Immune checkpoints in circulating and tumor-Infiltrating CD4 + T Cell Subsets in Colorectal cancer patients

Blockade of inhibitory immune checkpoints (ICs) is a promising therapeutic approach; however, it has shown limited success in some cancers including colorectal cancer (CRC). The tumor microenvironment (TME) is largely responsible for response to therapy, and its constituents may provide robust biomarkers for successful immunotherapeutic approaches. In this study, we performed phenotypical characterization and critical analyses of key inhibitory ICs and T regulatory cell (Treg)-related markers on CD4+ T cell subsets in CRC patients, and compared with normal colon tissues and peripheral blood from the same patients. We also investigated correlations between the levels of different CD4+ T cell subsets and the clinicopathologic features including disease stage and tumor budding. We found a significant increase in the levels of CD4+FoxP3+Helios+ T cells, which represent potentially highly immunosuppressive Tregs, in the CRC TME. Additionally, tumor-infiltrating CD4+ T cells upregulated programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), T cell immunoglobulin and mucin domain-3 (TIM-3) and lymphocyte-activation gene 3 (LAG-3). We also characterized the expression of PD-1, CTLA-4, TIM-3, and LAG-3 on different CD4+FoxP3−/+Helios−/+ T cell subsets. Interestingly, we found that CTLA-4, TIM-3, and LAG-3 were mainly co-expressed on FoxP3+Helios+ Tregs in the TME. Additionally, FoxP3high Tregs expressed higher levels of Helios, CTLA-4 and TIM-3 than FoxP3low T cells. These results highlight the significance of Tregs in the CRC TME and suggest that Tregs may hamper response to IC blockade in CRC patients, but effects of different IC inhibition regimes on Treg levels or activity warrants further investigations. We also found that CD4+CTLA-4+ T cells in circulation are increased in patients with advanced disease stage. This study simultaneously provides important insights into the differential levels of CD4+ T cell subpopulations and IC expression in CRC TME, compared to periphery and associations with clinicopathologic features, which could be used as potential biomarkers for CRC progression and response to therapy

Effect of Dimethoate Residues on Soil Micro-arthropods Population in the Valley of Zendan,Yemen

The present study aimed to screen the effect of overuse of the insecticide Dimethoate and its residues within 0 -15 cm soil layer on the soil micro-arthropods population, which play a very important role in maintenance of the fertility of the soil. The valley of Zendan is located in Arhab Destrict, Sana'a Governorate, Yemen. The valley is about 50 km north-east of Sana'a city and this geographical area is a large producer of Qat (Catha edulis Forsk); the plant which most of the people in the country chew. In this study the valley divided into three stations, each station contained three treated substations and three control substations. The control substations selected from the farms, which are historically free from pesticides. Due care has been taken to insure that the treated sub-stations were from the closest area to the control substations. The average use of Dimethoate 40% E.C is 1-2 ml/L water. The treatment replication was 1-3 times within the period of 2-3 weeks. In this study also, we analyzed the total concentration of Dimethoate, and its metabolite; Omethoate in the soil layer of 0-15 cm. The analysis of Dimethoate and Omethoate residues from representative soil samples covered the major part of the Zendan valley was done by the technique of LC-MS, and the results revealed that the detected levels ranged from 0.914 to 5.180 mg/kg air-dried soil for Dimethoate residues and 0.001 to 0.067 mg/kg air-dried soil for Omethoate residues. For studying the effect of Dimethoate residues on the soil micro-arthropods population, soil samples from each substation were collected with core sampler. The soil micro-arthropods were extracted for 48 hours and collected in vials containing 70% alcohol by using the extraction funnels technique. The number and type of soil micro-arthropods extracted from each substation were separated and recorded and the data obtained was analyzed. The outcomes of the study revealed that Dimethoate and Omethoate residues are beyond the Total Threshold Limit Concentration (TTLC) and significantly reduced the population of the non-target soil micro-arthropods that included mainly mites, collembolans, beetle larvae, trips, cutworms, symphyla and seed corn maggot. Mites and collembolans were the most affected micro-arthropods by Dimethoate and Omethoate residues. The reduction in the soil micro-arthropods population varied from 59% to 69% as compared to its population in control substations. The maximum decline of soil microarthropods was found to be in substations where the highest residues of Dimethoate and Omethoate were recorded. Thus indiscriminate use of pesticides such as Dimethoate will definitely affect the soil fertility in the area and that will reduce the crop yield in future. @JASE

Co-expression of PD-1 with TIGIT or PD-1 with TIM-3 on tumor-infiltrating CD8+ T cells showed synergistic effects on improved disease-free survival in treatment-naïve CRC patients

