Tissue-dependent T Cell Apoptosis and Transcriptional Regulation of Memory CD8+T Cell Differentiation During Viral Infections: A Dissertation

Activation and proliferation of antigen-specific T cells is the hallmark of an anti-viral immune response. Effector T cells generated during an immune response are heterogeneous in regards to their ability to populate the memory pool once the immune response has resolved. Initial T cell activation takes place in the lymphoid organs, after which T cells migrate into the non-lymphoid tissues. The presence of memory T cells at non-lymphoid tissue sites has been shown to be critical for protection against secondary virus challenge. Our lab has previously demonstrated that during and after the resolution of the immune response to Lymphocytic choriomeningitis virus (LCMV) CD8+T cells in the nonlymphoid tissues are more resistant to apoptosis than those in the lymphoid organs. This stability of T cells in the non-lymphoid tissues may be critical in ensuring protection against a secondary virus challenge. Mechanisms regulating tissue-dependent differences in CD8+T cell apoptosis were studied in an acute LCMV infection model. Virus-specific CD8+T cells from lymphoid (spleen, mesenteric lymph nodes (MLN), inguinal lymph nodes (ILN)) and non-lymphoid tissues (peritoneal exudate cells (PEC), fat-pads) were compared for expression of surface antigenic markers known to correlate with a memory phenotype. Non-lymphoid tissues were enriched in IL-7Rhi, KLRG-1lo, CD27hi and CXCR3hi virus-specific CD8+ T cells, and the presence of these antigenic markers correlated with increased memory potential and survival. Transcription factors in addition to cell surface antigens were assessed as correlates of resistance to apoptosis. Virus-specific CD8+T cells in the nonlymphoid tissues were enriched in cells expressing T cell factor-1 (TCF-1), which correlated with increased memory potential and survival. CD8+T cells in the peritoneum of TCF-1-deficient mice had decreased survival during resolution of the immune response to LCMV, suggesting a role for TCF-1 in promoting survival in the non-lymphoid tissues. As an additional mechanism, I investigated whether apoptosis-resistant CD8+T cells migrate to non-lymphoid tissues and contribute to tissue-dependent apoptotic differences. CXCR3+ CD8+T cells resisted apoptosis and accumulated in the lymph nodes of mice treated with FTY720, which blocks the export of lymph node cells into the peripheral tissues. The PECs expressed increased amounts of CXCR3 ligands, CXCL9 and CXCL10, which may have recruited the non-apoptotic cells from the lymph nodes. By adoptively transferring splenic T cells into the spleen or PEC environment I showed that the peritoneal environment through a yet undefined factor promoted survival of CD8+T cells. In this study I have elucidated the mechanisms by which CD8+T cells preferentially survive in the non-lymphoid tissues. I found that non-lymphoid tissues were enriched in memory-phenotype CD8+T cells which were intrinsically resistant to apoptosis irrespective of the tissue environment. Furthermore, apoptosisresistant CD8+T cells may preferentially migrate into the non-lymphoid tissues where the availability of tissue-specific factors may enhance memory cell survival. Few transcription factors have been identified that regulate CD8+T cell effector-memory differentiation during an immune response. In this thesis, I have also studied the mechanism by which the transcription factor Blimp-1 regulates the generation of effector and memory CD8+T cells. Blimp-1 is known to repress a large number of target genes, and ChIP (chromatin immunoprecipitation) sequencing analysis done by Dr. HyunMu Shin in the lab of Dr. Leslie J. Berg identified CD25 (IL-2Rα) and CD27 as potential targets of Blimp-1. I found that Blimp-1-deficient CD8+T cells had sustained expression of CD25 (IL-2Rα) and CD27 during peak and resolution of the immune response to LCMV. By performing adoptive transfers of CD25hi and CD27hi CD8+T cells I showed that CD25 and CD27 expression on CD8+T cells during resolution of the immune response correlates with enhanced survival. Silencing Il2rα and Cd27 expression reduced the Blimp-1-deficient CD8+T cell response, suggesting that sustained expression of CD25 and CD27 was in part responsible for the enhanced CD8+T cell response seen in the Blimp-1-deficient mice. Furthermore, our collaborator Dr. HyunMu Shin showed that CD25 and CD27 are direct targets of Blimp-1, and that Blimp-1 recruits histone modifying enzymes to Il2rα and Cd27 loci to suppress their expression during the peak of the anti-viral immune response. This study identifies one of the mechanisms by which Blimp-1 regulates the balance between generation of effector and memory CD8+T cells. In this thesis work I also studied the function of the transcription factor ROG (Repressor of GATA-3) in regulating in vivo T cell responses during both acute and chronic LCMV infection. ROG-deficient mice had increased CD8+T cell responses during an acute LCMV infection. ROG deficiency also led to the generation of memory T cells with an enhanced recall response compared to WT controls. By using LCMV-specific P14+ TCR transgenic ROG-deficient CD8+T cells these defects were shown to be T cell intrinsic. ROG-deficient mice had enhanced CD8+T cell responses and viral clearance during a persistent high dose LCMV Clone 13 infection. During chronic LCMV infection ROG-deficient mice also had increased lung pathology and mortality. The results indicate that ROG negatively regulates T cell responses and memory generation during both acute and chronic LCMV infection. The studies highlighted in this thesis elucidate the mechanisms promoting CD8+T cell survival in non-lymphoid tissues as well as transcription factormediated regulation of memory CD8+T cell differentiation. Knowledge of this will help us better understand T cell immunity after infections and may eventually help develop better vaccines

