PLASMACYTOID DENDRITIC CELLS SENSE SKIN INJURY AND PROMOTE WOUND HEALING THROUGH TYPE I INTERFERONS

Plasmacytoid dendritic cells (pDCs) are a rare population of circulating cells, which selectively express intracellular Toll-like receptors (TLR)-7 and TLR-9 and have the capacity to produce large amounts of type I IFNs (IFN-a/b) in response to viruses or host derived nucleic acid containing complexes. pDCs are normally absent in skin but accumulate in the skin of psoriasis patients where their chronic activation to produce IFN-a/b drives the disease formation. Whether pDCs and their activation to produce IFN-a/b play a functional role in healthy skin is unknown. Here we show that pDCs are rapidly and transiently recruited into healthy human and mouse skin upon epidermal injury. Infiltrating pDCs were found to sense nucleic acids in wounded skin via TLRs, leading to the production of IFN-a/b. The production of IFN-a/b was paralleled by a short lived expression of cathelicidins, which form complexes with extracellular nucleic acids and activated pDCs to produce IFN-a/b in vitro. In vivo, cathelicidins were sufficient but not necessary for the induction of IFN-a/b in wounded skin, suggesting redundancy of this pathway. Depletion of pDCs or inhibition of IFN-a/bR signaling significantly impaired the inflammatory response and delayed re-epithelialization of skin wounds. Thus we uncover a novel role of pDCs in sensing skin injury via TLR mediated recognition of nucleic acids and demonstrate their involvement in the early inflammatory process and wound healing response through the production of IFN-a/b.

THE ROLE OF MAP KINASES ON THE FUNCTIONAL HETEROGENEITY OF HUMAN CD8+ T CELL MATURATION SUBSETS

While prior studies have focused on naïve (CD45RA+CD27+) and early stage memory (CD45RA-CD27+) CD8+ T cells, late memory CD8+ T cells (CD45RA+CD27) have received less interest because this subset of T cells is generally recognized as effectors, which produce IFNγ (but no IL-2) and perforin. However, multiple studies suggest that late memory CD8+ T cells may provide inadequate protection in infectious diseases and cancer models. To better understand the unique function of late memory CD8+ T cells, I optimized multi-color flow cytometry techniques to assess the cytokine production of each human CD8+ T cell maturation subset. I demonstrated that late memory CD8+ T cells are the predominant producer of CC chemokines (e.g. MIP-1β), but rarely produce IL-2; therefore they do not co-produce IL-2/IFNγ (polyfunctionality), which has been shown to be critical for protective immunity against chronic viral infection. These data suggest that late memory CD8+ T cells are not just cytotoxic effectors, but may have unique functional properties. Determining the molecular signature of each CD8+ T cell maturation subset will help characterize the role of late memory CD8+ T cells. Prior studies suggest that ERK1 and ERK2 play a role in cytokine production including IL-2 in T cells. Therefore, I tested whether differential expression of ERK1 and ERK2 in CD8+ T cell maturation subsets contributes to their functional signature by a novel flow cytometry technique. I found that the expression of total ERK1, but not ERK2, is significantly diminished in late memory CD8+ T cells and that ERK1 expression is strongly associated with IL-2 production and CD28 expression. I also found that IL-2 production is increased in late memory CD8+ T cells by over-expressing ERK1. Collectively, these data suggest that ERK1 is required for IL-2 production in human CD8+ T cells. In summary, this dissertation demonstrated that ERK1 is down-regulated in human late memory CD8+ T cells, leading to decreased production of IL-2. The data in this dissertation also suggested that the functional heterogeneity in human CD8+ T cell maturation subsets results from their differential ERK1 expression.

