VTechWorks Repository :: Browsing by Author "Ahmed, S. Ansar"

Browsing by Author "Ahmed, S. Ansar"

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Adaptation of Three Different Apoptotic Methods in Equine Bronchoalveolar Cells and Comparison of Bronchoalveolar Lavage Cell Apoptosis in Normal and COPD Affected Horses Before and After Dexamethasone Administration
Leichner, Teri Lynn (Virginia Tech, 2001-06-21)
Recent studies suggest that lymphocyte apoptosis serves to regulate pulmonary inflammation. Equine COPD, an allergic disease of the lower airway, is likely due to dysregulation of the pulmonary immune response. In this study, the hypothesis tested was COPD affected horses would have less apoptotic airway lymphocytes than control horses during clinical disease. To achieve this, 3 methods of measuring apoptosis, Vindelov's propidium iodide with Triton-X (PI/Triton-X), 7-aminoactinomycin D (7-AAD), and Annexin V with propidium iodide (Annexin/PI) were evaluated in equine airway lymphocytes. A significant linear relationship was found for equine bronchoalveolar lavage (BAL) lymphocytes stained with 7-AAD and Annexin/PI . No relationship was identified with cells stained with PI/Triton-X and Annexin/PI, and 7-AAD and PI/Triton-X indicating that methods which preserve cell membrane characteristics are more comparable when measuring BAL lymphocytes apoptosis in a heterogeneous population of cells. Additionally, all stains appear to perform the same in COPD and normal horses in remission and disease. Comparison of predominately BAL lymphocyte apoptosis using the above methods were performed at baseline, after natural challenge, and after dexamethasone administration in nine horses, five of which were affected with COPD. No differences in bronchoalveolar lavage lymphocyte apoptosis between COPD and control horses were detected either before or after dexamethasone administration, although numerical trends in COPD horses identified less apoptosis after natural challenge indicating that defective apoptosis may play a role in equine COPD pathogenesis. Dexamethasone administration was associated with trends of improvement in the pulmonary gas exchange and increased apoptosis toward baseline in the COPD horses.
Altered Kinetics of Non-Homologous End Joining Mediated DNA Repair in Mouse Models of Aging and Leukemia
Puthiyaveetil Abdulkader, Abdul Gafoor (Virginia Tech, 2012-09-25)
DNA encodes the genetic instructions for the development and function of organisms and hence maintaining genomic integrity is essential for the propagation of life. However, DNA molecules are under constant threat of metabolic and environmental insults resulting in DNA damages including DNA double strand breaks (DSB), which are considered as a serious threat to cell survival. The majority of these DSB are repaired by Non-homologous end joining (NHEJ). Unrepaired DSB can lead to genomic instability resulting in cell cycle arrest, apoptosis, and mutations. Thus, delineating this DNA repair process is important in understanding the molecular mechanisms of aging and malignant progression. B lymphocytes undergo physiological DNA breaks and NHEJ-mediated DNA repair during their bone marrow differentiation and peripheral class switch recombination (CSR), thus lending them as a good model system in which to delineate the DNA repair mechanisms. To determine the effect of aging on NHEJ, B lymphocytes from old mice were analyzed. The results showed compromised DNA repair in cells from old mice compared to cells from adult mice. These results suggest that NHEJ is compromised during aging and might play critical roles in the aging process and age-associated conditions. To delineate the role of a CT in regulating the immune system, transgenic mice expressing NUP98-HOXD13 (NHD13) were analyzed for B lymphocyte differentiation, peripheral development, CSR, and antibody production. The results showed impaired B cell development and antibody production, which worsened with antigenic stimulation, suggesting the role of NHD13 in immune regulation. These studies explored the possibility of altered NHEJ-mediated DNA repair as a contributing reason for aging process and age-associated conditions. Also, the results from NHD13 study suggested that a primary CT can result in impaired NHEJ and regulate immune cell development and function. Furthermore, the results pointed to the possibility that a primary CT may lead to secondary mutations through altered NHEJ. Thus, these studies shed insight into the molecular mechanisms of altered NHEJ and may help in developing preventive or therapeutic strategies against accumulation of DNA damage, aging process and secondary mutations.
Amelioration of oxidative lung injury by antiarrhythmic agents
Das, Kumuda C. (Virginia Tech, 1992-10-15)
Class I antiarrhythmic drugs, such as lidocaine, quinidine and procainamide, are known to be effective membrane stabilizers. However, the mechanism of such "membrane stabilization" has not been elucidated. In the present study we found that all three drugs are powerful scavengers of hydroxyl! radical. In addition, lidocaine was found to be a quencher of singlet oxygen. These drugs are also found to inhibit NADPH-dependent lipid peroxidation in bovine lung microsomes in a dose dependent manner. Since oxyradicals are implicated in the lipid peroxidation process and antiarrhythmic drugs were found to scavenge/quench reactive oxygen species, we proposed that the membrane Stabilizing effects of antiarrhythmic drugs may, in part, be due to their antioxidant properties. Ischemia-reperfusion injury has been studied in many organs. Despite the evidence of functional, metabolic and structural abnormalities during reperfusion, the precise mechanism of reperfusion lung injury remains obscure. Data from the organ models suggest that toxic oxygen metabolites play an important role in the mechanism of reperfusion tissue injury. Lidocaine has also been shown to be clinically valuable for the treatment and prevention of ventricular arrhythmia occurring after surgical correction of myocardial infraction. We found that the class I antiarrhythmic drugs are effective in ameliorating post-ischemic lung reperfusion injury in an ex vivo perfused rat lung model exposed to both normoxic and hyperoxic conditions. Since phagocytes are known to generate reactive oxygen species and play an important role in causing irreversible oxidative tissue injury during reperfusion of organs, we examined the role of antiarrhythmic agents on macrophage function. We found that these drugs inhibit superoxide and hydrogen peroxide production in stimulated macrophages in a dose dependent manner. The diminished production of superoxide was found to be not due to the inactivation of superoxide generating NADPH-oxidase enzyme but by inhibition of the phagocytosis process by these drugs The results of these studies indicate that the antiarrhythmic drugs, such as, lidocaine, quinidine and procainamide, are effective antioxidants and can protect biomembranes against lipid peroxidation injury and post-ischemic reperfusion injury of the lung. We have investigated the mechanism(s) of action of these drugs in ameliorating oxidative tissue injury and found that these drugs are not only effective in removing reactive oxygen species but also cause inactivation of pulmonary macrophage from inappropriately generating reactive species of oxygen. The fundamental knowledge derived from these Studies could lead to enhanced functional improvement of patients following cardiopulmonary bypass, pulmonary arterial embolectomy and acute respiratory distress syndrome, all of which undergo a period of elective/induced ischemia and reperfusion or oxidative stress.
