliferative diseases (CNS-LPD) are aggressive clinical conditions with poor prognosis. We have previously reported use of a ganciclovir (GCV), zidovudine (AZT), rituximab and dexamethasone regimen (GARD) that induced complete and durable responses in patients with primary CNS post-transplant lymphoproliferative disease (PTLD). Responses were associated with detection of two GCV/AZT viral drug targets, the EBV protein kinases BGLF4 and BXLF1. These viral proteins are normally expressed in lytic phase EBV, and the molecular basis for expression in latently infected EBV+ CNS-LPD is unknown. Methods: RNA expression of EBV genes LMP1, BZLF1, BXLF1, and BGLF4 was measured in 12 CNS-LPD biopsies (n=8/12 DLBCL-type) via qRT-PCR. Mass spectrometry-based EpiTYPER assay was used to quantify DNA methylation in EBV gene promoters. Biopsies from 25 patients with systemic PTLD were used for comparison. The BGLF4 locus was investigated in detail using luciferase reporters and 5’RACE mapping of transcription start sites in 3 EBV-infected cell lines and 4 CNS-LPD biopsy samples. Overall EBV methylation was assessed in a screening assay covering 31 EBV loci. Mechanistic involvement of active demethylation via TET2 was studied in a HEK293 CRISPR-Cas9 gene knockout model under EBV (strain M81) infection. Results: Gene expression analysis of biopsy specimens showed expression of LMP1, BXLF1, and BGLF4, but not BZLF1. Expression of BXLF1 and BGLF4 was significantly higher in CNS-LPD when compared to systemic EBV+ PTLD (p=0.043 and p=0.00022, respectively). EpiTYPER data showed significantly decreased promoter methylation at BGLF4 (p=0.00013). Luciferase reporter analysis of the BGLF4 upstream sequence revealed 3 regions of promoter activity which were silenced by in vitro methylation of reporters. 5’RACE identified transcription start sites at all 3 BGLF4 promoters and detected their activity in EBV+ cell lines and CNS-LPD biopsies. By overlaying transcriptional activity, 5’RACE and DNA methylation data, we identified DNA methylation loss at single CpG dinucleotides correlated with BGLF4 expression (r2=0.73). These sites were specifically demethylated in CNS-LPD, while surrounding EBV methylation remained high. Knockout of TET2 followed by EBV infection revealed that active demethylation is required for demethylation at BGLF4 promoters. Conclusions: The molecular determinant for success of the GARD regimen in CNS-LPD has remained poorly understood, with immunohistochemical detection of BGLF4/BXLF1 as the only predictor of treatment response. Our results provide the molecular basis for expression of BGLF4 in CNS-LPD by showing decreased promotor DNA methylation. We identified specific BGLF4 methylation sites that hold promise as potential DNA biomarkers for EBV+ CNS-LPD. Citation Format: Christoph Weigel, Haley L. Klimaszewski, Selamawit Addissie, Sarah Schlotter, Fode Tounkara, James P. Dugan, Bradley M. Haverkos, Lynda Villagomez, Mark Lustberg, Pierluigi Porcu, Timothy Voorhees, Michael A. Caligiuri, Ginny Bumgardner, Christopher C. Oakes, Robert Baiocchi. Specific activation of the Epstein-Barr Virus BGLF4 locus via lowered DNA methylation in EBV-associated CNS lymphoproliferative disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB422.

Development and validation of a nomogram for predicting lymph node metastasis in ductal carcinoma in situ with microinvasion: A SEER population-based study.

