Passive transfer of IgG anti-GM1 antibodies impairs peripheral nerve repair.

Anti-GM1 antibodies are present in some patients with autoimmune neurological disorders. These antibodies are most frequently associated with acute immune neuropathy called Guillain-Barré syndrome (GBS). Some clinical studies associate the presence of these antibodies with poor recovery in GBS. The patients with incomplete recovery have failure of nerve repair, particularly axon regeneration. Our previous work indicates that monoclonal antibodies can inhibit axon regeneration by engaging cell surface gangliosides (Lehmann et al., 2007). We asked whether passive transfer of human anti-GM1 antibodies from patients with GBS modulate axon regeneration in an animal model. Human anti-GM1 antibodies were compared with other GM1 ligands, cholera toxin B subunit and a monoclonal anti-GM1 antibody. Our results show that patient derived anti-GM1 antibodies and cholera toxin beta subunit impair axon regeneration/repair after PNS injury in mice. Comparative studies indicated that the antibody/ligand-mediated inhibition of axon regeneration is dependent on antibody/ligand characteristics such as affinity-avidity and fine specificity. These data indicate that circulating immune effectors such as human autoantibodies, which are exogenous to the nervous system, can modulate axon regeneration/nerve repair in autoimmune neurological disorders such as GBS.

Suppression of peripheral blood mononuclear cell proliferation by a periodontopathic bacteria {\it Actinobacillus actinomycetemcomitans\/}

Actinobacillus actinomycetemcomitans (Aa) is a gram-negative coccobacillus implicated as a major pathogen in juvenile periodontitis. The immunosuppressive activity of a sonic extract (designated 100SN) derived from Aa was investigated. 100SN suppressed spontaneous proliferation as well as proliferative response to the mitogens, PHA and PWM, of human peripheral blood mononuclear cells (PBMC). 100SN-induced suppression of PHA-stimulated proliferation was heat-sensitive, inactivated by pronase and trypsin, dose-dependent and non-cytotoxic. There were no significant changes in the CD4$\sp+$ or CD8$\sp+$ subsets of PBMC after 7-day incubation with 100SN. There was a trend toward increased levels of the CD4$\sp+$CD45R$\sp{\rm hi}$CDw29$\sp{\rm lo}$ (naive cells, associated with suppressor-inducer activity) and CD4$\sp+$CDw29$\sp{\rm hi}$CD45R$\sp{\rm lo}$ (memory cells, associated with helper-inducer activity) subsets. The target of 100SN appeared to be the non-adherent cells and suppression by 100SN could not be reversed by indomethacin (IDM), the cyclo-oxygenase inhibitor of prostaglandin (PG) synthesis. The mechanism of 100SN-induced suppression was studied in terms of inhibition involving IL-2-regulated T cell proliferation and the results point to the possibility that suppression occurred subsequent to IL-2 receptor binding.^ The suppressive activity observed could occur through multiple mechanisms including cell-cell; contact or release of soluble factors. Supernatants derived from 7-day cultures of PBMC and 100SN (designated CSN-A) were able to suppress proliferative response of PBMC to PHA without affecting cell viability. Analysis of CSN-A showed that it contained PGE2 and soluble IL-2 receptors. Suppression by CSN-A could be partially overcome by either IDM or exogenous IL-2. Significant suppression was also maintained when both IDM and exogenous IL-2 were added at the same time. These findings suggest that PGE2 and soluble IL-2 receptors contribute to the suppression observed but other suppressive cytokine(s) may be involved. Collectively, the data indicate that a factor derived from oral bacteria associated with juvenile periodontitis have profound effects on cellular immune responses, and that these effects may be partially mediated by secondary factors produced by the host in response to the bacteria. ^

Lindane induced cellular dysfunction in MDCK cells: The role of the peripheral benzodiazepine receptor