Brucella abortus RB51 vaccine: Testing its Spectrum of Protective and Curative Characteristics
Contreras Rojas, Andrea Paz (Virginia Tech, 2004-07-30)
Brucella abortus (BA) are gram-negative, facultative intracellular bacteria that cause abortions in cattle and debilitating illness in humans. The US is now virtually free of bovine brucellosis, but the disease is endemic in wildlife. The official brucellosis vaccine in the US is strain RB51 (RB51). It elicits protective cell-mediated immunity (CMI) against BA infections. Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis in ruminants. It is a slow growing intracellular parasite that requires CMI for its control, belongs to the genus Mycobacterium, and is closely related to M. avium avium (MA). Using RB51 as a vector that induces strong protective CMI may be useful to protect against MAP if it expresses MAP protective antigens. Therefore, MAP 85A and 35kDa proteins were expressed at low levels in RB51, and the immune responses elicited by these vaccines in BALB/c mice were evaluated. Strong anti-Brucella immunity was generated, but the anti-mycobacterial response was low. To evaluate protective efficacy, a BALB/c model using MA was developed. When mice were challenged with MA, protection was obtained in some experiments but was inconsistent. This may be due to the low expression of MAP antigens in RB51. Another objective was to evaluate the effect of an ongoing Brucella-infection on the efficacy of RB51 vaccination, and whether vaccination of already infected animals could have a curative effect. Mice acutely or chronically infected with virulent BA, rapidly cleared the RB51 vaccine organisms, but there was no significant decrease in the number of virulent BA. Brucella spp. have been developed as biological weapons, but there are no vaccines to protect humans. The development of a very attenuated protective vaccine is necessary to prevent human infections, as well as to protect wildlife. To generate such a vaccine, RB51 based vaccines were irradiated to render them non-replicative, but metabolically active. We demonstrated that in general, irradiated and non-irradiated RB51 vaccines remain protective at levels similar to those elicited by the live vaccines. Therefore, irradiation of strain RB51 is an effective means of attenuating the strain without affecting its protective characteristics, and could eventually be used as a vaccine for wildlife and humans.
Comparative Immunological Effects of a Natural Estrogen (17β-estradiol) versus a Pharmacologic Synthetic Estrogen (17α-ethinyl estradiol)
Brummer, Tyson Peter Thomas (Virginia Tech, 2007-07-31)
Exposure to exogenous estrogens such as synthetic 17α-ethinyl estradiol (EE) occurs via multiple sources (i.e. hormonal contraceptives, environmental contamination, hormone replacement therapy). The natural estrogen, 17β-estradiol (E2), is a well-studied immunomodulatory hormone at both environmental and pharmacologic levels. Conversely, little data exist regarding the immune effects of EE at either environmentally-relevant exposure levels or at pharmacological levels. Further, EE is delivered to patients in a clinical setting via different routes of exposure (e.g. subcutaneous or oral). Many key questions in relation to potential immunological effects of EE are unanswered. Important variables in estrogen-modulation of the immune system include: (i) dose, (ii) age, (iii) gender, and (iv) route of exposure. Thus, pertinent questions emerge. Does exposure to EE at low concentrations for a subacute duration affect the immune or reproductive systems? Are the effects similar in both hormones and between sexes? Are these effects similar in juvenile and aged mice? How do the effects compare across two common routes of exposure (subcutaneous versus oral)? To address these questions, three separate studies were performed. In the first study, we investigated whether very low, but environmentally relevant, doses of EE, E2 (10 ng/kg body weight), or vehicle orally administered every other day for 21 days to young (6 week-old) and aged (>15 month-old) C57BL/6 mice had immunomodulatory effects. As expected, significant gender and age-related differences were noted with regard to thymus weight, thymocyte recovery, spleen weight, and splenocyte recovery. However, low dose treatment of either E2 or EE had no marked effects on the thymus or spleen organ to body weight ratios, cell numbers, or lymphocyte subsets. Low dose oral estrogens did not alter the ability of activated splenocytes to induce interferon-γ or nitric oxide. No effects on male reproductive organ to BW ratios of young or aged mice were found. Similarly, with the exception of E2-stimulating effects on the female reproductive tract of young mice, there were no pronounced effects in females. In separate studies, intact juvenile female and male C57BL/6 mice were given daily subcutaneous (second study) or oral (third study) doses of either EE or E2 (0.04, 0.4, or 4.0 μg per 25 g BW) for 21 days. In the subcutaneous exposure study, both EE and E2 morphologically altered uterine and seminal vesicle weights. However, EE had a more pronounced effect compared to E2, especially in males, even at the lowest dose administered. Additionally, like E2, EE induced thymic atrophy in both sexes. In female mice, thymic atrophy and thymic cellularity were significantly decreased by subcutaneous EE and E2 at doses of 0.4 and 4.0 μg/25 g body weights. EE elicited significantly more pronounced thymic atrophy-inducing effects compared to E2 at the 4.0 μg/25g dose. In males, thymocyte cellularity was decreased by both subcutaneous EE and E2 only at the highest dose tested (4.0 μg/25 g body weight), whereas only 4.0 EE significantly decreased thymus to body weight ratios. Neither splenic weights, splenic cellularity, nor splenic cell phenotype were affected by either estrogenic compound regardless of route of exposure. Oral exposure of EE or E2 did not induce marked immunological effects. Collectively, these data demonstrate that select thymic and reproductive endpoints are significantly altered following a 21-day subcutaneous exposure to either EE or E2 and that the thymus is a more sensitive target than the spleen with regard to subacute exposure to EE. In addition, EE at a comparable dose was more potent than E2 at exerting thymic and reproductive organ morphological alterations. Furthermore, route of administration is critical, as subcutaneous exposure induced far more dramatic thymic and reproductive morphological alterations than did oral administration. Future studies need to address the precise mechanism through which EE induces thymic atrophy and diminished thymus cellularity. Are these effects mediated directly through the thymus, perhaps through estrogen-induced increased thymocyte apoptosis or alterations to thymic epithelial cells? Or could EE be mediating alterations via bone marrow stem cells targeted for distribution to the thymus? Our novel findings regarding EE-induced effects on the thymus are of health significance and set the stage for future work.
