Claudin-2 upregulation enhances intestinal permeability, immune activation, dysbiosis, and mortality in sepsis.

Intestinal epithelial expression of the tight junction protein claudin-2, which forms paracellular cation and water channels, is precisely regulated during development and in disease. Here, we show that small intestinal epithelial claudin-2 expression is selectively upregulated in septic patients. Similar changes occurred in septic mice, where claudin-2 upregulation coincided with increased flux across the paracellular pore pathway. In order to define the significance of these changes, sepsis was induced in claudin-2 knockout (KO) and wild-type (WT) mice. Sepsis-induced increases in pore pathway permeability were prevented by claudin-2 KO. Moreover, claudin-2 deletion reduced interleukin-17 production and T cell activation and limited intestinal damage. These effects were associated with reduced numbers of neutrophils, macrophages, dendritic cells, and bacteria within the peritoneal fluid of septic claudin-2 KO mice. Most strikingly, claudin-2 deletion dramatically enhanced survival in sepsis. Finally, the microbial changes induced by sepsis were less pathogenic in claudin-2 KO mice as survival of healthy WT mice injected with cecal slurry collected from WT mice 24 h after sepsis was far worse than that of healthy WT mice injected with cecal slurry collected from claudin-2 KO mice 24 h after sepsis. Claudin-2 upregulation and increased pore pathway permeability are, therefore, key intermediates that contribute to development of dysbiosis, intestinal damage, inflammation, ineffective pathogen control, and increased mortality in sepsis. The striking impact of claudin-2 deletion on progression of the lethal cascade activated during sepsis suggests that claudin-2 may be an attractive therapeutic target in septic patients.

Mechanisms underlying distinct subcellular localization and regulation of epithelial long myosin light-chain kinase splice variants.

Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn's disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.

Intestinal Epithelial Digestive, Transport, and Barrier Protein Expression Is Increased in Environmental Enteric Dysfunction.

Environmental enteric dysfunction (EED) is characterized by malabsorption and diarrhea that result in irreversible deficits in physical and intellectual growth. We sought to define the expression of transport and tight junction proteins by quantitative analysis of duodenal biopsies from patients with EED. Biopsies from Pakistani children with confirmed EED diagnoses were compared to those from age-matched North American healthy controls, patients with celiac disease, and patients with nonceliac disease with villous atrophy or intraepithelial lymphocytosis. Expression of brush border digestive and transport proteins and paracellular (tight junction) proteins was assessed by quantitative multiplex immunofluorescence microscopy. EED was characterized by partial villous atrophy and marked intraepithelial lymphocytosis. Epithelial proliferation and enteroendocrine, tuft, and Paneth cell numbers were unchanged, but there was significant goblet cell expansion in EED biopsies. Expression of proteins involved in nutrient and water absorption and that of the basolateral Cl- transport protein NKCC1 were also increased in EED. Finally, the barrier-forming tight junction protein claudin-4 (CLDN4) was significantly upregulated in EED, particularly within villous enterocytes. In contrast, expression of CFTR, CLDN2, CLDN15, JAM-A, occludin, ZO-1, and E-cadherin was unchanged. Upregulation of a barrier-forming tight junction protein and brush border and basolateral membrane proteins that support nutrient and water transport in EED is paradoxical, as their increased expression would be expected to be correlated with increased intestinal barrier function and enhanced absorption, respectively. These data suggest that EED activates adaptive intestinal epithelial responses to enhance nutrient absorption but that these changes are insufficient to restore health.

Intelectin-1 binds and alters the localization of the mucus barrier-modifying bacterium Akkermansia muciniphila.

Intelectin-1 (ITLN1) is a lectin secreted by intestinal epithelial cells (IECs) and upregulated in human ulcerative colitis (UC). We investigated how ITLN1 production is regulated in IECs and the biological effects of ITLN1 at the host-microbiota interface using mouse models. Our data show that ITLN1 upregulation in IECs from UC patients is a consequence of activating the unfolded protein response. Analysis of microbes coated by ITLN1 in vivo revealed a restricted subset of microorganisms, including the mucolytic bacterium Akkermansia muciniphila. Mice overexpressing intestinal ITLN1 exhibited decreased inner colonic mucus layer thickness and closer apposition of A. muciniphila to the epithelial cell surface, similar to alterations reported in UC. The changes in the inner mucus layer were microbiota and A. muciniphila dependent and associated with enhanced sensitivity to chemically induced and T cell-mediated colitis. We conclude that by determining the localization of a select group of bacteria to the mucus layer, ITLN1 modifies this critical barrier. Together, these findings may explain the impact of ITLN1 dysregulation on UC pathogenesis.

