Permissive and protective roles for neutrophils in leishmaniasis.

Leishmania parasites are the causative agents of leishmaniasis, a neglected tropical disease that causes substantial morbidity and considerable mortality in many developing areas of the world. Recent estimates suggest that roughly 10 million people suffer from cutaneous leishmaniasis (CL), and approximately 76,000 are afflicted with visceral leishmaniasis (VL), which is universally fatal without treatment. Efforts to develop therapeutics and vaccines have been greatly hampered by an incomplete understanding of the parasite's biology and a lack of clear protective correlates that must be met in order to achieve immunity. Although parasites grow and divide preferentially in macrophages, a number of other cell types interact with and internalize Leishmania parasites, including monocytes, dendritic cells and neutrophils. Neutrophils appear to be especially important shortly after parasites are introduced into the skin, and may serve a dual protective and permissive role during the establishment of infection. Curiously, neutrophil recruitment to the site of infection appears to continue into the chronic phase of disease, which may persist for many years. The immunological impact of these cells during chronic leishmaniasis is unclear at this time. In this review we discuss the ways in which neutrophils have been observed to prevent and promote the establishment of infection, examine the role of anti-neutrophil antibodies in mouse models of leishmaniasis and consider recent findings that neutrophils may play a previously unrecognized role in influencing chronic parasite persistence.

The role of Rhipicephalus sanguineus sensu lato saliva in the dissemination of Rickettsia conorii in C3H/HeJ mice.

Animal models have been developed for the study of rickettsial pathogenesis. However, to understand what occurs during the natural route of rickettsial transmission via the tick bite, the role of tick saliva should be considered in these models. To address this, we analysed the role of tick saliva in the transmission of Rickettsia conorii (Rickettsiales: Rickettsiaceae) in a murine host by intradermally (i.d.) inoculating two groups of susceptible C3H/HeJ mice with this Rickettsia, and infesting one group with nymphal Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) ticks. Quantification of bacterial loads and mRNA levels of interleukin-1ß (IL-1ß), IL-10 and NF-κB was performed in C3H/HeJ lung samples by real-time quantitative polymerase chain reaction (PCR) and real-time reverse transcriptase PCR, respectively. Lung histology was examined to evaluate the pathological manifestations of infection. No statistically significant difference in bacterial load in the lungs of mice was observed between these two groups; however, a statistically significant difference was observed in levels of IL-1ß and NF-κB, both of which were higher in the group inoculated with rickettsiae but not infected with ticks. Lung histology in both groups of animals revealed infiltration of inflammatory cells. Overall, this study showed that i.d. inoculation of R. conorii caused infection in the lungs of C3H/HeJ mice and tick saliva inhibited proinflammatory effects.

Simple method to differentiate among Rickettsia species.

In this work we present a new option to identify 11 rickettsial species that cause human rickettsioses, with some advantages over the previous methods described. Using rickettsial isolates from 11 Rickettsia species as a sample, we used the polymerase chain reaction to amplify a 990- to 1,000-bp DNA fragment from the ompB gene, common for the 11 Rickettsia species analyzed in this study, which were digested with AluI restriction enzyme to obtain different digestion patterns. This restriction pattern can be visualized using a polyacrylamide gel electrophoresis technique. Using this method we could differentiate between the 11 Rickettsia species analyzed regardless of the group to which the Rickettsia belonged. We developed a simple method to identify 11 Rickettsia species which cause human rickettsioses using polymerase chain reaction and restriction fragment length polymorphism techniques with the advantage that it only needs one amplicon and only one restriction enzyme to obtain the restriction pattern. The identification of the species infecting vectors, reservoirs, and humans is essential to establish the ecological and behavioral ecosystem involved in its maintenance and transmission in nature in the specific region where the pathogen is circulating. This method is very helpful to identify Rickettsia species in a short time.

Molecular and clinical evidence of Ehrlichia chaffeensis infection in Cameroonian patients with undifferentiated febrile illness.

In the U.S.A., human monocytotropic ehrlichiosis (HME) caused by Ehrlichia chaffeensis is an emerging tick-transmitted zoonosis. In Cameroon, where E. canis, E. chaffeensis and E. ewingii have recently been detected in dogs and/or ticks (Rhipicephalus sanguineus), the potential exists for human infections. Patients from the coastal region of Cameroon who had acute fevers of unknown aetiology were therefore checked for ehrlichial infection, using a real-time PCR that amplifies part of a genus-specific gene (dsb) that codes for a disulphide-bond formation protein. Ehrlichial blood was detected in the peripheral blood from 12 (10%) of the 118 patients investigated by PCR. When the 12 amplicons from the positive cases were sequenced, they were found to be identical to each other and to the corresponding dsb sequence of an Arkansas strain of E. chaffeensis. The 12 patients who were PCR-positive for E. chaffeensis suffered from fever (100%), headache (67%), myalgia (42%), arthralgia (58%), pulmonary involvement (17%) and/or a diffuse rash (17%).