Cross-Species Infection and Characterization of Avian Hepatitis E Virus
Sun, Zhifeng (Virginia Tech, 2005-01-20)
As novel or variant strains of HEV continue to evolve rapidly both in humans and other animals, it is important to develop a rapid pre-sequencing screening method to select field isolates for further molecular characterization. Two heteroduplex mobility assays (HMA) were developed to genetically differentiate field strains of swine HEV and avian HEV from known reference strains. It was shown that the HMA profiles generally correlate well with nucleotide sequence identities and with phylogenetic clustering between field strains and the reference swine HEV or avian HEV strains. Therefore, by using different HEV isolates as references, the HMA developed in this study can be used as a pre-sequencing screening tool to identify variant HEV isolates for further molecular epidemiological studies. Our previous study showed that avian HEV antibody is prevalent in apparently healthy chickens. A prospective study was conducted on a known seropositive but healthy chicken farm. Avian HEV was identified from the healthy chicken flock. Avian HEV isolates recovered from the healthy chicken share 70-97% nucleotide sequence identities with those isolates which cause hepatitis-splenomegaly (HS) syndrome based on partial helicase and capsid gene regions. Recovery of identical viruses from the experimentally inoculated chickens in the subsequent transmission study further confirmed our field results. The capsid gene of avian HEV isolates from chickens with HS syndrome were also characterized and found to be heterogeneic, with 76-100% nucleotide sequence identities to each other. The study indicates that avian HEV is enzootic in chicken flocks and spread subclinically among chicken populations, and that the virus is heterogeneic. As HEV can not be propagated in vitro, in order to further characterize avian HEV, an infectious viral stock with a known infectious titer must be generated. Bile and feces collected from specific-pathogen-free (SPF) chickens experimentally infected with avian HEV were used to prepare an avian HEV infectious stock. The infectivity titer of this infectious stock was determined, by intravenously inoculating one-week old SPF chickens, to be 5 x 10^4.5 50% chicken infectious doses (CID₅₀) per ml. Seroconversion, viremia as well as fecal virus shedding were observed in the inoculated chickens. Contact control chickens also became infected via direct contact with inoculated ones. Avian HEV infection in chickens was found to be dose-dependent. To determine if avian HEV can infect across species, one-week old SPF turkeys were intravenously inoculated each with 10^4.5(CID₅₀) of avian HEV. The inoculated turkeys seroconverted to avian HEV antibodies at 4-8 weeks postinoculation (WPI). Viremia was detected at 2-6 WPI, and fecal virus shedding at 4-7 WPI in inoculated turkeys. This is the first demonstration of cross-species infection by avian HEV. Little is known regarding the characteristics of the small ORF3 protein largely due to the lack of a cell culture system for HEV. To characterize the small protein, the ORF3 proteins of avian HEV and swine HEV were expressed in Escherchia coli, and purified by BugBuster His-Bind Purification System. Western blot analysis showed that avian HEV ORF3 protein is unique and does not share common antigenic epitopes with those of swine HEV and human HEV. However, swine HEV (genotype 3) and human HEV (genotype 1) ORF3 proteins cross-react with each other antigenically. To determine if the ORF3 protein is a virion protein, infectious stocks of avian HEV and swine HEV were first generated in SPF chickens and pigs, respectively. Virions were subsequently purified by sucrose density gradient centrifugation and virion proteins were characterized by SDS-PAGE and Western blot analysis. Two major forms of ORF2 proteins of avian HEV were identified: a 56 kDa and an 80 kDa proteins. Multiple immunoreactive forms of ORF2 proteins of swine HEV were also observed: 40 kDa, 53 kDa, 56 kDa and 72 kDa. However, the ORF3 protein was not detected from the native virions of avian HEV or swine HEV. These findings provide direct evidence that ORF2 indeed encodes a structural protein of HEV, whereas ORF3 does not. To search for other potential animal reservoirs for HEV, the prevalence of IgG anti-HEV antibody was determined in field mice caught in chicken farms to assess the possibility of mice as a potential reservoir for HEV infection in chickens. Three different recombinant HEV antigens derived from avian HEV, swine HEV, and human HEV were used in the ELISA assays. The anti-HEV seropositive rates in wild field mice (Mus musculus), depending upon the antigen used, are 15/76 (20%), 39/74 (53%), and 43/74 (58%), respectively. HEV RNA was also detected from 29 fecal and/or serum samples of mice. The HEV sequences recovered from field mice shared 72-100% nucleotide sequence identities with each other, 73-99% sequence identities with avian HEV isolates, and 51-60% sequence identities with representative strains of swine and human HEVs. However, attempts to experimentally infect laboratory mice (Mus musculus) with the PCR-positive fecal materials recovered from the wild field mice were unsuccessful. We also attempted to experimentally infect 10 Wistar rats each with avian HEV, swine HEV, and an US-2 strain of human HEV, respectively. However, the inoculated rats did not become infected as evidenced by the lack of viremia, virus shedding in feces or seroconversion. These data suggest that mice caught in chicken farms are infected by a HEV-like virus, but additional work is needed to determine the origin of the mouse virus as well as the potential role of rodents in HEV transmission. In summary, we developed two HMAs which are useful for differentiation and identification of variant strains of swine and avian HEVs. We genetically identified and characterized an avian HEV strain from apparently healthy chickens in seropositive flocks. We showed that avian HEV can cross species barriers and infect turkeys. Our data indicated that avian and swine HEV ORF2 genes encode structural proteins, whereas ORF3 genes do not. Evidence in this study also showed that HEV or HEV-like agent exists in field mice on a chicken farm.
