Immunoblot cross-reactions among Rickettsia, Proteus spp. and Legionella spp. in patients with Mediterranean spotted fever

Abstract Sera from patients suffering from Mediterranean spotted fever (i.e. an infection due to Rickettsia conorii ) were studied by immunoblot to investigate cross-reactivity. A prevalence of IgM antibodies to Proteus OX 19, Proteus 0X 2, to the Rickettsia typhus group, to Legionella pneumophila serovars 4 and 5, to L. bozemanii Wiga and to L. micdadei Tatlock was found. Western blot confirmed that the antibodies were directed against the lipopolysaccharide as demonstrated by proteinase K digestion of the antigens. Cross-adsorptions showed that there is a common cross-reacting epitope among L. bozemanii Wiga, R. typhi and Proteus OX 19 but cross-reacting antibodies to L. micdadei and OX 2 were distinct and independent. This IgM cross-reaction could lead to a misdiagnosis.     

Immunological characterization of lipopolysaccharides from Proteus strains used in Weil-Felix test and reactivity with patient sera of tsutsugamushi diseases.

Immunological analyses of lipopolysaccharides (LPS) isolated from Proteus strains OX2, OX19, and OXK used as antigens of Weil-Felix (WF) test, were performed by quantitative agglutination, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Antisera against LPS and whole cells (WC) of the three Proteus strains reacted with homologous LPS but not with heterologous LPS, and the reaction was inhibited by the O-polysaccharide fraction isolated from the homologous LPS except OX19-LPS, which lacked O-polysaccharide moiety. The immunological data support the findings that the O-polysaccharide moieties of LPS from OX2 and OXK strains possess different chemical composition (Mizushiri, Amano, Fujii, Fukushi, and Watanabe, Microbiol. Immunol. 34: 121-133, 1990). Antisera against Proteus strains reacted weakly with WC of Rickettsia prowazekii, Rickettsia typhi, and Rickettsia tsutsugamushi. Antisera from patients with tsutsugamushi disease reacted with OXK-WC by WF test when the sera were obtained 13 days after onset of fever. The immunoperoxidase (IP) test titers of these antisera began to rise 6 days after the onset of fever. By ELISA tests these antisera reacted with OXK-WC and OXK-LPS independently of the titers of WF or IP tests.     

Serological Studies of the Antigenic Similarity between Typhus Group Rickettsiae and Weil-Felix Test Antigens

The sera from two patients with murine typhus reacted with whole cells of Rickettsia prowazekii, R. typhi, and Proteus vulgaris OX19, and with lipopolysaccharides (LPS) from the spotted fever group rickettsia strain TT-118 and P. vulgaris OX19 in the enzyme-linked immunosorbent assay. Sera from these patients reacted with ladder-like bands of LPS from R. prowazekii and R. typhi in the immunoblot, whereas the reactivity of these sera with LPS from P. vulgaris OX19 differed from each other. These results indicate that LPS from the typhus group rickettsiae and P. vulgaris OX19 contain similar epitopes.     

Serological Studies of an Acid-Labile O-Polysaccharide of Proteus vulgaris OX19 Lipopolysaccharide Using Human and Rabbit Antibodies

In a Weil-Felix test, sera from patients infected with Rickettsia sp. agglutinate Proteus OX types of bacteria and Proteus lipopolysaccharide (LPS) are responsible for the cross-reaction. Data on the character of LPS of one of the OX group strains, Proteus vulgaris OX19, are contradictory, and it remained unclear whether it has an O-polysaccharide (OPS) and is thus LPS of the smooth type (S) or not (rough-type LPS). Our studies showed that P. vulgaris OX19 (strain PZH-24) produces a smooth-type LPS that contains a long-chain OPS, but it undergoes depolymerization during mild acid hydrolysis conventionally used for LPS delipidation and loses the serological activity. An elucidation of the complete structure of OPS demonstrated the presence of a glycosyl phosphate linkage responsible for the acid-lability of the polysaccharide chain. In ELISA, both IgM type antibodies in a Weil-Felix test with human anti-Rickettsia typhi sera and rabbit anti-P. vulgaris OX19 antibodies reacted with OPS. Rabbit antibodies did not inhibit the cross-reaction with human antibodies and thus bind to different epitopes.     