The central nervous system GABAA/Benzodiazepine (GABAA/BZD) receptors are targets for many pharmaceutical agents and several classes of pesticides. Lindane is an organochlorine pesticide, although banned from production in the U.S. since 1977, still imported for use as an insecticide and pharmaceutically to control ectoparasites (ATSDR, 1994). Lindane functions as a GABA/BZD receptor antagonist within the central nervous system (CNS). Outside of the CNS, peripheral BZD receptors have been localized to the distal tubule of the kidney. Previous research in our laboratory has shown that incubation of renal cortical slices with lindane can produce an increase in kallikrein leakage, suggesting a distal tubular effect. In this study, Madin Darby Canine Kidney (MDCK) cells were used as an in vitro system to assess the toxicity of lindane. This purpose of this study was to determine if interactions between a renal distal tubular BZD-like receptor and lindane could lead to perturbations in renal distal cellular chloride (Cl−) transport and mitochondrial dysfunction and ultimately, cellular death. ^ Pertubations in renal chloride transport were measured indirectly by determining if lindane altered cell function responsiveness following osmotic stress. MDCK cells pre-treated with lindane and then subjected to osmotic stress remained swollen for up to 12 hours post-stress. Lindane-induced dysfunction was assessed through stress protein induction measured by Western Blot analysis. Lindane pretreatment delayed Heat Shock Protein 72 (HSP72) induction by 36 hours in osmotically stressed cells. Pretreatment with 1 × 10 −5 M LIN followed by osmotic stress elevated p38 and Stress Activated Protein Kinase (SAPK/JNK) at 15 minutes which declined at 30 minutes. Lindane appeared to have no effect on Endoplasmic Reticulum Related Kinase (ERK) induction. Lindane did not effect osmotically stressed LLC-PKI cells, a control cell line. ^ Lindane-treated MDCK cells did not exhibit necrosis. Instead, apoptosis was observed in lindane-treated MDCK cells in both time- and dose-dependent manners. LLC-PKI cells were not affected by LIN treatment. ^ To better understand the mechanism of lindane-induced apoptosis, mitochondrial function was measured. No changes in cytochrome c release or mitochondrial membrane potential were observed suggesting the mitochondrial pathway was not involved in lindane-induced apoptosis. ^ Further research will need to be conducted to determine the mechanism of lindane-induced adverse cellular effects. ^

The roles of costimulatory molecules in the cooperative effects of combination epinephrine and dexamethasone on type-1/type-2 cytokine production in tetanus-toxoid specific stimulated human peripheral blood mononuclear cells

A growing number of studies show strong associations between stress and altered immune function. In vivo studies of chronic and acute stress have demonstrated that cognitive stressors are strongly correlated with high circulating levels of catecholamines (CT) and corticosteroids (CS) that are associated with changes in type-1/type-2 cytokine expression. Although individual pharmacologic doses of CS and CT can inhibit the expression of T-helper 1 (Th1, type-1 like) and promote the production of T-helper 2 (Th2, type-2 like) cytokines in antigen-specific and mitogen stimulated human leukocyte cultures in vitro, little attention has been focused on the effects of combination physiologic-stress doses of CT and CS that may be more physiologically relevant. In addition, both in-vivo and in-vitro studies suggest that the differential expression of the B7 family of costimulatory molecules CD80 and CD86 may promote the expression of type-1 or type-2 cytokines, respectively. Furthermore, corticosteroids can influence the expression of β2-adrenergic receptors in various human tissues. We therefore investigated the combined effects of physiologic-stress doses of in vitro CT and CS upon the type-1/type-2 cytokine balance and expression of B7 costimulatory molecules of human peripheral blood mononuclear cells (PBMC) as a model to study the immunomodulatory effects of physiologic stress. Results demonstrated a significant decrease in type-1 cytokine expression and a significant increase in type-2 cytokine production in our CS+CT incubated cultures when compared to either CT or CS agents alone. In addition, we demonstrated the differential expression of CD80/CD86 in favor of CD86 at the cellular and population level as determined by flow cytometry in lipopolysaccharide stimulated human Monocytes. Furthermore, we developed flow cytometry based assays to detect total β2AR in human CD4+ T-lymphocytes that demonstrated decreased expression of β2AR in mitogen stimulated CD4+ T-lymphocytes in the presence of physiologic stress levels of CS and CT as single in vitro agents, however, when both CS and CT were combined, significantly higher expression of β2AR was observed. In summary, our in vitro data suggest that both CS and CT work cooperatively to shift immunity towards type-2 responses. ^

BLyS/BAFF-R signaling pathway in human aggressive non Hodgkin's lymphoma B cell and normal peripheral blood B lymphocytes