Non-surgical endodontics in retreatment of periapical lesions: two representative case reports

This article reports non-surgical endodontic retreatment of two patients with persistent or recurrent periapical lesions, who had previously undergone surgical and non-surgical endodontic therapy respectively. It further discusses and reviews the relevance of classification of periapical lesions, the explanation behind healing of periapical lesions by endodontic therapy alone, causes of persistence of periapical lesions, choice of treatment modalities (whether surgical or non ' surgical) and materials such as intracanal medicaments and irrigants for optimal healing

A novel technique for numerical approximation of 1D non-linear coupled Burgers' equation by using cubic Hyperbolic B-spline based Differential quadrature method

In this paper, a novel scheme, cubic Hyperbolic B-spline-based Differential Quadrature Method,is proposed for the solution of 1D non-linear viscous coupled Burgers' equation. The numericalapproximation of this mentioned equation is obtained by using the Hyperbolic B-spline-basedDifferential quadrature method. Hyperbolic B-spline is used as a basis function in DQM in orderto obtain weighting coefficients, then received a set of ODEs is solved by using Strong StabilityPreserving Runge Kutta-43 scheme(SSP-RK43 scheme). The accuracy and effectiveness of thisproposed scheme are tested by using three examples. The obtained results are matched with theprevious results present in the literature of other methods as well as with the exact solutions bymeans o

Model atomic systems in intense laser fields: Exact time-dependent density functional and Floquet theory

The ab initio solution of the time-dependent Schrödinger equation for a many-body system is feasible for only a few constituents. Success of TDDFT when it comes to highly correlated electron dynamics is very limited. In this thesis, the exact exchange-correlation potential for the highly correlated process of autoionization is constructed. One may try to employ the time-periodicity of external drivers. If this could also be applied to the time-dependent Kohn-Sham equation the time-dependent many-body problem could be reduced to a time-independent one. It is investigated if this is possible

Primary ovarian ectopic pregnancy: an unusual case study

Ovarian pregnancy is rare variant of ectopic pregnancy. An accurate preoperative diagnosis is very challenging various advances in diagnostic modalities like Transvaginal ultrasonography has evolved in identifying an ovarian pregnancy. It results in significant maternal morbidity, fetal loss, repeat ectopic, impairment of subsequent fertility. The case report a 34-year female who presented with pain and severe anemia and explored keeping in view ruptured tubal ectopic but intraoperatively diagnosed as primary ovarian pregnancy and managed conservatively by ovarian wedge resection

Efficacy of IGFBP7 for treatment of metastatic melanoma and other cancers in mouse models and human cell lines

