Biomedical subjects

Gary M Winslow

Publications and source records attributed to Gary M Winslow.

7 recordsLinked to original sources

PubMed2005

Immunity to the ehrlichiae: new tools and recent developments.

PURPOSE OF REVIEW: Discusses recent developments in the study of immunity and host defense against the monocytic ehrlichiae in 2003 and 2004. The review does not address anaplasmoses, as the anaplasmae were recently re-classified into the genus Anaplasma, and are distinct in cell tropism from the ehrlichiae. RECENT FINDINGS: The features of the immune responses against these emerging Gram-negative obligate intracellular pathogens are only beginning to be understood. Important advances in our ability to study host defense include the development of new experimental mouse models. Recent studies have defined possible mechanisms of innate immune subversion in human monocytes, as well as roles for lymphocyte subsets and type I cytokines during mouse infection. Other studies in the mouse suggest that cytokine production by CD8 T cells may contribute to immunopathology. New data also support a role for humoral immunity during host defense against these intracellular pathogens. SUMMARY: The use of new animal models will facilitate research of the mechanisms of innate, adaptive, and pathological immune responses, and will enhance our understanding of human immunity to the ehrlichiae as well as to other pathogenic intracellular bacteria.

Animals↗

PubMed2005

Susceptibility and resistance to monocytic ehrlichiosis in the mouse.

To address the role of cellular immunity during ehrlichia infection, we have utilized a model of monocytic ehrlichiosis that results from infection of mice by Ixodes ovatus ehrlichia (IOE). Although ehrlichiosis in humans is largely a disease of immunocompromised individuals, the use of the IOE model has allowed us to identify factors required for host defense in normal mice. Using a low-dose infection C57BL/6 mouse model, we have demonstrated that host defense requires immune mechanisms involving CD4 T cell-mediated, TNF-alpha-, IL-12-, and IFN-gamma-dependent, macrophage activation. We have also provided formal evidence that IFN-gamma produced by CD4 Th1 cells is sufficient for protective immunity. Our recent studies have demonstrated, in addition, an essential role for IL-10, which is probably important in inhibiting immunopathological responses, and for inducible nitric oxide synthase. The latter observation establishes an important role for reactive nitrogen intermediates in bacterial elimination in vivo. In contrast, evaluation of mice carrying wild-type and mutant alleles of Nramp1 revealed at most a modest role for this gene in resistance to fatal IOE infection. Other studies in low-dose infected mice have indicated that the generation of immunological memory may be impaired during low-dose IOE infection, possibly due to bacterial immune subversion. These studies highlight the utility of the IOE mouse model in identifying important parameters of the immune response during ehrlichiosis.

Animals↗

PubMed2004

Expression of members of the 28-kilodalton major outer membrane protein family of Ehrlichia chaffeensis during persistent infection.

The 28-kDa immunodominant outer membrane proteins (P28 OMPs) of Ehrlichia chaffeensis are encoded by a multigene family. As an indirect measure of the in vivo expression of the members of the p28 multigene family of E. chaffeensis, sera from two beagle dogs experimentally infected with E. chaffeensis were evaluated for the presence of specific antibodies to P28 OMPs by enzyme-linked immunosorbent assay. Antigenic peptides unique to each of the P28s were identified within the first hypervariable region of each P28 OMP. Serological responses to peptides derived from all P28 OMPs were detected from day 30 postinoculation to day 468 and from day 46 until day 159 in the two beagles. Although antibody titers to the peptides fluctuated, the peak response to all of the peptides appeared simultaneously in each dog. The antibody responses to another outer membrane protein of E. chaffeensis (GP120) showed similar temporal and quantitative changes. These data suggest that the P28 OMPs are expressed concurrently during persistent Ehrlichia infection.

Amino Acid Sequence↗

PubMed2003

Persistence and turnover of antigen-specific CD4 T cells during chronic tuberculosis infection in the mouse.

CD4 T cells are critical for resistance to Mycobacterium tuberculosis infection, but how effective T cell responses are maintained during chronic infection is not well understood. To address this question we examined the CD4 T cell response to a peptide from ESAT-6 during tuberculosis infection in the mouse. The ESAT-6(1-20)/IA(b)-specific CD4 T cell response in the lungs, mediastinal lymph nodes, and spleen reached maxima 3-4 wk postinfection, when the bacteria came under the control of the immune response. Once chronic infection was established, the relative frequencies of Ag-specific CD4 T cells were maintained at nearly constant levels for at least 160 days. ESAT-6(1-20)/IA(b)-specific CD4 T cells that responded in vitro expressed activation markers characteristic of chronically activated effector cells and used a limited Vbeta repertoire that was clonally stable in vivo for at least 12 wk. 5-Bromo-2-deoxyuridine incorporation studies indicated a relatively high rate of cell division among both total CD4 and ESAT-6(1-20)/IA(b)-specific CD4 T cells during acute infection, but the degree of 5-bromo-2-deoxyuridine incorporation by both the CD4 T cells and the Ag-specific cells declined at least 3-fold during chronic infection. The data indicate that the peripheral ESAT-6(1-20)/IA(b)-specific CD4 T cell response to M. tuberculosis is characterized during the acute phase of infection by a period of extensive proliferation, but once bacterial control is achieved, this is followed during chronic infection by an extended containment phase that is associated with a persistent response of activated, yet more slowly proliferating, T cells.