Deletion of microRNA-183-96-182 Cluster in Lymphocytes Suppresses Anti-DsDNA Autoantibody Production and IgG Deposition in the Kidneys in C57BL/6-Fas(lpr/lpr) Mice
Wang, Zhuang; Heid, Bettina; Lu, Ran; Sachdeva, Mohit; Edwards, Michael R.; Ren, JingJing; Cecere, Thomas E.; Khan, Deena; Jeboda, Taschua; Kirsch, David G.; Reilly, Christopher M.; Dai, Rujuan; Ahmed, S. Ansar (Frontiers, 2022-07-07)
Dysregulated miRNAs have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Our previous study reported a substantial increase in three miRNAs located at the miR-183-96-182 cluster (miR-183C) in several autoimmune lupus-prone mice, including MRL/lpr and C57BL/6-lpr (B6/lpr). This study reports that in vitro inhibition of miR-182 alone or miR-183C by specific antagomirs in activated splenocytes from autoimmune-prone MRL/lpr and control MRL mice significantly reduced lupus-related inflammatory cytokines, interferon-gamma (IFN gamma), and IL-6 production. To further characterize the role of miR-182 and miR-183C cluster in vivo in lupus-like disease and lymphocyte phenotypes, we used hCD2-iCre to generate B6/lpr mice with conditional deletion of miR-182 or miR-183C in CD2(+) lymphocytes (miR-182(-/-)B6/lpr and miR-183C(-/-)B6/lpr). The miR-182(-/-)B6/lpr and miR-183C(-/-)B6/lpr mice had significantly reduced deposition of IgG immunocomplexes in the kidney when compared to their respective littermate controls, although there appeared to be no remarkable changes in renal pathology. Importantly, we observed a significant reduction of serum anti-dsDNA autoantibodies in miR-183C(-/-)B6/lpr mice after reaching 24 weeks-of age compared to age-matched miR-183C(fl/fl)B6/lpr controls. In vitro activated splenocytes from miR-182(-/-)B6/lpr mice and miR-183C(-/-)B6/lpr mice showed reduced ability to produce lupus-associated IFN gamma. Forkhead box O1(Foxo1), a previously validated miR-183C miRNAs target, was increased in the splenic CD4(+) cells of miR-182(-/-)B6/lpr and miR-183C(-/-)B6/lpr mice. Furthermore, in vitro inhibition of Foxo1 with siRNA in splenocytes from miR-182(-/-)B6/lpr and miR-183C(-/-)B6/lpr mice significantly increased IFN gamma expression following anti-CD3/CD28 stimulation, suggesting that miR-182 and miR-183C miRNAs regulate the inflammatory IFN gamma in splenocytes via targeting Foxo1. The deletion of either miR-182 alone or the whole miR-183C cluster, however, had no marked effect on the composition of T and B cell subsets in the spleens of B6/lpr mice. There were similar percentages of CD4(+), CD8(+), CD19(+), as well as Tregs, follicular helper T (T-FH), germinal center B (GCB), and plasma cells in the miR-183C(-/-)B6/lpr and miR-182(-/-)B6/lpr mice and their respective littermate controls, miR-183C(fl/fl)B6/lpr and miR-182(fl/fl)B6/lpr mice. Together, our data demonstrate a role of miR-183C in the regulation of anti-dsDNA autoantibody production in vivo in B6/lpr mice and the induction of IFN gamma in in vitro activated splenocytes from B6/lpr mice.
Development of an antigen-specific ELISPOT to detect intestinal antibody responses to the swine whipworm, Trichuris suis
Kellman, Maxine Franchestcê (Virginia Tech, 1997-06-05)
The swine whipworm, Trichuris suis, is a parasite present throughout the United States and is of concern to the swine industry worldwide because it is very pathogenic to growing pigs. The economic threat posed by T. suis and other intestinal parasite infections has created a strong interest in the development of parasite vaccines for the swine industry. Use of a vaccine either alone or with anthelmintics should reduce the economic losses. However, before effective parasite vaccines can be created, the swine gastrointestinal immune response to parasite antigens must be understood. In this study, an enzyme-linked immunospot (ELISPOT) assay was developed to measure total and antigen-specific IgG and IgA antibody secreting cells (ASC) from gut-associated lymphoid tissues (GALT) [mesenteric lymph node explants from jejunal region of small intestine (SI-MLN) and cecum in large intestine (C-MLN); and ileocecal Peyer's patches (IC-PP)] and lamina propria from the proximal colon removed from T. suis infected pigs. Tbe local antibody responses were compared to peripheral antibody responses found in the spleen and submandibular lymph nodes. The hypotheses to be tested was that parasite antigen-specific antibody secreting cells would be greatest in lymphoid tissue draining the site of infection compared to peripheral lymphoid tissues and that 19A ASC would predominate over IgG ASC in the lamina propria of T. suis infected pigs. The total IgG and IgA ASC frequencies for the spleen, SI-MLN, and ICPP did not significantly change (P> 0.05) over time. For C-MLN, there was a significant increase (p< 0.05) of total IgG ASC during a primary infection with T. suis. Antigen-specific IgG ASC were greatest at the GALT site closest to the infection, CMLN, whereas, antigen-specific IgA ASC predominated in the proximal colonic: lamina propria. Host protection to T. suis develops after anthelmintic: treatment of a primary exposure to parasite. The ELISPOT assay provided valuable information on the localization and compartmentalization of the swine gastrointestinal immune response to T. suis which resides in the cecum and proximal colon. In the future, this technique may be useful for monitoring gastrointestinal immune parameters of pigs exposed to a T. sllis vaccine.
Developmental Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin: Induced and Exacerbated Autoimmunity in Adulthood
Mustafa, Amjad Issa (Virginia Tech, 2008-12-18)
Developmental 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure can permanently alter immune system ontogeny, resulting in the dysregulation of a number of vital immune pathways. We hypothesized that developmental exposure to TCDD may also impair the establishment of self-tolerance, resulting in an increased risk of autoimmunity. For example, we observed that a single prenatal TCDD exposure given to non-autoimmune-prone high affinity aryl hydrocarbon receptor (AhR) C57BL/6 mice resulted in an immune complex-mediated autoimmune disease during the adult stage. Further using a similar TCDD exposure protocol, autoimmune-prone low affinity AhR SNF1 mice exhibited acceleration and exacerbation of lupus-like nephritis in adulthood. Examination of these mice showed that perinatal TCDD exposure adversely affected both primary immune organs of the adaptive immune system. In the thymic compartment, prenatal TCDD affected thymocyte cellularity, differentiation and maturation as well as central tolerance as indicated by high levels of autoreactive VÎ² TCR T cells in the periphery. Prenatal TCDD also altered bone marrow B lymphopoiesis and B cell maturation and differentiation in the spleen. Functionally, these B cell changes resulted in high serum autoantibodies titers to dsDNA, ssDNA and cardiolipin suggesting a loss in central B cell tolerance. The functional assessment of T cells, via cytokine production showed that prenatal TCDD mice altered Th1/Th2 levels. As a result, significant changes were detected in the kidney characterized by increased immune complex deposition in the glomeruli, lymphocytic infiltration and general pathologic changes. This would suggest that multiple immune pathways are affected by prenatal TCDD and work either independently or synergistically to display immune-mediated disease during aging. Importantly, this study has also shown that the sex of an individual appears to influence both the type of immune pathways affected by TCDD as well as the progression and severity of the autoimmunity. In summary, these studies clearly demonstrate that postnatal immune system impairment due to prenatal TCDD exposure is not limited to immunosuppression but also can include inappropriate immune activation manifested as a hypersensitivity that can lead to the onset of autoimmune disease.