Ductal carcinoma in situ with microinvasion (DCIS-MI) is a special type of breast cancer. It is an invasive lesion less than 1.0 mm in size related to simple ductal carcinoma in situ (DCIS). Lymph node metastasis (LNM) in DCIS-MI often indicates a poor prognosis. Therefore, the management of lymph nodes plays a vital role in the treatment strategy of DCIS-MI. Since DCIS-MI is often diagnosed by postoperative paraffin section and immunohistochemical detection, to obtain the best clinical benefits for such patients, we aim to establish and verify a nomogram to predict the possibility of lymph node metastasis in DCIS-MI patients and help preoperative or intraoperative clinical decision-making. A retrospective analysis of patients with DCIS-MI in the Surveillance, Epidemiology, and End Results (SEER) database from 2010 to 2019 was performed. The study cohort was randomly divided into a training cohort and a validation cohort at a ratio of 7:3. The risk factors were determined by univariate and multivariate logistic regression analyses in the training cohort, and a nomogram was constructed. The receiver operating characteristic (ROC) curve, calibration curve, and decision curve analysis (DCA) were used to evaluate the nomogram in the training set and validation set. An independent data cohort was obtained from the Shanghai Jiao Tong University Breast Cancer Database (SJTU-BCDB) for external validation. This study included 3951 female patients from SEER with DCIS-MI, including 244 patients with regional lymph node metastasis, accounting for 6.18% of the total. An independent test set of 323 patients from SJTU-BCDB was used for external validation. According to the multifactorial logistic regression analysis results, age at diagnosis, ethnicity, grade, and surgical modality were included in the prediction model. The areas under the ROC curves (AUCs) were 0.739 (95% CI: 0.702~0.775), 0.732 (95% CI: 0.675~0.788), and 0.707 (95%CI: 0.607-0.807) in the training, validation and external test groups, suggesting that the column line graphs had excellent differentiation. The calibration curves slope was close to 1, and the model's predicted values were in good agreement with the actual values. The DCA curves showed good clinical utility. In this study, we constructed accurate and practical columnar maps with some clinical benefit to predict the likelihood of lymph node metastasis in patients with postoperatively diagnosed DCIS-MI and provide a reference value for specifying treatment strategies.

Diagnostic value of tuberculosis-specific antigens ESAT-6 and CFP10 in lymph node tuberculosis

ObjectiveTo assess the diagnostic value of immunohistochemical (IHC) staining for detecting the tuberculosis-secreted antigens ESAT-6 and CFP10 in lymph node tuberculosis. MethodsArchived, paraffin-embedded lymph node specimens from 72 patients diagnosed with lymph node tuberculosis and 68 patients with lymphoma were retrospectively collected from the Department of Pathology at the Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan Province, China between January 2016 and March 2023. These specimens were subjected to acid-fast and immunohistochemical staining to compare the effectiveness of these methods, with their sensitivity and specificity evaluated against a comprehensive reference standard. ResultsAcid-fast staining demonstrated a sensitivity of 12.3% and a specificity of 100%. IHC staining for ESAT-6 showed a sensitivity of 87.5% and a specificity of 85.3%, whereas IHC staining for CFP10 exhibited a sensitivity of 75.0% and a specificity of 89.7%. ConclusionThe study indicates that IHC detection of ESAT-6 and CFP10 in paraffin-embedded lymph node tuberculosis tissues has a markedly higher sensitivity compared to acid-fast staining. Thus, IHC staining may serve as a supplementary diagnostic tool for the pathological evaluation of lymph node tuberculosis.

Harmine alleviated STZ-induced rat diabetic nephropathy: A potential role via regulating AMPK/Nrf2 pathway and deactivating ataxia-telangiectasia mutated (ATM) signaling

Diabetic nephropathy (DN) is a serious kidney disorder driven by diabetes and affects people all over the world. One of the mechanisms promoting NF-κB-induced renal inflammation and injury has been theorized to be ATM signaling. On the other hand, AMPK, which can be activated by the naturally occurring alkaloid harmine (HAR), has been proposed to stop that action. As a result, the goal of this study was to evaluate the therapeutic effectiveness of HAR against streptozotocin (STZ)-induced DN in rats through AMPK-mediated inactivation of ATM pathways. Twenty male Wistar rats were grouped into 4 groups, as follow: CONT, DN, HAR (10 mg/kg), DN + HAR, where HAR was daily administered I.P. once for 2 weeks. The renal AMPK and PGC-1α expressions, as well as Sirt1 levels, were assessed. To ascertain the oxidative reactions, renal Nrf2 expression, HO-1, MDA, and TAC concentrations were measured. As parts of ATM pathways, ATM and p53 expressions, in addition to GSK-3β levels were determined. Renal expression of NEMO, TNF-α, and IL-6 levels were also estimated. Moreover, histopathological and immunohistochemical detection of Bcl-2, Bax, and caspase 3 were reported. Results indicated that HAR intake notably alleviated STZ-induced kidney damage by triggering AMPK and Sirt1, which in turn boosted PGC-1α, improved NRf2/HO-1 axis, and lowered ROS production. As a consequence, HAR blocked the ATM-triggered renal inflammation and minimized caspase-3 expression by repressing the Bax/Bcl2 ratio. Because of its ability to activate AMPK/Nrf2 axis, HAR may represent an emerging avenue for future DN therapy by blocking ATM pathways.

