Deletion of the Braun Lipoprotein-Encoding Gene and Altering the Function of Lipopolysaccharide Attenuate the Plague Bacterium

Braun (murein) lipoprotein (Lpp) and lipopolysaccharide (LPS) are major components of the outer membranes of Enterobacteriaceae family members that are capable of triggering inflammatory immune responses by activating Toll-like receptors 2 and 4, respectively. Expanding on earlier studies that demonstrated a role played by Lpp in Yersinia pestis virulence in mouse models of bubonic and pneumonic plague, we characterized an msbB in-frame deletion mutant incapable of producing an acyltransferase that is responsible for the addition of lauric acid to the lipid A moiety of LPS, as well as a Δlpp ΔmsbB double mutant of the highly virulent Y. pestis CO92 strain. Although the ΔmsbB single mutant was minimally attenuated, the Δlpp single mutant and the Δlpp ΔmsbB double mutant were significantly more attenuated than the isogenic wild-type (WT) bacterium in bubonic and pneumonic animal models (mouse and rat) of plague. These data correlated with greatly reduced survivability of the aforementioned mutants in murine macrophages. Furthermore, the Δlpp ΔmsbB double mutant was grossly compromised in its ability to disseminate to distal organs in mice and in evoking cytokines/chemokines in infected animal tissues. Importantly, mice that survived challenge with the Δlpp ΔmsbB double mutant, but not the Δlpp or ΔmsbB single mutant, in a pneumonic plague model were significantly protected against a subsequent lethal WT CO92 rechallenge. These data were substantiated by the fact that the Δlpp ΔmsbB double mutant maintained an immunogenicity comparable to that of the WT strain and induced long-lasting T-cell responses against heat-killed WT CO92 antigens. Taken together, the data indicate that deletion of the msbB gene augmented the attenuation of the Δlpp mutant by crippling the spread of the double mutant to the peripheral organs of animals and by inducing cytokine/chemokine responses. Thus, the Δlpp ΔmsbB double mutant could provide a new live-attenuated background vaccine candidate strain, and this should be explored in the future.     

Fine-Tuning Synthesis of Yersinia pestis LcrV from Runaway-Like Replication Balanced-Lethal Plasmid in a Salmonella enterica Serovar Typhimurium Vaccine Induces Protection against a Lethal Y. pestis Challenge in Mice