Dynamic Programming of Innate Immunity in Health and Disease
Yuan, Ruoxi (Virginia Tech, 2016-11-02)
Whether innate immune cells may be adapted into potential memory states has becoming an important question in the field of immunity. Although previous conceptual paradigm failed to acknowledge this important question, emerging clinical and basic observations have started to shed intriguing clues to shake the previous dogma regarding innate immunity of being "simple", "raw", "first-line defense with no memory". We have aimed to further address this fundamental issue in this dissertation work, under the close guidance of Dr. Liwu Li. We have chosen to use the model system of Toll-Like-Receptor (TLR) signaling networks within primary monocytes. TLRs play fundamental roles in sensing pathogen-associated molecular patterns (PAMPs) and modulation of innate immunity. Lipopolysaccharide (LPS), an endotoxin found on the cell membrane of gram-negative bacteria, is the ligand of TLR4 and induces a range of inflammatory as well as anti-inflammatory responses. Higher dosages of LPS were known to cause robust yet transient expression of pro-inflammatory mediators. On the other hand, the effects of super-low dose LPS, commonly manifested in humans with adverse health conditions, have been largely ignored in the basic research field. Super-low dose LPS may skew host immune environment into a mild non-resolving pro-inflammatory state, which is a risk factor for inflammatory diseases such as atherosclerosis, compromised wound healing, and elevated risks for sepsis. Our central hypothesize is that monocytes may be adapted by super-low dose LPS into a non-resolving low-grade inflammatory state conducive for the pathogenesis of inflammatory diseases. We have employed both in vitro cell culture system as well as in vivo disease models to test this hypothesis. For the in vitro system, we have cultured primary murine monocytes with increasing signal strength of LPS. Monocyte phenotypes such as the expression of key inflammatory mediators including cytokines, chemokines, and cellular surface markers were studied. Potential molecular and cellular mechanisms were examined. We revealed a novel low-grade inflammatory monocyte phenotype termed ML adapted by super-low dose LPS, mediated through IRF5. For the in vivo system, we have employed both acute and chronic models of inflammation. For the chronic model, we have tested the effects of super-low dose LPS on monocyte polarization in vivo, as well as its contribution to the pathogenesis of atherosclerosis. Furthermore, we have tested the effects of programmed monocytes on wound healing. For the acute model, we have tested the effects of pre-conditioning with super-low dose LPS on the subsequence risks of sepsis elicited by cecal ligation and puncture. We have demonstrated aggravated atherosclerosis, compromised wound healing, and increased sepsis mortality in mice pre-conditioned with super-low dose LPS. Taken together, our findings reveal that monocytes can be differentially programmed into distinct states, depending on the signal strength of LPS. The differential programming and adaptation of monocytes can occur both in vitro and in vivo, and may bear profound pathological consequences.
The Effect of Social Stress and Vitamin C on Immunity and Response to Hemorrhagic Enteritis Virus in Turkeys
Meade, Sharonda Madrica (Virginia Tech, 2004-12-07)
Hemorrhagic Enteritis (HE) vaccine is perhaps the most commonly used vaccine in the turkey industry. Although it provides protection against clinical disease, the vaccine is still thought to produce transient immunosuppression. In the field, HE still remains a significant concern for turkey producers. Research conducted over the years has shown that management stressors such as movement of turkeys from brooding to finishing environments and the timing of these stressors may influence the short-term response to vaccination. Strategic stress application may be of benefit in the optimization of protective responses and the development of vaccination protocols without detrimental effects on performance. Ascorbic acid may also have important implications on social stress and may play a role in immunity and response to HE vaccination in turkeys. Trials were conducted to examine the interrelationship among social stress, nutrition (vitamin C), immunity and their influence on response to hemorrhagic enteritis virus (HEV) vaccination. Stress is unavoidable, however if it is managed properly, it can be beneficial. In this dissertation, it was first demonstrated that stress in the form of social disruption can have negative physiological and immunological effects on turkey poults and that these effects can be alleviated with the addition of 300mg/kg vitamin C to the diet. Secondly, it was also demonstrated that when stress is applied on the day of vaccination, response to HEV vaccination can be improved. Thirdly, vitamin C supplementation at 300mg/kg can improve responses to HEV vaccination. However, it was concluded that vitamin C supplementation during periods of simultaneous stress application and vaccination does not provide benefit to response to vaccination.
