Gary A. Splitter

The development and analysis of species specific and cross reactive monoclonal antibodies to leukocyte differentiation antigens and antigens of the major histocompatibility complex for use in the study of the immune system in cattle and other species

We examined the potential of developing a set of species specific and cross reactive monoclonal antibodies (MoAbs) for use in the study of the phylogenetic and functional relation of class I and class II antigens of the major histocompatibility complex (MHC) and leukocyte differentiation antigens in cattle and other species. Comparing immunization strategies demonstrated the number of hybrids producing cross reactive antibodies can be increased by hyperimmunization of mice with lymphoid cells from multiple species. Comparing various methods of assay (antibody-complement mediated cytotoxicity [CT], enzyme linked immunosorbent assay [ELISA] and flow microfluorimetry [FMF]), revealed FMF is the most useful technique for the primary assay of hybridomas producing MoAbs of potential interest. By using dual parameter and dual fluorescence analysis, we could determine whether a given MoAb reacted with mononuclear cells (lymphocytes and monocytes) and/or granulocytes, and also whether any two MoAbs of different isotype and specificity recognized antigens present on identical or separate populations of leukocytes. Comparing the patterns of MoAb reactivity with leukocytes obtained from cows, goats, sheep, pigs, horses and humans, as well as comparing the patterns of reactivity with a panel of lymphoid cell lines derived from cattle (with enzootic bovine leukemia) and humans (with various forms of leukemia), revealed sets of MoAbs reactive with unique antigenic determinants present on BoLA class I (15 MoAbs) and class II (9 MoAbs) antigens, and also MoAbs reactive with determinants present on leukocyte differentiation antigens (36 MoAbs). Dual fluorescence analysis demonstrated the antigens detected by some MoAbs are predominantly expressed on one lineage of leukocytes while others are expressed on two or more lineages of leukocytes. Dual and single fluorescence analysis also demonstrated the PNA receptor(s) is: expressed on T cells, granulocytes and class II antigen monocytes and absent or expressed in low amount on sIgM+ B cells and a newly defined Non T/Non B population of cells. The strategies described for identifying and analyzing the specificity of MoAbs demonstrate the feasibility of developing a set of cross reactive MoAbs for identifying homologous molecules in multiple species and delineating their functional and phylogenetic relation.

Molecular Host-Pathogen Interaction in Brucellosis: Current Understanding and Future Approaches to Vaccine Development for Mice and Humans

SUMMARY Brucellosis caused by Brucella spp. is a major zoonotic disease. Control of brucellosis in agricultural animals is a prerequisite for the prevention of this disease in human beings. Recently, Brucella melitensis was declared by the Centers for Disease Control and Prevention to be one of three major bioterrorist agents due to the expense required for the treatment of human brucellosis patients. Also, the economic agricultural loss due to bovine brucellosis emphasizes the financial impact of brucellosis in society. Thus, vaccination might efficiently solve this disease. Currently, B. abortus RB51 and B. melitensis REV.1 are used to immunize cattle and to immunize goats and sheep, respectively, in many countries. However, these genetically undefined strains still induce abortion and persistent infection, raising questions of safety and efficiency. In fact, the REV.1 vaccine is quite virulent and apparently unstable, creating the need for improved vaccines for B. melitensis. In addition, Brucella spp. may or may not provide cross-protection against infection by heterologous Brucella species, hampering the acceleration of vaccine development. This review provides our current understanding of Brucella pathogenesis and host immunity for the development of genetically defined efficient vaccine strains. Additionally, conditions required for an effective Brucella vaccine strain as well as the future research direction needed to investigate Brucella pathogenesis and host immunity are postulated.

Blue-light-activated histidine kinases: two-component sensors in bacteria.

Histidine kinases, used for environmental sensing by bacterial two-component systems, are involved in regulation of bacterial gene expression, chemotaxis, phototaxis, and virulence. Flavin-containing domains function as light-sensory modules in plant and algal phototropins and in fungal blue-light receptors. We have discovered that the prokaryotes Brucella melitensis, Brucella abortus, Erythrobacter litoralis, and Pseudomonas syringae contain light-activated histidine kinases that bind a flavin chromophore and undergo photochemistry indicative of cysteinyl-flavin adduct formation. Infection of macrophages by B. abortus was stimulated by light in the wild type but was limited in photochemically inactive and null mutants, indicating that the flavin-containing histidine kinase functions as a photoreceptor regulating B. abortus virulence.