[Comparative study on the degradation rate and regulatory effects of two resorbable collagen membranes during the in vivo implantation].

Objective: To explore the differences in the performance and tissue repair promotion effects of small intestinal submucosa membrane (SIS membrane) and Bio-Gide resorbable collagen membrane (Bio-Gide membrane) by performing the subcutaneous implantation models in mice. Methods: For in vivo studies, we stablished membrane implantation models using 6-8 week-old male C57BL/6 mice. The degradation rates were explored through HE staining analysis at different time points (1 d, 3 d, 5 d, 7 d, 14 d, 28 d, 3 mice/group/time point). The influences of the two membranes on local macrophages and neovasculum were evaluated by immunofluorescence detection of F4/80 and CD31, and the mobilization effects of the two membranes on local stem cells were evaluated by immunohistochemical detection of Ki67 and CD146. For in vitro studies, mice periodontal ligament stem cells (mPDLSCs) were co-cultured with these two membrane materials, and the cell morphologies were observed by scanning electron microscopy. In addition, the gene expressions of Ki67, Cxcl1, Ccl1, Tnfa were investigated by real-time fluorescence quantitative PCR (RT-qPCR). Results: The results of in vivo studies showed that by day 28, there was no significant difference in degradation rate between these two membrane materials [SIS degradation rate (16.84±4.00) %, Bio-Gide degradation rate (24.07±3.97) %, P=0.090], illustrating that both of them could maintain the barrier effects for more than one month. In addition, there was no significant difference in the infiltration number of local F4/80 positive macrophages between these two groups by the day 3 after implantation [SIS: (20.67±5.69) cell/visual field, Bio-Gide: (25.33±2.52) cell/visual field, P=0.292]. However, compared with the Bio-Gide membrane, SIS membrane significantly promoted local CD31+vascular regeneration [SIS (4.67±1.15)cell/visual field, Bio-Gide: (1.00±1.00) cell visual field, P=0.015] and CD146+stem cell recruitment [SIS: (22.33±4.16, Bio-Gide: (11.33±2.52) cell/visual field, P=0.025]. The RT-qPCR results also showed that SIS membrane promoted the gene expression of Cxcl1 (SIS vs Bio-Gide P<0.001) in mPDLSCs, but had no effect on the gene expression of Tnfa (SIS vs Bio-Gide P=0.885). Conclusions: SIS membrane showed a similar degradation rate compared with Bio-Gide membrane, and there was no significant difference in the effects of these two membranes on local inflammation or macrophages. Therefore, both of these membranes could meet the barrier effects required by guided tissue regeneration.

Immunohistochemical analysis of PDGFR-α for wound age determination.

Immunohistochemical analysis of platelet-derived growth factor receptor-α (PDGFR-α) was performed on human skin wounds obtained from forensic autopsy cases. Thirty human skin wounds were collected at different post-infliction intervals as follows: Group I, 4h to 3 days (n = 16); Group II, 4 to 7 days (n = 7); Group III, 9 to 10 days (n = 3); and Group IV, 14 to 20 days (n = 4). Immunopositive reactions for PDGFR-α were not observed in the uninjured human skin specimens. In a semi-quantitative morphometrical analysis, the number of PDGFR-α-positive cells was observed increased in Group II, with the average number of PDGFR-α-positive cells being the highest in Group II. Additionally, in Group II, all specimens showed PDGFR-α-positive cells, with an average number of > 200 cells in five fields of view, suggesting a wound age of 4 to 7 days. Taken together, the immunohistochemical detection of PDGFR-α in human skin wounds can be a useful tool for wound age determination.

