An effective HIV-1 vaccine will likely elicit broadly neutralizing antibodies (bnAbs). However, development of vaccine immunogens that induce bnAbs remains a challenging goal. Understanding the somatic maturation pathways of known broadly neutralizing HIV-1 human monoclonal antibodies (bnmAbs) may help vaccine immunogen design. All known HIV-1 bnmAbs are highly somatically matured, and the putative germline antibodies of the known HIV-1 bnmAbs lack measurable binding activity to HIV-1 envelope glycoprotein (Env). Based on these observations, we hypothesize that somatic maturation of known HIV-1 bnmAbs may be initiated by primary immunogens which may not be related to HIV-1 Env; such primary immunogens trigger the somatic maturation of the germline antibodies and induce intermediate antibodies that bind to HIV-1 Env and further mature to bnAbs upon HIV-1 infection or Env vaccination. The main objective of my study is to identify intermediate antibodies of bnmAb b12 in uninfected and infected human individuals, as well as in uninfected rhesus macaques, the model animals for vaccine development. I constructed two nonimmune cDNA antibody VH1 scFv libraries using the mRNAs isolated from pooled PBMCs of 200 uninfected healthy human individuals and one uninfected rhesus macaque, respectively, and identified 5 and 10 possible b12 intermediate immunoglobulin heavy chain V-segments (IGHVs) from the human and macaque nonimmune libraries, respectively. 454 deep sequencing of the VHs and VLs in the nonimmune and two immune human cDNA Fab libraries confirmed the existence of b12 intermediate IGHVs, but we did not find further maturation of the b12 intermediate IGHVs in HIV-1-infected human individuals. Further sequence analysis revealed the extremely low frequency of the VHs with exactly the same V(D)J recombination as b12, which may explain the lack of further maturation of the intermediate IGHVs of b12 in HIV-1-infected humans. Characterization of HIV-1 Env trimer may aid in Env-based vaccine immunogen design. Therefore, I investigated the importance of Env variable loops in Env-mediated viral function. A panel of gp160JRFL loop deletion/replacement mutants were constructed and tested. The results indicate that, besides the CD4 binding loop and V3, V1V2 and loop D are also critical for virus entry into permissive cells. Deletion of variable V4 or V5 loop or replacement of V4 or V5 loop with a flexible linker of the same length abolish Env cell surface display, which may result from the conformational changes of the V4 or V5 loop deletion or replacement Env proteins. V4 or V5 deletion or replacement knocks out the CD4 binding site and CD4-induced site on Env, but enhances the exposure of the membrane-proximal external region (MPER) and N-trimer structure. In summary, my study demonstrated the existence of intermediate b12 IGHVs in uninfected and HIV-1-infected humans and rhesus macaques. These intermediate antibody fragments may be used to identify primary immunogens that initiate b12 somatic maturation. My study also showed the importance of Env variable loops for Env structural integrity and Env-mediated viral function. The enhanced exposure of the MPER in gp160JRFL ΔV4 or ΔV5 may be further explored for vaccine development to induce MPER-specific bnAbs. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Non-small cell lung cancer (NSCLC) causes most of the cancer deaths worldwide. Tyrosine kinase inhibitors (TKIs), like erlotinib and crizotinib, are commonly used as specific treatments targeting epidermal growth factor receptor (EGFR)-mutated and anaplastic lymphoma kinase (ALK)-rearranged NSCLC. Autophagy is a highly conserved cellular process in response to stress. Tumor microenvironment (TME) is composed of both tumor cells and stromal cells. This study aimed to investigate whether autophagy could confer intrinsic and acquired resistance to TKIs in NSCLC, and its role in the presence of TME or in animal models. In the first part of this study, the effect of EGFR TKI or ALK TKI on sensitive NSCLC cells to generate autophagy was investigated, and manipulation of autophagy in these cell lines was performed. Autophagy inhibition was shown to enhance apoptotic effect of TKIs in sensitive NSCLC cells. This part provided strong evidence that TKIs and autophagy inhibitor chloroquine (CQ) work synergistically in sensitive NSCLC cells. Autophagy induction by erlotinib treatment was observed in a HCC827 (lung adenocarcinoma, EGFR exon 19 del) xenograft model, which was in line with the in vitro observation. Correspondingly, the combination of erlotinib (12.5 mg/kg) with CQ (50 mg/kg) in the HCC827 xenograft model achieved greater tumor growth suppression, compared with single drug treatments. In the second part of this study, a model of TME was established to allow study of autophagy under such circumstances. An activated TME with cytokine production, autophagy induction and epithelial-to-mesenchymal transition (EMT) was generated by co-culturing NSCLC cells and human fibroblasts. Sensitivity to TKI under TME was not affected, and combination of chloroquine with TKI under TME remained synergistic compared with single treatments. In the third part of this study, erlotinib-resistant (ER) HCC827 cells were acquired by stepwise exposure to increasing concentrations of erlotinib in cell culture. Common acquired resistance mechanisms to EGFR TKI (EGFR T790M or c-MET amplification) were excluded in this ER HCC827 model, except EMT. Autophagy status in ER HCC827 cells was studied and autophagy manipulation was performed. It was found that CQ and erlotinib worked synergistically to induce cell death even in ER HCC827 cells. In an ER HCC827 xenograft model, significant degree of autophagy and EMT was evident. Interestingly, combining erlotinib (25 mg/kg) with CQ (50 mg/kg) showed better inhibitory effect on tumor growth compared with single treatments. In summary, TKIs induced both apoptosis and autophagy in EGFR-mutated and ALK-rearranged NSCLC cells. Autophagy inhibition by CQ enhanced TKI-induced cell death in sensitive cells. The presence of TME did not confer TKI resistance. Autophagy was highly activated in EGFR-mutated NSCLC cells with acquired resistance to erlotinib. Combination of CQ with erlotinib remained synergistic in the presence of TME and acquired resistance, both in vitro and in vivo. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The aging process is strongly associated with decreased activity in the immune system. Dysregulation of T-lymphocyte function, such as reduced proliferation, is one problem faced by most elder people, which prevents them from successfully dealing with exogenous pathogens. Effective regulation of T-lymphocyte activity depends on the proper and prompt transduction of both positive and negative signals within Tlymphocytes and reflects the balance between positive and negative effects. Decline of positive signaling in aging has been studied and reported, while mechanisms concerning up-regulation of negative signaling with age and its role in immune senescence are still unclear. Cbl-b, an E3 ubiquitin ligase, was studied by our lab since it regulates the ubiquitin process, a protein modification process that has suppressive effects on signaling pathways. We first determined the reaction of Cbl-b to different stimuli in young rat splenic T-lymphocytes, and showed that there is a decrease in Cbl-b protein expression upon CD28 stimulation and such protein degradation is proteasome-dependent only. We also showed the mechanism of Cbl-b expression regulation involves the intracellular movement of Nedd4 toward Cbl-b and an up-regulation of Nedd4 expression. Then we proved that in old splenic T-lymphocytes, decreased proteasome activity was unable to down-regulate the Cbl-b protein. High levels of Cbl-b in old T-lymphocytes are functional in preventing PI3K activity and are associated with reduced T-lymphocyte proliferation upon regular stimulation. T-lymphocytes from old Cbl-b knock-out mice show similar proliferative reaction to CD3 stimulation as T-lymphocytes from young wild-type, which establishes the causeeffect relationship between sustained Cbl-b expression and decreased T-lymphocyte proliferation. In summary, these data suggest a unique role of Cbl-b in regulating Tlymphocyte signal transduction and provide critical preliminary data for extending Cbl-b studies into other fields, such as carcinogenesis.