Immunization of mice with recombinant L7L12 ribosomal protein confers protection against Brucella abortus infection

BALB/c mice were immunized with the recombinant Brucella abortus L7/L12 ribosomal protein fused to maltose binding protein (MBP). Vaccinated animals mounted a specific immune response to the recombinant fusion protein as demonstrated by immunoblot analyses. Additionally, B. abortus L7/L12 ribosomal protein conferred a significant degree of protection when compared to mice vaccinated with adjuvant alone, adjuvant plus MBP or B. abortus. These results indicate that a recombinant B. abortus protein, previously identified as T-cell-reactive, engendered protective immunity to mice against brucellosis.

Brucella TIR Domain-containing Protein Mimics Properties of the Toll-like Receptor Adaptor Protein TIRAP

Toll-like receptors (TLRs) play essential roles in the activation of innate immune responses against microbial infections. TLRs and downstream adaptor molecules contain a conserved cytoplasmic TIR domain. TIRAP is a TIR domain-containing adaptor protein that recruits the signaling adaptor MyD88 to a subset of TLRs. Many pathogenic microorganisms subvert TLR signaling pathways to suppress host immune responses to benefit their survival and persistence. Brucella encodes a TIR domain-containing protein (TcpB) that inhibits TLR2- and TLR4-mediated NF-κB activation. Sequence analysis indicated a moderate level of similarity between TcpB and the TLR adaptor molecule TIRAP. We found that TcpB could efficiently block TIRAP-induced NF-κB activation. Subsequent studies revealed that by analogy to TIRAP, TcpB interacts with phosphoinositides through its N-terminal domain and colocalizes with the plasma membrane and components of the cytoskeleton. Our findings suggest that TcpB targets the TIRAP-mediated pathway to subvert TLR signaling. In vivo mouse studies indicated that TcpB-deficient Brucella is defective in systemic spread at the early stages of infection.

Role of Toll-Like Receptor 4 in Induction of Cell-Mediated Immunity and Resistance to Brucella abortus Infection in Mice

ABSTRACT Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) during Brucella abortus infection. CHO reporter cell lines transfected with CD14 and TLRs showed that B. abortus triggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived from Brucella rough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-α) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A from Rhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers of Brucella were monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks following B. abortus infection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance to B. abortus infection and that S lipid A has potent adjuvant activity.

Comparative Whole-Genome Hybridization Reveals Genomic Islands in Brucella Species

Brucella species are responsible for brucellosis, a worldwide zoonotic disease causing abortion in domestic animals and Malta fever in humans. Based on host preference, the genus is divided into six species. Brucella abortus, B. melitensis, and B. suis are pathogenic to humans, whereas B. ovis and B. neotomae are nonpathogenic to humans and B. canis human infections are rare. Limited genome diversity exists among Brucella species. Comparison of Brucella species whole genomes is, therefore, likely to identify factors responsible for differences in host preference and virulence restriction. To facilitate such studies, we used the complete genome sequence of B. melitensis 16M, the species highly pathogenic to humans, to construct a genomic microarray. Hybridization of labeled genomic DNA from Brucella species to this microarray revealed a total of 217 open reading frames (ORFs) altered in five Brucella species analyzed. These ORFs are often found in clusters (islands) in the 16M genome. Examination of the genomic context of these islands suggests that many are horizontally acquired. Deletions of genetic content identified in Brucella species are conserved in multiple strains of the same species, and genomic islands missing in a given species are often restricted to that particular species. These findings suggest that, whereas the loss or gain of genetic material may be related to the host range and virulence restriction of certain Brucella species for humans, independent mechanisms involving gene inactivation or altered expression of virulence determinants may also contribute to these differences.

