Browsing by Subject "T cells"
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Item Investigation of Proteasome Chymotryptic Activities and Effects on their Inhibition in Rat and Human Natural Killer Cells(2003-04-01) Lu, Min; Goldfarb, Ronald H.; Borejdo, Julian; Easom, RichardLu, Min, Investigation of Proteasome Chymotryptic Activites and Effects of Their Inhibition in Rat and Human Natural Killer Cells. Doctor of Philosophy (Biochemistry and Molecular Biology), April, 2003, 185 pp., 3 tables, 32 illustrations, bibliography, 158 titles. The proteasome is a multicatalytic proteinase complex that is involved in the major extralysosomal pathway responsible for intracellular protein degradation in mammalian cells. This dissertation focuses on investigating proteasome chymotryptic activities and the effects of selective inhibitors of these activities on the function of natural killer (NK) cells. In this dissertation, 20S proteasomes derived from rat RNK16 cells were purified and some of their biochemical and biophysical properties were investigated extensively. The results indicated that RNK16 cell-derived proteasome differ from the proteasome of other origins in many aspects including substrate selectivity, inhibitor specificity, and kinetic regulation, although they may share some common biochemical properties with others. To investigate the effects of proteasomal inhibition on the function of NK cells, several proteasome inhibitors were used including MG115, MG132, clasto-lactacystin-β-lactone, EGCG and LLnL. MG115 and MG 132 were shown to induce apoptosis of RNK16 cells, as evidenced by DNA fragmentation, caspase-3 activation and the appearance of sub-G1 cell populations. Activation of multiple caspases and increased expression of cell surface Fas (CD95) protein were also observed following the treatment of RNK16 cells by these two inhibitors. This dissertation also tested the hypothesis that different cell types could respond differentially to proteasome inhibitors. The effects of several proteasome inhibitors were determined on the purified 20S proteasomal and 26S proteasomal chymotrypsin-like activity in whole cell extracts and intact YT and Jurkat cells, human NK and T cell lines respectively. Following such treatment, caspase-3 activation occurred much earlier in Jurkat cells than YT cells; cell cycle analysis indicated a sub-G1 apoptotic cell population in Jurkat cells and G2/M arrest in YT cells. In addition, accumulation of p27 and IκB-α was detected only in Jurkat cells, but not YT cells. Therefore, proteasome inhibitors appear to act differentially in cell cycle progression and apoptosis signaling pathways between human NK and T cells. These studies indicate that the generation of ideal proteasome inhibitors for the treatment of malignancies could be screened or designed to specifically induce cancer cells to undergo programmed cell death, while having little or no apoptosis-inducing abilities for natural killer cells and other cells of the immune response, thus enhancing the selectivity and specificity of the anti-cancer, apoptosis-inducing capabilities of proteasome inhibitors.Item Of Cross-immunity, Herd Immunity and Country-specific Plans: Experiences from COVID-19 in India(JKL International, 2020-12-01) Chakrabarti, Sankha Shubhra; Kaur, Upinder; Singh, Anup; Chakrabarti, Suddhachitta; Krishnatreya, Manigreeva; Agrawal, Bimal Kumar; Mittal, Amit; Singh, Amit; Khanna, Rahul; Gambhir, Indrajeet Singh; Jin, Kunlin; Chakrabarti, SasankaIndia has witnessed a high number of COVID-19 cases, but mortality has been quite low, and most cases have been asymptomatic or mild. In early April, we had hypothesized a low COVID-19 mortality in India, based on the concept of cross-immunity. The presence of cross-immunity is presumed to lead to a milder course of disease and allow the time necessary for the development of adaptive immunity by the body to eliminate the virus. Evidence supporting our hypothesis has started showing up. Multiple studies have shown the generation of different T cell subsets and B cells responding to epitopes of viral proteins, especially of the spike protein, as a part of adaptive immunity against SARS-CoV-2. Cross-reactive T-cells have been demonstrated in patients who have been previously exposed to endemic coronaviruses. The interplay of cross-immunity and herd immunity is apparent in the COVID-19 scenario in India from the presence of a large number of asymptomatic or mild cases, a low infection-fatality ratio and a generally flat curve of percentage positivity of cases with respect to total testing, both in periods of strict lock-down and step-wise unlocking. It seems that cross-immunity resulted in faster generation of herd immunity. Although the initial restrictive measures such as lockdown prevented the rapid spread of the outbreak, further extension of such measures and overly expensive ones such as enhanced testing in India will result in a huge burden on the health economics as well as the society. Hence, we propose a restructuring of the health services and approach to COVID-19. The restructured health services should move away from indiscriminate testing, isolation and quarantine, and