CXCR3 expression in regulatory T cells drives interactions with type I dendritic cells in tumors to restrict CD8+ T cell antitumor immunity.

Infiltration of regulatory T (Treg) cells, an immunosuppressive population of CD4+ T cells, into solid cancers represents a barrier to cancer immunotherapy. Chemokine receptors are critical for Treg cell recruitment and cell-cell interactions in inflamed tissues, including cancer, and thus are an ideal therapeutic target. Here, we show in multiple cancer models that CXCR3+ Treg cells were increased in tumors compared with lymphoid tissues, exhibited an activated phenotype, and interacted preferentially with CXCL9-producing BATF3+ dendritic cells (DCs). Genetic ablation of CXCR3 in Treg cells disrupted DC1-Treg cell interactions and concomitantly increased DC-CD8+ T cell interactions. Mechanistically, CXCR3 ablation in Treg cells increased tumor antigen-specific cross-presentation by DC1s, increasing CD8+ T cell priming and reactivation in tumors. This ultimately impaired tumor progression, especially in combination with anti-PD-1 checkpoint blockade immunotherapy. Overall, CXCR3 is shown to be a critical chemokine receptor for Treg cell accumulation and immune suppression in tumors.

Two epilepsy-associated variants in KCNA2 (KV 1.2) at position H310 oppositely affect channel functional expression.

Two KCNA2 variants (p.H310Y and p.H310R) were discovered in paediatric patients with epilepsy and developmental delay. KCNA2 encodes KV 1.2-channel subunits, which regulate neuronal excitability. Both gain and loss of KV 1.2 function cause epilepsy, precluding the prediction of variant effects; and while H310 is conserved throughout the KV -channel superfamily, it is largely understudied. We investigated both variants in heterologously expressed, human KV 1.2 channels by immunocytochemistry, electrophysiology and voltage-clamp fluorometry. Despite affecting the same channel, at the same position, and being associated with severe neurological disease, the two variants had diametrically opposite effects on KV 1.2 functional expression. The p.H310Y variant produced 'dual gain of function', increasing both cell-surface trafficking and activity, delaying channel closure. We found that the latter is due to the formation of a hydrogen bond that stabilizes the active state of the voltage-sensor domain. Additionally, H310Y abolished 'ball and chain' inactivation of KV 1.2 by KV ß1 subunits, enhancing gain of function. In contrast, p.H310R caused 'dual loss of function', diminishing surface levels by multiple impediments to trafficking and inhibiting voltage-dependent channel opening. We discuss the implications for KV -channel biogenesis and function, an emergent hotspot for disease-associated variants, and mechanisms of epileptogenesis. KEY POINTS: KCNA2 encodes the subunits of KV 1.2 voltage-activated, K+ -selective ion channels, which regulate electrical signalling in neurons. We characterize two KCNA2 variants from patients with developmental delay and epilepsy. Both variants affect position H310, highly conserved in KV channels. The p.H310Y variant caused 'dual gain of function', increasing both KV 1.2-channel activity and the number of KV 1.2 subunits on the cell surface. H310Y abolished 'ball and chain' (N-type) inactivation of KV 1.2 by KV ß1 subunits, enhancing the gain-of-function phenotype. The p.H310R variant caused 'dual loss of function', diminishing the presence of KV 1.2 subunits on the cell surface and inhibiting voltage-dependent channel opening. As H310Y stabilizes the voltage-sensor active conformation and abolishes N-type inactivation, it can serve as an investigative tool for functional and pharmacological studies.

Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.

Cytogenetic endpoints and Xenobiotic gene polymorphism in lymphocytes of hospital workers chronically exposed to ionizing radiation in Cardiology, Radiology and Orthopedic Laboratories.

Ionizing radiation (IR) is known as a classical mutagen capable of inducing various kinds of stable and unstable chromosomal aberrations (CA) including the possibility of increasing the incidence of DNA damage. This study aims to assess occupationally induced CA in workers chronically exposed to low doses of IR in Radiology (RL), Cardiology (CL) and Orthopedic (OL) Laboratories in hospitals of Tamil Nadu. We performed the analysis of CA by trypsin G-banding, micronucleus (MN) assay, Comet assay and Xenobiotic-metabolizing gene polymorphisms (GSTM1, GSTT1 and GSTP1) in 56 exposed and 56 control subjects who were matched for gender and age (± 2 years). Higher degree of CA and MN frequencies were observed in exposed groups, especially in CL subjects compared to other exposed groups and controls (p<0.05). Higher frequency of DNA tail length and tail moment was observed in the CL exposed subjects compared to the RL and OL subjects. The frequencies of GSTM1 and GSTT1 null genotypes were 39.3 percent and 14.3 percent, respectively. No significant difference in allele frequencies between exposed subjects and controls were observed (p=0.0128). Using multiple linear regression analysis, statistical significance was determined for work duration and age for the CL, RL and OL workers and the examination of the possible impact by confounding factors showed few significant influences on the radiation exposure, as a specific biomarker. However, the findings from the present study suggest that, awareness should be created among the personnel exposed to radiations in hospital laboratories, highlighting the necessity of applying radiation protection principles against medical radiation exposure.

