Prevalence and risk factors of lymphatic dysfunction in cirrhosis patients with refractory ascites: An often unconsidered mechanism.

BACKGROUND: The lymphatic system is crucial in maintaining the body fluid homeostasis. A dysfunctional lymphatic system may contribute to the refractoriness of ascites and edema in cirrhosis patients. Therefore, assessment of lymphatic dysfunction in cirrhosis patients with refractory ascites (RA) can be crucial as it would call for using different strategies for fluid mobilization. AIM: To assessing the magnitude, spectrum, and clinical associations of lymphatic dysfunction in liver cirrhosis patients with RA. METHODS: This observational study included 155 consecutive cirrhosis patients with RA. The presence of clinical signs of lymphedema, such as peau d'orange appearance and positive Stemmer sign, intestinal lymphangiectasia (IL) on duodenal biopsy seen as dilated vessels in the lamina propria with strong D2-40 immunohistochemistry, and chylous ascites were used to diagnose the overt lymphatic dysfunctions. RESULTS: A total of 69 (44.5%) patients out of 155 had evidence of lymphatic dysfunction. Peripheral lymphedema, found in 52 (33.5%) patients, was the most common manifestation, followed by IL in 42 (27.0%) patients, and chylous ascites in 2 (1.9%) patients. Compared to patients without lymphedema, those with lymphedema had higher mean age, median model for end-stage liver disease scores, mean body mass index, mean ascitic fluid triglyceride levels, and proportion of patients with hypoproteinemia (serum total protein < 5 g/dL) and lymphocytopenia (< 15% of total leukocyte count). Patients with IL also had a higher prevalence of lymphocytopenia and hypoproteinemia (28.6% vs. 9.1%, P = 0.004). Seven (13%) patients with lymphedema had lower limb cellulitis compared to none in those without it. On multivariate regression analysis, factors independently associated with lymphatic dysfunction included obesity [odds ratio (OR): 4.2, 95% confidence intervals (95%CI): 1.1-15.2, P = 0.027], lymphocytopenia [OR: 6.2, 95%CI: 2.9-13.2, P < 0.001], and hypoproteinemia [OR: 3.7, 95%CI: 1.5-8.82, P = 0.003]. CONCLUSION: Lymphatic dysfunction is common in cirrhosis patients with RA. Significant indicators of its presence include hypoproteinemia and lymphocytopenia, which are likely due to the loss of lymphatic fluid from the circulation. Future efforts to mobilize fluid in these patients should focus on methods to improve lymphatic drainage.

Single-cell antigen-specific landscape of CAR T infusion product identifies determinants of CD19-positive relapse in patients with ALL.

A notable number of acute lymphoblastic leukemia (ALL) patients develop CD19-positive relapse within 1 year after receiving chimeric antigen receptor (CAR) T cell therapy. It remains unclear if the long-term response is associated with the characteristics of CAR T cells in infusion products, hindering the identification of biomarkers to predict therapeutic outcomes. Here, we present 101,326 single-cell transcriptomes and surface protein landscape from the infusion products of 12 ALL patients. We observed substantial heterogeneity in the antigen-specific activation states, among which a deficiency of T helper 2 function was associated with CD19-positive relapse compared with durable responders (remission, >54 months). Proteomic data revealed that the frequency of early memory T cells, rather than activation or coinhibitory signatures, could distinguish the relapse. These findings were corroborated by independent functional profiling of 49 patients, and an integrative model was developed to predict the response. Our data unveil the molecular mechanisms that may inform strategies to boost specific T cell function to maintain long-term remission.

The RAG1 N-terminal region regulates the efficiency and pathways of synapsis for V(D)J recombination.

Immunoglobulin and T cell receptor gene assembly depends on V(D)J recombination initiated by the RAG1-RAG2 recombinase. The RAG1 N-terminal region (NTR; aa 1-383) has been implicated in regulatory functions whose influence on V(D)J recombination and lymphocyte development in vivo is poorly understood. We generated mice in which RAG1 lacks ubiquitin ligase activity (P326G), the major site of autoubiquitination (K233R), or its first 215 residues (Δ215). While few abnormalities were detected in R1.K233R mice, R1.P326G mice exhibit multiple features indicative of reduced recombination efficiency, including an increased Igκ+:Igλ+ B cell ratio and decreased recombination of Igh, Igκ, Igλ, and Tcrb loci. Previous studies indicate that synapsis of recombining partners during Igh recombination occurs through two pathways: long-range scanning and short-range collision. We find that R1Δ215 mice exhibit reduced short-range Igh and Tcrb D-to-J recombination. Our findings indicate that the RAG1 NTR regulates V(D)J recombination and lymphocyte development by multiple pathways, including control of the balance between short- and long-range recombination.