Differentiating Between Tight Junction-Dependent and Tight Junction-Independent Intestinal Barrier Loss In Vivo.

The intestinal barrier is an essential component of innate host defense. The single layer of epithelial cells that line the intestine must balance barrier function with both active, transcellular and diffusive, paracellular transport. Tight junctions, which link adjacent cells, form a selectively permeable seal that defines both paracellular transport and barrier properties. Molecules can cross tight junctions by either of two distinct routes, termed pore and the leak pathways, that differ in capacity, charge-selectivity, size-selectivity, and responses to physiological and pathophysiological stimuli. A third intestinal permeability route, the unrestricted pathway, reflects loss of the epithelial barrier, as occurs with mucosal damage, is independent of paracellular and transcellular pathways, and is neither charge- nor size-selective.The most commonly used approach for measuring intestinal permeability in vivo involves gavage of FITC-4 kDa dextran and analysis of the quantity recovered in serum. Unfortunately, this method cannot distinguish between leak and unrestricted pathways, as 4 kDa dextran can cross both. Moreover, 4 kDa dextran is too large to cross the pore pathway and, therefore, provides no information regarding this paracellular flux route. Here we describe a multiplex method that allows simultaneous, independent analysis of each pathway.

A Simple Method for Creating a High-Content Microscope for Imaging Multiplexed Tissue Microarrays.

High-throughput, high-content imaging technologies and multiplex slide scanning have become widely used. Advantages of these approaches include the ability to archive digital copies of slides, review slides as teams using virtual microscopy software, and standardize analytical approaches. The cost and hardware and software inflexibility of dedicated slide scanning devices can, however, complicate implementation. Here, we describe a simple method that allows any microscope to be used for slide scanning. The only requirements are that the microscope be equipped with a motorized filter turret or wheels (for multi-channel fluorescence) and a motorized xyz stage. This example uses MetaMorph software, but the same principles can be used with any microscope control software that includes a few standard functions and allows programming of simple command routines, or journals. The series of journals that implement the method perform key functions, including assistance in defining an unlimited number of regions of interest (ROI) and imaging parameters. Fully automated acquisition is rapid, taking less than 3 hr to image fifty 2.5-mm ROIs in four channels. Following acquisition, images can be easily stitched and displayed using open-source or commercial image-processing and virtual microscope applications. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Hardware and software configuration Basic Protocol 2: Create a preliminary scan Basic Protocol 3: Select, save, and position ROIs Basic Protocol 4: Determine and set autofocus parameters Basic Protocol 5: Acquire tiled images Basic Protocol 6: Review the scans Basic Protocol 7: Reimage ROIs as needed Basic Protocol 8: Stitch, stack, and assemble images Basic Protocol 9: Repeat scanning for multiplex immunostaining or background subtraction.

Investigation of TNF-α and IL-6 Levels in the Sera of Non-Melanoma Skin Cancer Patients.

Background: TNF-α and IL-6 are both pleiotropic cytokines playing major roles in cancer-associated cytokine networks. They have previously been investigated for their function in skin malignancies, mostly melanomas, and studies on non-melanoma skin cancer (NMSC) patients are relatively rara. In this study, we aimed to investigate the associations of serum levels of IL-6 and TNF-α with NMSCs and its clinicopathological features. Methods: This cases-control study was carried out to assess the serum levels of TNF-α and IL-6 in 70 NMSC patients, in comparison with 30 healthy individuals, by means of flow cytometric bead-based immuneoassay. Results: Serum levels of both TNF-α and IL-6 were significantly higher in NMSC patients (6.470 vs. 4.355 pg/ml; p = 0.0468, respectively), compared to healthy individuals (3.205 vs. 0.000 pg/ml; p = 0.0126, respectively). In the subgroup analysis, squamous cell carcinomas patients had higher serum levels of IL-6 compared to healthy individuals (3.445 vs. 0.000 pg/ml; p = 0.0432). No other significant differences were observed in the serum levels of these two cytokines among different clinicopathological subgroups of the patients. Conclusion: The increased levels of TNF-α and IL-6 in NMSC patients can be introduced as an epiphenomenon of a complex cancer-induced cytokine cascade.