Abstract 3769: Spatial biology in situ hybridization and multiplexed immunohistochemistry detection compatibility using a modified protocol

Abstract In the field of spatial-omics imaging, combining mRNA in situ hybridization (ISH) detection with multiplexed protein immunohistochemistry detection (IHC) is desirable. We present a simplified workflow in FFPE tissue sections where ISH is detected using branched ssDNA signal amplification visible by brightfield imaging combined with multiplexed IHC using directly labeled antibodies detected by fluorescence. In multiple studies mRNA expression of PCNA (Proliferating Cell Nuclear Antigen) shows moderate to high nuclear positivity observed in most cancer cells, with gliomas and lymphomas being the few negative exceptions. In contrast, expression of PCNA in adult non-cancerous tissue has low to no expression, with tonsil and bone marrow being the exceptions. We first show the relationship between proliferation as measured by EdU detection, PCNA antibody and PCNA mRNA expression in the embryonic mouse model where PCNA expression is prevalent in developing tissue. Then we demonstrate a simplified working protocol for combining ISH and IHC in FFPE human cancer tissue sections for the co-detection of PCNA mRNA together with IHC using a panel of six protein markers relevant to the tumor microenvironment. For IHC, we directly labeled validated unconjugated antibodies with fluorophores using amine-reactive chemistry and select the degree of labeling (DOL) and titer for optimal signal/background most useful for multiplexing on FFPE tissue. We found important protocol steps to consider for compatibility between IHC and ISH detection method. Loss of IHC protein epitope due to ISH protocol use of proteases and loss of ISH signal due to presence of RNase commonly present in IHC antibody relevant reagents can prevent combining IHC with ISH. Stabilizing IHC through refixation prior to ISH workflow is also required. Modifying the protocols to avoid these incompatible treatments and using directly labeled antibodies provides useful strategies for multiplexing IHC with ISH. Citation Format: Scott T. Clarke, Mae Voeun, Leticia Montoya, Chris Vonnegut, Bhaskar Mandavilli. Spatial biology in situ hybridization and multiplexed immunohistochemistry detection compatibility using a modified protocol [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3769.

Skin Biopsy Detection of Phosphorylated α-Synuclein in Patients With Synucleinopathies.

Finding a reliable diagnostic biomarker for the disorders collectively known as synucleinopathies (Parkinson disease [PD], dementia with Lewy bodies [DLB], multiple system atrophy [MSA], and pure autonomic failure [PAF]) is an urgent unmet need. Immunohistochemical detection of cutaneous phosphorylated α-synuclein may be a sensitive and specific clinical test for the diagnosis of synucleinopathies. To evaluate the positivity rate of cutaneous α-synuclein deposition in patients with PD, DLB, MSA, and PAF. This blinded, 30-site, cross-sectional study of academic and community-based neurology practices conducted from February 2021 through March 2023 included patients aged 40 to 99 years with a clinical diagnosis of PD, DLB, MSA, or PAF based on clinical consensus criteria and confirmed by an expert review panel and control participants aged 40 to 99 years with no history of examination findings or symptoms suggestive of a synucleinopathy or neurodegenerative disease. All participants completed detailed neurologic examinations and disease-specific questionnaires and underwent skin biopsy for detection of phosphorylated α-synuclein. An expert review panel blinded to pathologic data determined the final participant diagnosis. Skin biopsy for detection of phosphorylated α-synuclein. Rates of detection of cutaneous α-synuclein in patients with PD, MSA, DLB, and PAF and controls without synucleinopathy. Of 428 enrolled participants, 343 were included in the primary analysis (mean [SD] age, 69.5 [9.1] years; 175 [51.0%] male); 223 met the consensus criteria for a synucleinopathy and 120 met criteria as controls after expert panel review. The proportions of individuals with cutaneous phosphorylated α-synuclein detected by skin biopsy were 92.7% (89 of 96) with PD, 98.2% (54 of 55) with MSA, 96.0% (48 of 50) with DLB, and 100% (22 of 22) with PAF; 3.3% (4 of 120) of controls had cutaneous phosphorylated α-synuclein detected. In this cross-sectional study, a high proportion of individuals meeting clinical consensus criteria for PD, DLB, MSA, and PAF had phosphorylated α-synuclein detected by skin biopsy. Further research is needed in unselected clinical populations to externally validate the findings and fully characterize the potential role of skin biopsy detection of phosphorylated α-synuclein in clinical care.