A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal ΔasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain χ9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/P_cro) (P_R), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC P_BAD c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of β-lactamase, and cloned into pYA4534 under the control of the P_trc promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain χ9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.Live, attenuated bacteria have been developed to generate safe and immunogenic vaccine strains (50). Attenuated Salmonella enterica has been used as both a homologous vaccine and a delivery system for recombinant heterologous antigens from bacterial, parasitic, viral, and tumor sources (8, 40). The oral administration of Salmonella allows the infection of Peyer''s patches via the M cells and colonization of the mesenteric lymph nodes, liver, and spleen, generating a range of humoral and cellular immune responses against Salmonella and the heterologous antigens (8) at local and distal sites such as the mucosa. Because most systems for the expression of heterologous antigenic proteins in Salmonella use plasmids, several approaches have been developed for the antibiotic-free maintenance of plasmid vectors (29, 48). However, a number of factors may affect the immune response to protective antigens, such as the ability of the vaccine strain to invade and colonize the host and the stability of the plasmid expression system. High levels of bacterial protein synthesis specified by multiple-copy plasmids often result in either the rapid loss of the foreign plasmid or a reduction in bacterial growth and the ability to colonize lymphoid tissues due to the demand of the extrametabolic burden. Both of these factors result in a reduction of immunogenicity. The insertion of genes into the bacterial chromosome by homologous recombination can achieve a high degree of stability, but this approach sometimes limits the level of protein synthesis due to the single gene copy and, thus, may lessen the production of a protective immune response with the live vaccine (29).To overcome some of these problems, we have constructed a balanced-lethal vaccine vector with its copy number regulated by arabinose (pYA4534) that switches to runaway-like high-copy-number replication regulating the delivery and dosage of heterologous antigens. In previous work, the switch from a low to a high copy number of the plasmid was mediated by a temperature change from 30°C to higher than 35°C and is called uncontrolled replication or runaway replication of the plasmid in Escherichia coli (62).Yersinia pestis is a Gram-negative bacterium that causes plague in humans and is transmitted from rodents to humans by fleas (26, 51). Y. pestis infections present three different clinical forms: bubonic, pneumonic, or septicemic (59). Widespread aerosol dissemination of the bacterium combined with high mortality rates make Y. pestis a deadly pathogen (31). LcrV is a multifunctional protein that forms part of a type III secretion system (T3SS) encoded on Y. pestis 70-kb virulence plasmid pCD1 (16, 52). LcrV along with LcrG helps regulate the expression of Yersinia outer proteins (YOPs) that are injected into the cytosol of the host cell, where they interfere with the cellular signaling involved in phagocytosis and inhibit proinflammatory cytokine production (28, 43, 47). Experimental evidence indicates that antibody responses to LcrV offer protection against plague. Thus, the passive transfer of LcrV monoclonal antibodies (MAbs) or polyclonal-specific serum to LcrV protects animals against bubonic and pneumonic plague (25, 46). Antibodies against LcrV apparently block the translocation of effector YOPs, allowing the phagocytosis of Y. pestis bacilli by macrophages, but the exact mechanism of this protection remains to be determined (19).In addition to the direct role of LcrV in the formation of the T3SS needle, LcrV has an immunomodulatory function mediated by interleukin-10 (IL-10) induction, which blocks the host protective inflammatory responses and suppresses the proinflammatory cytokines (7). Partial deletions of LcrV and the use of synthetic peptides allowed the identification of two LcrV regions involved in the production of IL-10, which are located from amino acid residues 37 to 57 and from amino acid residues 271 to 285 (34, 49). The induction of IL-10 by LcrV is through the interaction of Toll-like receptor 2 (TLR-2) as well as TLR-6 and cluster of differentiation 14 (CD14) (1, 15, 58). Immunization with full-length LcrV elicited protective immunity (41), but truncated LcrV forms were also able to elicit an immune response that was protective against a lethal challenge with Y. pestis. These variants included rV10 (lacking amino acid residues 271 to 300) (49), the major protective LcrV region (amino acid residues 135 to 275) (25), LcrV196 (amino acid residues 131 to 326) (5), and a small fragment of LcrV (amino acids 135 to 262) (64).In this work, we describe the construction of pYA4534, a balanced-lethal plasmid expression system containing an arabinose-regulated genetic switch to shift to runaway-like high-copy-number replication in vivo for the regulated delayed dosage of heterologous antigens. The derivative pYA4535, encoding the T2SS β-lactamase N- and C-terminal domains for the export of the lcrV196-encoded fused antigen, was used to transform S. enterica serovar Typhimurium strain χ9447, a new generation of live recombinant attenuated Salmonella vaccines (RASVs) that is phenotypically similar to the wild type at the time of oral vaccination but displays a regulated delayed in vivo attenuation (14), a regulated delayed in vivo synthesis of recombinant antigen (66), and regulated delayed in vivo lysis to release a bolus of protective antigen and confers complete biological containment (35) after host tissue colonization. These RASV strains are able to colonize and persist in the lymphoid tissue without causing disease symptoms, giving an advantageous alternative when carrying heterologous antigens that induce higher protective mucosal and systemic immunity responses. The immune responses of mice immunized orally with this live RASV strain synthesizing an optimized LcrV protein were evaluated for protection against a lethal challenge with virulent Y. pestis CO92 (4). Thus, we offer an alternative for the development of vaccines against clinical forms of plague.     

A Live Recombinant Avirulent Oral Salmonella Vaccine Expressing Pneumococcal Surface Protein A Induces Protective Responses against Streptococcus pneumoniae

A live oral recombinant Salmonella vaccine strain expressing pneumococcal surface protein A (PspA) was developed. The strain was attenuated with Δcya Δcrp mutations. Stable expression of PspA was achieved by the use of the balanced-lethal vector-host system, which employs an asd deletion in the host chromosome to impose an obligate requirement for diaminopimelic acid. The chromosomal Δasd mutation was complemented by a plasmid vector possessing the asd⁺ gene. A portion of the pspA gene from Streptococcus pneumoniae Rx1 was cloned onto a multicopy Asd⁺ vector. After oral immunization, the recombinant Salmonella-PspA vaccine strain colonized the Peyer’s patches, spleens, and livers of BALB/cByJ and CBA/N mice and stimulated humoral and mucosal antibody responses. Oral immunization of outbred New Zealand White rabbits with the recombinant Salmonella strain induced significant anti-PspA immunoglobulin G titers in serum and vaginal secretions. Polyclonal sera from orally immunized mice detected PspA on the S. pneumoniae cell surface as revealed by immunofluorescence. Oral immunization of BALB/cJ mice with the PspA-producing Salmonella strain elicited antibody to PspA and resistance to challenge by the mouse-virulent human clinical isolate S. pneumoniae WU2. Immune sera from orally immunized mice conferred passive protection against otherwise lethal intraperitoneal or intravascular challenge with strain WU2.     

Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD₅₀) equivalent to 6,800 LD₅₀s of WT CO92. The mutant-infected animals developed balanced T_H1- and T_H2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD₅₀s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant''s sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection.     

Immunogenicity of a Live Recombinant Salmonella enterica Serovar Typhimurium Vaccine Expressing pspA in Neonates and Infant Mice Born from Na?ve and Immunized Mothers

We are developing a Salmonella vectored vaccine to prevent infant pneumonia and other diseases caused by Streptococcus pneumoniae. One prerequisite for achieving this goal is to construct and evaluate new recombinant attenuated Salmonella vaccine (RASV) strains suitable for use in neonates and infants. Salmonella enterica serovar Typhimurium strain χ9558(pYA4088) specifies delivery of the pneumococcal protective antigen PspA and can protect adult mice from challenge with S. pneumoniae. This strain is completely safe for oral delivery to day-old and infant mice. Here we assess the colonizing ability, immunogenicity, and protective efficacy of χ9558(pYA4088) in neonatal mice. Colonization was assessed in mice 0, 2, 4, or 7 days of age after oral inoculation. In the presence of maternal antibodies, the colonization of lymphoid tissues was delayed, but the immune responses were enhanced in mice born to immunized mothers. Both oral and intranasal routes were used to assess immunogenicity. All orally or intranasally immunized neonatal and infant mice born to either immunized or naïve mothers developed PspA-specific mucosal and systemic immune responses. Mice born to immunized mothers produced higher titers of PspA-specific antibodies in the blood and mucosa and greater numbers of PspA-specific interleukin-4 (IL-4)-secreting cells than mice born to naïve mothers. More importantly, mice born to immune mothers showed a significant increase in protection against S. pneumoniae challenge. These results suggest that strain χ9558(pYA4088) can circumvent some of the limitations of the immature immune system in neonatal and infant mice, generating enhanced protective immune responses in the presence of maternal antibodies.Streptococcus pneumoniae is a respiratory pathogen that enters the body through the respiratory mucosa (45) and may cause diseases such as pneumonia, otitis media, meningitis, and bacteremia, especially in young children and the elderly (24). Moreover, it is a leading cause of pneumonia in young children, resulting in an estimated 1 to 3 million deaths each year (16, 40). An increase in the incidence of antibiotic-resistant S. pneumoniae is a growing problem worldwide (1, 10), and infants are colonized at an early age in countries where resistant strains are prevalent (31). Fortunately, the use of antipneumococcal vaccines can prevent antibiotic-resistant infections and limit the development of drug resistance. A 7-valent pneumococcal conjugate vaccine (Prevnar) was licensed in 2000 by Wyeth and has been used for children under the age of 2 years (5). Although this vaccine has proven useful, capsular types not covered by the vaccine have emerged (18, 44), leaving young children once again vulnerable to S. pneumoniae infection and disease. Prevnar 13, which includes five additional serotypes, is currently under review by the FDA (34).In our laboratory, we have been developing a vaccine for the prevention of S. pneumoniae infections based on surface protein antigens, such as PspA and PspC (7, 9). Our strategy has been to use live attenuated Salmonella vectors to deliver the relevant antigens (23, 27, 33, 48, 49). One challenge of early-life immunization arises as a consequence of the limited immune responses in neonates and infants (43). Successful induction of a protective response must circumvent the typically weak and short-lived antibody response of the immature immune system and the inhibitory influence of maternal antibodies (42). In a previous study, a live attenuated Salmonella vaccine was used to induce a strong immune response in the face of an immature immune system and maternal antibodies (11).While safety and immunogenicity are the two most important factors to consider in developing a live recombinant attenuated Salmonella vaccine (RASV), when the vaccine is targeted toward infants and young children, safety becomes paramount. We have recently reported the development of several new strategies to enhance both RASV safety and immunogenicity, including regulated delayed attenuation (12, 13, 27), regulated delayed antigen synthesis (49), programmed cell lysis (25), and a constellation of other mutations, such as ΔsopB, which enhances the immune response against a vectored antigen (28, 29) and reduces fluid accumulation in the intestines (16a, 50). Salmonella enterica serovar Typhimurium strain χ9558 (16a) has many of these new features.We have taken a balanced approach to our strain construction strategy, adding features to improve both immunogenicity and safety. As a result, strain χ9558 has demonstrated an improved safety profile in adult mice, with a reduced ability to cause meningitis when administered orally, intranasally (i.n.), or intraperitoneally (i.p.) (6), and it is totally safe and noninflammatory in newborn mice at doses equal to 10⁷ times the 50% lethal dose (LD₅₀) of the wild-type parent (16a). Plasmid pYA4088 is an Asd⁺ balanced-lethal plasmid that carries the gene for an immunogenic portion of the protective PspA antigen fused to a type 2 secretion signal for β-lactamase, directing secretion of the fusion protein to the periplasm and outside the cell (21, 23, 49). When χ9558 carrying a plasmid nearly identical to pYA4088 was used to immunize adult mice, the mice were significantly protected against challenge with 200 times the LD₅₀ of virulent S. pneumoniae (27). The high level of protection was comparable to the protection observed in mice immunized with an RASV lacking many of these new-generation vaccine safety features and was significantly greater than the protection afforded by a Δcya Δcrp RASV lacking any of the new-generation features.In this work we confirmed the safety of χ9558(pYA4088) for young mice and examined the immunogenicity and protective efficacy of χ9558(pYA4088) for neonatal and infant mice born to naïve and immunized mothers. In a previous study, Capozzo et al. demonstrated both the safety and the immunogenicity of a live attenuated Salmonella strain when it was administered by the intranasal route (11). Our ultimate goal is to produce a vaccine that can be given orally, and we therefore immunized mice by the oral route. For comparison, we also evaluated immunity in mice immunized by the intranasal route. Surprisingly, we found that maternal immunization enhanced the protective efficacy of the vaccine when it was administered by either route.     