Antigenic protein modifications in Ehrlichia.

To develop effective vaccination strategies against Ehrlichia, we have previously reported developing an animal model of cross-protection in which C57BL/6 mice primed with E. muris were resistant to lethal infection with Ixodes ovatus ehrlichia (IOE). Polyclonal antibody produced in mice after priming with E. muris and later injected with IOE-detected antigenic proteins in E. muris and IOE cell lysates. Cross-reaction of antigenic proteins was observed when we probed both the E. muris and IOE cell lysates with IOE and E. muris-specific polyclonal antibody. Analysis of the total proteins of E. muris and IOE by two dimensional electrophoresis showed that both E. muris and IOE have the same antigenic proteins. Finally, studies on post-translational protein modifications using a novel technique, Eastern blotting, showed that E. muris proteins are more lipoylated and glycosylated than those of IOE.

Regulatory roles of CD1d-restricted NKT cells in the induction of toxic shock-like syndrome in an animal model of fatal ehrlichiosis.

CD1d-restricted NKT cells are key players in host defense against various microbial infections. Using a murine model of fatal ehrlichiosis, we investigated the role of CD1d-restricted NKT cells in induction of toxic shock-like syndrome caused by gram-negative, lipopolysaccharide-lacking, monocytotropic Ehrlichia. Our previous studies showed that intraperitoneal infection of wild-type (WT) mice with virulent Ehrlichia (Ixodes ovatus Ehrlichia [IOE]) results in CD8+ T-cell-mediated fatal toxic shock-like syndrome marked by apoptosis of CD4+ T cells, a weak CD4+ Th1 response, overproduction of tumor necrosis factor alpha and interleukin-10, and severe liver injury. Although CD1d-/- mice succumbed to high-dose IOE infection similar to WT mice, they did not develop signs of toxic shock, as shown by elevated bacterial burdens, low serum levels of tumor necrosis factor, normal serum levels of liver enzymes, and the presence of few apoptotic hepatic cells. An absence of NKT cells restored the percentages and absolute numbers of CD4+ and CD8+ T cells and CD11b+ cells in the spleen compared to WT mice and was also associated with decreased expression of Fas on splenic CD4+ lymphocytes and granzyme B in hepatic CD8+ lymphocytes. Furthermore, our data show that NKT cells promote apoptosis of macrophages and up-regulation of the costimulatory molecule CD40 on antigen-presenting cells, including dendritic cells, B cells, and macrophages, which may contribute to the induction of pathogenic T-cell responses. In conclusion, our data suggest that NKT cells mediate Ehrlichia-induced T-cell-mediated toxic shock-like syndrome, most likely via cognate and noncognate interactions with antigen-presenting cells.

Rickettsia felis in the Americas.

The authors describe their work in the Americas in Rickettsia felis cases in humans and the presence of Rickettsia felis in vectors.

Revisiting brazilian spotted fever focus of Caratinga, Minas Gerais State, Brazil.

We revisited a Brazilian spotted fever focal area in Minas Gerais state, Brazil, in 2002, and performed a serologic survey in dogs and cats. The results of this survey are compared with the survey made 10 years before. The possible efficacy of vector control measures adopted in this area and the role of dogs and horses as sentinels of infection by Rickettsia are discussed.

The genome of the obligately intracellular bacterium Ehrlichia canis reveals themes of complex membrane structure and immune evasion strategies.

Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, alpha-proteobacterium, is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, 17 putative pseudogenes, and a substantial proportion of noncoding sequence (27%). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences and a unique serine-threonine bias associated with the potential for O glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly(G-C) tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Genes associated with pathogen-host interactions were identified, including a small group encoding proteins (n = 12) with tandem repeats and another group encoding proteins with eukaryote-like ankyrin domains (n = 7).

Ehrlichia under our noses and no one notices.