instead, the emphasis should be on improving facilities for testing and management of only critical COVID cases and the replacement of complete lockdowns by the selective isolation and quarantine of susceptible persons such as the aged and those with co-morbidities. In the process of describing India-specific plans, we emphasize why the development of country-specific plans for tackling epidemics is important, instead of adopting a "one policy fits all" approach.Item Setting Us Up to Fail: Pulmonary Dendritic Cells Promote Immunopathology during Mycoplasma Respiratory Disease(2010-08-01) Dobbs, Nicole A.; Jerry SimeckaThe purpose of these studies was to define the contributions of T helper 2 cells and dendritic cells toward the development of immunopathology during mycoplasma respiratory disease. IFN-γ+ CD8+ T cells, IFN-γ+ Th1 cells and IL-13+ Th2 cells developed over the course of mycoplasma infection. By day 14 post-infection, the results demonstrated a significant and preferential increase of an IL-13+ Th2 cell sub-population in the LRNs. Additional studies using STAT4-/- animals, which have a Th2 polarized environment, demonstrated no difference in disease compared to the wild-type animals. Absence of STAT6, which strongly contributes to a Th1 polarized environment, conveyed significantly more protection from mycoplasma disease in immunized mice compared to STAT4-/- and WT mice. By day 14 post-infection, all mice had significantly more IL-13+ Th2 cells than IFN-γ+ Th1 in the LRN compared to STAT6-/- immunized mice, thus suggesting that the reduction in the IL-13+ Th2 population leads to protection, while an increase in Th2 is pathogenic. Additional studies demonstrated that pulmonary dendritic cells support the mycoplasma-specific CD4+ and CD8+ T cell activation when stimulated with mycoplasma antigen. Knowing that T cells and DCs have an intimate relationship during mycoplasma disease, sub-classes of cytokine differentiated BMDCs were created to attempt to skew to the protective arm of immunity against mycoplasma disease. However, in vivo adoptive transfer studies demonstrated antigen pulsed DCs accelerated and exacerbated the pathological effects of mycoplasma disease. The exacerbation was antigen-specific and lymphocyte dependent. Mice that received antigen pulsed DCs demonstrated a significant increase in IL-13+ Th2 cell sub-population in the LRNs with a similar trend found in the lungs prior to infection. The same exacerbation was seen when antigen pulsed pulmonary DCs were adoptively transferred into mice, but not with antigen pulsed splenic DCs. Prior to infection, mice that received antigen-pulsed pulmonary DC, not splenic DC, had a significant increase in a IL-13+ Th2 population in the LRNs. Taken collectively, these studies demonstrate two key players in the development of the detrimental response against mycoplasma disease. This knowledge will assist in the development of targeted vaccines that will promote protection over pathology.Item The Role of Regulatory T Cells in Mycoplasma Respiratory Infection(2011-05-01) Odeh, Adam N.; Jerry SimeckaThe purpose of these studies was to examine the role of regulatory T cells (Tregs) in mycoplasma respiratory disease. Depletion of Tregs resulted in increased disease severity. Tregdepleted mice lost significantly more weight over the course of the experiment, and displayed a significantly higher incidence of both gross lung lesions and histological lung lesions at day 14 post-infection. Treg depletion resulted in increased cell infiltration into the lungs by day 14 postinfection, and significant increases in the serum levels of mycoplasma-specific antibodies. Treg depletion also led to an increase in the percentage of IL-13+ T cells in the LRNs, meaning that the immune response was skewed towards a Th2 phenotype. There were no differences observed in lung CFU. These data demonstrate that Tregs in mycoplasma respiratory disease play a role in inflammation and disease severity, but have no effect on bacterial clearance. Importantly, depletion of Tregs causes a Th2-directed shift in the immune response. Additional studies demonstrated that Tregs from mycoplasma-infected mice secreted IFN-γ or IL-17. IFN-γ + and IL-17+ Treg populations both preferentially expanded in response to M. pulmonis infection. Depletion of Tregs resulted in decreased secretion of IFN-γ and IL-17 by CD4+ non-Treg cells. Cocultures of Tregs and T helper cells from mycoplasmainfected mice secreted large amounts of IFN-γ and IL-17 when stimulated with mycoplasma membrane antigen. Levels of IL-4, IL-10, and IL-13 did not significantly increase in response to antigen. Together these studies demonstrate that mycoplasma respiratory disease is influenced by Tregs. These data further suggest that mycoplasma-specific Tregs include two unique subpopulations that express either IFN-γ or IL-17, and that these Tregs may promote the secretion of IFN-γ and IL-17 by T helper cells. This may represent a novel mechanism of Tregmediated immune suppression. This knowledge can assist in the development of treatments for mycoplasma respiratory disease and in the development of Treg-mediated therapies for a number of diseases.