The intestine is the largest lymphoid organ in the body, challenged constantly by an enonnous quantity and diversity of antigens. Distinct from peripheral lymphocytes, intestinal lymphocytes have evolved unique mechanisms of tolerance and appear to govern mucosal processes such as "chronic physiologic inflammation" and oral tolerance. Failure of mucosal tolerance has been implicated in the pathogenesis of several diseases, including inflammatory bowel disease, celiac disease, and even autoimmune diabetes. One population of intestinal lymphocytes, intraepithelial lymphocytes (IELs), exists within the intestinal epithelium itself and remains poorly characterized. IELs respond to unique activation signals and appear to be in part responsible for the maintenance of epithelial integrity and mucosal tolerance. Type 1 diabetes is one of the most common chronic childhood illnesses and causes significant morbidity and mortality. Type 1 diabetes mellitus is an autoimmune disease that results from immune-mediated destruction of insulin-producing pancreatic beta cells and is characterized by an absolute insulin deficiency. Several animal models are used to study the immunopathogenesis of type 1 diabetes, including the BB rat and NOD mouse. BBDP rats spontaneously develop autoimmune diabetes mellitus and are severely deficient in peripheral T cells. BBDR rats do not spontaneously develop autoimmune diabetes, have nonnal numbers of peripheral T cells, and can be induced to become diabetic by injections of a cytotoxic anti-ART2a mAb and low doses of poly I:C. The cause of autoimmune diabetes in BB rats and humans is still unknown, but both genetic and environmental factors appear to participate. I hypothesize that one important class of environmental factors--diet and enteromicrobial agents--participates in this pathogenic process through the mediation of the gut immune system. In this dissertation, I report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The yield of rat IELs using this method is 5-10 fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrate that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyer's patch lymphocytes. Phenotypic analysis reveals 5 major subsets of IELs, including populations of γδ T and natural killer (NK) cells present at levels not previously detected. I also report that rat intraepithelial NK (IENK) and peripheral NK cells are similar in morphology, in their ability to lyse NK-sensitive targets, and in their ability to suppress a one-way mixed lymphocyte culture. In contrast, IENK cells differ from splenic NK cells phenotypically, and a substantial fraction of IENK cells appear to spontaneously secrete IL-4 and/or IFN-γ. I conclude that rat IELs harbor a large population of NKR-P1A+ CD3-cells that function as NK cells but display an activated phenotype and unusual cytokine profile that clearly distinguish them from splenic NK cells. Their phenotypic and functional characteristics suggest that these distinctive intraepithelial NK cells may participate in the regulation of mucosal immunity. I next demonstrate that, prior to diabetes, both BBDP and ART2a-depleted BBDR rats have a reduced total number of IELs and exhibit a selective deficiency of IENK cell number and function as compared to control BBDR rats. The deficiency of BBDP rat IELs can be corrected by engraftment of bone marrow from histocompatible WF donors. These results suggest 1) that the peripheral lymphopenia in BBDP rats extends to the IEL compartment, particularly to IENK cells, 2) that in BBDR rats the diabetes-inducing treatment depletes IELs, particularly IENK cells, and 3) that the defect in BBDP rat IELs is intrinsic to hematopoietic cells, not intestinal stromal cells. I also establish that, unlike BBDR and WF rats, BBDP rats are also deficient in γδTCR+IELs, a population of T cells that may play a role in normal mucosal tolerance. In addition, I report preliminary data supporting the hypothesis that systemic autoreactivity may be initiated in the intestine; peripheral autoreactive lymphocyte populations appear to emanate first from mesenteric lymph nodes that drain the intestine, and such cells may initiate a type 2 autoimmune phenomenon driven by IL-4. Collectively, my findings support the hypothesis that a failure of mucosal tolerance in BBDP rats, perhaps secondary to deficiencies in one or more IEL subpopulations, participates in the pathogenesis of autoimmune diabetes in these animals by activating peripheral autoreactive T cells. The nature of the autoimmune response in BB rats (driven by IL-4) appears to be distinct from that of NOD mice. Despite the differences between these two well-accepted animal models of autoimmune diabetes, until more is known about the pathogenesis of type 1 DM in humans, lessons learned from both the BB rat and NOD mouse continue to be of tremendous benefit to our understanding of human disease.