Ehrlichia chaffeensis, an obligately intracellular bacterium, resides within a cytoplasmic vacuole in macrophages, establishes persistent infection in natural hosts such as white-tailed deer and canids, and is transmitted transstadially and during feeding by ticks, particularly Amblyomma americanum. Ehrlichial cell walls contain glycoproteins and a family of divergent 28 kDa proteins, but no peptidoglycan or lipopolysaccharide. The dense-cored ultrastructural form preferentially expresses certain glycoproteins, including a multiple repeat unit-containing adhesin. Ehrlichiae attach to L-selectin and E-selectin, inhibit phagolysosomal fusion, apoptosis, and JAK/STAT activation, and downregulate IL-12, IL-15, IL-18, TLR2 and 3, and CD14. Mouse models implicate overproduction of TNF-alpha by antigen-specific CD8 T lymphocytes in pathogenesis and strong type 1 CD4 and CD8 T lymphocyte responses, synergistic activities of IFN-gamma and TNF-alpha, and IgG2a antibodies in immunity. Human monocytotropic ehrlichiosis (HME) manifests as a flu-like illness that progresses in severity to resemble toxic shock-like syndrome, with meningoencephalitis or adult respiratory distress syndrome in some patients, and requires hospitalization in half. In immunocompromised patients, HME acts as an overwhelming opportunistic infection. In one family physician's practice, active surveillance for three years revealed an incidence of 1000 cases per million population. Diagnosis employs serology or polymerase chain reaction, which are not utilized sufficiently to establish the true impact of this emerging virus-like illness.

Ehrlichial infection in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii.

Ehrlichia chaffeensis and Ehrlichia ewingii are agents of emerging human ehrlichioses in North America and are transmitted primarily by Amblyomma americanum ticks, while Ehrlichia canis is the globally distributed cause of canine monocytic ehrlichiosis (CME) and is transmitted by the brown dog tick, Rhipicephalus sanguineus. Although E. canis and Ehrlichia ruminantium are endemic in Africa, the presence of ehrlichial agents in dogs and ticks in Cameroon has not been investigated. The objective of this study was to determine the prevalence of ehrlichial infections in Cameronian dogs using a combination of serologic and molecular methods. Peripheral blood was collected, clinical signs and the presence or absence of ticks on dogs (n=104) presenting for various reasons at local veterinary clinics around the Mount Cameroon region were noted. IFA identified 33 dogs (32%) with antibodies reactive with E. canis, and reactivity of these sera with all major E. canis antigens (200, 140, 95, 75, 47, 36, 28, and 19-kDa) was confirmed by immunoblotting. Multicolor real-time PCR detected ehrlichial DNA (E. canis (15) and E. ewingii (2)) in 17 dogs (16.3%), all of which had attached ticks at time of presentation. The dsb amplicons (378 bp) from E. canis and E. ewingii were identical to gene sequences from North American isolates. This study identifies canine ehrlichiosis as a prevalent unrecognized cause of disease in Cameroonian canines.

Identification of Ctenocephalides felis fleas as a host of Rickettsia felis, the agent of a spotted fever rickettsiosis in Yucatan, Mexico.

In search for the vector of the recently recognized spotted fever rickettsiosis of the Yucatán, ticks, fleas, and lice were collected from vegetation and dogs in localities where seropositive persons had been found. The arthropods were examined by polymerase chain reaction (PCR) using primers for the genus-specific 17-kDa protein gene followed by restriction fragment length polymorphism (RFLP) and DNA sequencing. Eleven (20%) of 54 pools of Ctenocephalides felis fleas contained DNA of Rickettsia felis. None of 219 Amblyomma cajennense, 474 Rhiphicephalus sanguineus, 258 Boophilus sp. ticks, and 33 Poliplax species lice contained DNA of Rickettsia. The identity of the rickettsial DNA was confirmed as R. felis by PCR/RFLP for the citrate synthase and outer membrane protein A genes and by DNA sequencing. The results indicate that the host of R. felis in Yucatán is C. felis and suggest that the spotted fever rickettsiosis that has infected >5% of the population of the Yucatán and can present as a dengue-like illness is likely to be caused by R. felis.

Immunization with a portion of rickettsial outer membrane protein A stimulates protective immunity against spotted fever rickettsiosis.

Two approaches for presentation of a part of the rickettsial outer membrane protein A (OmpA) of Rickettsia rickettsii, namely (1) recombinant Mycobacterium vaccae (rMV) or (2) recombinant DNA vaccine, stimulated protective immunity against a lethal challenge with the closely related bacterium, R. conorii, in mice. After primary immunization with rMV and booster immunization with homologous recombinant protein, 67 and 55% of mice were protected against challenge in two experiments. DNA vaccination with booster recombinant protein immunization protected six out of eight animals from a lethal challenge. Production of IFN-gamma by antigen-exposed T-lymphocytes of DNA vaccine recipients indicated that cellular immunity had been stimulated.

Oxindole-based inhibitors of cyclin-dependent kinase 2 (CDK2): design, synthesis, enzymatic activities, and X-ray crystallographic analysis.