B-lymphocyte stimulator (BLyS also called BAFF), is a potent cell survival factor expressed in many hematopoietic cells. BLyS levels are elevated in the serum of non-Hodgkin lymphoma (NHL) patients, and have been reported to be associated with disease progression, and prognosis. To understand the mechanisms involved in BLyS gene expression and regulation, we examined expression, function, and regulation of the BLyS gene in B cell non-Hodgkin's lymphoma (NHL-B) cells. BLyS is constitutively expressed in aggressive NHL-B cells including large B cell lymphoma (LBCL) and mantle cell lymphoma (MCL) contributing to survival and proliferation of malignant B cells. Two important transcription factors, NF-κB and NFAT, were found to be involved in regulating BLyS expression through at least one NF-κB and two NFAT binding sites in the BLyS promoter. Further study indicates that the constitutive activation of NF-κB and BLyS in NHL-B cells forms a positive feedback loop contributing to cell survival and proliferation. In order to further investigate BLyS signaling pathway, we studied the function of BAFF-R, a major BLyS receptor, on B cells survival and proliferation. Initial study revealed that BAFF-R was also found in the nucleus, in addition to its presence on plasma membrane of B cells. Nuclear presentation of BAFF-R can be increased by anti-IgM and soluble BLyS treatment in normal peripheral B lymphocytes. Inhibition of BLyS expression decreases nuclear BAFF-R level in LBCL cells. Furthermore, we showed that BAFF-R translocated to nucleus through the classic karyopherin pathway. A candidate nuclear localization sequence (NLS) was identified in the BAFF-R protein sequence and mutation of this putative NLS can block BAFF-R entering nucleus and LBCL cell proliferation. Further study showed that BAFF-R co-localized with NF-κB family member, c-rel in the nucleus. We also found BAFF-R mediated transcriptional activity, which could be increased by c-rel. We also found that nuclear BAFF-R could bind to the NF-κB binding site on the promoters of NF-κB target genes such as BLyS, CD154, Bcl-xL, Bfl-1/A1 and IL-8. These findings indicate that BAFF-R may also promote survival and proliferation of normal B cells and NHL-B cells by directly functioning as a transcriptional co-factor with NF-κB family member. ^

Selective elimination of cancer cells by PEITC: Biological basis and therapeutic implications

Toxic side effect is a major problem in cancer chemotherapy. Therefore, identification and development of new agents that can selectively remove cancer with low toxicity to normal cells would have significant clinical impact. Compared to normal cells, cancer cells are under intrinsic stress with elevated reactive oxygen species (ROS) production. My research aimed to exploit this biochemical alteration as a novel basis to develop a selective agent. The goal of my dissertation research was to test the hypothesis that since most cancer cells are under higher oxidative stress than normal cells, compounds which modulate oxidative stress such as pphenylethyl isothiocyanate (PEITC) may preferentially impact cancer cells through ROS-mediated mechanisms and have implications in cancer therapeutics. Using H-RasV1-transformed ovarian cells and their immortalized non-tumorigenic counterparts, I discovered that the transformed cells exhibited increased ROS generation and this intrinsic stress rendered them highly dependent on glutathione antioxidant system to maintain redox balance. Abolishing this system by PEITC through depletion of glutathione and inhibition of GPX activity led to a preferential ROS increase in the transformed cells. The severe ROS accumulation caused oxidative damage to the mitochondria membranes and impaired the membrane integrity leading to massive cell death. In contrast, PEITC caused only a modest increase of ROS insufficient to cause significant cell death in non-transformed cells. Promisingly, PEITC exhibited anticancer activity in vivo by prolonging survival of mice bearing the Ras-transformed ovarian xenograft with minimal toxic side effect. Further study in chronic lymphocytic leukemia (CLL) cells isolated from the blood samples of CLL patients revealed that PEITC not only exhibits promising selectivity against primary CLL cells compared to normal lymphocytes, but it is also effective in removing CLL cells resistant to standard anti-cancer drug Fludarabine. In conclusion, the data implicate that intrinsic oxidative stress in cancer cells could serve as a biochemical basis to develop selective novel anticancer agents such as PEITC, with significant therapeutic implications. ^

Effects of thymus size and involution on the contribution of recent thymic emigrants to the peripheral T cell pool

The contribution of recent thymic emigrants (RTEs) to the peripheral naïve T cell population is necessary to maintain diversity of the T cell receptor (TCR) repertoire and produce immune responses against newly encountered antigens. The thymus involutes with age, after irradiation or chemotherapy, and due to severe viral infections. Thymus involution results in decreased thymopoiesis and RTE output leading to a reduced diversity of peripheral T cells. This increases susceptibility to disease and impairs immune responsiveness to vaccines. Therefore, studies aimed at maintaining or regenerating thymic function are integral for maintaining and restoring peripheral TCR diversity. Mice that express a K5.CyclinD1 transgene expression have a severely hyperplastic thymus that fails to undergo involution. Both thymocyte and TEC development appear normal in these mice. We have used the K5.CyclinD1 transgenic model to test the hypothesis that preventing thymus involution will sustain RTE output and incorporation into the peripheral T cell pool to prevent naïve T cell depletion with age. The K5.CyclinD1 transgene was crossed to the RAG2p-GFP transgenic model so that RTEs could be tracked by the intensity of the GFP signal. The frequency and number of RTEs in naïve CD4 splenic T cells was analyzed at monthly intervals to 5 months of age. Using this double transgenic approach, we determined that preventing thymus involution does maintain or enhance the number of RTEs in the peripheral T cell pool before and after thymus involution.