Detection of antibodies to Rickketsia prowazekii by using the antigen neutralization test]

The authors studied a possibility of using the antigen neutralization test with dry immunoglobulin typhus erythrocytic diagnostic agent for the purpose of detection of Rickettsia prowazeki antibodies. Blood sera of 315 healthy persons, 24 patients with sporadic typhus, and 18 laboratory animals immunized with R. sibirica and R. burneti, as well as with Proteus OX19 were examined. The results obtained pointed to the high specificity and sensitivity of the given serological test. A possibility of its use for antibody detection both in the typhus patients and in persons who sustained this infection in the past was demonstrated. In difference from the complement fixation test it permits to study anticomplementary sera.     

Experimental forms of infection and serological analysis of the antigenic structure of Rickettsia canada

Experimental forms of Rickettsia canada infection were characterized and serological analysis of the antigenic structure of R. canada was carried out. According to its pathogenicity for experimental animals, R. canada can be characterized as a poorly virulent species of rickettsiae, similar to R. prowazekii (for guinea pigs). The complement-fixing, haemagglutinating and agglutinating antigens of R. canada are fairly similar to those of the typhus group rickettsiae. The region of antigenic activity common to or identical in R. canada and the typhus group rickettsiae, is larger in R. canada than in the typhus group rickettsiae. R. canada has common antigens with Proteus OX19. R. canada has active toxic substances similar to those of R. prowazekii which, however, are detectable only with sera of Brill's disease convalescents. The position of R. canada in the taxonomy of rickettsiae is discussed.     

Molecular phylogeny and rearrangement of rRNA genes in Rickettsia species.

It has previously been observed that Rickettsia prowazekii has an unusual arrangement of the rRNA genes. In this species, the three rRNA genes, 16S (rrs), 23S (rrl), and 5S (rrf), are not linked in the typical arrangements for bacteria. Rather, the 16S rRNA gene has been separated from the 23S and 5S rRNA gene cluster, and the 23S rRNA gene is preceded by a gene which codes for methionyl-tRNAf(Met) formyltransferase (fmt). In this study, we screened the genus Rickettsia for the fmt-rrl motif in order to examine the phylogenetic depth of this unusual rRNA gene organization. A rearranged operon structure was observed in Rickettsia conorii, Rickettsia parkeri, Rickettsia sibirica, Rickettsia rickettsii, Rickettsia amblyomii, Rickettsia montana, Rickettsia rhipicephali, Rickettsia australis, Rickettsia akari, Rickettsia felis, Rickettsia canada, and Rickettsia typhi. There is also evidence for a divided operon in Rickettsia belli, but in this species, the fmt gene could not be identified upstream of the 23S rRNA gene. In order to place the rearrangement event in the evolutionary history of the Rickettsia, phylogenetic analyses were performed based on the fmt-rrl spacer regions and the 23S rRNA genes. Based on these phylogenies, we suggest that the genomic rearrangement of the rRNA genes preceded the divergence of the typhus group and the spotted fever group Rickettsia. The unique organization of the 23S rRNA genes provides a simple diagnostic tool for identification of Rickettsia species.     

Spotted fever rickettsioses in southern and eastern Europe

Mediterranean spotted fever due to Rickettsia conorii conorii was thought, for many years, to be the only tick-borne rickettsial disease prevalent in southern and eastern Europe. However, in recent years, six more species or subspecies within the spotted fever group of the genus Rickettsia have been described as emerging pathogens in this part of the world. Tick-borne agents include Rickettsia conorii israelensis, Rickettsia conorii caspia, Rickettsia aeschlimannii, Rickettsia slovaca, Rickettsia sibirica mongolitimonae and Rickettsia massiliae. Many Rickettsia of unknown pathogenicity have also been detected from ticks and could represent potential emerging pathogens to be discovered in the future. Furthermore, a new spotted fever rickettsia, Rickettsia felis, was found to be associated with cat fleas and is an emerging human pathogen. Finally, the mite-transmitted Rickettsia akari, the agent of rickettsialpox, is also known to be prevalent in Europe. We present here an overview of these rickettsioses, focusing on emerging diseases.     

