Experimental and molecular pathology最新文献

Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a highly reactive bifunctional alkylating agent synthesized for chemical warfare. The eyes are particularly sensitive to SM where it causes irritation, pain, photophobia, and blepharitis, depending on the dose and duration of exposure. In these studies, we examined the effects of SM vapor on the corneas of New Zealand white male rabbits. Edema and hazing of the cornea, signs of acute injury, were observed within one day of exposure to SM, followed by neovascularization, a sign of chronic or late phase pathology, which persisted for at least 28 days. Significant epithelial-stromal separation ranging from ~8–17% of the epithelial surface was observed. In the stroma, there was a marked increase in CD45⁺ leukocytes and a decrease of keratocytes, along with areas of disorganization of collagen fibers. SM also disrupted the corneal basement membrane and altered the expression of perlecan, a heparan sulfate proteoglycan, and cellular fibronectin, an extracellular matrix glycoprotein. This was associated with an increase in basement membrane matrix metalloproteinases including ADAM17, which is important in remodeling of the basement membrane during wound healing. Tenascin-C, an extracellular matrix glycoprotein, was also upregulated in the stroma 14–28 d post SM, a finding consistent with its role in organizing structural components of the stroma necessary for corneal transparency. These data demonstrate that SM vapor causes persistent alterations in structural components of the cornea. Further characterization of SM-induced injury in rabbit cornea will be useful for the identification of targets for the development of ocular countermeasures.

Background

Plasma leakage is a major pathogenic manifestation of severe dengue and is a precursor of life-threatening complications associated with dengue. Accumulating evidence indicates the role of Matrix Metalloproteinases (MMPs) in mediating vascular permeability and plasma leakage following induction by the dengue virus. This study aims to investigate the utility of MMP-2, MMP-3, and MMP-9 in predicting the severity of dengue infection and further explore the relationship of these markers with the pathogenic factors associated with plasma leakage.

Methods

The dengue-positive subjects were classified into mild and severe dengue groups based on the manifestation of warning signs. The samples in each group and healthy controls were quantified for basic laboratory characteristics. The levels of MMP-2, MMP-3, MMP-9, and Macrophage migration inhibitory factor (MIF) were estimated in all serum samples using a multiplex bead-based assay.

Results

MMP-2 and MMP-9 were markedly elevated in severe dengue patients compared to mild dengue patients and healthy controls. No alteration in the circulating levels of MMP-3 was observed between the study groups. ROC curve analysis indicated that MMP-2 and MMP-9 exhibited good potential for predicting severe dengue. Notably, an increase in MMP-9 was associated with increased MIF and Hematocrit levels in severe dengue patients.

Conclusion

MMP-2 and MMP-9 could serve as prognostic biomarkers for severe dengue. These findings also identify the association of MMP-9 with markers of plasma leakage, thereby encouraging further studies to explore the therapeutic potential of targeting MMP-9 in managing plasma leakage in severe dengue.

Purpose

Glioblastoma (GBM) patients currently face poor survival outcomes with an average survival period of <15 months, while only 3–5% of patients survive longer than 36 months. Although the mechanisms of tumorigenesis are still being elucidated, miRNAs are promising candidates to explore as novel and prognostic biomarkers in GBM. In this study, we identified the association between miR-575 expression and overall survival (OS) of primary GBM patients and undertook functional studies to discern the contribution of miR-575 to GBM tumorigenesis.

Methods

Total RNAs were isolated from 254 FFPE GBM tumor samples and miR expression was assayed (simultaneously) using NanoString Technologies. To determine the association between miR-575 and patients' prognosis, Kaplan-Meier, univariable and multivariable Cox regression analyses were performed. Cell proliferation, colony formation, migration assays were conducted to investigate the function of miR-575 in vitro and in vivo. In silico target gene network analysis was performed to identify the putative targets of miR-575 in GBM, which were further verified by luciferase reporter assay, as well as qPCR and immunoblotting.

Results

Our clinical data (n = 254) show that miR-575 is associated with worse GBM OS by univariable analysis (UVA, HR = 1.27, p-value<0.001) and multivariable (MVA, HR = 1.23, p = 0.007) analysis incorporating critical clinical variables. Functional studies indicated that overexpression of miR-575 significantly increased cell proliferation and migration of GBM cells in vitro, as well as tumor growth in vivo. Subsequent in silico target gene network and mechanistic studies identified CDKN1B/p27 and PTEN, as potential targets of miR-575 in GBM. MicroRNA-575 can also regulate the activity of AKT and ERK pathways in GBM.

Conclusion

miR-575 has prognostic value in GBM, with higher expression associating with worse OS of patients, and contributes to GBM tumorigenesis by regulating multiple signaling pathways in GBM.