NOD mice model human type 1 diabetes and have been used to investigate tolerance induction protocols for islet transplantation in a setting of autoimmunity. Costimulation blockade-based tolerance protocols that induce prolonged skin and permanent islet allograft survival in non-autoimmune mice have failed in NOD mice. To investigate the underlying mechanisms, we generated NOD hematopoietic chimeras. We were able to show that dendritic cell maturation defects seen in NOD mice are partially corrected in mixed hematopoietic chimeras. Furthermore, skin allograft survival was dependent upon the phenotype of the bone marrow donor, demonstrating that in the NOD the resistance to tolerance induction resides in the hematopoietic compartment. In addition, we studied congenic NOD mice bearing insulin dependent diabetes (Idd) loci that reduce diabetes incidence. The incidence of diabetes is reduced in NOD.B6 Idd3 mice, and virtually absent in NOD.B6 Idd3Idd5 mice. Islet allograft survival in NOD.B6 Idd3 mice is prolonged as compared to NOD mice, and in NOD.B6 Idd3Idd5 mice islet allograft survival is similar to that achieved in C57BL/6 mice. Alloreactive CD8 T cell depletion in NOD mice treated with costimulation blockade is impaired, but is partially restored in NOD.B6 Idd3 mice, and completely restored in NOD.B6 Idd3Idd5 mice. Idd3 results from variations in Il2 gene transcription. We hypothesized insufficient levels of IL-2 in NOD mice contributes to impaired deletion of alloreactive CD8 T cells and shortened islet allograft survival. We observed using synchimeric mice that co-administration of exogenous IL-2 to NOD mice treated with costimulation blockade led to deletion of alloreactive CD8 T cells comparable to that in C57BL/6 mice and prolonged islet allograft survival. However, some Idd loci impaired the induction of transplantation tolerance. These data suggest that Idd loci can facilitate or impair induction of transplantation tolerance by costimulation blockade, and that Idd3 (IL-2) is critical component in this process.

The JNK signal transduction pathway mediates a broad, complex biological process in response to inflammatory cytokines and environmental stress. These responses include cell survival and apoptosis, proliferation, tumorigenesis and the immune response. The divergent cellular responses caused by the JNK signal transduction pathway are often regulated by spatial and cell type contexts, as well as the interaction with other cellular processes. The discovery of additional components of the JNK signal transduction pathway are critical to elucidate the stress response mechanisms in cells. This thesis first discusses the cloning and characterization of two novel members of the JNK signal transduction pathway. JIP1 and JMP1 were initially identified from a murine embryo library through a yeast Two-Hybrid screen to identify novel JNK interacting proteins. Full length cDNAs of both genes were cloned and analyzed. JIP1 represents the first member of the JIP group of JNK scaffold proteins which were characterized. The JNK binding domain (JBD) of JIP1 matches the D-domain consensus of other JNK binding proteins, and it demonstrates JNK binding both in vitro and in vivo. This JNK binding was demonstrated to inhibit JNK signal transduction and over-expression of JIP1 inhibits the JNK mediated pre-B cell transformation by bcr-abl. Over-expressed JIP1 also sequesters JNK in the cytoplasm, which may be a mechanism of the inhibition of JNK signaling. A new, high-resolution digital imaging microscopy technique using deconvolution demonstrated the absence of JNK1 in the nucleus of co-transfected JIP1 and JNK1 cells. The other protein discussed in this thesis is JMP1, a novel JNK binding, microtubule co-localized protein. There is a JBD in the JMP1 carboxyl end and a consensus D-domain within this region. The JMP1 JBD demonstrates an increased association with phospho-JNK from UV irradiated cells compared to un-irradiated cells in vivo. JMP1 also has 12 WD-repeat motifs in its amino terminal end which are required for microtubule co-localization. JMP1 demonstrates a cell cycle specific localization at the mitotic spindle poles. This co-localization is dependent on intact microtubules and the amino-terminal WD-repeats are required for this localization. JMP1 mRNA is highly expressed in testis tissues. Immunocytochemistry on murine testis sections using an affinity purified anti-JMP1 antibody demonstrates JMP1 protein in the lumenal compartment of the seminiferous tubules. JMP1 protein is expressed in primary and secondary spermatocytes, cells which are actively undergoing meiosis. The results obtained from the localization of JMP1 in meiotic spermatocytes led to an investigation of the roles of JNK signal transduction in the testis. The testis is an active region of cellular proliferation, apoptosis and differentiation, which make it an appealing model for studying JNK signal transduction. However, the roles JNK signaling have in the testis are poorly understood. I investigated the reproduction capability of Jnk3-/- male mice and discovered older Jnk3-/- males had a reduced capacity to impregnate females compared to younger animals and age-matched wild type controls. The testis morphology and sperm motility of these animals were similar to wild-type animals, and there was no alteration of apoptosis in the testis. The final section of this thesis involves the study of this breeding defect and investigating for cellular defects that might account for this age-related Jnk3-/- phenotype.