We recently identified the secreted protein IGFBP7 as a factor required for an activated BRAF oncogene to induce senescence or apoptosis in primary human cells. In human melanomas containing an activating BRAF mutation (BRAF-positive melanomas), IGFBP7 is epigenetically silenced, which seems to be a critical step in melanoma genesis. Restoration of IGFBP7 function by the addition of recombinant IGFBP7 (rIGFBP7) induces apoptosis in BRAF-positive human melanoma cell lines, and systemically administered rIGFBP7 markedly suppresses the growth of BRAF-positive primary tumors in xenografted mice. Here we further evaluate the role of IGFBP7 in the treatment of BRAF-positive melanoma and other malignancies. We find that in human metastatic melanoma samples IGFBP7 is epigenetically silenced and at an even higher frequency than that found in primary melanomas. Using a murine experimental metastasis assay, we show that systemic administration of rIGFBP7 markedly suppresses the growth of metastatic disease and prolongs survival. An analysis of the NCI60 panel of human cancer cell lines reveals that in addition to melanoma, IGFBP7 induces apoptosis in several other cancer types, in particular colorectal cancer cell lines. In general, IGFBP7 induces apoptosis in human cancer cell lines that have an activating mutation in BRAF or RAS, and that are sensitive to chemical inhibition of BRAF-MEK-ERK signaling. Significantly, systemically administered rIGFBP7 blocks the growth of colorectal tumors containing an activating RAS or BRAF mutation in mouse xenografts. The results presented here, in conjunction with those from previous studies, justify the further development of IGFBP7 as an anticancer agent

MTrack: Automated Detection, Tracking, and Analysis of Dynamic Microtubules

Microtubules are polar, dynamic filaments fundamental to many cellular processes. In vitro reconstitution approaches with purified tubulin are essential to elucidate different aspects of microtubule behavior. To date, deriving data from fluorescence microscopy images by manually creating and analyzing kymographs is still commonplace. Here, we present MTrack, implemented as a plug-in for the open-source platform Fiji, which automatically identifies and tracks dynamic microtubules with sub-pixel resolution using advanced objection recognition. MTrack provides automatic data interpretation yielding relevant parameters of microtubule dynamic instability together with population statistics. The application of our software produces unbiased and comparable quantitative datasets in a fully automated fashion. This helps the experimentalist to achieve higher reproducibility at higher throughput on a user-friendly platform. We use simulated data and real data to benchmark our algorithm and show that it reliably detects, tracks, and analyzes dynamic microtubules and achieves sub-pixel precision even at low signal-to-noise ratios.V.K. was supported by the IRI Life Sciences postdoc fellowship in the labs of S.R. and S.P. C.H. and S.R. acknowledge funding by the IRI Life Sciences (Humboldt-Universität zu Berlin, Excellence Initiative/DFG). W.H. was supported by the Alliance Berlin Canberra “Crossing Boundaries: Molecular Interactions in Malaria”, which is co-funded by a grant from the Deutsche Forschungsgemeinschaf (DFG) for the International Research Training Group (IRTG) 2290 and the Australian National University. S.P. was supported by the MDC Berlin

Regulation of tissue-dependent differences in CD8+ T cell apoptosis during viral infection

Virus-specific CD8+ T cells in the lymphoid organs contract at the resolution of virus infections by apoptosis or by dissemination into peripheral tissues, and those residing in nonlymphoid organs, including the peritoneal cavity and fat pads, are more resistant to apoptosis than those in the spleen and lymph nodes. This stability of memory T cells in the nonlymphoid tissues may enhance protection to secondary challenges. Here, we show that lymphocytic choriomeningitis virus (LCMV)-specific CD8+ T cells in nonlymphoid tissues were enriched for memory precursors (expressing high levels of interleukin-7 receptor and low levels of killer cell lectin-like receptor G1 [IL-7Rhi KLRG1lo]) and had higher expression of CD27, CXCR3, and T cell factor-1 (TCF-1), each a marker that is individually correlated with decreased apoptosis. CD8+ T cells in the peritoneal cavity of TCF-1-deficient mice had decreased survival, suggesting a role for TCF-1 in promoting survival in the nonlymphoid tissues. CXCR3+ CD8+ T cells resisted apoptosis and accumulated in the lymph nodes of mice treated with FTY720, which blocks the export of lymph node cells into peripheral tissue. The peritoneal exudate cells (PEC) expressed increased amounts of CXCR3 ligands, CXCL9 and CXCL10, which may normally recruit these nonapoptotic cells from the lymph nodes. In addition, adoptive transfer of splenic CD8+ T cells into PEC or spleen environments showed that the peritoneal environment promoted survival of CD8+ T cells. Thus, intrinsic stability of T cells which are present in the nonlymphoid tissues along with preferential migration of apoptosis-resistant CD8+ T cells into peripheral sites and the availability of tissue-specific factors that enhance memory cell survival may collectively account for the tissue-dependent apoptotic differences. IMPORTANCE: Most infections are initiated at nonlymphoid tissue sites, and the presence of memory T cells in nonlymphoid tissues is critical for protective immunity in various viral infection models. Virus-specific CD8+ T cells in the nonlymphoid tissues are more resistant to apoptosis than those in lymphoid organs during the resolution and memory phase of the immune response to acute LCMV infection. Here, we investigated the mechanisms promoting stability of T cells in the nonlymphoid tissues. This increased resistance to apoptosis of virus-specific CD8+ T cells in nonlymphoid tissues was due to several factors. Nonlymphoid tissues were enriched in memory phenotype CD8+ T cells, which were intrinsically resistant to apoptosis irrespective of the tissue environment. Furthermore, apoptosis-resistant CD8+ T cells preferentially migrated into the nonlymphoid tissues, where the availability of tissue-specific factors may enhance memory cell survival. Our findings are relevant for the generation of long-lasting vaccines providing protection at peripheral infection sites