Ovarian cancer (OC) is the fifth most common type of cancer in women and the fourth most common cause of cancer death in women. Identification of pathogenic variants in OC tissues has an important clinical significance for therapeutic and prevention purposes. This study aims to evaluate the mutational profile of a patient cohort, negative for BRCA1/2 germinal variants and Mismatch Repair defects, using next-generation sequencing (NGS) approach on DNA from formalin-fixed paraffin-embedded samples. We used a custom NGS panel, targeting 34 cancer-related genes, mainly of the BRCA and PARP pathways, and analyzed NGS data to identify somatic and germline variants in Italian patients affected by primary epithelial ovarian cancer. We analyzed 75 epithelial ovarian cancer tissues and identified 54 pathogenic variants and 56 variants of unknown significance. TP53 was characterized by the highest mutational rate, occurring in 55% of tested epithelial ovarian cancers (EOCs). Interestingly, a subset of 8 EOCs showed pathogenic variants of homologous recombination pathway, which could be sensitive to PARP-inhibitor therapies. Germline analysis of actionable genes revealed 4 patients carrier of pathogenic germline variants respectively of RAD51C (2 patients), RAD51D, and PALB2. Molecular profiling of EOCs using our custom NGS panel has enabled the detection of both somatic and germline variants, allowing the selection of patients suitable for targeted therapies, and the identification of high-risk OC families that can benefit from genetic counseling and testing.

Background

Sickle cell disease (SCD) is an inherited genetic disorders of hemoglobin that causes multisystem morbidity. The pathophysiology of SCD is complex and includes HbS polymerization/sickling, hemolysis, endothelial dysfunction and inflammation. Chemokines are proteins playing an important role in the inflammation process and could be involved the context of pro-inflammatory SCD. Some chemokine polymorphism were found to be associated with clinical complication in SCD.

Aim of the study

Was to explore the frequency and the possible effect of Monocyte Chemo-attractant Protein 1–2518A/G (MCP1–2518A/G) and Chemokine Receptor 2-V64I (CCR2-V64I) genetic polymorphisms on clinical and laboratory disease-related variables in Egyptian Sickle cell disease patients.

Patients and methods

Genotyping of the two genes were performed by PCR-RFLP technique for 80 SCD patients as well as 50 healthy control group.

Results

The study revealed that the MCP1–2518 polymorphic genotypes (AG & GG) showed no statistically significant difference in the distribution of the polymorphic genotypes between SCD patients and the controls (p = 0.164). While a significantly higher frequency of the mutant variants CCR2-V64I GA/AA among SCD patients than the control subjects were found (p = 0.032). Regarding the clinic-pathological features, the frequency of recurrent infections, vaso-occlusive crisis, severe vaso-occlusive crisis and number of hospitalization/year were higher in patients harbouring the MCP1–2518A/G and CCR2-V64I polymorphic genotypes than the wild genotype, and gall bladder complications were higher in MCP1–2518 G allele patients, whereas surgical splenectomy were higher in CCR2-V64I A allele patients (p < 0.05).

In conclusion

MCP1–2518A/G and CCR2-V64I genetic polymorphisms may influence the clinical severity of sickle cell disease.

Introduction

Colorectal cancer (CRC) has become one of the most common cancers in recent years. Given the importance that non-coding RNAs have recently acquired in various diseases including cancers, we decided to design this study to evaluate the expression levels of circ0001955/miR-145-5p/ONECUT2 axis in CRC.

Methods

After bioinformatics analysis of GEO datasets related to CRC, a putative circ0001955/ miR-145-5p/ ONECUT2 pathway was assumed. Then, the expression levels of these genes were measured in 50 CRC samples and adjacent tissues by qRT- PCR. Also, correlation coefficients, receiver operating characteristic (ROC) curves, and correlation between circ0001955 levels with clinicopathological parameters of patients were analyzed.

Results

Circ0001955 and ONECUT2 were considerably up-regulated, while the expression level of miR-145-5p was decreased in CRC samples compared with adjacent tissues (p < 0.05). Moreover, statistically significant correlations were observed between expression levels of circ0001955, miR-145-5p, and ONECUT2. We did not find any significant correlation between circ0001955 expression and clinicopathological parameters.

Conclusion

Our study showed that circ0001955 is dysregulated in CRC. This finding can open a new window for researchers for a better understanding of the potential pathways involved in CRC pathogenesis and, consequently, to find new treatment pathways.

Background and aims

In this study ten mouse strains representing ~90% of genetic diversity in laboratory mice (B6C3F1/J, C57BL/6J, C3H/HeJ, A/J, NOD.B1oSnH2/J, NZO/HILtJ, 129S1/SvImJ, WSB/EiJ, PWK/PhJ, CAST/EiJ) were examined to identify the mouse strain with the lowest incidence of cancer. The unique single polymorphisms (SNPs) associated with this low cancer incidence are reported.