ROLE OF VITAMIN-D3 AND RETINOIC ACID IN A HUMAN THP-1 MACROPHAGE MODEL OF MYCOBACTERIUM TUBERCULOSIS INFECTION

Mycobacterium tuberculosis (Mtb) replicates within the human macrophages and we investigated the activating effects of retinoic acid (RA) and vitamin D3 (VD) on macrophages in relation to the viability of Mtb. A combination of these vitamins (RAVD) enhanced the receptors on THP-1 macrophage (Mannose receptor and DC-SIGN) that increased mycobacterial uptake but inhibited thesubsequent intracellular growth of Mtb by inducing reactive oxygen species (ROS) and autophagy. RAVD also enhanced antigen presenting and homing receptors in THPs that suggested an activated phenotype for THPs following RAVD treatment. RAVD mediated activation was also associated with a marked phenotypic change in Mtb infected THPs that fused with adjacent cells to formmultinucleate giant cells (MNGCs). Typically MNGCs occurred over 30 days of in vitro culture and contained non-replicating persisting Mtb for as long as 60 days in culture. We propose that the RAVD mediated inhibition of replicating Mtb leading to persistence of non-replicating Mtb within THPs may provide a novel human macrophage model simulating formation of MNGCs in humanlungs.

The use of second-generation antipsychotics and the changes in physical growth in children and adolescents with perinatally acquired HIV.

Second-generation antipsychotics (SGAs) are increasingly prescribed to treat psychiatric symptoms in pediatric patients infected with HIV. We examined the relationship between prescribed SGAs and physical growth in a cohort of youth with perinatally acquired HIV-1 infection. Pediatric AIDS Clinical Trials Group (PACTG), Protocol 219C (P219C), a multicenter, longitudinal observational study of children and adolescents perinatally exposed to HIV, was conducted from September 2000 until May 2007. The analysis included P219C participants who were perinatally HIV-infected, 3-18 years old, prescribed first SGA for at least 1 month, and had available baseline data prior to starting first SGA. Each participant prescribed an SGA was matched (based on gender, age, Tanner stage, baseline body mass index [BMI] z score) with 1-3 controls without antipsychotic prescriptions. The main outcomes were short-term (approximately 6 months) and long-term (approximately 2 years) changes in BMI z scores from baseline. There were 236 participants in the short-term and 198 in the long-term analysis. In linear regression models, youth with SGA prescriptions had increased BMI z scores relative to youth without antipsychotic prescriptions, for all SGAs (short-term increase = 0.192, p = 0.003; long-term increase = 0.350, p < 0.001), and for risperidone alone (short-term = 0.239, p = 0.002; long-term = 0.360, p = 0.001). Participants receiving both protease inhibitors (PIs) and SGAs showed especially large increases. These findings suggest that growth should be carefully monitored in youth with perinatally acquired HIV who are prescribed SGAs. Future research should investigate the interaction between PIs and SGAs in children and adolescents with perinatally acquired HIV infection.

IMMUNE MODULATION OF THE MYCOBACTERIUM TUBERCULOSIS GRANULOMATOUS RESPONSE

Tuberculosis (TB) remains a major public health burden. The immunocompetant host responds to Mycobacterium tuberculosis (MTB) infection by the formation of granulomas, which initially prevent uncontrolled bacterial proliferation and dissemination. However, increasing evidence suggests that granuloma formation promotes persistence of the organism by physically separating infected cells from effector lymphocytes and by inducing a state of non-replicating persistence in the bacilli, making them resistant to the action of antibiotics. Additionally, immune-mediated tissue destruction likely facilitates disease transmission. The granulomatous response is in part due to mycobacterial glycolipid antigens. Therefore, studies were first undertaken to determine the innate mechanisms of mycobacterial cord factor trehalose-6’6-dimycolate (TDM) on granuloma formation. Investigations using knock-out mice suggest that TNF-a is involved in the initiation of the granulomatous response, complement factor C5a generates granuloma cohesiveness, and IL-6 is necessary for maintenance of an established granulomatous responses. Studies were next performed to determine the ability of lactoferrin to modulate the immune response and pathology to mycobacterial cord factor. Lactoferrin is an iron-binding glycoprotein with immunomodulatory properties that decrease tissue damage and promote Th1 responses. Mice challenged with TDM and treated with lactoferrin had decreased size and numbers of granulomas at the peak of the granulomatous response, accompanied by increased IL-10 and TGF-b production. Finally, the ability of lactoferrin to serve as a novel therapeutic for the treatment of TB was performed by aerosol challenging mice with MTB and treating them with lactoferrin added to the drinking water. Mice given tap water had lung log10 CFUs of 7.5 ± 0.3 at week 3 post-infection. Lung CFUs were significantly decreased in mice given lactoferrin starting the day of infection (6.4 ± 0.7) and mice started therapeutically on lactoferrin at day 7 after established infection (6.5 ± 0.4). Total lung inflammation in lactoferrin treated mice was significantly decreased, with fewer areas of macrophages, increased total lymphocytes, and increased numbers of CD4+ and CD8+ cells. The lungs of lactoferrin treated mice had increased CD4+ IFN-g+ cells and IL-17 producing cells on ELISpot analysis. It is hypothesized that lactoferrin decreases bacterial burden during MTB infection by early induction of Th1 responses.