Widespread use of real-time PCR for rickettsial diagnosis

We report 2?years of experience with rickettsial molecular diagnosis using real-time PCR at the French National Reference Center. All Rickettsia genomes available were compared to discover specific sequences to design new sets of primers and probes. The specificity was verified in silico and against a panel of 30 rickettsial species. Sensitivity was determined using 10-fold serial dilutions. Finally, primers and probes that were both specific and sensitive were routinely used for the diagnosis of rickettsial infections from clinical specimens. We retained sets of primers and probes to detect spotted fever group Rickettsia, typhus group Rickettsia,Rickettsia conorii,Rickettsia slovaca,Rickettsia africae and Rickettsia australis; 643 clinical samples were screened for the presence of Rickettsia DNA. Overall, 45 positive samples were detected, including 15 Rickettsia africae, nine R.?conorii, five Rickettsia sibirica mongolitimonae, four R.?slovaca, two R.?australis, four Rickettsia massiliae, one Rickettsia honei, one Rickettsia typhi and eight Rickettsia sp. Positive samples were detected mainly from cutaneous biopsies and swabs (31/45). Widespread use of real-time PCR is inexpensive and reduces delay in the diagnosis of rickettsial infections. These real-time PCR assays could be implemented easily in laboratories that have molecular facilities and may be added to existing molecular tools as a point-of-care strategy.     

Critical involvement of OX40 ligand signals in the T cell priming events during experimental autoimmune encephalomyelitis

OX40 ligand (OX40L) expressed on APCs, and its receptor, OX40 present on activated T cells, are members of the TNF/TNFR family, respectively, and have been located at the sites of inflammatory conditions. We have observed in OX40L-deficient mice (OX40L(-/-)) an impaired APC capacity and in our recently constructed transgenic mice expressing OX40L (OX40L-Tg), a markedly enhanced T cell response to protein Ags. Using these mice, we demonstrate here the critical involvement of the OX40L-OX40 interaction during the T cell priming events in the occurrence of experimental autoimmune encephalomyelitis (EAE). In OX40L(-/-) mice, abortive T cell priming greatly reduced the clinical manifestations of actively induced EAE, coupled with a reduction in IFN-gamma, IL-2, and IL-6 production in vitro. Adoptive transfer experiments however revealed an efficient transfer of disease to OX40L(-/-) mice using wild-type donor T cells, indicating an intact capacity of OX40L(-/-) mice to initiate effector responses. On the other hand, OX40L(-/-) donor T cells failed to transfer disease to wild-type recipient mice. Furthermore, OX40L-Tg mice developed a greater severity of EAE despite a delayed onset, while both OX40L-Tg/CD28(-/-) and OX40L-Tg/CD40(-/-) mice failed to develop EAE demonstrating a requisite for these molecules. These findings indicate a pivotal role played by OX40L in the pathogenesis of EAE.     

Constitutive OX40/OX40 ligand interaction induces autoimmune-like diseases

The interaction between OX40 and OX40 ligand (OX40L) is suggested to provide T cells with an effective costimulatory signals during T cell-APC interaction. To examine the in vivo effect of constitutive OX40/OX40L interaction during immune regulation, we report the establishment of OX40L-transgenic (OX40L-Tg) mice that constitutively express OX40L on T cells. Markedly elevated numbers of effector memory CD4(+) T cells, but not CD8(+) T cells, were observed in the secondary lymphoid organs of OX40L-Tg mice. Upon immunization with keyhole limpet hemocyanin in the absence of adjuvant, profound T cell proliferative responses and cytokine productions were seen in the OX40L-Tg mice as compared with wild-type mice. Furthermore, in OX40L-Tg mice administrated with superantigen, this constitutive OX40/OX40L interaction on CD4(+) T cells completely prevented normal in vivo clonal T cell deletion. Interestingly, OX40L-Tg mice on the C57BL/6 background spontaneously developed interstitial pneumonia and inflammatory bowel disease that was accompanied with a significant production of anti-DNA Ab in the sera. Surprisingly, these diseases were not evident on the OX40L-Tg mice on the BALB/c strain. However, such inflammatory diseases were successfully reproducible in recombination-activating gene (RAG)2-deficient mice upon transfer of OX40L-Tg CD4(+) T cells. Blockade of OX40/OX40L interaction in the recipient RAG2-deficient mice completely prevented disease development. The present results orchestrated in this study indicate that OX40/OX40L interaction may be a vital link in our understanding of T cell-mediated organ-specific autoimmunity.     

