Background & Aims: Human intestinal epithelial cells inducibly express neutrophil and monocyte chemoattractants, yet little is known about the regulated production of T-cell chemoattractants by the ...intestinal epithelium. IP-10, Mig, and I-TAC are 3 CXC chemokines that are known to act as CD4+ T-cell chemoattractants. Methods: We studied constitutive chemokine expression in human colon, and defined the regulated expression of these chemokines by reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistology using cultured human intestinal epithelial cell lines and a novel adaptation of an in vivo human intestinal xenograft model. Results: IP-10 and Mig were constitutively expressed by normal human colon epithelium, and their cognate receptor, CXCR3, was expressed by mucosal mononuclear cells. Interferon (IFN)-γ stimulation increased mRNA expression and the polarized basolateral secretion of these chemokines by human colon epithelial cell lines; infection with enteroinvasive bacteria, or stimulation with the proinflammatory cytokines tumor necrosis factor α and interleukin 1α, strongly potentiated IFN-γ—induced epithelial cell IP-10, Mig, and I-TAC production. Epithelial cell mRNA and protein expression of IP-10, Mig, and I-TAC were rapidly up-regulated in human intestinal xenografts in response to stimulation with IFN-γ alone or in combination with IL-1. Conclusions: The constitutive and regulated production of the IFN-γ—inducible chemokines IP-10, Mig, and I-TAC by human intestinal epithelium, and the expression of their cognate receptor, CXCR3, by mucosal mononuclear cells, suggest that the intestinal epithelium can play a role in modulating physiologic and pathologic T cell-mediated mucosal inflammation.
Rat models have been used to investigate physiological and pathophysiological mechanisms for decades. With the availability of the rat genome and other online resources, tools to identify rat models ...that mimic human disease are an important step in translational research. Despite the large number of papers published each year using rat models, integrating this information remains a problem. Resources for the rat genome are continuing to grow rapidly, while resources providing access to rat phenotype data are just emerging. An overview of rat models of disease, tools to characterize strain by phenotype and genotype, and steps being taken to integrate rat physiological data is presented in this article. Integrating functional and physiological data with the rat genome will build a solid research platform to facilitate innovative studies to unravel the mechanisms resulting in disease.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Abnormal ventilatory responses to increased levels of inspired CO2 during postnatal development may pose a risk for Sudden Infant Death Syndrome, primarily during periods of vulnerability. ...The purpose of this study was to test the hypothesis that in awake piglets the ventilatory response to hypercapnia would be attenuated between 10 and 15 days of age relative to younger and older ages. To test this hypothesis, we measured the ventilatory response to 5% inspired CO2 in piglets from postnatal (PN) days 1 through PN28. Piglets were divided into groups and exposed to 5% CO2 daily, every 3rd day or on and after PN20–21 only to avoid any plasticity that may result from repeated exposure to CO2 . Room air ventilation normalized to body weight ( V ˙ E , ml/min/kg) declined with postnatal age in piglets from all groups. The ventilatory response to 5% inspired CO2 (expressed as % change from control) was present at birth, and we did not find an age-dependent change from PN1 to PN28 ( p > 0.1). In addition, we did not find that repeated exposure (daily or every 3rd day) to 5% inspired CO2 altered the ventilatory response during this period of development. We conclude that the previously documented apparent critical period of development in piglets between 10 and 15 days of age is not associated with attenuation of the ventilatory response to 5% inspired CO2.
Many clinically important enteric pathogens initiate disease by invading and passing through the intestinal epithelium, a process accompanied by increased epithelial expression of proinflammatory ...cytokines. To further define the role intestinal epithelial cells play in initiating and modulating the host response to infection with invasive bacteria, hybrid selection on high density cDNA arrays was used to characterize the mRNA expression profile of approximately 4,300 genes in human intestinal epithelial cells after infection with the prototypic invasive bacteria, Salmonella. Selected findings were further evaluated by reverse transcription-polymerase chain reaction, Northern blot analysis, and protein assays. Epithelial infection with Salmonella significantly up-regulated mRNA expression of a relatively small fraction of all genes tested. Of these, several cytokines (granulocyte colony-stimulating factor, inhibin A, Epstein-Barr virus-induced gene 3, interleukin-8, macrophage inflammatory protein-2alpha), kinases (TKT, Eck, HEK), transcription factors (interferon regulatory factor-1), and HLA class I were the most prominent. Furthermore, the transcription factor NF-kappaB is shown to be important for inducible mRNA expression for a broad group of genes tested. These findings expand the repertoire of known epithelial cell responses to infection with an invasive enteric pathogen. The results also show that evaluation of mRNA expression profiles by cDNA array analysis is a powerful approach to characterizing and understanding host-pathogen interactions.