Determining the roles of dendritic cells and ICAM-1 in the transpresentation of IL-15 to CD8 T cells

The maintenance and generation of memory CD8 T cells is dependent on the cytokine IL-15. IL-15 is delivered by a novel mechanism termed transpresentation: IL-15 is presented by a cell expressing IL-15Ralpha to the CD8 T cell which responds via IL-2Rbeta/gammac. The identity of what cells transpresent IL-15 to support the survival and homeostatic proliferation of memory CD8 T cells is unknown. Using a transgenic mouse model that limits IL-15 transpresentation to DCs, I have demonstrated that DCs transpresent IL-15 to CD8 T cells. DCs transpresent IL-15 to CD8 T cells during the contraction of an immune response and also drive homeostatic proliferation of memory CD8 T cells. Additionally, I identified a role for ICAM-1 in promoting homeostatic proliferation. Wt memory CD8 T cells displayed impaired homeostatic proliferation in ICAM-1-/- hosts but not in models of acute IL-15-driven proliferation. In this way, the role of ICAM-1 in IL-15 transpresentation resembles the role for ICAM-1 in antigenpresentation: where antigen or IL-15 is limited, adhesion molecules are important for generating maximal responses. In vitro cultures between CD8 T cells and bone marrowdifferentiated DCs (BMDC) activated with a TLR agonist established a model of proliferation and signaling in CD8 T cells that was dependent on IL-15 transpresentation and required ICAM-1 expression by BMDCs. Regarding the expression of IL-15, I demonstrated that in normal mice it is undetectable without stimulation but is elevated in lymphopenic mice, suggesting a role for T cells in regulating IL-15 expression. Overall, these studies have identified many novel aspects of the interaction between DCs and CD8 T cells that were previously unknown. The study of adhesion molecules in IL-15 transpresentation describes a novel role for these well-known adhesion molecules and it will be interesting for future studies to further characterize this relationship for other IL-15-dependent cell types.

Enforced expression of Tbx1 in fetal thymic epithelial cells antagonizes thymus organogenesis

Enforced expression of Tbx1 in fetal thymic epithelial cells antagonizes thymus organogenesis Kim T. Cardenas The thymus and parathyroid glands originate from organ-specific domains of 3rd pharyngeal pouch (PP) endoderm. At embryonic day 11.5 (E11.5), the ventral thymus and dorsal parathyroid domains can be identified by Foxn1 and Gcm2 expression respectively. Neural crest cells, (NCCs) play a role in regulating patterning of 3rd PP endoderm. In addition, pharyngeal endoderm influences fate determination via secretion of Sonic hedgehog (Shh), a morphogen required for Gcm2 expression and generation of the parathyroid domain. Gcm2 is a downstream target of the transcription factor Tbx1, which in turn is positively regulated by Shh. Although initially expressed throughout pharyngeal pouch endoderm, Tbx1 expression is excluded from the thymus-specific domain of the 3rd PP by E10.5, but persists in the parathyroid domain. Based on these observations, we hypothesized that Tbx1 expression is non-permissive for thymus fate specification and that enforced expression of Tbx1 in the fetal thymus would impair thymus development. To test this hypothesis, we generated knock-in mice containing a Cre-inducible allele that allows for tissue-specific Tbx1 expression. Expression of the R26iTbx1 allele in fetal and adult thymus using Foxn1Cre resulted in severe thymus hypoplasia throughout ontogeny that persisted in the adult. Thymic epithelial cell (TEC) development was impaired as determined by immunohistochemical and FACS analysis of various differentiation markers. The relative level of Foxn1 expression in fetal TECs was significantly reduced. TECs in R26iTbx1/+ thymi assumed an almost universal expression of Plet-1, a marker associated with a TEC stem/progenitor cell fate. In addition, embryonic R26iTbx1/+ mice develop a perithymic mesechymal capsule that appears expanded compared to control littermates. Interestingly, thymi from neonatal and adult R26iTbx1/+ but not R26+/+ mice were encased in adipose tissue. This thymic phenotype also correlated with a decrease in thymocyte cellularity and aberrant thymocyte differentiation. The results to date support the conclusion that enforced expression of Tbx1 in TECs antagonizes their differentiation and prevents normal organogenesis via both direct and indirect effects.