Comparing massa medicata fermentata before and after charred in terms of digestive promoting effect via metabolomics and microbiome analysis

Ethnopharmacological relevanceMassa Medicata Fermentata, a fermented Chinese medicine, is produced by the fermentation of six traditional Chinese medicines. Liu Shenqu (LSQ) and charred Liu Shenqu (CLSQ) have been used for strengthening the spleen and enhancing digestion for over a thousand years, and CLSQ is commonly used in clinical practice. However, it is unclear whether there is a difference in the spleen strengthening and digestion effects between LSQ and CLSQ, as well as their mechanisms of action. Aim of studyThis study aims to compare the effects of LSQ and CLSQ on the digestive function of functional dyspepsia (FD) rats and reveal their mechanisms of action. Materials and methodsSPF grade SD rats were randomly divided into 6 groups: control group, model group, Liu Shenqu decoction low-dosage (LSQ LD) group, Liu Shenqu decoction high-dosage (LSQ HD) group, charred Liu Shenqu decoction low-dosage (CLSQ LD) group, and charred Liu Shenqu decoction high-dosage (CLSQ HD) group. Rats were injected intraperitoneally with reserpine to create an FD model and then treated by intragastric administration. During this period, record the weight and food intake of the animals. After 18 days of treatment, specimens of the gastric antrum, spleen, and duodenum of rats were taken for pathological staining and immunohistochemical detection of Ghrelin protein expression. Enzyme linked immunosorbent assay (ELISA) was used to determine the concentration of relevant gastrointestinal hormones in serum. The 16 S rDNA sequencing method was used to evaluate the effect of cecal contents on the structure of the gut microbiota in experimental rats. Plasma metabolomics analysis was performed using ultra high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) to further reveal their mechanism of action. ResultsLSQ and CLSQ improved the pathological tissue histological structure of FD rats and increased the levels of MTL and GAS hormones in serum and the levels of ghrelin in the gastric antrum, spleen, and duodenum, while reducing VIP, CCK, and SP hormone levels. The above results showed that the therapeutic efficacy of CLSQ is better than that of LSQ. Futhermore, the mechanism of action of LSQ and CLSQ were revealed. The 16 S rDNA sequencing results showed that both LSQ and CLSQ can improve the composition and diversity of the gut microbiota. And metabolomic analysis demonstrated that 20 metabolites changed after LSQ treatment, and 16 metabolites underwent continuous changes after CLSQ treatment. Further analysis revealed that LSQ mainly intervened in the metabolic pathways of glycerol phospholipid metabolism and arginine and proline metabolism, but CLSQ mainly intervened in the metabolic pathways of ether lipid metabolism, sphingolipid metabolism, and glycerophospholipid metabolism. ConclusionsBoth LSQ and CLSQ can improve functional dyspepsia in FD rats, but CLSQ has a stronger improvement effect on FD. Although their mechanisms of action are all related to regulating gastrointestinal hormone secretion, significantly improving intestinal microbiota disorders, and improving multiple metabolic pathways, but the specific gut microbiota and metabolic pathways they regulate are different.