Association of OX40 gene polymorphisms (rs17568G/A and rs229811A/C) with head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading incident cancer worldwide. In this study, we aimed to investigate the possible association of OX40 gene polymorphisms, rs17568G/A and rs229811A/C, with susceptibility to HNSCC and its clinicopathological features. Two hundred and two HNSCC patients and 200 healthy age-sex matched individuals were enrolled. rs17568G/A and rs229811A/C polymorphisms in OX40 gene were genotyped using RFLP-PCR method. We observed more than 2 times increased risk for squamous cell carcinoma development in nose and paranasal sinuses among individuals who inherited GG genotype at rs17568 region (OR 2.29; CI 1.01-5.20; P = 0.035). Considering rs2298211 SNP, AA genotype was also observed with higher frequency, in comparison with other two genotypes (AC or CC), among patients with HNSCC originated from these regions (P = 0.003). Besides, we observed that patients with C allele at this locus (AC and CC genotypes) have tumors with significantly higher histological grade (P = 0.042). Our findings suggest the possible association of rs17568 GG genotype, as well as rs2298211 AA genotype with susceptibility to develop squamous cell carcinoma in the nose and sinonasal cavities.

Investigating the Levels of Soluble Extracellular Domain of HER2 Protein in the Sera of Meningioma Patients.

AIM: Bearing in mind the association of breast cancer and meningioma, the present study aimed to investigate the levels of the soluble extracellular domain of HER2 protein in meningioma cases and control group. Besides, in the present research, its associations with pathological features and prognostic indicators of meningioma were examined. MATERIAL AND METHODS: A total of 68 meningioma patients along with 20 healthy age-sex matched individuals, as controls, were selected. Levels of HER2 in the sera were measured by a quantitative enzyme-linked immunosorbent assay (ELISA). RESULTS: The observations showed that Serum HER2 levels in meningioma patients were significantly lower than normal controls. However, outlier quantities were mostly observed in the cases. Furthermore, in meningiomas with higher histological grade (grade II, III), statistically significant elevated serum levels of HER2 were observed compared to patients with low-grade meningiomas (grade I). CONCLUSION: Serum HER2 levels were a poor biomarker for determination of pathological and prognostic characteristics of meningiomas and coupling serum HER2 levels with immunohistochemistry examination of HER2 in meningioma tissue samples would be helpful in future studies.

Mesenchymal Stem Cells, Immune Cells and Tumor Cells Crosstalk: A Sinister Triangle in the Tumor Microenvironment.

Mesenchymal Stem Cells [MSCs] are a heterogeneous population of fibroblast-like cells which maintain self-renewability and pluripotency. Many studies have demonstrated the immunomodulatory effects of MSCs on the innate and adaptive immune cells. As a result of interactions with tumor cells, microenvironment and immune-stimulating milieu, MSCs contribute to tumor progression by several mechanisms, including sustained proliferative signal in cancer stem cells [CSCs], inhibition of tumor cell apoptosis, transition to tumor-associated fibroblasts [TAFs], promotion of angiogenesis, stimulation of epithelial-mesenchymal transition [EMT], suppression of immune responses, and consequential promotion of tumor metastasis. Here, we present an overview of the latest findings on Janusfaced roles that MSCs play in the tumor microenvironment [TME], with a concise focus on innate and adaptive immune responses.

Investigation of Interleukin-27 in the Sera of Nonmelanoma Skin Cancer Patients.

IL-27 has been shown to have both tumor promoting and suppressing functions. IL-27, with its diverse influences on immune responses, has not been studied extensively in nonmelanoma skin cancers (NMSC), including Squamous and Basal Cell Carcinomas (SCC and BCC), and its roles in tumor initiation, progression, and its probable use in NMSC treatment have yet to be unveiled. A cross-sectional analytical study was designed to investigate the serum levels of IL-27 in NMSC patients in comparison to normal individuals. Levels of IL-27 in the sera of 60 NMSC patients along with 28 healthy controls were measured by means of quantitative enzyme-linked immunosorbent assay (ELISA). In this study we observed that IL-27 serum levels were significantly higher in NMSC patients in comparison to healthy individuals (0.0134 versus 0.0008 ng/ml; P<0.001). Furthermore, when subcategorized based on pathological diagnosis, both BCC and SCC patients had higher levels of IL-27 in their sera compared to controls (P=0.002 and P=0.033; respectively). However, these levels were not different among SCC and BCC patients. According to our results, it seems that IL-27 is involved in antitumor immune responses in NMSCs. On the other hand, these observations might be indicative of this cytokine involvement in NMSC tumorigenesis and progression. Therefore, administration of this cytokine for therapeutic purposes in patients with such conditions should be erred on the side of caution.