The intestinal mucosa contains a subset of lymphocytes that produce Th2 cytokines, yet the signals responsible for the recruitment of these cells are poorly understood. Macrophage-derived chemokine ...(MDC/CCL22) is a recently described CC chemokine known to chemoattract the Th2 cytokine producing cells that express the receptor CCR4. The studies herein demonstrate the constitutive production of MDC/CCL22 in vivo by human colon epithelium and by epithelium of human intestinal xenografts. MDC/CCL22 mRNA expression and protein secretion was upregulated in colon epithelial cell lines in response to proinflammatory cytokines or infection with enteroinvasive bacteria. Inhibition of nuclear factor (NF)-kappaB activation abolished MDC/CCL22 expression in response to proinflammatory stimuli, demonstrating that MDC/CCL22 is a NF-kappaB target gene. In addition, tumor necrosis factor-alpha-induced MDC/CCL22 secretion was differentially modulated by Th1 and Th2 cytokines. Supernatants from the basal, but not apical, side of polarized epithelial cells induced a MDC/CCL22-dependent chemotaxis of CCR4-positive T cells. These studies demonstrate the constitutive and regulated production by intestinal epithelial cells of a chemokine known to function in the trafficking of T cells that produce anti-inflammatory cytokines.
Ventilatory sensitivity to hypercapnia is greater in Dahl salt-sensitive (SS) rats than in Fawn Hooded hypertensive (FHH) and Brown Norway (BN) inbred rats. Since pH-sensitive potassium ion (K(+)) ...channels are postulated to contribute to the sensing and signaling of changes in CO(2)-H(+) in chemosensitive neurons, we tested the hypothesis that there are more pH-sensitive K(+) channel-immunoreactive (ir) neurons within the medullary raphé nuclei of the highly chemosensitive SS rats than in the other two strains. Medullary tissues from male and female BN, FHH, and SS rats were stained with cresyl violet or with antibodies targeting TASK-1, K(v)1.4, and Kir2.3 channels. K(+) channel-ir neurons were quantified and compared with the total neurons in the region. The total number of neurons in the medullary raphé 1) was greater in male FHH than the other male rats, 2) did not differ among the female rats, and 3) did not differ between sexes. The average number of K(+) channel-ir neurons per section was 30-60 neurons higher in the male SS than in the other rat strains. In contrast, for the females, the number of K(+) channel-ir neurons was greatest in the BN. We also found significant differences in the number of K(+) channel-ir neurons between sexes in SS (males > females) and BN (females > males) rats, but not the FHH strain. Our findings support the hypothesis for males but not for females, suggesting that both genetic background and sex are determinants of K(+) channel immunoreactivity of medullary raphé neurons, and that the expression of pH-sensitive K(+) channels in the medullary raphé does not correlate with the ventilatory sensitivity to hypercapnia.
Cryopreservation of pharmaceutical based CAR-T cell products prior to administration is standard practice, yet the impact of cryopreservation on CAR-T functionality and clinical outcomes remains ...uncertain. At the Medical College of Wisconsin, utilizing a point-of-care CAR-T manufacturing system (Miltenyi CliniMACS Prodigy®), the goal has been to administer fresh, non-cryopreserved bispecific anti-CD20, anti-CD19 (LV20.19) CAR-T to all patients when clinically feasible as part of an ongoing clinical trial (NCT04186520). To understand the impact of cryopreservation, we initiated a dedicated arm mandating cryopreservation of CAR-T cells prior to infusion for patients with relapsed, refractory B-cell non-Hodgkin's lymphoma.
To fully characterize whether cryopreservation impacted the final CAR-T products, we transcriptionally profiled representative samples of LV20.19 CAR-T cells from the same patients at two timepoints: at harvest and immediately post-thaw for a total of 12 patients using single-cell RNA sequencing. Cells were loaded into the Chromium Controller (10x Genomics) for barcoding and sequenced on Illumina NextSeq500. Raw sequencing data were demultiplexed and converted to gene-barcode matrices using Cell Ranger v7.2.0. Analysis was performed in R v4.3.1 using the package Seurat v5. The number of genes detected per cell, number of UMIs, and percentage of mitochondrial genes were plotted, and outliers were removed to filter doublets and dead cells. Based on expression of lineage-specific markers and immunophenotyping using Seurat's module score feature with GSEA gene sets, the analysis revealed distinct CD8 and CD4 subsets (Fig.1). These clusters showed higher expression of IFNG, GZMB, GZMA, PRF1, and NKG7 in fresh compared to cryopreserved cells (Fig.2). Genes indicative of apoptosis and senescence were elevated in cryopreserved CAR-Ts while proliferation and cytotoxicity markers were reduced (p<0.05) (Fig.3). This indicates cryopreservation negatively impacts the proliferative and cytotoxic capacity and viability of CAR-T cells. Results further suggest impaired functional efficacy and quality of cryopreserved CAR-T cells. However, the clinical significance of these findings remains unknown. We will confirm findings by performing comparative in vitro function and in vivo expansion kinetics analyses of CAR-T cells and outcomes (time to CRS and ICANS, overall response, and duration of response) of patients receiving fresh versus cryopreserved products.