Intestinal Secretory Factor Released by Macrophages Stimulated with Clostridium difficile Toxin A: Role of Interleukin 1β

Clostridium difficile toxin A is associated with enterocolitis in animals and humans. However, the mechanisms of its secretory and damaging effects are not totally understood. In this work, we examined the intestinal secretion of electrolytes and water caused by supernatants from macrophages stimulated with toxin A in rabbit ileal mucosa mounted in Üssing chambers. We also investigated the mechanism by which the intestinal secretory factor (ISF) is released from stimulated macrophages. Supernatants from macrophages stimulated with toxin A caused potent intestinal secretion (change in short-circuit current [ΔIsc], 76 μA · cm⁻²; P < 0.01). The release of the ISF was pertussis toxin sensitive (reduction, 61%; P < 0.01) and was also reduced (P < 0.05) by a protein synthesis inhibitor (67%), protease inhibitors (57%), a phospholipase A₂ inhibitor (54%), a cyclo-oxygenase inhibitor (62%), a dual cyclo- and lipoxygenase inhibitor (48%), a platelet-activating factor (PAF) receptor antagonist (55%), and tumor necrosis factor alpha (TNF-α) synthesis inhibitors (48%). However, this release was not inhibited by a lipo-oxygenase inhibitor. Monoclonal anti-interleukin 1β (IL-1β) but not anti-IL-1α antibody blocked (72%; P < 0.01) the secretory action of the ISF, as did recombinant human IL-1 receptor antagonist (80%; P < 0.01). High levels of IL-1β (3,476 pg/ml) were detected by an enzyme-linked immunosorbent assay in the above supernatants. Furthermore, the addition of IL-1β to the serosal side caused a potent secretory effect (ΔIsc, 80 μA · cm⁻²; P < 0.01). These results show that macrophages stimulated with toxin A release an ISF capable of provoking intestinal secretion. The regulation of this factor is dependent upon the activation of the G protein. In addition, prostaglandins, PAF, and TNF-α are involved in the release of the ISF. We conclude that IL-1β is probably the ISF released by macrophages in response to toxin A.     