Association between CTLA-4 + 49A > G and - 318C > T single-nucleotide polymorphisms and susceptibility to thyroid neoplasm.

PURPOSE: Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4 or CD152) is among the immune checkpoint molecules and its abnormal expression in cancer predisposed individuals might make the person more susceptible to tumor initiation and progression. Considering the implication of single-nucleotide polymorphisms (SNPs) in CTLA-4 gene expression and probably protein function, one can assume the involvement of these SNPs in neoplastic diseases. rs5742909 ( - 318C > T) and rs231775 ( + 49 A > G) are among the most commonly studied SNPs and have been considered as genetic factors related to thyroid diseases. In this cross-sectional study, we aimed to elucidate the association between these SNPs and susceptibility to various types of thyroid cancers. METHODS: We investigated the genetic polymorphisms of - 318C > T and + 49 A > G in the CTLA-4 gene by means of ARMS-PCR and RFLP-PCR, respectively, in 167 patients with thyroid carcinomas (papillary, follicular, and anaplastic). The results were then compared with a group of 100 age-sex matched healthy individuals. RESULTS: A statistically significant association was observed between the presence of G allele in + 49 A > G locus and thyroid carcinoma, when comparing cases and controls (OR = 2.10; 95% CI = 1.35-3.28; P = 0.001) and the frequency of heterozygotes (AG) was higher than homozygotes for allele A (AA), in patients with and papillary thyroid carcinoma (PTC). Regarding Hashimoto's thyroiditis, the frequencies of A allele and AA genotype in PTC patients were higher than Hashimoto patients with no history of cancer (OR = 4.67, 95% CI = 2.70-8.08, P < 0.0001; and, OR = 4.68, 95% CI = 1.84-11.91, P = 0.0012; respectively). However; we observed no difference among allele/genotype frequencies in regards to locus - 318C > T. CONCLUSION: In this study, we observed that G allele in + 49 A > G and possibly lower expression of mutated CTLA-4 molecule, is associated with higher susceptibility to thyroid carcinoma. Although the G allele frequency was higher in thyroid cancer patients, when justified for the presence of lymphocytic thyroiditis, the presence of A allele seemed to increase the odds for PTC in patients with Hashimoto's disease.

Mesenchymal Stem Cells: Miraculous Healers or Dormant Killers?

Mesenchymal Stem Cells (MSCs) are a heterogeneous population of fibroblast-like cells which maintain self-renewability and pluripotency to differentiate into mesodermal cell lineages. The use of MSCs in clinical settings began with high enthusiasm and the number of MSC-based clinical trials has been rising ever since. However; the very unique characteristics of MSCs that made them suitable to for therapeutic use, might give rise to unwanted outcomes, including tumor formation and progression. In this paper, we present a model of carcinogenesis initiated by MSCs, which chains together the tissue organization field theory, the stem cell theory, and the inflammation-cancer chain. We believe that some tissue resident stem cells could be leaked cells from bone marrow MSC pool to various injured tissue, which consequently transform and integrate in the host tissue. If the injury persists or chronic inflammation develops, as a consequence of recurring exposure to growth factors, cytokines, etc. the newly formed tissue from MSCs, which still has conserved their mesenchymal and stemness features, go through rapid population expansion, and nullify their tumor suppressor genes, and hence give rise to neoplastic cell (carcinomas, sarcomas, and carcino-sarcomas). Considering the probability of this hypothesis being true, the clinical and therapeutic use of MSCs should be with caution, and the recipients' long term follow-up seems to be insightful.

Measurement of Lung Cancer Tumor Markers in a Glass Wool Company Workers Exposed to Respirable Synthetic Vitreous Fiber and Dust.