CAR-T cell therapy continues to be a powerful approach for treating relapsed, refractory hematological malignancies. Cryopreservation of CAR-T cell products prior to administration is standard ...practice in the delivery process between third-party manufacturing sites. At the Medical College of Wisconsin, utilizing a point of care CAR-T manufacturing system (CliniMACS Prodigy ®), the goal has been to administer fresh, non-cryopreserved bispecific, lentiviral anti-CD20, anti-CD19 (LV20.19) CAR-T to all patients when clinically feasible. To determine if cryopreservation affects the final CAR product we initiated a new arm on an ongoing clinical trial (NCT04186520) that mandates cryopreservation prior to infusion.
We initiated a 24-patient arm within an ongoing trial utilizing LV20.19 CAR-T cells for patients with relapsed, refractory B-cell DLBCL, FL, and MZL. The manufacturing of this arm mirrored our fresh cohorts but mandated cryopreservation of the final product before infusion. To assess whether cryopreservation affected the activation or bioenergetic capacity of administered CAR-T cells, we examined energy metabolism and activation status of T cells prior to CAR manufacturing, of CAR-T cells immediately before cryopreservation (“fresh”), and of CAR-T cells after thawing on the day of infusion (“post-thaw”) from the same patient, using Seahorse Pro analyzer to measure oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and ATP production.
Analysis of 15 intra-patient comparisons indicated statistically significant decreases in both the OCR and ECAR in the post-thaw CAR-T cells. Moreover, maximal respiratory capacity and spare respiratory capacity (SRC) were significantly decreased in the post-thaw CAR-T cells. Total glycolytic and mitochondrial ATP production decreased in post-thaw CAR-T cells compared to fresh CAR-T cells.
Together these data suggest that cryopreservation impacts CAR-T-cell energy metabolism and activation. The decrease in total ATP production in post-thaw samples suggests impaired CAR-T cell functionality. However, to date, the clinical significance of these findings remains unknown. We continue to enroll patients to this cryopreservation cohort with plans to perform comparative analyses with patients infused with fresh product. Specifically, we will compare in vivo expansion, severity, and time to CRS and ICANS, along with overall response and duration of response.
Inflammatory cells infiltrate the liver in response to microbial infection or hepatic injury. To assess the potential role hepatocytes may play in initiating or amplifying the acute inflammatory ...response in the liver, we used three human hepatocyte cell lines and primary human hepatocyte cultures to characterize the repertoire of cytokines that can be expressed and regulated in hepatocytes in response to agonist stimulation or bacterial infection. As reported herein, a proinflammatory cytokine gene program that includes C-X-C and C-C chemokines interleukin-8(IL-8), growth related (GRO)-alpha, GRO-beta, GRO-gamma, epithelial neutrophil activating peptide-78 (ENA-78), and RANTES and the cytokines tumor necrosis factor-alpha (TNF-alpha) and macrophage colony stimulating factor was upregulated in human hepatocytes after stimulation with IL-1 alpha or TNF-alpha or bacterial invasion. In contrast, expression of hematopoietic/ lymphoid growth factors by the same cells was either down-regulated (erythropoietin and stem cell factor) or unchanged (IL-7 and IL-15) in response to the identical stimuli. Hepatocytes did not express cytokines that often are associated with the regulation of antigen-specific immune responses (IL-2, IL-4, IL-5, IL-10, IL-12p40, IL-13, and interferon-gamma) or genes for several other proinflammatory cytokines IL-1 alpha, IL-6, monocyte chemotactic protein-1 (MCP-1), and MCP-3 or hematopoietic growth factors (granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, IL-3, and IL-11). Together, these studies suggest that hepatocytes can both initiate and amplify acute inflammatory responses in the liver through the regulated expression and secretion of a specific array of proinflammatory cytokines.
Introduction: DNA excision repair protein (ERCC1) and thymidylate synthase (TYMS) are important pancreatic ductal adenocarcinoma (PDAC) biomarkers: low TYMS & low ERCC1 are associated with clinical ...benefit from 5-FU and cisplatin, respectively.
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