Effects of Long-Term Exposure of Normal C57BL/6J Inbred Mice to 17β-Estradiol on Gene Expression in Lymphocytes: mRNA Analysis of Lymphokines and bcl-2/fas
Yin, Zhi-Jun (Virginia Tech, 1997-08-05)
It is now clear that human and animal exposure to estrogenic compound occurs through several sources. This include: i) naturally occurring endogenous estrogens, ii) exogenous or intentional estrogens for prophylactic (e.g. oral contraceptive) and therapeutic (e.g. as replacement therapy for ovulation in nulliparous women and in menopausal women, and in some men suffering from prostate cancer) purposes, iii) accidental via estrogenic chemical exposure (e.g. pesticides, industrial byproducts) and phytoestrogens (e.g. soybeans). It has long been recognized that estrogen, a female sex hormone, functions not only on the reproductive system, but also on various other systems including the immune system. Estrogens are thought to be of both physiologic and pathologic importance. Female in general, have better immune capabilities than males, a phenomenon attributed to the action of sex hormones on the immune system. There is also a female-gender bias in susceptibility to autoimmune diseases. Estrogens have been linked either directly or indirectly to the etiology and pathogenesis of various female-predominant autoimmune diseases. Estrogens have also been linked to the onset of cancer, and conditions where the immune system often malfunctions. Estrogen affects the functions of both B and T cells, possibly by regulating such factors as lymphokine gene expression and/or cellular death by apoptosis. However, the functioning of both B and T cells under the influence of long-term exposure to estrogen has not been fully understood. The primary aim of this thesis was to investigate the effect of long-term exposure to 17β-estradiol on lymphokine and bcl-2/fas (proto-oncogenes) mRNA expression. We evaluated the effects of estrogen on the expression of genes for lymphokines, which are essential for the immune response. It is hypothesized that estrogen may regulate the immune system by modifying the expression of lymphokine genes and/or genes that regulate apoptosis. The results demonstrated that long-term 17β-estradiol exposure reduced the viability of lymphocytes when compared to lymphocytes from placebo-treated mice. IL-2 and IFN-g mRNA was consistently higher in ConA-stimulated lymphocytes from estrogen-treated mice (P < 0.05). The mRNA for TGF-β₁ lymphokine was also increased but was not consistent at all time points of incubation. The expression of IL-4 mRNA was not noticeably affected by estrogen treatment of mice. Long-term exposure to 17β-estradiol appear to have some influence on the mRNA expression of proto-oncogenes fas and bcl-2 in splenic and thymic T lymphocytes. There was a trend of increased bcl-2 mRNA expression in estrogen-treated mice compared to placebo-treated mice, whereas the mRNA expression of fas gene appeared to be lower compared to controls. Overall, these findings suggest that 17β-estradiol may selectively influence lymphokine and proto-oncogene mRNA expression. These results suggest that the one mode of modulation of the immune response by 17β-estradiol may be through alterations in the lymphokine and proto-oncogene expression. Since estrogen-treatment markedly induces atrophy of the thymus and diminishes the cellularity of the lymphoid organs (e.g. Spleen), it became necessary to perform multiple assays on the same cells, particularly lymphokine and apoptosis gene expression. A secondary objective of this thesis was to investigate whether lymphocytes, which have undergone proliferation in Lympho-Pro™ assay (Alamar Blue assay), could be utilized for further analysis. In this regard, we found that a non-radioactive assay that utilizes Alamar Blue had significant advantages over the conventional ³H-thymidine incorporation assay. By using cells from estrogen and placebo-treated mice in the Alamar Blue assay, we found that this assay not only allowed determination of lymphocyte proliferation, but also the assessment of mRNA expression, cytogenetics, apoptosis and immunophenotyping of the same lymphocytes.
Effects of Short-Term Exposure to Octylphenol and Genistein on the Immune System of C57BL/6 and (NZBxNZW)F1 Mice
Becker, Kelcey Manae (Virginia Tech, 1999-08-30)
Octylphenol and genistein are two of the growing list of endocrine disrupting chemicals found in the environment that mimic estrogen in reproductive tissue both in vitro and in vivo. It is well established that endogenous estrogens modulate not only the reproductive system, but also the immune system. However, the effects of many endocrine disrupting chemicals, such as octylphenol and genistein, on the immune system have yet to be determined. Preliminary studies on short-term treatment with genistein (0.6 mg) and octylphenol (10 mg) showed that the thymus of orchiectomized (NZBxNZW)F1 males is sensitive to these agents. Further studies focused on the effects of short-term treatment of octylphenol on the morphology and function of the thymus in adult, reproductively intact non-autoimmune C57BL/6 and pre-autoimmune (NZBxNZW)F1 males. Oral dosing of 0.1 mg, 1 mg, or 10 mg of octylphenol 3 times a week for 3 weeks did not affect the morphology or function of the thymus as assessed by its weight, thymocyte cellularity, proportion of immature and mature thymocytes, level of apoptosis, apoptotic rates of stimulated thymocytes, and proportion of mature T cells in the spleen. Furthermore, oral dosing of 0.1 mg, 1 mg, or 10 mg of octylphenol did not result in estrogenic changes in the reproductive tract in our model. Subcutaneous injection of 10 mg of octylphenol resulted in skin lesions that confounded the assessment of its affects on the thymus. Further studies are needed to definitively determine the effects of octylphenol on the immune system of both males and females of various ages and to determine the effect of long-term exposure.
EGR2 Deletion Suppresses Anti-DsDNA Autoantibody and IL-17 Production in Autoimmune-Prone B6/lpr Mice: A Differential Immune Regulatory Role of EGR2 in B6/lpr Versus Normal B6 Mice
Dai, Rujuan; Wang, Zhuang; Heid, Bettina; Eden, Kristin; Reilly, Christopher M.; Ahmed, S. Ansar (Frontiers, 2022-06-15)
Previous studies have reported that deletion of the transcription factor, early growth response protein 2 (EGR2), in normal C57BL/6 (B6) resulted in the development of lupus-like autoimmune disease. However, increased EGR2 expression has been noted in human and murine lupus, which challenges the notion of the autoimmune suppressive role of EGR2 in B6 mice. In this study, we derived both conditional EGR2-/-B6/lpr and EGR2-/-B6 mice to elucidate the immune and autoimmune regulatory roles of EGR2 in autoinflammation (B6/lpr) versus physiologically normal (B6) conditions. We found that conditional EGR2 deletion increased spleen weight, enhanced T cell activation and IFNγ production, and promoted germinal center B cells and LAG3+ regulatory T cells development in both B6/lpr and B6 mice. Nevertheless, EGR2 deletion also showed strikingly differential effects in these two strains on T lymphocyte subsets profile, Foxp3+ Tregs and plasma cell differentiation, anti-dsDNA autoantibodies and immunoglobulins production, and on the induction of IL-17 in in vitro activated splenocytes. Specifically, EGR2 deletion in B6/lpr mice significantly decreased serum levels of anti-dsDNA autoantibodies, total IgG, IgM, IgG1, and IgG2a with reduced plasma cells differentiation. Furthermore, EGR2 deletion in B6/lpr mice had no obvious effect on IgG immunocomplex deposition, medium caliber vessel, and glomeruli inflammation but increased complement C3 immunocomplex deposition and large caliber vessel inflammation in the kidneys. Importantly, we demonstrated that EGR2 deletion in B6/lpr mice significantly reduced pathogenic CD4-CD8-CD3+B220+ double negative T cells, which correlated with the reduced anti-dsDNA autoantibodies in serum and decreased IL-17 production in splenocytes of EGR2-/-B6/lpr mice. Together, our data strongly suggest that the role of EGR2 is complex. The immunoregulatory role of EGR2 varies at normal or autoinflammation conditions and should not be generalized in differential experimental settings.