Animals↗

PubMed2003

Mechanisms of humoral immunity during Ehrlichia chaffeensis infection.

Our laboratory has been investigating the mechanisms of host defense during Ehrlichia chaffeensis infection in the mouse. Although major roles are clearly played by T cells, we found that antibodies could also control infection in both normal and immunocompromised SCID mice, and could protect the latter from lethal infection. Antibodies are not generally effective during such intracellular infections, so we would like to understand exactly how antibodies can mediate immunity in this model. We have found that much of the humoral immune response is directed at the bacterial outer membrane proteins (OMPs), and that highly effective OMP antibodies (mostly IgG2a) exhibited picomolar affinities and very long binding half-lives. These antibodies, which could mediate bacterial clearance from tissues as early as 24 hours after administration, require host Fc receptors for their function(s). In contrast, we have failed to find any role for complement or reactive nitrogen intermediates, or for neutrophils, or for NK cells. One possible mechanism is that antibodies or immune complexes trigger microbiocidal activities in infected macrophages that lead to the elimination of bacteria residing inside host macrophages. Alternatively, it is proposed that antibodies opsonize bacteria exposed during intercellular transfer. This notion is supported by studies that have demonstrated the presence of bacteria in the extracellular milieu during infection, and suggests that our understanding of the behavior of the bacterium in the host may be key to our understanding of its susceptibility to antibody-mediated host defenses.

Animals↗

PubMed2003

Survival, replication, and antibody susceptibility of Ehrlichia chaffeensis outside of host cells.

Ehrlichia chaffeensis, an obligate intracellular, tick-transmitted bacterium, is susceptible to antibody-mediated host defense, but the mechanism by which this occurs is not understood. One possible explanation is that antibodies directly access the bacteria in the extracellular environment of the host, perhaps during bacterial intercellular transfer. Accordingly, we investigated whether bacteria could be found outside of host cells during infection. Host cell-free plasma obtained from infected mice was found to contain ehrlichiae, and the host cell-free ehrlichiae readily transferred disease to susceptible SCID recipients. The host cell-free ehrlichiae were found during infection of both immunocompetent BALB/c and immunocompromised BALB/c-scid mice and reached levels as high as 10(8)/ml in plasma during persistent infection in SCID mice. Approximately 10% of the blood-borne bacteria were found outside of host cells. Although it is generally accepted that replication of ehrlichiae occurs only within host cells, the cell-free bacteria were shown to undergo DNA replication and cell division in vitro for 3 to 5 days when incubated at 37 degrees C in plasma. Paradoxically, both infectivity and virulence were lost after 24 h of ex vivo culture. The data indicate that E. chaffeensis is exposed to the extracellular milieu during infection, presumably during intercellular transfer, and reveal that these intracellular bacteria do not require the environment of the host cell for replication. Our findings reveal a possible mechanism by which antibodies can access the intracellular bacteria upon their release into the extracellular milieu and mediate host defense and also have implications for understanding the replication and transmission of this vector-borne pathogen.

Animals↗

PubMed2002

Antibodies highly effective in SCID mice during infection by the intracellular bacterium Ehrlichia chaffeensis are of picomolar affinity and exhibit preferential epitope and isotype utilization.

Although often considered to be ineffective against intracellular bacteria, Abs, in the absence of lymphocytes, have been shown previously to protect SCID mice from lethal infection by the obligate intracellular bacterium Ehrlichia chaffeensis, even when administered well after infection has been established. To identify characteristics of Abs that are critical for host defense during this intracellular infection, a panel of Ehrlichia-specific mAbs was generated and analyzed. Among 100 Abs recovered, 39 recognized an amino-terminal hypervariable region of an outer membrane protein (OMP), demonstrating that the OMPs are both antigenically variable and immunodominant. A subset of 16 representative OMP-specific Abs was further examined to identify characteristics that were essential for in vivo efficacy. The highly effective Abs recognized a linear epitope within the first hypervariable region of OMP-1g. Only IgG were found to be effective, and among the effective IgG, the following hierarchy was observed: IgG2a > IgG3 = IgG2b. The most striking characteristics of the highly effective Abs were their picomolar binding affinities and long binding t(1/2). Thus, although epitope recognition and isotype use may contribute to efficacy, high affinity may be a critical characteristic of Abs that can act effectively during this intracellular bacterial infection.

Animals↗