Activity of soluble OX40 ligand is enhanced by oligomerization and cell surface immobilization

OX40 ligand (OX40L) and OX40 are typical members of the tumor necrosis factor ligand family and the tumor necrosis factor receptor superfamily, respectively, and are involved in the costimulation and differentiation of T cells. Like other tumor necrosis factor ligands, OX40L is a type II transmembrane protein. Recombinant soluble human OX40L assembles into trimers and is practically inactive despite binding to OX40. However, oligomerization of soluble OX40L trimers by cross-linking with antibodies or by expression as a hexameric fusion protein strongly increased the activity of the ligand. Moreover, a fusion protein of OX40L with a single chain fragment recognizing the tumor stroma antigen fibroblast activation protein showed a cell surface antigen-dependent increase in the activity of the ligand domain of the molecule and thus mimicked the activity of membrane OX40L upon antigen binding. Trimeric single chain OX40L fusion proteins therefore represent a novel type of OX40L-derived immunostimulatory molecule with potentially reduced systemic side effects.     

Expression of an epitope-tagged virulence protein in Rickettsia parkeri using transposon insertion

Despite recent advances in our ability to genetically manipulate Rickettsia, little has been done to employ genetic tools to study the expression and localization of Rickettsia virulence proteins. Using a mariner-based Himar1 transposition system, we expressed an epitope-tagged variant of the actin polymerizing protein RickA under the control of its native promoter in Rickettsia parkeri, allowing the detection of RickA using commercially-available antibodies. Native RickA and epitope-tagged RickA exhibited similar levels of expression and were specifically localized to bacteria. To further facilitate protein expression in Rickettsia, we also developed a plasmid for Rickettsia insertion and expression (pRIE), containing a variant Himar1 transposon with enhanced flexibility for gene insertion, and used it to generate R. parkeri strains expressing diverse fluorescent proteins. Expression of epitope-tagged proteins in Rickettsia will expand our ability to assess the regulation and function of important virulence factors.     

Functional CD4 T cells after intercellular molecular transfer of 0X40 ligand.

OX40/OX40 ligand (OX40L) proteins play critical roles in the T cell-B cell and T cell-dendritic cell interactions. Here we describe the intercellular transfer of OX40L molecules by a non-Ag specific manner. After 2-h coculture of activated CD4(+) T cell (OX40L(-), OX40(+)) with FLAG peptide-tagged OX40L (OX40L-flag) protein-expressing COS-1 cells, the OX40L-flag protein was detected on the cell surface of the CD4(+) T cells by both anti-OX40L and anti-FLAG mAbs. The intercellular OX40L transfer was specifically abrogated by pretreatment of the COS-1 cells with anti-OX40L mAb, 5A8. The OX40L transfer to OX40-negative cells was also observed, indicating an OX40-independent pathway of OX40L transfer. HUVECs, allostimulated monocytes, and human T cell leukemia virus type I-infected T cells, which all express OX40L, can potentially act as the donor cells of OX40L. The entire molecule of OX40L was transferred and stabilized on the recipient cell membrane with discrete punctate formation. The transferred OX40L on normal CD4(+) T cells was functionally active as they stimulated latent HIV-1-infected cells to produce viral proteins via OX40 signaling. Therefore, these findings suggest that the intercellular molecular transfer of functional OX40L may be involved in modifying the immune responses.     

Detection of spotted fever group Rickettsia in Haemaphysalis longicornis from Hebei Province, China

DNA samples from 737 tick pools, representing 6,850 Haemaphysalis longicornis and 51 Dermacentor nuttalli collected from Hebei Province, China, were analyzed by polymerase chain reaction (PCR) for the presence of spotted fever group Rickettsia. Fifty (6.9%) of 724 H. longicornis in the tick pool were positive, but no positive samples were found in 13 D. nuttalli. Sequence analysis of the partial outer membrane protein A (ompA) genes from the 10 positive samples showed 97.4-99.8% identity, but were different from the homologous sequence of Rickettsia previously deposited in GenBank. Phylogenetic analysis of ompA genes indicated that the Rickettsia detected in this study belonged to a novel haplotype, and formed a clade distinct from Rickettsia heilongjiangii, Rickettsia sibirica, and Rickettsia hulinii in China. The new strain, named Candidatus Rickettsia hebeiii, appears to represent a distinct lineage and could constitute a new species with a minimum prevalence of about 0.7% in H. longicornis from Hebei Province, China.     