Endotoxin-induced microRNA expression in equine peripheral blood mononuclear cells
Parkinson, Nicholas J. (Virginia Tech, 2016-07-22)
The innate immune response to lipopolysaccharide (LPS) mediated by toll-like receptor 4 (TLR4) contributes substantially to the morbidity of equine gastrointestinal disease, neonatal sepsis and other diseases. MicroRNAs (miRNAs), small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in TLR4 signaling regulation in other species. The central hypothesis of this study was that LPS induces differential expression of miRNAs in equine peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy adult horses and cultured with LPS or medium only for 2, 4 and 8 hours. Concentrations of inflammatory cytokines were measured in supernatants by immunoassay. Illumina Next-Generation Sequencing of the miRNA transcriptome was performed in PBMCs at 0, 2 and 4 hours. Selected expression changes were verified by qRT-PCR. 327 mature miRNAs were detected in equine PBMCs. Only miR-155 was significantly upregulated by LPS. 9 miRNAs showed statistically significant expression changes with time. Tumor necrosis factor-α concentration was significantly higher in supernatants from LPS-treated cells than controls from 2 hours, while interleukin-10 and interferon-γ were increased at 8 hours. miR-155 expression was correlated to all three cytokines. These data provide a foundation for future research into miRNA involvement in equine inflammatory responses. miR-155 is the principal LPS-induced miRNA in horses. Bioinformatic target predictions support roles in regulation of innate and adaptive immune responses including TLR4 signaling, as in humans. It is thus likely to influence the acute inflammatory response to LPS. Further research will be necessary to establish its role in naturally occurring disease.
Equine Protozoal Myeloencephalitis. Preliminary Investigation of Protozoan-Host interactions in the horse
Goehring, Lutz Steffen (Virginia Tech, 1998-08-14)
Equine Protozoal Myeloencephalitis is the most frequently diagnosed neurologic disorder of horses in the united states, which is caused by the protozoan organism Sarcocystis neurona. The disease has a profound impact on the American Horse Industry. This impact includes prolonged and expensive treatment without a guaranteed return to a previous level of use for the individual horse. Poor respponse to and prolonged duration of treatment may suggest an immune mediated impariement of host response. There is limited information about the direct interaction between the pathogen and the host. In two in vitro experiments we investigated a) whether the presence of the protozoan organism can influence mitogen-stimulated peripheral blood mononuclear cells (PBMCs), suggesting a direct influence of the protozoan organism on cells of the immune system, and b) if cerebrospinal fluid (CSF) from horses with EPM has an effect on mitogen-stimulated PBMCs, suggesting that the microenvironment of the site of infection influences the course of disease. Experiment 1: Mitogen simulated PBMCs from EPM affected and control horses were co-cultured with fragments of freeze thawed bovine turbinate cells that were infected with S. neurona merozoites. Compared to controls PBMCs co-cultured with S. neurona fragments were the only cells that showed a decreased proliferation (p<0.05). A difference between EPM affected and control horses could not be detected (p>0.05). These results may imply that the persistence of S. neurona infection in the horses CNS is, in part, due to a pathogen-derived mechanism that attentuates the hosts immune response. Experiment 2: Mitogen stimulated PBMCs from a horse affected with EPM and a control were co-cultured n the presence of CSF from EPM affected and uninfected controls. Prior to co-culture the CSF was fractionated by a filtration process over two microfilter units. An identical volume of NaCl (0.9%) served as a control for the volume of CSF that was added. The proliferation assay revealed a deviation of the response depending on cell donor and CSF fraction used. The effect was independant of the protein concentration of the CSF fraction, and a decrease in lymphocyte proliferation was not caused by increased cellular death. This suggests the presence of subsets within the CSF which have a stimulatory of suppressive influence on the cells in culture. The effect was cell donor dependant which implies a difference in lymphocyte subsets between the two horses that were used.
Estrogen Regulates Interferon-gamma (IFN-g) and IFN-g-Inducible iNOS Gene Expression: Implications to Immunity and Autoimmunity
Sahin, Ebru Karpuzoglu (Virginia Tech, 2005-04-07)
It is now clear that estrogen not only modulates the differentiation and function of reproductive systems, but it also profoundly regulates the immune system of normal and autoimmune individuals. An important mechanism by which estrogen regulates the immune system is by altering the secretion and/or response to cytokines. We hypothesized that estrogen may alter the levels and/or response to IFN-g, a prototype Th1 cytokine, that plays a pivotal role in immunity against intracellular infections and in many autoimmune and inflammatory disorders. We found that estrogen treatment tended to upregulate the secretion of IFN-g protein and mRNA expression from Concanavalin-A (Con-A)-activated splenic lymphocytes. Impressively, we found that splenocytes from estrogen-treated mice when activated with Con-A also resulted in increased release of nitric oxide compared to placebo-treated mice. Furthermore, Con-A-activated splenocytes from estrogen-treated mice also had upregulated iNOS mRNA, iNOS protein, and nitric oxide-regulated COX-2 protein when compared to control mice. Blocking co-stimulatory signals mediated through interactions of CD28 and B7 molecules by using CTLA-4Ig markedly decreased not only IFN-g, but also nitric oxide, thereby implying an important role for CD28/B7 interactions in IFN-g/nitric oxide. Estrogen-induced upregulation of iNOS/nitric oxide is mediated through IFN-g since: (i) Estrogen alone did not upregulate iNOS/nitric oxide in IFN-g knockout mice; (ii) addition of rIFN-g to activated splenocytes from estrogen-treated mice further upregulated nitric oxide levels. We next investigated whether estrogen also upregulated IFN-g-inducing cytokines and select IFN-g-inducing transcription factors. Estrogen treatment resulted in increased mRNA and/or protein expression of IFN-g inducing cytokines and their receptors, including: IL-18, IL-15, IL-27, IL-12Rb2, and IL-18Rb. We also found that T-bet, a critical Th1 transcription factor, and STAT-4 phosphorylation, a key molecule in IL-12 signaling were both increased, while IRF-4, an important player in Th2 differentiation, was diminished in Con-A-activated splenocytes from mice treated with estrogen. Altogether, these studies are the first to demonstrate that estrogen regulates IFN-g-dependent iNOS and describes the potential mechanisms of how estrogen alters IFN-g-inducible genes, IFN-g inducing cytokines, and transcription factors in normal C57BL/6 mice. These studies may have profound implications to many autoimmune and inflammatory disorders, where estrogen is known to regulate the course of these diseases. Since estrogen may promote inflammatory disorders by upregulating pro-inflammatory biomolecules including IFN-g, nitric oxide, and COX-2, these studies may help in the design of therapeutic agents that regulate or block secretion and/or response to these inflammatory molecules.