BACKGROUND: Occupational exposures to respirable synthetic vitreous fiber (SVF) and dust are associated with many lung diseases including lung cancer. Low-dose computed tomography is used for screening patients who are highly suspicious of having lung carcinoma. However, it seems not to be cost-effective. Serum biomarkers could be a useful tool for the surveillance of occupational exposure, by providing the possibility of diagnosing lung cancer in its early stages. OBJECTIVE: To determine if serum carcinoembryonic antigen (CEA) and cytokeratin fragment (CYFRA) 21-1 levels in workers exposed more than normal population to respirable SVF and dust may be used as indicators of progression towards lung cancer. METHODS: An analytic cross-sectional study, including 145 personnel of a glass wool company, along with 25 age-matched healthy individuals, was conducted to investigate the relationship between occupational exposure to respirable SVFs and dust and serum levels of two lung/pleura serum tumor markers, CEA and CYFRA 21-1, measured by ELISA. RESULTS: Individuals exposed to higher than the recommended levels of respirable SVF had higher serum concentrations of CEA and CYFRA 21-1, compared to controls (p=0.008 and 0.040, respectively), as well as in comparison to those exposed to lower than recommended OSHA levels (p=0.046 and 0.033, respectively). Workers with >9 years work experience, had significantly (p=0.045) higher levels of serum CYFRA 21-1 than those with ≤9 years of experience. CONCLUSION: It seems that working for >9 years in sites with detectable levels of respirable SVF and dust would increase the levels of known lung cancer serum tumor markers. Transferring these workers to sites with respirable SVF concentrations lower than the limit of detection in the air is recommended.

Dual association of serum interleukin-10 levels with colorectal cancer.

BACKGROUND: Interleukin 10 (IL-10) is considered an immune modulator cytokine, showing both antitumor and pro-tumor characteristics. Its role in the pathogenesis and progression of colorectal cancer depends on microenvironmental milieu. MATERIALS AND METHODS: A case-control study with 58 newly diagnosed colorectal cancer (CRC) patients, and 30 healthy individuals was conducted to compare the serum IL-10 levels between patients and controls. Furthermore, the correlation of the cytokine levels with the pathological features and prognosis of the CRC was investigated. IL-10 levels in the sera of patients and controls were measured by Enzyme-linked immunosorbent assay. RESULTS: Mean serum IL-10 levels were significantly lower in CRC patients than in controls (P = 0.04). CRC patients with worse prognosis at the time of diagnosis tend to have higher levels of circulating IL-10 than those with better prognosis (P = 0.008). Receiver operating characteristics curve analysis demonstrated that IL-10 levels in the sera of CRC patients can be used as a prognostic biomarker in CRC patients (area under the curve = 0.71; P = 0.01). CONCLUSIONS: Our results demonstrated a dual association of serum IL-10 levels in the initiation and progression of CRC. While lower IL-10 levels were associated with higher risk of the disease, its higher levels were associated with a poorer prognosis.

Transforming Growth Factor ß1 (TGF-ß1) in the Sera of Postmenopausal Osteoporotic Females.

BACKGROUND: Postmenopausal osteoporosis is a major cause of morbidity in postmenopausal females. Transforming growth factor ß1 (TGF-ß1) and interleukin 18 (IL-18) play complex roles in normal bone metabolism, and in pathophysiology of postmenopausal osteoporosis. OBJECTIVES: The aim of this study was to design an analytic cross sectional study in order to further clarify the role of TGF-ß1 and IL-18 in osteoporosis of postmenopausal females. METHODS: A cross sectional study including 65 postmenopausal osteoporotic females as cases and 69 postmenopausal females of similar age without osteoporosis as controls was conducted. Dual energy X-ray absorptiometry (DXA) was used to determine bone mass density (BMD) of participants and T-scoring was applied to establish whether the patient has osteoporosis or not. Serum TGF-ß1 and IL-18 levels were measured by quantitative sandwich Enzyme linked immunosorbent assay (ELISA). RESULTS: Serum TGF-ß1 levels were significantly higher in osteoporotic postmenopausal females than non-osteoporotic individuals (23.8 vs. 15.8 ng/mL; P = 0.009). There was no difference between IL-18 levels in the sera of osteoporotic and non-osteoporotic postmenopausal females in this study. There was a positive correlation between body mass index (BMI) and serum level of TGF-ß1 (P = 0.04). CONCLUSIONS: Our study demonstrated that TGF-ß1 serum levels is higher in osteoporotic postmenopausal females than non-osteoporotic ones, and probably aberrant increase in TGF-ß1 in postmenopausal females can result in uncoupled bone resorption and formation, which leads to osteoporosis.