Intranasal insulin attenuates hypoxia-ischemia-induced short-term sensorimotor behavioral disturbances, neuronal apoptosis, and brain damage in neonatal rats.

There is a significant need for additional therapy to improve outcomes for newborns with acute Hypoxic-ischemic (HI) encephalopathy (HIE). New evidence suggests that insulin could be neuroprotective. This study aimed to investigate whether intranasal insulin attenuates HI-induced brain damage and neurobehavioral dysfunction in neonatal rats. Postnatal day 10 (P10), Sprague-Dawley rat pups were randomly divided into Sham + Vehicle, Sham + Insulin, HI + Vehicle, and HI + Insulin groups with equal male-to-female ratios. Pups either had HI by permanent ligation of the right common carotid artery followed by 90 min of hypoxia (8% O2) or sham surgery followed by room air exposure. Immediately after HI or Sham, pups were given fluorescence-tagged insulin (Alex-546-insulin)/vehicle, human insulin (25 µg), or vehicle in each nare under anesthesia. Shortly after administration, widespread Alex-546-insulin-binding cells were detected in the brain, primarily co-localized with neuronal nuclei-positive neurons on double-immunostaining. In the hippocampus, phospho-Akt was activated in a subset of Alex-546-insulin double-labeled cells, suggesting activation of the Akt/PI3K pathway in these neurons. Intranasal insulin (InInsulin) reduced HI-induced sensorimotor behavioral disturbances at P11. InInsulin prevented HI-induced increased Fluoro-Jade C+ degenerated neurons, cleaved caspase 3+ neurons, and volume loss in the ipsilateral brain at P11. There was no sex-specific response to HI or insulin. The findings confirm that intranasal insulin provides neuroprotection against HI brain injury in P10 rats associated with activation of intracellular cell survival signaling. If further pre-clinical research shows long-term benefits, intranasal insulin has the potential to be a promising non-invasive therapy to improve outcomes for newborns with HIE.

Evolving Applications of Circulating Tumor DNA in Merkel Cell Carcinoma.

Circulating tumor DNA (ctDNA) is a subset of circulating cell-free DNA released by lysed tumor cells that can be characterized by its shorter strand length and tumor genome-specific information. The relatively short half-life of ctDNA allows it to provide a real-time measure of tumor burden which has potential prognostic and surveillance value as a tumor biomarker. Merkel cell carcinoma (MCC) is a rare neuroendocrine skin cancer that requires close monitoring due to the high risk of relapse. There are currently no good tumor biomarkers for MCC patients, especially those who are negative for Merkel cell polyomavirus. ctDNA shows promise for improving the prognoses of MCC patients by monitoring tumor burden, identifying minimal residual disease (MRD), and stratifying patients by their likelihood of response to immune checkpoint inhibition or risk of relapse. In particular, bespoke ultra-sequencing platforms allow for the creation of patient-specific mutation panels that improve ctDNA detection, especially for patients with rare or uncharacteristic mutations. Leveraging bespoke ctDNA assays may improve physicians' ability to alter treatment plans for non-responsive or high-risk patients. In addition, ctDNA MRD monitoring may allow physicians to treat relapses early before clinically evident disease is present.

Evaluation of Uric Acid to Albumin Ratio as a Marker of Coronary Artery Disease Severity in Acute Coronary Syndrome: A Cross-Sectional Study.