Serum vitamin D deficiency/insufficiency, Chronic Kidney Disease (CKD) and elevated blood pressure are important health concerns especially among minorities with type 2 diabetes. The effect of vitamin D3 supplementation (cholecalciferol) at 6,000 IU/day (d) vs. 4,000 IU/d on kidney function and cardiovascular disease markers among Hispanics and African Americans with type 2 diabetes and hypovitaminosis D (/ml) was evaluated. Subjects (n=63) were recruited from two clinics in Miami-Dade County, FL. Fasting venous blood and fresh, single-voided first morning urine samples were collected from each participant by a certified phlebotomist and analyzed by Solstas Lab Partners, Davie, FL. Linear mixed models were used to compare the interaction between time and intervention. Least Significant Difference (LSD) comparisons were used to detect significant differences within and between 4,000 IU/d and 6,000 IU/d groups from baseline, 3 and 6 months. In the 4,000 IU/d and 6,000 IU/d groups, a significant increase in serum 25-hydroxy vitamin D [25(OH)D] levels were observed from baseline [(19.9±1.1 ng/mL) and (21.4±1.3 ng/mL)] to 3 months [(36.1±2.2 ng/mL, p3 longer than 6 months may be needed to determine sustained long term effects in kidney and cardiovascular disease markers. Further research could provide more information for translation of these findings into recommendations for individuals with CKD, hypertension and type 2 diabetes. The efficacy of vitamin D3 supplementation as complementary therapy for CKD and blood pressure in minority and other ethnic groups needs further investigation in larger and longer duration randomized controlled trials.

The observations that substance P (SP) is localized in the anterior pituitary gland (AP) and is regulated by the hormonal status of the animal, as well as the demonstration of SP binding sites in the AP, have led to the idea that SP may participate in the regulation of AP function. Numerous and sometimes contradictory reports of SP effects on AP hormone secretion, particularly on luteinizing hormone (LH), left the question of whether SP acts directly at the level of the AP to regulate LH secretion still unanswered. To investigate a possible physiological function of SP in the AP, the effects of exogenous SP on LH secretion from AP cells from adult and prepubertal male and female rats in short term culture were studied. It was found that SP (100nM-1μM) significantly stimulates LH release in cultured AP cells and that this effect varies as a function of age and sex. SP has no significant effect on LH release from AP cells of male and female prepubertal rats. After day 30 a sharp increase in the response to SP occurs in both sexes. This level of responsiveness continues through adulthood in AP cells from the female rat. In contrast, AP cells from male rats failed to respond during adulthood (over 50 days of age) but were highly responsive during the peripubertal period (30-35 days). The possibility that the responsiveness to SP is influenced by the endocrine status of the animal was investigated by exposing AP cells from responding animals to androgens in vivo and in vitro. It was found that AP cells from female rats treated with androgen were less responsive to 100nM SP but did respond at higher doses of SP. SP effects on AP function were further analyzed in experiments using radioligand binding assays to assess possible changes in SP receptor number or affinity as related to age and sex. In AP membranes from female rats, maximum binding is 8-fold higher (Bmax=4.2 pmo1/mg membrane protein) than in AP membranes from male rats (Bmax=560fmo1/ mg membrane protein). These studies suggest a role for SP as a secondary regulator of LH secretion with possible physiological significance for reproductive function.