No description available.

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

No description available.

Gerbils, experimentally infected with Giardia lamblia trophozoites, had trophozoites and encysting trophozoites in all 3 equal sections of the small intestine and in the colon at necropsy. Cysts were found in the second and third sections of the small intestine and in the colon. WB strain derived trophozoites (WB, D1, WB-C6, and V1) differed in levels of encystation in vitro but not in gerbils. Passage in gerbils increased the in vitro encystation levels of WB and D1 but decreased that of WB-C6 and V1. No differences were found in the total protein profiles or isoenzyme patterns of these G. lamblia populations. Immunization of mice with in vitro cysts produced monoclonal antibodies (mAbs) recognizing cyst protein antigens. In Immunofluorescence (IFA), mAb 5A4.G6 recognized cyst walls. This mAb reacted with a 38 kD band on Western blots. IFA results showed that mAb 8C5.C11 reacts with vesicles in encysting trophozoites and with cyst walls. It recognized 26, 28, 42 and 46 kD bands in Western blots. When mAb 8C5.C11 and Guinea pig complement were added to 0-9 hour encysting cultures, the numbers of cysts produced were significantly reduced compared to control. MAb 5A4.G6 did not affect in vitro encystation.

The immunological responses in hosts infected with Trypanosoma lewisi were examined during the course of infection and after recovery. Peak antibody levels coincided with the time of parasite elimination, but remained significantly elevated for over one year after the end of the infection The antigen repertoire recognized by antibodies demonstrated that some were revealed only by sera taken during the infection, and other antigens were revealed for the first time only by post-recovery sera. Immunomodulatory protective and suppressive factors were demonstrated in the plasma of irradiated, infected rats. These factors were identified as parasite-derived exoantigens which are shed in vivo and in vitro; exoantigens are complexes of proteins, lipids and polysaccharides and are membrane-surface coat associated, as shown by phase-partitioning and surface-labeling studies. The suppressive activity of the exoantigens was dose-dependent, probably mediated by a suppressor substance(s) produced by macrophages that subsequently inhibits production of interleukin 2 by T helper cells.