The HTLV-I tax protein transcriptionally modulates OX40 antigen expression

OX40 is a member of the TNF receptor family, expressed on activated T cells. It is the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukemia virus type-I (HTLV-I)-producing cells. In a T cell line, OX40 surface expression was shown to be induced by HTLV-I Tax alone. To understand molecular mechanisms of OX40 gene regulation and modulation by HTLV-I Tax, we have cloned the human OX40 gene and analyzed its 5'-flanking region. By reporter gene analysis with progressive 5' deletions from nucleotides -1259 to -64, we have defined a 157-bp DNA fragment as a minimal promoter for constitutive expression. In addition, we show that in the OX40+ cell line, Co, Tax is able to further increase OX40 surface expression. Up-regulation of OX40 promoter activity by Tax requires two upstream NF-kappaB sites, which are not active in the constitutive OX40 expression. Their deletion abrogates Tax responsiveness in reporter gene analysis. The site-directed mutagenesis of each NF-kappaB site demonstrates that cooperative NF-kappaB binding is a prerequisite for Tax-directed activity as neither site alone is sufficient for a full Tax responsiveness of the OX40 promoter. Upon Tax expression, both sites bind p65 and c-Rel. These data provide new insight into the direct regulation of OX40 by Tax and add to our understanding of the possible role of the OX40/OX40 ligand system in the proliferation of HTLV-I+ T cells.     

Multiple levels of activation of murine CD8(+) intraepithelial lymphocytes defined by OX40 (CD134) expression: effects on cell-mediated cytotoxicity, IFN-gamma, and IL-10 regulation.

The involvement of OX40 (CD134) in the activation of CD8(+) intestinal intraepithelial lymphocytes (IELs) has been studied using freshly isolated IELs and in vitro CD3-stimulated IELs. Although freshly isolated CD8(+) IELs exhibited properties of activated T cells (CD69 expression and ex vivo cytotoxicity), virtually all CD8(+) IELs from normal mice were devoid of other activation-associated properties, including a lack of expression of OX40 and the ligand for OX40 (OX40L) and an absence of intracellular IFN-gamma staining. However, OX40 and OX40L expression were rapidly up-regulated on CD8 IELs following CD3 stimulation, indicating that both markers on IELs reflect activation-dependent events. Unlike IELs, activated lymph node T cells did not express OX40L, thus indicating that OX40-OX40L communication in the intestinal epithelium is part of a novel CD8 network. Functionally, OX40 expression was exclusively associated with IELs with active intracellular IFN-gamma synthesis and markedly enhanced cell-mediated cytotoxicity. However, OX40 costimulation during CD3-mediated activation significantly suppressed IL-10 synthesis by IELs, whereas blockade of OX40-OX40L by anti-OX40L mAb markedly increased IL-10 production. These findings indicate that: 1) resident CD69(+)OX40(-) IELs constitute a population of partially activated T cells poised for rapid delivery of effector activity, 2) OX40 and OX40L expression defines IELs that have undergone recent immune activation, 3) OX40(+) IELs are significantly more efficient CTL than are OX40(-) IELs, and 4) the local OX40/OX40L system plays a critical role in regulating the magnitude of cytokine responses in the gut epithelium.     

The crystal structure of the costimulatory OX40-OX40L complex

OX40 is a T cell costimulator activated by OX40L. Blockade of the OX40L-OX40 interaction has ameliorative effects in animal models of T cell pathologies. In order to better understand the interaction between OX40 and OX40L, we have determined the crystal structure of murine OX40L and of the human OX40-OX40L complex at 1.45 and 2.4 A, respectively. These structures show that OX40L is an unusually small member of the tumor necrosis factor superfamily (TNFSF). The arrangement of the OX40L protomers forming the functional trimer is atypical and differs from that of other members by a 15 degrees rotation of each protomer with respect to the trimer axis, resulting in an open assembly. Site-directed changes of the interfacial residues of OX40L suggest this interface lacks a single "hot spot" and that instead, binding energy is dispersed over at least two distinct areas. These structures demonstrate the structural plasticity of TNFSF members and their interactions with receptors.     