Identification and validation of a novel cuproptosis signature for stratifying different prognostic, immune, metabolic, and therapeutic landscapes in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is a highly malignant cancer that urgently needs more effective therapeutic strategies. The discovery of cuproptosis brings great inspiration for the treatment and clinical assessment of cancers. A novel cuproptosis-related (CR) risk signature was constructed using the Lasso regression analysis. Its prognostic value was assessed via a series of survival analyses and validated in four GEO cohorts. The effects of CR risk signature on tumor immune microenvironment (TIM) were explored through CIBERSORT, ESTIMATE, and ssGSEA algorithms. Using GESA, we investigated its associations with various patterns of programmed cell death (PCD) and the metabolism process. The somatic mutation features of each CR-risk group were also probed using 'maftools' R package and cBioPortal database. The potential linkages between CR risk score and the efficacy of multiple therapeutic approaches were elucidated using tumor mutation burden, the expressions of immune checkpoints, the TIDE score, and the GDSC database. Finally, we ascertained the biofunctions of LIPT1 (Lipoyltransferase 1) in pancreatic cancer (PC) cells through immunohistochemistry, qPCR (quantitative polymerase chain reaction), colony formation, and Transwell assays. LIPT1, LIAS (lipoyl synthase), PDP1 (Pyruvate dehydrogenase phosphatase1), and GCSH (Glycine cleavage system H protein) constituted the CR risk signature. The CR risk signature possessed a high prognostic value and could improve the traditional prognostic model. Moreover, the CR risk score was indicative of the changes in infiltration levels of CD8+T cells and macrophages, whereas it was not associated with the enrichment of various PCD patterns and multiple metabolic processes. As for therapeutic correlation, CR risk score was a potential biomarker for predicting the efficacy of ICBs but failed in targeted drugs and chemotherapeutic agents. Through qPCR and immunohistochemistry detection in clinical samples, we confirmed that LIPT1 was significantly downregulated in pancreatic adenocarcinoma (PAAD) samples. Experiments in vitro revealed that silencing LIPT1 promoted the proliferation, migration, and invasion of PANC-1 and SW1990 cells. The novel CR risk signature contributed to the risk stratification of PAAD patients. Cuproptosis regulatory genes, well represented by LIPT1, provided new insights into PAAD treatment and assessment.

Colonic epithelial hypoxia remains constant during the progression of diabetes in male UC Davis type 2 diabetes mellitus rats

IntroductionColonocyte oxidation of bacterial-derived butyrate has been reported to maintain synergistic obligate anaerobe populations by reducing colonocyte oxygen levels; however, it is not known whether this process is disrupted during...

Immunohistochemical Detection of CD3 and MAC387 Antibodies in the Mesenteric Lymph Nodes and the Small Intestine of 20 Dogs that Died of Canine Parvoviral Enteritis

Abstract There is little data on the use of the immunohistochemical method in the observation of the T lymphocytes and macrophages distribution within the mesenteric lymph nodes in canine parvovirus (CPV) enteritis. The virus initially replicates in the systemic lymphoid tissue and causes highest changes in the small intestine. This current study aimed to demonstrate the CD3 and MAC387 antibodies detection and distribution in mesenteric lymph nodes and small intestines in dogs which had positive clinical, pathological, and histological findings of CPV. Twenty dogs that were clinically confirmed on CPV, had been autopsied following onset of death. Tissue samples (lymph nodes and small intestines) from each dog was processed for histology and immunohistochemistry utilizing hematoxylin-eosin and peroxidase/DAB (3,3′-diaminobenzidine) staining methods, respectively. The virus distribution was greatest in the tissue areas of the small intestine and the lymph nodes where the virus had made the most severe tissue damage. The distribution of macrophage was highest in the necrotic areas of the small intestine and the mesenteric lymph nodes. The amount of CD3 positive lymphocytes was greatly reduced in the mesenteric lymph nodes and the lymphoid tissue of the small intestine.

Extracellular vesicles derived from mesenchymal stem cells mediate extracellular matrix remodeling in osteoarthritis through the transport of microRNA-29a.