Upregulation of Reactive Oxygen Species During the Retrovirus Life Cycle and Their Roles in a Mutant of Moloney Murine Leukemia Virus, ts1-Mediated Neurodegeneration

Viral invasion of the central nervous system (CNS) and development of neurological symptoms is a characteristic of many retroviruses. The mechanism by which retrovirus infection causes neurological dysfunction has yet to be fully elucidated. Given the complexity of the retrovirus-mediated neuropathogenesis, studies using small animal models are extremely valuable. Our laboratory has used a mutant moloney murine leukemia retrovirus, ts1-mediated neurodegneration. We hypothesize that astrocytes play an important role in ts1-induced neurodegeneration since they are retroviral reservoirs and supporting cells for neurons. It has been shown that ts1 is able to infect astrocytes in vivo and in vitro. Astrocytes, the dominant cell population in the CNS, extend their end feet to endothelial cells and neuronal synapse to provide neuronal support. Signs of oxidative stress in the ts1-infected CNS have been well-documented from previous studies. After viral infection, retroviral DNA is generated from its RNA genome and integrated into the host genome. In this study, we identified the life cycle of ts1 in the infected astrocytes. During the infection, we observed reactive oxygen species (ROS) upregulations: one at low levels during the early infection phase and another at high levels during the late infection phase. Initially we hypothesized that p53 might play an important role in ts1-mediated astrocytic cell death. Subsequently, we found that p53 is unlikely to be involved in the ts1-mediated astrocytic cell death. Instead, p53 phosphorylation was increased by the early ROS upregulation via ATM, the protein encoded by the ataxia-telangiectasia (A-T) mutated gene. The early upregulation of p53 delayed viral gene expression by suppressing expression of the catalytic subunit of NADPH oxidase (NOX). We further demonstrated that the ROS upregulation induced by NOX activation plays an important role in establishing retroviral genome into the host. Inhibition of NOX decreased viral replication and delayed the onset of pathological symptoms in ts1-infected mice. These observations lead us to conclude that suppression of NOX not only prevents the establishment of the retrovirus but also decreases oxidative stress in the CNS. This study provides us with new perspectives on the retrovirus-host cell interaction and sheds light on retrovirus-induced neurodegeneration as a result of the astrocyte-neuron interaction.