Improving and Maintaining Responses in Pediatric B-Cell Acute Lymphoblastic Leukemia Chimeric Antigen Receptor-T Cell Therapy.

ABSTRACT: Chimeric antigen receptor T therapy has heralded a new era in the treatment of acute lymphoblastic leukemia (ALL) and other hematologic malignancies. In this autologous immunotherapy, patient-derived T cells are genetically engineered and then infused back to kill the leukemia cells. The observed response rates in ALL are a testament to the success of this therapy. However, there have been instances where the patients either did not respond or relapsed after initial response. Emergence of resistance due to antigen loss and T-cell exhaustion has been observed. This poses a challenge in making this therapy successful for every ALL patient and warrants deeper understanding of emergence of resistance and potential approaches to overcome them. Here we discuss current perspectives and advances in this area.

The Lineage-Defining Transcription Factors SOX2 and NKX2-1 Determine Lung Cancer Cell Fate and Shape the Tumor Immune Microenvironment.

The major types of non-small-cell lung cancer (NSCLC)-squamous cell carcinoma and adenocarcinoma-have distinct immune microenvironments. We developed a genetic model of squamous NSCLC on the basis of overexpression of the transcription factor Sox2, which specifies lung basal cell fate, and loss of the tumor suppressor Lkb1 (SL mice). SL tumors recapitulated gene-expression and immune-infiltrate features of human squamous NSCLC; such features included enrichment of tumor-associated neutrophils (TANs) and decreased expression of NKX2-1, a transcriptional regulator that specifies alveolar cell fate. In Kras-driven adenocarcinomas, mis-expression of Sox2 or loss of Nkx2-1 led to TAN recruitment. TAN recruitment involved SOX2-mediated production of the chemokine CXCL5. Deletion of Nkx2-1 in SL mice (SNL) revealed that NKX2-1 suppresses SOX2-driven squamous tumorigenesis by repressing adeno-to-squamous transdifferentiation. Depletion of TANs in SNL mice reduced squamous tumors, suggesting that TANs foster squamous cell fate. Thus, lineage-defining transcription factors determine the tumor immune microenvironment, which in turn might impact the nature of the tumor.

V(D)J Recombination Exploits DNA Damage Responses to Promote Immunity.

It has been recognized for 40 years that the variable (diversity) joining [V(D)J] recombination-mediated assembly of diverse B and T lymphocyte antigen receptor (AgR) genes is not only essential for adaptive immunity, but also a risk for autoimmunity and lymphoid malignancies. Over the past few years, several studies have revealed that recombination-activating gene (RAG) endonuclease-induced DNA double-strand breaks (DSBs) transcend hazardous intermediates during antigen receptor gene assembly. RAG cleavage within the genomes of lymphocyte progenitors and immature lymphocytes regulates the expression of ubiquitous and lymphocyte-specific gene transcripts to control the differentiation and function of both adaptive and innate immune cell lineages. These unexpected discoveries raise important new questions that have broad implications for basic immunology research and the screening, diagnosis, and treatment of human immunological disease.

Lymphocyte lineage-specific and developmental stage specific mechanisms suppress cyclin D3 expression in response to DNA double strand breaks.

Mammalian cells are thought to protect themselves and their host organisms from DNA double strand breaks (DSBs) through universal mechanisms that restrain cellular proliferation until DNA is repaired. The Cyclin D3 protein drives G1-to-S cell cycle progression and is required for proliferation of immature T and B cells and of mature B cells during a T cell-dependent immune response. We demonstrate that mouse thymocytes and pre-B cells, but not mature B cells, repress Cyclin D3 protein levels in response to DSBs. This response requires the ATM protein kinase that is activated by DSBs. Cyclin D3 protein loss in thymocytes coincides with decreased association of Cyclin D3 mRNA with the HuR RNA binding protein that ATM regulates. HuR inactivation reduces basal Cyclin D3 protein levels without affecting Cyclin D3 mRNA levels, indicating that thymocytes repress Cyclin D3 expression via ATM-dependent inhibition of Cyclin D3 mRNA translation. In contrast, ATM-dependent transcriptional repression of the Cyclin D3 gene represses Cyclin D3 protein levels in pre-B cells. Retrovirus-driven Cyclin D3 expression is resistant to transcriptional repression by DSBs; this prevents pre-B cells from suppressing Cyclin D3 protein levels and from inhibiting DNA synthesis to the normal extent following DSBs. Our data indicate that immature B and T cells use lymphocyte lineage- and developmental stage-specific mechanisms to inhibit Cyclin D3 protein levels and thereby help prevent cellular proliferation in response to DSBs. We discuss the relevance of these cellular context-dependent DSB response mechanisms in restraining proliferation, maintaining genomic integrity, and suppressing malignant transformation of lymphocytes.