Elimination of racial/ethnic Hepatitis B vaccination disparities in the US in the 2007--2008 National Health and Nutrition Examination Survey

Viral hepatitis is a significant public health problem worldwide and is due to viral infections that are classified as Hepatitis A, B, C, D, and E. Hepatitis B is one of the five known hepatic viruses. A safe and effective vaccine for Hepatitis B was first developed in 1981, and became adopted into national immunization programs targeting infants since 1990 and adolescents since 1995. In the U.S., this vaccination schedule has led to an 82% reduction in incidence from 8.5 cases per 100,000 in 1990 to 1.5 cases per 100,000 in 2007. Although there has been a decline in infection among adolescents, there is still a large burden of hepatitis B infection among adults and minorities. There is very little research in regards to vaccination gaps among adults. Using the National Health and Nutrition Examination Survey (NHANES) question "{Have you/Has SP (Study Participant)} ever received the 3-dose series of the hepatitis B vaccine?" the existence of racial/ethnic gaps using a cross-sectional study design was explored. In this study, other variables such as age, gender, socioeconomic variables (federal poverty line, educational attainment), and behavioral factors (sexual practices, self-report of men having sex with men, and intravenous drug use) were examined. We found that the current vaccination programs and policies for Hepatitis B had eliminated racial and ethnic disparities in Hepatitis B vaccination, but that a low coverage exists particularly for adults who engage in high risk behaviors. This study found a statistically significant 10% gap in Hepatitis B vaccination between those who have and those who do not have access to health insurance.^

Expression of TNF-$\alpha$ protein and C -FOS and TNF mRNA during human peripheral blood monocyte maturation and activation

Human peripheral blood monocytes (HPBM) were isolated by centrifugal elutriation from mononuclear cell enriched fractions after routine plateletapheresis and the relationship between maturation of HPBM to macrophage-like cells and activation for tumoricidal activity determined. HPBM were cultured for various times in RPMI 1640 supplemented with 5% pooled human AB serum and cytotoxicity to $\sp{125}$IUDR labeled A375M, a human melanoma cell line, and TNF-$\alpha$ release determined by cytolysis of actinomycin D treated L929 cells. Freshly isolated HPBM or those exposed to recombinant IFN-$\gamma$(1.0 U/ml) were not cytolytic and did not release TNF-$\alpha$ into culture supernatants. Exposure to bacterial lipopolysaccharide (LPS, 1.0 $\upsilon$g/ml) stimulated cytolytic activity and release of TNF-$\alpha$. Maximal release of TNF-$\alpha$ protein occurred at 8 hrs and returned to baseline by 72 hrs. Expression of TNF-$\alpha$ protein was determined by Western blotting. Neither freshly isolated nor IFN-$\gamma$ treated HPBM expressed TNF protein at any time during in vitro culture. LPS treated HPBM maximally expressed the 17KD TNF-$\alpha$ protein at 8 hrs, and protein was not detected after 36 hrs of in vitro culture. Expression of TNF-$\alpha$ mRNA was determined by Northern blotting. Freshly isolated HPBM express TNF-$\alpha$ mRNA which decays to basal levels by 6 hrs of in vitro culture. IFN-$\gamma$ treatment maintains TNF-$\alpha$ mRNA expression for up to 48 hrs of culture, after which it is undetectable. LPS induces TNF-$\alpha$ mRNA after 30 minutes of exposure with maximal accumulation occurring between 4 to 8 hrs. TNF mRNA was not detected in control HPBM at any time after 6 hrs or IFN-$\gamma$ treated HPBM after 48 hrs of in vitro culture. A pulse of LPS the last 24 hrs of in vitro culture induces the accumulation of TNF-$\alpha$ mRNA in HPBM cultured for 3, 5, and 7 days, with the magnitude of induction decreasing approximately 10 fold between 3 and 7 days. Induction of TNF-$\alpha$ mRNA occurred in the absence of detectable TNF-$\alpha$ protein or supernatant activity. Maturation of HPBM to macrophage-like cells controls competence for activation, magnitude and duration of the activation response. ^