BACKGROUND: Coronary artery disease (CAD) is a widespread cause of morbidity and mortality. Serum uric acid, a mediator of endothelial dysfunction and inflammation in vascular disease, can increase the risk of atherosclerosis, contributing to CAD. As serum albumin inhibits platelet activation and aggregation, low levels of it can contribute to platelet-induced coronary artery stenosis. Limited studies have been conducted worldwide in evaluating the role of uric acid to albumin ratio (UAR) in predicting severity or poor outcomes in acute coronary syndrome (ACS) patients. This study was undertaken to assess the role of UAR as a predictor of CAD severity, which can facilitate the identification of high-risk patients. METHODOLOGY: A hospital-based analytical cross-sectional study was conducted in an urban tertiary healthcare center for a period of two months between June and August of 2022. A total of 100 ACS patients were included in the study. The study population included patients above the age of 18 years diagnosed with ACS who underwent a coronary angiography. Coronary angiograms were used to diagnose the presence of CAD, and its severity was assessed using Synergy between Percutaneous Coronary Intervention with Taxus and Cardiac Surgery (SYNTAX) scores (SS). The correlation of UAR with CAD severity using SS was studied and compared between three varieties of ACS: ST-elevation myocardial infarction (STEMI), non-ST-elevation myocardial infarction (NSTEMI), and unstable angina (UA). STATISTICS: Chi-squared tests were used to determine statistical significance for qualitative data. Independent t-tests were used to identify the mean difference between two quantitative variables. Receiver operating characteristic (ROC) curves were constructed for UAR and high SS. A comparison between UAR and neutrophil to lymphocyte ratio (NLR) as a predictor of disease severity was done. ROC and optimal cutoff points were chosen to calculate sensitivity, specificity, and positive and negative predictive values. Microsoft Excel (Microsoft, Redmond, WA, USA) and SPSS V22.0 (IBM Corp., Armonk, NY, USA) were used to analyze the data. RESULTS: A total of 100 ACS patients were included in the study and divided into two groups on the basis of SS, with 74% showing low severity and 26% showing intermediate-high severity. There was a statistically significant difference found between older age and SS (p=0.017). Our study showed 74% (n=74) of the patients were male and 26% (n=26) were female. It also revealed that 75.7% (n=56) of the male patients were in the low-severity group, and 24.3% (n=18) of males were in the intermediate-high severity group. 69.2% (n=18) of the female patients were in the low-severity group, and 30.8% (n=8) were in the intermediate-high severity group. Of the 100 patients, 55% were diagnosed with STEMI, of which 69.1% were in the low-severity group, and 30.9% were in the intermediate-high severity group. Among all the patients 33% of the patients were diagnosed as NSTEMI, of which 72.7% were in the low-severity group, and 27.3% were in the intermediate-high severity group. Twelve percent of the patients were diagnosed with UA, and 100% of these patients were in the low-severity group. The mean UAR was 1.40 ± 0.38 in the low-severity group and 1.29 ± 0.46 in the intermediate-high severity group (p=0.22). CONCLUSION: Our study yielded no statistically significant difference in UAR among varying severities of CAD.

Ventilation-Induced Lung Injury (VILI) in Neonates: Evidence-Based Concepts and Lung-Protective Strategies.

Supportive care with mechanical ventilation continues to be an essential strategy for managing severe neonatal respiratory failure; however, it is well known to cause and accentuate neonatal lung injury. The pathogenesis of ventilator-induced lung injury (VILI) is multifactorial and complex, resulting predominantly from interactions between ventilator-related factors and patient-related factors. Importantly, VILI is a significant risk factor for developing bronchopulmonary dysplasia (BPD), the most common chronic respiratory morbidity of preterm infants that lacks specific therapies, causes life-long morbidities, and imposes psychosocial and economic burdens. Studies of older children and adults suggest that understanding how and why VILI occurs is essential to developing strategies for mitigating VILI and its consequences. This article reviews the preclinical and clinical evidence on the pathogenesis and pathophysiology of VILI in neonates. We also highlight the evidence behind various lung-protective strategies to guide clinicians in preventing and attenuating VILI and, by extension, BPD in neonates. Further, we provide a snapshot of future directions that may help minimize neonatal VILI.

DFSP of the Breast: Histomorphological, Immunohistochemical, and Molecular Features of a Rare Case in an Unusual Location.

We present a case of a 21-year-old female with a vague nontender mass in the lower inner quadrant of the left breast discovered incidentally on chest imaging following trauma. A breast ultrasound demonstrated an 8×6×8 mm irregular hyperechoic mass at the 7 o'clock position of the left breast, 9 cm from the nipple. The mass was graded Breast Imaging Reporting and Data System (BI-RADS) category 4 (suspicious finding). An ultrasound-guided biopsy of the mass showed a proliferation of monotonous spindled cells in a storiform pattern with tapered nuclei with infiltration into the adipose tissue. No normal breast elements were identified in the biopsy. Myofibroblastoma was the first differential diagnosis; however, the characteristic infiltrative pattern of the tumor mandated additional tests including fluorescence in situ hybridization to rule out a dermatofibrosarcoma protruberance (DFSP). Immunohistochemical staining showed positive staining for CD34, which can be positive in myofibroblastoma also. However, fluorescence in situ hybridization demonstrated a platelet-derived growth factor B (22q13.1) gene rearrangement confirming a diagnosis of DFSP. The patient underwent a wide local excision of the DFSP for definitive treatment. She is doing well with no recurrence reported so far, after 15 months of follow-up. Conventional DFSP does not metastasize but is prone to recurrence making wide margins imperative for definitive treatment.