Knee osteoarthritis (KOA) is a common orthopedic condition with an uncertain etiology, possibly involving genetics and biomechanics. Factors like changes in chondrocyte microenvironment, oxidative stress, inflammation, and immune responses affect KOA development. Early-stage treatment options primarily target symptom relief. Mesenchymal stem cells (MSCs) show promise for treatment, despite challenges. Recent research highlights microRNAs (miRNAs) within MSC-released extracellular vesicles that can potentially promote cartilage regeneration and hinder KOA progression. This suggests exosomes (Exos) as a promising avenue for future treatment. While these findings emphasize the need for effective KOA progression management, further safety and efficacy validation for Exos is essential. To explore miR-29a's role in KOA, we'll create miR-29a-loaded vesicles, testing for early treatment in rat models. Extraction of bone marrow MSC-derived extracellular vesicles, preparation of engineered vesicles loaded with miR-29a using ultrasonication, and identification using quantitative reverse transcription polymerase chain reaction; after establishing a rat model of KOA, rats were randomly divided into three groups: Blank control group injected with saline, normal extracellular vesicle group injected with normal extracellular vesicle suspension, and engineered extracellular vesicle group injected with engineered extracellular vesicle suspension. The three groups were subjected to general behavioral observation analysis, imaging evaluation, gross histological observation evaluation, histological detection, and immunohistochemical detection to compare and evaluate the progress of various forms of arthritis. General behavioral observation results showed that the extracellular vesicle group and engineered extracellular vesicle group had better performance in all four indicators of pain, gait, joint mobility, and swelling compared to the blank control group. Additionally, the engineered extracellular vesicle group had better pain relief at 4 wk and better knee joint mobility at 8 wk compared to the normal extracellular vesicle group. Imaging examination results showed that the blank control group had the fastest progression of arthritis, the normal extracellular vesicle group had a relatively slower progression, and the engineered extracellular vesicle group had the slowest progression. Gross histological observation results showed that the blank control group had the most obvious signs of arthritis, the normal extracellular vesicle group showed signs of arthritis, and the engineered extracellular vesicle group showed no significant signs of arthritis. Using the Pelletier gross score evaluation, the engineered extracellular vesicle group had the slowest progression of arthritis. Results from two types of staining showed that the articular cartilage of rats in the normal extracellular vesicle and engineered extracellular vesicle groups was significantly better than that of the blank control group, and the engineered extracellular vesicle group had the best cartilage cell and joint surface condition. Immunohistochemical detection of type II collagen and proteoglycan showed that the extracellular matrix of cartilage cells in the normal extracellular vesicle and engineered extracellular vesicle groups was better than that of the blank control group. Compared to the normal extracellular vesicle group, the engineered extracellular vesicle group had a better regulatory effect on the extracellular matrix of cartilage cells. Engineered Exos loaded with miR-29a can exert anti-inflammatory effects and maintain extracellular matrix stability, thereby protecting articular cartilage, and slowing the progression of KOA.

Acetylated oligopeptide and N-acetylcysteine protect against iron overload-induced dentate gyrus hippocampal degeneration through upregulation of Nestin and Nrf2/HO-1 and downregulation of MMP-9/TIMP-1 and GFAP.

Iron accumulation in the brain causes oxidative stress, blood-brain barrier (BBB) breakdown, and neurodegeneration. We examined the preventive effects of acetylated oligopeptides (AOP) from whey protein on iron-induced hippocampal damage compared to N-acetyl cysteine (NAC). This 5-week study used 40 male albino rats. At the start, all rats received 150 mg/kg/day of oral NAC for a week. The 40 animals were then randomly divided into four groups: Group I (control) received a normal diet; Group II (iron overload) received 60 mg/kg/day intraperitoneal iron dextran 5 days a week for 4 weeks; Group III (NAC group) received 150 mg/kg/day NAC and iron dextran; and Group IV (AOP group) received 150 mg/kg/day AOP and iron dextran. Enzyme-linked immunosorbent assay, spectrophotometry, and qRT-PCR were used to measure MMP-9, tissue inhibitor metalloproteinase-1 (TIMP-1), MDA, reduced glutathione (GSH) levels, and nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) gene expression. Histopathological and immunohistochemical detection of nestin, claudin, caspase, and GFAP was also done. MMP-9, TIMP-1, MDA, caspase, and GFAP rose in the iron overload group, while GSH, Nrf2, HO-1, nestin, and claudin decreased. The NAC and AOP administrations improved iron overload-induced biochemical and histological alterations. We found that AOP and NAC can protect the brain hippocampus from iron overload, improve BBB disruption, and provide neuroprotection with mostly no significant difference from healthy controls.