Microarray Analysis of mRNA Levels from RAW264.7 Macrophages Infected with Brucella abortus

ABSTRACT Identification of host responses at the gene transcription level provides a molecular profile of the events that occur following infection. Brucella abortus is a facultative intracellular pathogen of macrophages that induces chronic infection in humans and domestic animals. Using microarray technology, the response of macrophages 4 h following B. abortus infection was analyzed to identify early intracellular infection events that occur in macrophages. Of the >6,000 genes, we identified over 140 genes that were reproducibly differentially transcribed. First, an increase in the transcription of a number of proinflammatory cytokines and chemokines, such as tumor necrosis factor alpha, interleukin-1β (IL-1β), IL-1α, and members of the SCY family of proteins, that may constitute a general host recruitment of antibacterial defenses was evident. Alternatively, Brucella may subvert newly arriving macrophages for additional intracellular infection. Second, transcription of receptors and cytokines associated with antigen presentation, e.g., major histocompatibility complex class II and IL-12p40, were not evident at this 4-h period of infection. Third, Brucella inhibited transcription of various host genes involved in apoptosis, cell cycling, and intracellular vesicular trafficking. Identification of macrophage genes whose transcription was inhibited suggests that Brucella utilizes specific mechanisms to target certain cell pathways. In conclusion, these data suggest that B. abortus can alter macrophage pathways to recruit additional macrophages for future infection while simultaneously inhibiting apoptosis and innate immune mechanisms within the macrophage, permitting intracellular survival of the bacterium. These results provide insights into the pathogenic strategies used by Brucella for long-term survival within a hostile environment.

The Proteasome as a Lipopolysaccharide-Binding Protein in Macrophages: Differential Effects of Proteasome Inhibition on Lipopolysaccharide-Induced Signaling Events

We have developed a novel LPS probe using a highly purified and homogenous preparation of [3H] Escherichia coli LPS from the deep rough mutant, which contains a covalently linked, photoactivable 4-p-(azidosalicylamido)-butylamine group. This cross-linker was used to identify the LPS-binding proteins in membranes of the murine-macrophage-like cell line RAW 264.7. The α-subunit (PSMA1 C2, 29.5 kDa) and the β-subunit (PSMB4 N3, 24.36 kDa) of the 20S proteasome complex were identified as LPS-binding proteins. This is the first report demonstrating LPS binding to enzymes such as the proteasome subunits. Functionally, LPS enhanced the chymotrypsin-like activity of the proteasome to degrade synthetic peptides in vitro and, conversely, the proteasome inhibitor lactacystin completely blocked the LPS-induced proteasome’s chymotrypsin activity as well as macrophage TNF-α secretion and the expression of multiple inflammatory mediator genes. Lactacystin also completely blocked the LPS-induced expression of Toll-like receptor 2 mRNA. In addition, lactacystin dysregulated mitogen-activated protein kinase phosphorylation in LPS-stimulated macrophages, but failed to inhibit IL-1 receptor-associated kinase-1 activity. Importantly, lactacystin also prevented LPS-induced shock in mice. These data strongly suggest that the proteasome complex regulates the LPS-induced signal transduction and that it may be an important therapeutic target in Gram-negative sepsis.

Susceptibility of IFN Regulatory Factor-1 and IFN Consensus Sequence Binding Protein-Deficient Mice to Brucellosis

IFN-γ is a key cytokine controlling Brucella infection, and the diverse functions of this cytokine are mediated by IFN regulatory factors (IRFs) such as IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). However, the roles of these three IRFs in Brucella infection have not been investigated. The infection of each IRF-deficient mouse strain provides an opportunity to determine not only the significance of each IRF molecule but also the crucial immune components necessary for host defense during in vivo infection, because respective IRF-deficient mouse strains contain unique immunodeficient phenotypes. Brucella abortus S2308-infected IRF-1−/− mice were dead within 2 wk postinfection, while IRF-2−/− mice contained less splenic Brucella CFU than wild-type mice at the early stage of infection. Infected ICSBP−/− mice maintained a plateau of splenic Brucella CFU throughout the infection. Additional infection of IL-12p40-, NO synthase 2-, and gp91phox-deficient mice indicates that these immune components are crucial for Brucella immunity and may contribute to the susceptibility of IRF-1−/− and ICSBP−/− mice. Immunologic and histopathological analyses of infected IRF-1−/− mice indicate that the absence of IL-12p40 induction and serious hepatic damage are involved in the death of IRF-1−/− mice. These results indicate that 1) IRF-1 and ICSBP are essential transcriptional factors for IFN-γ-mediated protection against Brucella; 2) IL-12, reactive nitrogen intermediates, and reactive oxygen intermediates are crucial immune components against Brucella, and their absence may contribute to the susceptibility of IRF-1−/− and ICSBP−/− mice; and 3) hepatic damage caused by Brucella virulence contributes to the death of IRF-1−/− mice.