Immune checkpoints (ICs) are highly expressed on tumor-infiltrating immune cells (TIICs) in different malignancies, including colorectal cancer (CRC). T cells play crucial roles in shaping CRC, and their presence in the tumor microenvironment (TME) has proven to be one of the best predictors of clinical outcomes. A crucial component of the immune system is cytotoxic CD8+ T cells (CTLs), which play decisive roles in the prognosis of CRC. In this study, we investigated associations of immune checkpoints expressed on tumor-infiltrating CD8+ T cells with disease-free survival (DFS) in 45 naïve-treatment CRC patients. First, we examined the associations of single ICs, and found that CRC patients with higher levels of T-cell immunoglobulin and ITIM-domain (TIGIT), T-cell immunoglobulin and mucin domain-3 (TIM-3) and programmed cell death-1 (PD-1) CD8+ T cells tended to have longer DFS. Interestingly, when PD-1 expression was combined with other ICs, there were more evident and stronger associations between higher levels of PD-1+ with TIGIT+ or PD-1+ with TIM-3+ tumor-infiltrating CD8+ T cells and longer DFS. Our findings for TIGIT were validated in The Cancer Genome Atlas (TCGA) CRC dataset. This study is the first to report on the association of co-expression of PD-1 with TIGIT and PD-1 with TIM-3 in CD8+ T cells and improved DFS in treatment-naïve CRC patients. This work highlights the significance of immune checkpoint expression on tumor-infiltrating CD8+ T cells as critical predictive biomarkers, especially when co-expression of different ICs is considered

Congenital myopathy with "corona" fibres, selective muscle atrophy, and craniosynostosis associated with novel recessive mutations in SCN4A

We describe two brothers with lower facial weakness, highly arched palate, scaphocephaly due to synostosis of the sagittal and metopic sutures, axial hypotonia, proximal muscle weakness, and mild scoliosis. The muscle MRI of the younger sibling revealed a selective pattern of atrophy of the gluteus maximus, adductor magnus and soleus muscles. Muscle biopsy of the younger sibling revealed myofibres with internalized nuclei, myofibrillar disarray, and “corona” fibres. Both affected siblings were found to be compound heterozygous for c.3425G>A (p.Arg1142Gln) and c.1123T>C (p.Cys375Arg) mutations in SCN4A on exome sequencing, and the parents were confirmed carriers of one of the mutations. Electrophysiological characterization of the mutations revealed the Cys375Arg confers full and Arg1142Gln mild partial loss-of-function. Loss of function of the Nav1.4 channel leads to a decrement of the action potential and subsequent reduction of muscle contraction. The unusual muscle biopsy features suggest a more complex pathomechanism, and broaden the phenotype associated with SCN4A mutations

Transcriptome of tumor-Infiltrating T cells in colorectal cancer patients uncovered a unique gene signature in CD4 + T cells associated with poor disease-specific survival

Colorectal cancer (CRC) is influenced by infiltration of immune cell populations in the tumor microenvironment. While elevated levels of cytotoxic T cells are associated with improved prognosis, limited studies have reported associations between CD4+ T cells and disease outcomes. We recently performed transcriptomic profiling and comparative analyses of sorted CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) from bulk tumors of CRC patients with varying disease stages. In this study, we compared the transcriptomes of CD4+ with CD8+ TILs. Functional annotation pathway analyses revealed the downregulation of inflammatory response-related genes, while T cell activation and angiogenesis-related genes were upregulated in CD4+ TILs. The top 200 deregulated genes in CD4+ TILs were aligned with the cancer genome atlas (TCGA) CRC dataset to identify a unique gene signature associated with poor prognosis. Moreover, 69 upregulated and 20 downregulated genes showed similar trends of up/downregulation in the TCGA dataset and were used to calculate “poor prognosis score” (ppScore), which was significantly associated with disease-specific survival. High ppScore patients showed lower expression of Treg-, Th1-, and Th17-related genes, and higher expression of Th2-related genes. Our data highlight the significance of T cells within the TME and identify a unique candidate prognostic gene signature for CD4+ TILs in CRC patients

Central Role of Pyrophosphate in Acellular Cementum Formation

Background: Inorganic pyrophosphate (PPi) is a physiologic inhibitor of hydroxyapatite mineral precipitation involved in regulating mineralized tissue development and pathologic calcification. Local levels of PPi are controlled by antagonistic functions of factors that decrease PPi and promote mineralization (tissue-nonspecific alkaline phosphatase, Alpl/TNAP), and those that increase local PPi and restrict mineralization (progressive ankylosis protein, ANK; ectonucleotide pyrophosphatase phosphodiesterase-1, NPP1). The cementum enveloping the tooth root is essential for tooth function by providing attachment to the surrounding bone via the nonmineralized periodontal ligament. At present, the developmental regulation of cementum remains poorly understood, hampering efforts for regeneration. To elucidate the role of PPi in cementum formation, we analyzed root development in knock-out ((-/-)) mice featuring PPi dysregulation. Results: Excess PPi in the Alpl(-/-) mouse inhibited cementum formation, causing root detachment consistent with premature tooth loss in the human condition hypophosphatasia, though cementoblast phenotype was unperturbed. Deficient PPi in both Ank and Enpp1(-/-) mice significantly increased cementum apposition and overall thickness more than 12-fold vs. controls, while dentin and cellular cementum were unaltered. Though PPi regulators are widely expressed, cementoblasts selectively expressed greater ANK and NPP1 along the root surface, and dramatically increased ANK or NPP1 in models of reduced PPi output, in compensatory fashion. In vitro mechanistic studies confirmed that under low PPi mineralizing conditions, cementoblasts increased Ank (5-fold) and Enpp1 (20-fold), while increasing PPi inhibited mineralization and associated increases in Ank and Enpp1 mRNA. Conclusions: Results from these studies demonstrate a novel developmental regulation of acellular cementum, wherein cementoblasts tune cementogenesis by modulating local levels of PPi, directing and regulating mineral apposition. These findings underscore developmental differences in acellular versus cellular cementum, and suggest new approaches for cementum regeneration