Hospital acquired acute renal failure

Background: Acute renal failure has continued to attract interest and stimulate investigators .This is in part, a reflection of many clinical entities that can result in an acute renal failure. HAARF is an important cause of morbidity and mortality and is associated with the ten fold increase in the risk of death during the hospitalization.14 Despite advances in diagnosis and management it still carries a high mortality. HAARF is associated with grave consequences. Some of the clinical setting leading to HAARF can be limited by monitoring of renal function, better control of infection, avoiding nephrotoxic drugs and initiation of therapy at the earliest. Present study is proposed to evaluate the incidence, etiological factors and measures to reduce the incidence of HAARF.Methods:All patients were admitted to RPGMC from Dec. 2010 to APRIL 2014. They were screened for the development of the ARF during their hospital stay. The parameters of Prakash et al. were taken for the diagnosis of HAARF.  Results:In our present study HAARF was diagnosed in 88 patients of 56904 admission during 40 month period, representing 0.15% of the admission. Predisposing factors were present in 64 % of the patients. They were elderly age >60 years (22.72%), DM (13.64%), pre-existing renal disease (13.64%) and HTN (4.54%). It was due to nephrotoxic dugs in 45.45%, decreased renal perfusion in 22.72%, infections in 13.64%, hepatorenal syndrome 9.09% surgery in 4.54% and Weil’s disease in 4.54 %. Among the nephrotoxic drugs antibiotics were the most common and NSAIDs were the second most common etiological factors. Decrease renal perfusion secondary to volume depletion and heart failure were equally responsible for HAARF in 9.09% each. Septicemia was responsible for 4.54% of cases. Oliguric renal failure was seen in the 31.82% where as nonoliguric renal failure was seen in 50% of cases. Great majority of non oliguric renal failure was due to nephrotoxic drugs. Oliguric patients have longer duration of hospital stay and high mortality as compared to the non oliguric renal failure. A high s. creatinine and high urea levels at the time of admission were associated with the earlier development of HAARF. Oliguria and anuric patients had a longer duration of hospital stay. Overall mortality of HAARF was 18.18% and nephrotoxic drugs responsible for one half of the total mortality.Conclusion:High risk group patients for HAARF needs meticulous monitoring during hospital stay. Hospitalized patients on nephrotoxic drugs should have frequent renal function tests. Proper fluid and electrolyte balance in hospitalized patients needs special emphasis to avoid HAARF.   

Assessment of pulmonary function in patients with type 2 diabetes mellitus: a case-control study

Background:As other microvascular complications, respiratory involvement is far less studied among patients with type-2 Diabetes Mellitus (DM). Objective: to study the extent of pulmonary function limitation among patients with type-2 DM.  Methods:Hospital based matched case-control study.Results:Total of 90 cases and 90 controls matched for age, sex, height and weight were recruited. Patients with DM had neuropathy [63.3% (57; male=27: Female: 30)], retinopathy [44.4% (40; male=22: Female: 18)], nephropathy [41.1% (37; male=17: Female: 20)] and microalbuminuria [14.4% (13; male=5: Female: 8)]. All cases and 88 controls observed with FEV1:FVC ratio of >70.0%, further assessment for delineation of normal and restrictive pattern patients with high level of predicted values of FEV1 as compare to FVC showed that significantly (P = 0.00) more (Cases: 76.6%; Controls: 42.2%) cases had FEV1 >FVC predicted levels as compare to controls, means among diabetics odds of restrictive pattern of lung abnormality is four times (OR: 4.4; CI: 2.3-8.5) more as compare to non-diabetics. In addition a long duration of DM was significantly (r: 0.39; P = 0.00) positively correlated with lung dysfunction.  Conclusion: Patients with type 2 DM patients as compare to its controls observed with restrictive pattern of lung dysfunction.