AUTOIMMUNE RESPONSES TO ATHEROSCLEROTIC

Atherosclerosis is a chronic, complex arterial disease characterized by intimal lipid accumulation and inflammation. A unique lipid-binding molecule, namely cluster of differentiation 1d (CD1d), may impact atherosclerosis. Structurally, CD1d acts as a nonpolymorphic cell-surface receptor, resembling the major histocompatibility complex-I (MHC-I). While MHC-I restricts peptide antigen presentation to T cells, CD1d presents lipid antigens to T cells named CD1d-restrictedd T cells. Although increased expression of CD1d has been found in human plaques, the exact nature of CD1d-recognized lipids in atherosclerosis remains to be determined. Three groups of lipids may undergo oxidation in atherosclerosis producing atherogenic lipids: phospholipids, fatty acids, and cholesterol. The central hypothesis is that CD1d recognizes and present oxidative lipids to activate CD1d-restricted T cells, and trigger proinflammatory signal transduction In the first part of this study, oxidative phospholipids were identified and characterized as potential autoantigen for CD1d-restricted T cells. Derived from phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine by oxidization, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) is commonly found in atherosclerotic plaques. Upon stimulation with PGPC, spleen-derived CD1d-restricted T cells produced higher levels of cytokines and proliferated at higher rates than those without PGPC stimulation. CD1d deficiency compromised the PGPC-triggered T cell activation, suggesting that PGPC may function as a potentially novel autoantigen for T cells in atherosclerosis. In the second part of this study, CD1d-mediated proinflammatory signaling was evaluated in murine models. Enhanced CD1 expression occurred in spleens of db/db mice with hyperlipidemia. Tumor necrosis factor-alpha (TNF-α) was increased in db/db spleen, while TNF-α receptor expression augmented in the db/db murine heart, in comparison with those in normal mice. The nuclear factor-κ B (NF-κB) expression was enhanced in the db/db heart, whereas CD1d-null mice showed lower NF-κB, implying the involvement of CD1d in inflammation of the spleen and heart tissues in the mice with hyperlipidemia. The current study has identified PGPC as a novel lipid antigen recognized by CD1d-restricted T cells in atherosclerosis. The animal study has also provided evidence that CD1d regulates NF-κB-mediated proinflammatory signaling. Hence, CD1d-restricted T cell responses to autolipid antigen and mediated inflammatory signal may represent a new molecular pathway that triggers cardiovascular tissue injury in atherosclerosis and hyperlipidemia.

Eluding antibiotic resistance: capitalizing on antimicrobial peptides interaction with the lipid bilayer

It is widely accepted that the emergence of drug-resistant pathogens is the result of the overuse and misuse of antibiotics. Infectious Disease Society of America, Center for Disease Control and World Health Organization continue to view, with concern, the lack of antibiotics in development, especially those against Gram-negative bacteria. Antimicrobial peptides (AMPs) have been proposed as an alternative to antibiotics due to their selective activity against microbes and minor ability to induce resistance. For example, the Food and Drug Administration approved Daptomycin (DAP) in 2003 for treatment of severe skin infections caused by susceptible Gram-positive organisms. Currently, there are 12 to 15 examples of modified natural and synthetic AMPs in clinical development. But most of these agents are against Gram-positive bacteria. Therefore, there is unmet medical need for antimicrobials used to treat infections caused by Gram-negative bacteria. In this study, we show that a pro-apoptotic peptide predominantly used in cancer therapy, (KLAKLAK)2, is an effective antimicrobial against Gram-negative laboratory strains and clinical isolates. Despite the therapeutic promise, AMPs development is hindered by their susceptibility to proteolysis. Here, we demonstrate that an all-D enantiomer of (KLAKLAK)2, resistant to proteolysis, retains its activity against Gram-negative pathogens. In addition, we have elucidated the specific site and mechanism of action of D(KLAKLAK)2 through a repertoire of whole-cell and membrane-model assays. Although it is considered that development of resistance does not represent an obstacle for AMPs clinical development, strains with decreased susceptibility to these compounds have been reported. Staphylococci resistance to DAP was observed soon after its approval for use and has been linked to alterations of the cell wall (CW) and cellular membrane (CM) properties. Immediately following staphylococcal resistance, Enterococci resistance to DAP was seen, yet the mechanism of resistance in enterococci remains unknown. Our findings demonstrate that, similar to S. aureus, development of DAP-resistance in a vancomycin-resistant E. faecalis isolate is associated with alterations of the CW and properties of the CM. However, the genes linked to these changes in enterococci appear to be different from those described in S. aureus.

Amyotrophic lateral sclerosis and Parkinsonism-dementia complex of Guam: Descriptive epidemiology, secular trends, and birth cohort effects on incidence, 1950--1989