Complex Case of Aggressive Intra-abdominal Desmoid-type Fibromatosis Status Post Cholecystectomy.

Desmoid-type fibromatosis (DF), also known as desmoid tumor, is an extremely rare, benign, mesenchymal fibrous tumor with no potential for metastasis. It can arise from any part of the body, most commonly extra-abdominally. Intra-abdominal DF can present sporadically, in sites of previous trauma, surgical scars and irradiation, or in association with familial adenomatous polyposis and Gardner syndrome. Intra-abdominal DF is uncommon and especially rare after a common surgery like cholecystectomy. We report a rare case of a 67-year-old male who presented with a locally aggressive intra-abdominal DF in the gallbladder fossa, status post cholecystectomy. This progressively enlarging infiltrative enhancing solid mass in the gallbladder fossa on serial computed tomography and magnetic resonance imaging demonstrated gastric outlet obstruction, biliary obstruction, portal vein narrowing and encasement of hepatic artery. Diagnosis of DF in this postoperative setting was delayed and challenging due to uncharacteristic clinical presentation. Radiologists should be aware of this unusual diagnosis and spectrum of imaging findings to help in timely surgical management and planning.

His bundle pacing-is it the final frontier of physiological pacing ?-A single centre experience from the Indian sub-Continent.

BACKGROUND: Long term right ventricular pacing can have deleterious effects on left ventricular (LV) function. His bundle pacing (HBP), a novel procedure can probably circumvent this setback. We investigated if (1) HBP is associated with pacing induced LV dysfunction by using LV global longitudinal strain (GLS) and (2) intermediate term performance of the Select Secure (3830) lead in the His bundle location. This report is probably the first on HBP in the Indian population. METHODS: 61 patients, with normal LV ejection fraction (EF) with a guideline based indication for permanent pacing underwent a HBP pacemaker implantation using the His Select Secure 3830 lead; with lead guided mapping for locating the His bundle. The patients underwent GLS assessment; evaluation of the His lead parameters - sensing, impedance and capture thresholds immediately after implantation and at 6 months in addition to the standard follow up. RESULTS: At 6 month follow up, the average GLS did not show significant variation from baseline in patients requiring ventricular pacing more than 40% and was similar, irrespective of selective or non selective His bundle pacing. All the patients had stable pacemaker parameters - with little change in capture threshold, lead impedance or sensing of the His bundle lead - implying electrical and mechanical stability on intermediate term follow-up. CONCLUSION: HBP is a feasible procedure in the hands of an experienced operator, with stable lead performance. It does not appear to be associated with pacing mediated left ventricular dysfunction at intermediate term follow up. It should probably become the default method of permanent pacing.

Ribosome biogenesis during cell cycle arrest fuels EMT in development and disease.

Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ERα) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.

Evaluation of genetic alterations in inhabitants of a naturally high level background radiation and Kudankulam nuclear power project site in India.

Evaluation of genetic alterations in inhabitants of an area of Tamil Nadu, India, chronically exposed to high background radiation (HBRA), was the major purpose of the present study. A total of 216 samples (exposed inhabitants, 108; control subjects, 108) were selected based on the confirmation of radiation dose level using thermoluminescence dosimetry (TLD). After signing a consent form, volunteers provided blood samples (5 ml each) to establish cell cultures at 52 h. One hundred complete metaphase cells from each subject were evaluated for karyotyping. The frequencies of chromosomal alterations (CA) were found to be higher in the exposed groups and the aberrations predominately observed were of chromatid-type. Smoking was found to have considerable effect on the frequency of CA in exposed subjects. With the comet assay for DNA damage, a significant increase in comet tail frequency was also observed in exposed subjects compared to controls. At present there are no radioepidemiological data regarding the cytogenetic studies in these areas. Furthermore, the Kudankulam nuclear power plant nuclear power plant is being constructed in the same area. The study gives potentially important information on the general health effects due to radiation exposure and increases people's understanding of the hazardous nature of chronic low level natural radiation exposure. However, we may conclude that the HBRA by itself does not pose any significant risk of genetic damage as measured by conventional cytogenetic analysis.