Study of human Orexin-1 and -2 G-protein-coupled receptors with novel and published antagonists by modeling, molecular dynamics simulations, and site-directed mutagenesis

The class A G-protein-coupled receptors (GPCRs) Orexin-1 (OX1) and Orexin-2 (OX2) are located predominantly in the brain and are linked to a range of different physiological functions, including the control of feeding, energy metabolism, modulation of neuro-endocrine function, and regulation of the sleep-wake cycle. Site-directed mutagenesis (SDM) and domain exchange (chimera) studies have provided important insight into key features of the OX1 and OX2 binding sites. However, the precise determinants of antagonist binding and selectivity are still not fully known. In this work, we used homology modeling of OX receptors to direct further SDM studies. These SDM studies were followed by molecular dynamics (MD) simulations to rationalize the full scope of the SDM data and to explain the role of each mutated residue in the binding and selectivity of a set of OX antagonists: Almorexant (dual OX1 and OX2 antagonist), SB-674042 (OX1 selective antagonist), EMPA (OX2 selective antagonist), and others. Our primary interest was focused on transmembrane helix 3 (TM3), which is identified as being of great importance for the selectivity of OX antagonists. These studies revealed conformational differences between the TM3 helices of OX1 and OX2, resulting from differences in amino acid sequences of the OX receptors that affect key interhelical interactions formed between TM3 and neighboring TM domains. The MD simulation protocol used here, which was followed by flexible docking studies, went beyond the use of static models and allowed for a more detailed exploration of the OX structures. In this work, we have demonstrated how even small differences in the amino acid sequences of GPCRs can lead to significant differences in structure, antagonist binding affinity, and selectivity of these receptors. The MD simulations allowed refinement of the OX receptor models to a degree that was not possible with static homology modeling alone and provided a deeper rationalization of the SDM data obtained. To validate these findings and to demonstrate that they can be usefully applied to the design of novel, very selective OX antagonists, we show here two examples of antagonists designed in house: EP-109-0092 (OX1 selective) and EP-009-0513 (OX2 selective).     

Impact of proestrous milieu on expression of orexin receptors and prepro-orexin in rat hypothalamus and hypophysis: actions of Cetrorelix and Nembutal

Orexins and their receptors OX1 and OX2 regulate energy balance and the sleep-wake cycle. We studied the expression of prepro-orexin (PPO), OX1, and OX2 in brain and pituitary under the influence of the hormonal status in adult rats. Primarily, PPO, OX1, and OX2 expression was determined in Sprague-Dawley female cycling rats during proestrus and in males. Animals were killed at 2-h intervals. Anterior (AH) and mediobasal (MBH) hypothalamus, anterior pituitary (P), and frontoparietal cortex (CC) were homogenized in TRIzol, and mRNAs were obtained for screening of PPO, OX1, OX2 expression by semiquantitative RT-PCR. Main findings were confirmed and extended to all days of the cycle by quantitative real-time RT-PCR. Hormones and food consumption were determined. Finally, OX1, OX2, and PPO were measured by real-time RT-PCR in tissues collected at 1900 of proestrus after treatments at 1400 with ovulation-blocking agents Cetrorelix or pentobarbital. OX1 and OX2 expression increased at least threefold in AH, MBH, and P, but not in CC, between 1700 and 2300 of proestrus, without variations in estrus, diestrus, or in males. PPO in AH and MBH showed a fourfold or higher increase only during proestrus afternoon. Cetrorelix or pentobarbital prevented increases of OX1 and OX2 only in the pituitary and blunted gonadotropin surges, but left OX1, OX2, and PPO brain expression unchanged. Reproduction, energy balance, and sleep-wake cycle are integrated. Here, we demonstrate that, in the physiological neuroendocrine condition leading to ovulation, information to the orexinergic system acts in hypothalamus and pituitary by different mechanisms.