This study describes the patterns of occurrence of amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC) of Guam during 1950-1989. Both ALS and PDC occur with high frequency among the indigenous Chamorro population, first recognized in the early 1950's. Reports in the early 1980's indicated that both ALS and PDC were disappearing, due to a purported reduction in exposure to harmful environmental factors as a result of the dramatic changes in lifestyle that took place after World War II. However, this study provides compelling evidence that ALS and PDC have not disappeared on Guam and that rates for both are higher during 1980-1989 than previously reported.^ The patterns of occurrence for both ALS and PDC overlap in most respects: (1) incidence and mortality are decreasing; (2) median age at onset is increasing; (3) males are at increased risk for developing disease; (4) risk is higher for those residing in the south compared to the non-south; and (5) age-specific incidence is decreasing over time except in the oldest age groups.^ Age-specific incidence of ALS and PDC, separately and together, is generally higher for cohorts born before 1920 than for those born after 1920. A significant birth cohort effect on the incidence of PDC for the 1906-1915 birth cohort was found, but not for ALS and for ALS and PDC together. Whether or not a cohort effect, period effect, or both are associated with incidence of ALS and PDC cannot be determined from the data currently available and will require additional follow-up of individuals born after 1920.^ The epidemiological data amassed over this 40-year period provide evidence that supports an environmental exposure model for disease occurrence as opposed to a simple genetic or infectious disease model. Whether neurodegenerative disease in this population occurs as a consequence of a single exposure or is explained by a multifactorial model such as a genetic predisposition with some environmental interaction is yet to be determined. However, descriptive studies such as this can provide clues concerning timing and location of potential adverse exposures but cannot determine etiology, underscoring the urgent need for analytic studies of ALS and PDC to further investigate existing etiologic hypotheses and to test new hypotheses. ^

Pathogenesis of bacterial meningitis.

Bacterial meningitis is fatal in 5% to 40% of patients and causes neurologic sequelae in up to 30% of survivors. Much has been learned recently about the mechanisms that lead to brain injury during meningitis. Once bacteria have gained access to the central nervous system, their multiplication triggers a complex host response consisting of humoral and cellular immune mediators, reactive oxygen intermediates, matrix-metalloproteinases, and other host-derived factors. Alterations of the cerebral vasculature, with disruption of the blood brain barrier and global and focal ischemia, ultimately lead to functional and structural brain damage. This article reviews current concepts of the pathophysiology of bacterial meningitis and emphasizes possible therapeutic strategies to prevent its harmful consequences.

Infections de prothèse articulaire : aspects pratiques à l’attention du médecin de premier recours

L’infection de prothèse articulaire est une complication rare mais redoutée. Sa prise en charge nécessite une collaboration entre médecin de premier recours, orthopédiste et infectiologue. Une méconnaissance du diagnostic peut avoir pour conséquences des traitements chirurgicaux lourds. L’identification du germe responsable de l’infection est essentielle. Elle guide le choix de l’antibiothérapie et est aussi un critère décisif de la stratégie chirurgicale. Une antibiothérapie ne devrait jamais être instaurée sans prélèvement microbiologique adéquat préalable. Ici, le frottis de plaie superficielle n’est d’aucune utilité, car il reflète tout au plus la colonisation par des germes de la flore cutanée. Cette revue se veut un aperçu pratique des infections de prothèse articulaire à l’attention du médecin de premier recours.

Regulation of NAMPT in human gingival fibroblasts and biopsies

Adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), are molecules, which are produced in adipose tissue. Recent studies suggest that NAMPT might also be produced in the tooth-supporting tissues, that is, periodontium, which also includes the gingiva. The aim of this study was to examine if and under what conditions NAMPT is produced in gingival fibroblasts and biopsies from healthy and inflamed gingiva. Gingival fibroblasts produced constitutively NAMPT, and this synthesis was significantly increased by interleukin-1β and the oral bacteria P. gingivalis and F. nucleatum. Inhibition of the MEK1/2 and NFκB pathways abrogated the stimulatory effects of F. nucleatum on NAMPT. Furthermore, the expression and protein levels of NAMPT were significantly enhanced in gingival biopsies from patients with periodontitis, a chronic inflammatory infectious disease of the periodontium, as compared to gingiva from periodontally healthy individuals. In summary, the present study provides original evidence that gingival fibroblasts produce NAMPT and that this synthesis is increased under inflammatory and infectious conditions. Local synthesis of NAMPT in the inflamed gingiva may contribute to the enhanced gingival and serum levels of NAMPT, as observed in periodontitis patients. Moreover, local production of NAMPT by gingival fibroblasts may represent a possible mechanism whereby periodontitis may impact on systemic diseases.