Assessment of oxidative stress and tissue damage in Echinococcus granulosus naturally infected bovine liver

Echinococcus granulosus is a zoonotic parasite infects many livestock species, especially cattle, sheep, goat and buffalo, causing cystic echinococcosis. The aim of this study was to demonstrate the presence of the parasite and parasitic tissue damage histopathologically and to determine the role of oxidative stress in the tissue damage through the immunohistochemical detection of the oxidative damage-marker malondialdehyde (MDA) and the antioxidant response-marker superoxide dismutase (SOD). The material of the study consisted of 20 liver samples with Echinococcus cysts and 10 E.granulosus- negative healthy liver samples obtained from different cattle at various times from slaughterhouses in Kırıkkale province, Turkey. Histopathologically, Echinococcus cysts of various sizes were observed along with the surrounding fibrous connective tissue. Giant cells, mononuclear cells, and eosinophilic leukocytes were found between the fibrous connective tissue and the cyst. In the parenchymal tissue distant from the cyst, inflammatory changes were observed, including vacuolation and necrosis in hepatocytes, congestion and dilation sinusoidal capillaries. Immunohistochemically, MDA immunopositivity was observed in both hepatocytes surrounding the cyst and areas distant from the cyst, while SOD immunopositivity was mainly detected in fibrous connective tissue and hepatocytes surrounding the Echinococcus cysts. A significant increase in MDA immunoreactivity was observed in E.granulosus s.l.-infected livers. Although no statistically significant change was observed in SOD immunopositivity in the liver tissues with cystic echinococcosis, regional variations were noted. Germinal layer (GL) of Echinococcus cyst showed immunopositive staining for MDA, while laminated layer (LL) exhibited immunonegative staining. To the authors' best understanding, this study represents a pioneering effort in showcasing and evaluating the immunoreactivities of MDA and SOD within the liver tissue afflicted with Echinococcus cysts. Simultaneously, the examination extends to encompass tissue damage and the infiltration of inflammatory cells. This study highlights the role of oxidative stress in the pathogenesis of Cystic Echinococcosis (CE) and the need for further investigation of antioxidant defense mechanisms and their regional variations.

Clinical symptoms, biochemistry, and liver histology during the native liver period of progressive familial intrahepatic cholestasis type 2

BackgroundProgressive familial intrahepatic cholestasis type 2 (PFIC2) is an ultra-rare disease caused by mutations in the ABCB11 gene. This study aimed to understand the course of PFIC2 during the native liver period.MethodsFrom November 2014 to October 2015, a survey to identify PFIC2 patients was conducted in 207 hospitals registered with the Japanese Society of Pediatric Gastroenterology, Hepatology, and Nutrition. Investigators retrospectively collected clinical data at each facility in November 2018 using pre-specified forms.ResultsBased on the biallelic pathogenic variants in ABCB11 and/or no hepatic immunohistochemical detection of BSEP, 14 Japanese PFIC2 patients were enrolled at seven facilities. The median follow-up was 63.2 [47.7–123.3] months. The median age of disease onset was 2.5 [1–4] months. Twelve patients underwent living donor liver transplantation (LDLT), with a median age at LDLT of 9 [4–57] months. Two other patients received sodium 4-phenylbutyrate (NaPB) therapy and survived over 60 months with the native liver. No patients received biliary diversion. The cases that resulted in LDLT had gradually deteriorated growth retardation, biochemical tests, and liver histology since the initial visit. In the other two patients, jaundice, growth retardation, and most of the biochemical tests improved after NaPB therapy was started, but pruritus and liver fibrosis did not.ConclusionsJapanese PFIC2 patients had gradually worsening clinical findings since the initial visit, resulting in LDLT during infancy. NaPB therapy improved jaundice and growth retardation but was insufficient to treat pruritus and liver fibrosis.