Congenital Adrenal Hyperplasia (CAH) encompasses several disorders related to disruptions in the adrenal steroid production pathway. These disruptions may cause virilization of the external female sex organs, incorrect gender assignment, precocious puberty, and in the most severe form, may cause life-threatening salt wasting and adrenal crisis if not detected and treated early in the newborn period. 17α-Hydroxyprogesterone (17-OHP) is the primary target for immunofluorescence detection of CAH from dried blood spots in newborn screening (NBS). Unfortunately, current immunoassay techniques for the detection of CAH suffer from high false positive rates. The primary factors contributing to false positive determinations can include the natural increase of 17-OHP due to stress stimuli as well as cross-reactivity of the immunoassay antibody with other hormones and endogenous compounds in blood. Analysis of the adrenal steroid profile and corresponding analyte ratios using high performance liquid chromatography (HPLC)or ultra-high pressure liquid chromatography (UHPLC)combined with tandem mass spectrometry (MS/MS) has been shown to be a sensitive and selective technique for the significant reduction of the false positive reporting rate for CAH in newborn screening. In working toward optimization, validation, and implementation of an HPLC-MS/MS steroid profile for use by Virginia’s Newborn Screening laboratory as a 2nd tier analysis for CAH screening, a commercially-available core-shell HPLC column with a biphenyl stationary phase was determined to offer adequate retention and selectivity to achieve baseline resolution of isobaric target analytes under rapid reversed phase gradient conditions. Method linearity, precision, and accuracy were assessed using enriched dried blood spot materials. Double-blinded analyses of over 300 newborn dried blood spot specimens were used to determine clinical sensitivity and specificity of the assay, which is projected to substantially reduce the false positive reporting rate for CAH screening while meeting target sample turnaround times.

Aging is associated with a loss of proliferation of the insulin-secreting beta cell, a possible contributing factor to the greatly increased rate of type-2 diabetes in the elderly. A landmark study from our lab previously illustrated that mild endoplasmic reticulum (ER) stress drives beta cell proliferation specifically through ATF6α, one arm of the tripartite Unfolded Protein Response (UPR). It is unknown if old beta cells differ from young beta cells in UPR signaling or proliferative response to ER stress or ATF6α activation. To investigate, young and old mouse islets were cultured ex vivo in high glucose, and beta cell proliferation was quantified by BrdU incorporation after treatment with low dose thapsigargin or activation of overexpressed ATF6α. In addition, levels of UPR signaling were compared by semi-quantitative Xbp1 splicing assay. Interestingly, although old beta cells displayed reduced proliferation in glucose compared to young beta cells, their proliferative response to low-dose thapsigargin and ATF6α activation were nearly identical, and no difference was found in Xbp1 splicing under high glucose or high ER stress conditions. These results suggest that the aged mouse beta cell does not have impaired UPR-responsive proliferation or aberrant UPR signaling when cultured ex vivo

The increasing healthcare burden of type 1 diabetes (T1D) makes finding preventive or therapeutic strategies a global priority. This chronic disease is characterized by the autoimmune destruction of the insulin-producing β cells. This destruction leads to poorly controlled blood glucose and accompanying life threatening acute and chronic complications. The role of viral infections as initiating factors for T1D is probable, but contentious. Therefore, my goal is to better characterize the effects of viral infection on human β cells in their function of producing insulin and to define innate immune gene responses in β cells upon viral infection. These aspects were evaluated in various platforms including mice engrafted with primary human islets, cultured primary human islets, β cells derived from human stem cells, and a human β cell line. Furthermore, the contributions of cell-type specific innate immune responses are evaluated in flow cytometry-sorted primary human islet cells. Taken together, the results from these studies provide insights into the mechanisms of the loss of insulin production in β cells during virus infection, and characterize the antiviral innate immune responses that may contribute to the autoimmune destruction of these cells in T1D.