The Humoral Immune Response of Elks (Cervus elaphus nelsoni) and Mice to Vaccination with Brucella abortus Strain RB51
Colby, Lesley A. (Virginia Tech, 1997-02-04)
Vaccine Brucella abortus strain RB51, unlike the wild strain 2308 and another vaccine strain (strain 19) does not induce anti-O-chain antibodies. An efficacious vaccine strain that fails to produce an O-chain and thus a lack of an anti-O-chain humoral response greatly simplifies identification of vaccinated versus field strain infected animals. The three primary objectives of this research were the following: 1) to develop a serological assay to detect anti-RB51 antibodies in vaccinated elk (Cervus elaphus nelsoni), 2) to identify potential antigenic alterations in RB51 after vaccination of elk and BALB/c mice, and 3) to confirm the general stability of RB51. Elk were divided into four groups based upon gender and the route of inoculation (subcutaneous or ballistic) of RB51 bacteria. This study developed a highly reliable ELISA (using a monoclonal anti-bovine IgG 1 antibody and acetone killed whole RB51 bacteria) which can identify RB51-vaccinated elk. Also, isolates recovered from RB51-vaccinated elk were inoculated into female BALB/c mice whose spleens were then cultured. All elk and mice isolates were bacteriologically, biochemically, and serologically evaluated. This study showed that RB51 is a highly stable strain, which does not revert to smooth morphology or initiate synthesis of LPS-O-chain, maintains it biochemical characteristics, does not undergo detectable antigenic variations, and remains attenuated even after successive passages in elk and mice. Overall, this research indicates that RB51 is a vaccine candidate for the prevention of brucellosis in elk. Further studies are needed to determine the protective capabilities of RB51 in elk.
Immunomodulatory Effects of Diethylstilbestrol During Prenatal and Adult Life
Fenaux, Jillian Beth (Virginia Tech, 2003-02-28)
For nearly forty years diethylstilbestrol (DES) was administered to pregnant women to maintain healthy pregnancies. During this time, it is estimated that several million men and women have been exposed to DES during sometime of their life. The most common period of exposure was during fetal development. Although rarely used for the maintenance of pregnancy now, its current medical use is restricted to certain clinical situations such as breast and prostate cancer therapies in adults. Thus, DES exposure spans the entire lifetime, from prenatal to geriatric age. Since the early 1950s, health risks were beginning to be associated with prenatal DES treatment. So far only reproductive problems such as infertility, neoplastic diseases of the cervix and vagina and testicular cancers have been well-documented in DES cases. Immunological abnormalities associated with DES are only now beginning to be recognized. Self-reported cases and questionnaire-based studies have revealed increased incidence of infections and autoimmune diseases in DES exposed people. Animal studies that have examined the immunological effects of DES treatment are largely restricted to one gender, or to one dose of DES or to the developmental period. This is an important issue since human exposure to DES occurred in both men and women, at all ages and, at a wide-range of doses. The purpose of these studies was to investigate the immunological consequences resulting from the exposure to DES. Since sensitivity can vary between genders, dose and at the time of exposure, it is critical to investigate the DES-induced immunological changes during all stages of life in both genders. To address these critical gaps in the literature, we examined the immunomodulatory effects of adult and prenatal exposure to DES in males and females. Our findings show that DES effects were evident in both the thymus and spleen. DES markedly affected the apoptosis of thymocytes and the ability of splenic lymphocytes to proliferate in response to stimulants and secrete vital cytokines such as interferon-gamma. Our notable findings were that in-utero exposure to DES resulted in profound alterations in lymphocyte functionality, which were noticed as late as one-year of age. This suggests that alterations to the in utero environment can have deleterious consequences that may be long lasting. These studies have profound implications to the humans and animals exposed to DES, and indirectly to a whole range of other estrogenic compounds.
Immunoteratological Studies of Diabetic Embryopathy Using Gene Expression Analysis
Punareewattana, Korawuth (Virginia Tech, 2003-04-18)
Diabetic embryopathy is a major complication of pregnant women with type I diabetes. Immune defects in the pathogenesis of diabetic embryopathy have been suggested. We hypothesized that activated immune system can counteract diabetic effect and result in prevention of diabetic embryopathy. Diabetes was induced in pregnant ICR mice by streptozocin injection. Three different techniques of maternal immune stimulation, complete Freund's adjuvant (CFA), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interferon-gamma (IFN-g), were used to stimulate the maternal immune system. Approximately 50% of fetuses from hyperglycemic (>27 mM/L) dams were malformed, with neural tube defects predominating. Maternal immune stimulation during the time of normoglycemia, i.e. prior to onset of hyperglycemia, was necessary for reducing teratogenic effects associated with hyperglycemia. The immune-stimulated diabetic mice then produced significantly lower numbers of malformed fetuses: CFA 20.9%, GM-CSF 23.3%, IFN-g 13.9%. A gene microarray was then used to examine a selected panel of placental and splenic genes. We hypothesized that a shared profile of placental or splenic gene expression changes may correlate to the reduced birth defect outcome induced by the different immune stimulation procedures. Diabetes did not cause significant changes in placenta or spleen gene expression profile. In placenta, CFA and GM-CSF changed placental gene expression relative to control or diabetes, but differentially affected such genes relative to each other; further, IFN-g did not affect gene expression relative to control or diabetes. Thus no common pattern of improved placental cytokine, cell-cycle, apoptotic, transcription factor, or other gene expression was identified in the immune-stimulated mice. In spleen, all 3 immune activators produced a common altered gene expression profile. The overall gene expression profile after all immune stimulation procedures suggested increased splenocyte activity and cytokine production. The cytokine GM-CSF, in particular, was up-regulated in splenic leukocytes. This cytokine has previously been associated with reduced cleft palate in urethane-exposed mice after immune stimulation, and with reduced limb malformations in cyclophosphamide-treated mice after intra-uterine administration. In contrast, the TGF-beta3 gene was down-regulated in immune-stimulated diabetic mice. This gene was up-regulated in urethane-exposed mice, an effect that may be associated with reduced cleft palate. Thus unlike urethane, TGF-beta3 gene expression did not show a relationship with reduced diabetes-induced birth defects. Taken together, these data prove our hypotheses and suggest that mechanistically diverse forms of immune activation result in protection against diabetes-related teratogenesis, but only if given prior to onset of hyperglycemia. Such immune stimulation in mice may act through systemic immune organs, i.e. spleen, over-riding adverse effects of diabetes on development.

Browsing by Author "Ahmed, S. Ansar"

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