Two closely related classes of oxindole-based compounds, 1H-indole-2,3-dione 3-phenylhydrazones and 3-(anilinomethylene)-1,3-dihydro-2H-indol-2-ones, were shown to potently inhibit cyclin-dependent kinase 2 (CDK2). The initial lead compound was prepared as a homologue of the 3-benzylidene-1,3-dihydro-2H-indol-2-one class of kinase inhibitor. Crystallographic analysis of the lead compound bound to CDK2 provided the basis for analogue design. A semiautomated method of ligand docking was used to select compounds for synthesis, and a number of compounds with low nanomolar inhibitory activity versus CDK2 were identified. Enzyme binding determinants for several analogues were evaluated by X-ray crystallography. Compounds in this series inhibited CDK2 with a potency approximately 10-fold greater than that for CDK1. Members of this class of inhibitor cause an arrest of the cell cycle and have shown potential utility in the prevention of chemotherapy-induced alopecia.

Identification of protective components of two major outer membrane proteins of spotted fever group Rickettsiae.

Fragments representing the genes of the two major outer membrane proteins of spotted fever group rickettsiae (rOmpA and rOmpB) were tested as DNA vaccines. Immunizations with each of three fragments (rompA4999-6710, rompB1550-2738, and rompB2459-4123) conferred a degree of protection on vaccinated mice against virulent rickettsial challenge. Protection was achieved when DNA immunizations were followed by booster immunizations with the homologous recombinant protein. Proliferation and gamma-interferon secretion were detected after in vitro stimulation of lymphocytes from immunized animals with whole Rickettsia conorii antigen. The data validate particular segments of rOmpA and rOmpB as potent immunogens and hence as sources of immunostimulatory elements with specificity for T lymphocytes, which are the key effectors of protective immunity against rickettsial infections.

Hemostatic/fibrinolytic protein changes in C3H/HeN mice infected with Rickettsia conorii--a model for Rocky Mountain spotted fever.

Changes in plasma hemostatic and fibrinolytic proteins were determined during courses of a murine model of fatal and non-fatal Rocky Mountain spotted fever. C3H/HeN mice were infected with Rickettsia conorii and coagulation and histopathologic studies were performed at prescribed periods of time. A significant decrease in plasma factor VIII activity and rise in plasma factor V procoagulant activity correlated with a fatal infection. Factor VII levels were unchanged; factor XI levels dropped early in the course in the lethally infected animals, but returned to normal. Factor XII, high molecular weight kininogen, and prekallikrein levels were unchanged by the sublethal infection. Prekallikrein levels fell during the lethal infection. Antithrombin concentrations were decreased significantly in all animals, but plasma plasminogen levels did not change in either group of animals. Nonocclusive thrombi were microscopically observed rarely and only in animals surviving a sublethal infection. A fall in tissue plasminogen activator activity and a rise in plasminogen activator inhibitor activity highly correlated with a lethal outcome. Lethal infection with R. conorii is associated with primary endothelial cell injury resulting in decreased tissue plasminogen activator and increased plasminogen activator inhibitor.

Rickettsial infection in murine models activates an early anti-rickettsial effect mediated by NK cells and associated with production of gamma interferon.

Natural killer (NK) cell activity was significantly increased on days 2-6 of infection in the Rickettsia conorii-infected C3H/HeN mice and on day 2 in the Rickettsia typhi-infected C57BL/6 mice. Depletion of NK cell activity utilizing anti-NK1.1 monoclonal antibody enhanced the susceptibility of normally resistant C57BL/6 mice to infection with R. typhi, and depletion of NK cell activity with antibody to asialo GM1 enhanced the susceptibility of C3H/HeN mice to infection with R. conorii. Serum gamma interferon was increased in R. conorii-infected C3H/HeN mice compared with NK cell-depleted, infected mice during the early course of infection. Additionally, the NK cell activating cytokine IL-12 was elevated in the sera of infected mice during the time period representing enhanced NK cell activity compared with uninfected mice. Thus, it appears that NK cells contribute to the early anti-rickettsial immune response, likely via a mechanism involving gamma interferon.

Identification of the target cells of Orientia tsutsugamushi in human cases of scrub typhus.

Orientia tsutsugamushi is the etiologic agent of scrub typhus, a chigger-borne zoonosis that is a highly prevalent, life-threatening illness of greatest public health importance in tropical Asia and the islands of the western Pacific Ocean. The target cell of this bacterium is poorly defined in humans. In this study, O. tsutsugamushi were identified by immunohistochemistry using a rabbit polyclonal antibody raised against O. tsutsugamushi Karp strain in paraffin-embedded archived autopsy tissues of three patients with clinical suspicion of scrub typhus who died during World War II and the Vietnam War. Rickettsiae were located in endothelial cells in all of the organs evaluated, namely heart, lung, brain, kidney, pancreas, and skin, and within cardiac muscle cells and in macrophages located in liver and spleen. Electron microscopy confirmed the location of rickettsiae in endothelium and cardiac myocytes.