Methods

Evaluations of cancer incidence in the 10 mouse strains were based on gross and microscopic diagnosis of tumors. Single nucleotide polymorphisms (SNPs) in the coding regions of the genome were derived from the respective mouse strains located in the Sanger mouse sequencing database and the B6C3F1/N genome from the National Toxicology Program (NTP).

Results

The WSB strain had an overall lower incidence of both benign and malignant tumors compared to the other mouse strains. At 2 years, the incidence of total malignant tumors (Poly-3 incidence rate) ranged from 2% (WSB) to 92% (C3H) in males, and 14% (WSB) to 93% (NZO) in females, and the total incidence of benign and malignant tumor incidence ranged from 13% (WSB) to 99% (C3H) in males and 25% (WSB) to 96% (NOD) in females. Single nucleotide polymorphism (SNP) patterns were examined in the following strains: B6C3F1/N, C57BL/6J, C3H/HeJ, 129S1/SvImJ, A/J, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ. We identified 7519 SNPs (involving 5751 Ensembl transcripts of 3453 Ensembl Genes) that resulted in a unique amino acid change in the coding region of the WSB strain.

Conclusions

The inherited genetic patterns in the WSB cancer-resistant mouse strain occurred in genes involved in multiple cell functions including mitochondria, metabolic, immune, and membrane-related cell functions. The unique SNP patterns in a cancer resistant mouse strain provides insights for understanding and developing strategies for cancer prevention.

Objective

Joint destruction in Charcot neuroarthropathy (CNA) is accompanied with abundant hyperplastic synovium. This study aimed to characterize the expression patterns of a group of neuropeptides in the CNA synovium.

Methods

Synovial specimens were collected during surgery from the CNA (n = 6) and non-CNA joints (n = 14). Tissue samples were processed for protein extraction and western blot for vasoactive intestinal peptide (VIP), galanin, and calcitonin gene-related peptide (CGRP). Immunohistochemistry was performed to localize CGRP in the CNA synovium. Additionally, CGRP was applied to fibroblast-like synoviocytes (FLS) isolated from CNA synovium for its effects on cell proliferation and collagenolysis in vitro.

Results

Western blot detected light bands of VIP in the CNA samples but abundant galanin in both CNA and non-CNA samples. Most of the CNA samples (5/6) increased expression of CGRP, with an average band density about 2 times that in the non-CNA group (p < .05). Immunohistochemistry of CGRP demonstrated intense staining in the intimal layer of the CNA synovium. In tissue culture, adding CGRP (10 nM) in the medium promoted FLS proliferation. In combination with TNF-α, CGRP enhanced FLS-mediated collagenolysis in vitro.

Conclusion

This study revealed an increased expression of CGRP in the CNA synovium and demonstrated that CGRP regulates FLS proliferation and collagenolytic activity, suggesting CGRP may contribute to the bone and cartilage destruction in CNA.

Introduction

Although wound refers to simple cut in the skin, most wounds don't heal because of the various local and systemic factors that lead to its complexity and chronicity. Thus, prior understanding of the status of the wound is necessary and methods that can differentiate between the healing and non-healing wounds at a much earlier stage is crucial for a successful treatment.

Methods

The current study aims at differentiating Acute Wound Fibroblasts (AWFs) and Chronic Wound Fibroblasts (CWFs) based on differential expression of fibroblast specific markers such as Vimentin and Alpha Smooth Muscle Actin (α-SMA) and compare its cell cycle and proliferation.

Results

Immunostaining and western blotting analysis showed that, AWFs and CWFs differentially expressed vimentin and α-SMA, with AWFs and CWFs showing higher expression of vimentin and α-SMA respectively. AWFs showed higher distributions in G0/G1 (67.43% vs. 62.16%), S phase (22.61% vs. 8.51%) compared to CWFs. However, AWFs showed decreased distributions compared to CWFs in G2 + M phase (8.14% vs. 10.6%). Thus, it was observed that CWFs showed cell cycle arrest in the G1/G0 phase and inhibited DNA synthesis, which was further confirmed by reduced proliferation of CWFs. We suggest that, differential expression of the cell specific markers can be attributed to its pathophysiological status and chronicity of the wound and reduced proliferation rate of CWFs is due to lesser expression of vimentin, which is a key protein for in vitro cell proliferation.

Conclusions

Outcome of the study serve as an immunological tool to guide the chronicity of the wound, which helps to understand the wound towards design of personalized care. The findings also represent a promising opportunity to gain insight into how cell cycle arrest can impact on wound healing and clinical outcomes.