Niche Applications of Irreversible Electroporation.

Irreversible electroporation (IRE) induces cell death by exposing it to high-voltage, low-energy DC current pulses. The mechanism of cell death and healing is a departure from the other existing technologies such as radiofrequency ablation, microwave ablation, and cryoablation. These thermal ablative technologies have several applications in oncology but have limitations that have also been established. IRE has shown promise to overcome some of these limitations and has enabled the use of an ablative technology in treating lesions close to the bile ducts and vasculature and in organs such as the pancreas. This review highlights some of the niche applications of IRE and the data so far.

Mycobacterium tuberculosis and Human Immunodeficiency Virus Type 1 Cooperatively Modulate Macrophage Apoptosis via Toll Like Receptor 2 and Calcium Homeostasis.

The emergence of drug resistant strains of Mycobacterium tuberculosis (M. tuberculosis) together with reports of co-infections with the human immunodeficiency virus (HIV) has renewed interest to better understand the intricate mechanisms prevalent during co-infections. In this study we report a synergistic effect of M. tuberculosis and HIV-1, and their antigens Rv3416 and Nef, respectively, in inhibiting apoptosis of macrophages. This inhibition involves the TLR2 pathway and second messengers that play complementing and contrasting roles in regulating apoptosis. Interestingly, the route of calcium influx into cells differentially regulates apoptosis during antigenic co-stimulation. While calcium released from intracellular stores was anti-apoptotic, calcium influx from the external milieu was pro-apoptotic. Further, molecular sensors of intracellular calcium release aid in antigen mediated inhibition of apoptosis. A cross-regulation between oxidative burst and differential routing of calcium influx governed apoptosis. Interestingly, the HIV-1 Nef supported anti-apoptotic responses in macrophages whereas Vpu had no significant effect. These results point to a synergistic liaison between M. tuberculosis and HIV-1 in regulating macrophage apoptosis.

Clinico-pathology, diagnosis and management of Cysticercus fasciolaris and Hymenolepis diminuta co-infection in wistar rats.

AIM: The present study was undertaken to study the pathology and control of sudden unexplained mortality in wistar rats. MATERIALS AND METHODS: This study was conducted in a colony of 25 male wistar rats where there was mortality of nine rats. The dead rats were subjected to thorough post-mortem examination and necropsy samples were processed for hematoxylin and eosin staining for histopathological studies. Faecal samples of live rats were studied for the presence of parasitic eggs. Treatment with anthelmintics was given to manage the mortality and infections. RESULTS: The investigation revealed a natural co-infection of Cysticercus fasciolaris and Hymenolepis diminuta in wistar rats, which were pathogenic enough to cause mortality. Typical lesions associated with the parasites were found in the dead rats. The mortality and infection were managed with common anthelmintics. CONCLUSION: C. fasciolaris and H. diminuta infection can cause mortality in wistar rats even when individually they cause asymptomatic infection. The mortality and infection can be managed with common anthelmintics.

Measuring glutathione redox potential of HIV-1-infected macrophages.

Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E(GSH); Grx1-roGFP2) and measured subcellular changes in E(GSH) during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E(GSH) (approximately -310 mV), active viral replication induces substantial oxidative stress (E(GSH) more than -240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular E(GSH) between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ∼25 mV in E(GSH) is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E(GSH). Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.

AID: a riddle wrapped in a mystery inside an enigma.

To combat the ever-changing pool of pathogens we face, B cells generate highly optimized antibodies in two distinct steps. A large variety of antibodies are first generated randomly by V(D)J recombination, and then, upon encountering an antigen, antibodies are fine-tuned by somatic hypermutation and class switch recombination--both of which are initiated by the same protein, activation-induced cytidine deaminase (AID). All three processes are highly mutagenic, and mistargeting of each of these has been shown to contribute to tumorigenesis. We study these processes because they provide an excellent model to understand how highly mutagenic reactions are channeled into productive use by cells and the consequent risk this carries. In this review, we will discuss many of the outstanding questions in the field that we grapple with while developing a consistent model for AID action. We will also discuss the complexity added to these models by the recent finding that AID might be part of a demethylase complex.