Background. Diabetes requires significant disease management, patient-provider communication, and interaction between patients, family members, caregivers, and care teams. Emerging patient-facing technologies, such as cellular-enabled glucose meters, can facilitate additional care support and improve diabetes self-management. This study evaluated patient acceptability, feasibility, and efficacy of a diabetes care support program facilitated by cellular-enabled glucose meters. Methods. A two-phase study approach was taken. Get In Touch – Phase 1 (GIT-1) was a 1-month pilot involving patients with type 1 and type 2 diabetes. Get In Touch – Phase 2 (GIT-2) was a 12-month randomized controlled crossover trial involving patients with poorly-controlled type 2 diabetes. Results from GIT-1 and preliminary results from GIT-2 are presented. Results. GIT-1 participants with type 1 (n=6) and type 2 (n=10) diabetes reported the intervention and cellular-enabled glucose meter were easy to use and useful while identifying potential areas of improvement. GIT-2 participants in both the intervention (n=60) and control (n=60) groups saw significant improvements in treatment satisfaction and A1c change, with intervention participants experiencing slightly greater improvements in each after 6 months (p=0.09 and p=0.16, respectively) compared to control participants. Conclusions. Patients reported favorable acceptability of the intervention. Preliminary results from a randomized trial demonstrated potential of intervention to improve patient-reported and physiological health outcomes. Future studies should evaluate feasibility and efficacy over a longer period of time, with a greater number of participants, and targeting different populations of patients with diabetes. Provider perspectives and changes in provider behavior, clinical work flow, and caregiver burden should also be assessed.

Pancreatic cancer is the 4th leading cause of cancer related death in the United States with a median survival time of less than 6 months. Pancreatic ductal adenocarcinoma (PDAC) accounts for greater than 85% of all pancreatic cancers, and is marked by early and frequent mutation of the KRAS oncogene, with activating KRAS mutations present in over 90% of PDAC. To date, though, targeting activated KRAS for cancer treatment has been very difficult, and targeted therapies are currently being sought for the downstream effectors of activated KRAS. Activation of KRAS stimulates multiple signaling pathways, including the MEK-ERK and PI3K-AKT signaling cascades, but the role of downstream effectors in pancreatic tumor initiation and progression remains unclear. I therefore used primary pancreatic ductal epithelial cells (PDECs), the putative cell of origin for PDAC, to determine the role of specific downstream signaling pathways in KRAS activated pancreatic tumor initiation. As one third of KRAS wild type PDACs harbor activating mutations in BRAF , and KRAS and BRAF mutations appear to be mutually exclusive, I also sought to determine the effect of activated BRAF (BRAF V600E ) expression on PDECs and the signaling requirements downstream of BRAF. I found that both KRAS G12D and BRAF V600E expressing PDECs displayed increased proliferation relative to GFP expressing controls, as well as increased PDEC survival after challenge with apoptotic stimuli. This survival was found to depend on both the MEK-ERK and PI3K-AKT signaling cascades. Surprisingly, I found that this survival is also dependent on the IGF1R, and that activation of PI3K/AKT signaling occurs downstream of MEK/ERK activation, and is dependent on signaling through the IGF1R. Consistent with this, I find increased IGF2 expression in KRAS G12D and BRAF V600E expressing PDECs, and show that ectopic expression of IGF2 rescues survival in PDECs with inhibited MEK, but not PI3K. Finally, I showed that the expression of KRAS G12D or BRAF V600E in PDECs lacking both the Ink4a/Arf and Trp53 tumor suppressors is sufficient for tumor formation following orthotopic transplant of PDECs, and that IGF1R knockdown impairs KRAS and BRAF-induced tumor formation in this model. In addition to these findings within PDECs, I demonstrate that KRAS G12D or BRAF V600E expressing tumor cell lines differ in MEK-ERK and PI3K-AKT signaling from PDECs. In contrast to KRAS G12D or BRAF V600E expressing PDECs, activation of AKT at serine 473 in the KRAS G12D or BRAF V600E expressing tumor cell lines does not lie downstream of MEK, and only the inhibition of PI3K alone or both MEK and the IGF1R simultaneously results in loss of tumor cell line survival. However, inhibition of MEK, PI3K, or the IGF1R in KRAS G12D or BRAF V600E expressing tumor cell lines also resulted in decreased proliferation relative to DMSO treated cells, demonstrating that all three signaling cascades remain important for tumor cell growth and are therefore viable options for pancreatic cancer therapeutics.