Tumor growth reduction is regulated at the gene level in Walker 256 tumor-bearing rats supplemented with fish oil rich in EPA and DHA

We investigated the effect of fish oil (FO) supplementation on tumor growth, cyclooxygenase 2 (COX-2), peroxisome proliferator-activated receptor gamma (PPARγ), and RelA gene and protein expression in Walker 256 tumor-bearing rats. Male Wistar rats (70 days old) were fed with regular chow (group W) or chow supplemented with 1 g/kg body weight FO daily (group WFO) until they reached 100 days of age. Both groups were then inoculated with a suspension of Walker 256 ascitic tumor cells (3×10⁷ cells/mL). After 14 days the rats were killed, total RNA was isolated from the tumor tissue, and relative mRNA expression was measured using the 2^-ΔΔCT method. FO significantly decreased tumor growth (W=13.18±1.58 vs WFO=5.40±0.88 g, P<0.05). FO supplementation also resulted in a significant decrease in COX-2 (W=100.1±1.62 vs WFO=59.39±5.53, P<0.001) and PPARγ (W=100.4±1.04 vs WFO=88.22±1.46, P<0.05) protein expression. Relative mRNA expression was W=1.06±0.022 vs WFO=0.31±0.04 (P<0.001) for COX-2, W=1.08±0.02 vs WFO=0.52±0.08 (P<0.001) for PPARγ, and W=1.04±0.02 vs WFO=0.82±0.04 (P<0.05) for RelA. FO reduced tumor growth by attenuating inflammatory gene expression associated with carcinogenesis.     

Toll-Like Receptor Activation by Generalized Modules for Membrane Antigens from Lipid A Mutants of Salmonella enterica Serovars Typhimurium and Enteritidis

Invasive nontyphoidal Salmonella (iNTS) disease is a neglected disease with high mortality in children and HIV-positive individuals in sub-Saharan Africa, caused primarily by Africa-specific strains of Salmonella enterica serovars Typhimurium and Enteritidis. A vaccine using GMMA (generalized modules for membrane antigens) from S. Typhimurium and S. Enteritidis containing lipid A modifications to reduce potential in vivo reactogenicity is under development. GMMA with penta-acylated lipid A showed the greatest reduction in the level of cytokine release from human peripheral blood monocytes from that for GMMA with wild-type lipid A. Deletion of the lipid A modification genes msbB and pagP was required to achieve pure penta-acylation. Interestingly, ΔmsbB ΔpagP GMMA from S. Enteritidis had a slightly higher stimulatory potential than those from S. Typhimurium, a finding consistent with the higher lipopolysaccharide (LPS) content and Toll-like receptor 2 (TLR2) stimulatory potential of the former. Also, TLR5 ligand flagellin was found in Salmonella GMMA. No relevant contribution to the stimulatory potential of GMMA was detected even when the flagellin protein FliC from S. Typhimurium was added at a concentration as high as 10% of total protein, suggesting that flagellin impurities are not a major factor for GMMA-mediated immune stimulation. Overall, the stimulatory potential of S. Typhimurium and S. Enteritidis ΔmsbB ΔpagP GMMA was close to that of Shigella sonnei GMMA, which are currently in phase I clinical trials.     

High-Throughput,Signature-Tagged Mutagenic Approach To Identify Novel Virulence Factors of Yersinia pestis CO92 in a Mouse Model of Infection

The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD₅₀) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD₅₀ when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD₅₀. Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD₅₀ in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD₅₀ in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the Δlpp single or the Δlpp ΔmsbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD₅₀. The mice infected with the Δlpp ΔmsbB ΔrbsA triple mutant at 50 LD₅₀ were 90% protected upon subsequent challenge with 12 LD₅₀ of WT CO92, suggesting that this mutant or others carrying combinational deletions of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s).     

Human Immunodeficiency Virus Type 1 Disease Progression, CCR5 Genotype, and Specific Immune Responses

The correlation among the presence of a 32-bp deletion in the CC-chemokine receptor 5 (CCR5) gene, disease progression, and human immunodeficiency virus type 1 (HIV-1)-specific immune responses was analyzed for a cohort of 79 Caucasian HIV-1-infected patients. The CCR5 genotype (CCR5/CCR5 = wild type/wild type or Δ32CCR5/CCR5 = 32-bp deletion/wild type) in peripheral blood mononuclear cells was determined by PCR, followed by sequencing of both wild-type and Δ32CCR5 gene fragments. HIV-1-specific humoral responses to gp41 and V3_MN peptides were determined by enzyme immunoassays. The prevalence of the Δ32CCR5 allele was lower among 37 patients with rapid progression (progression to AIDS or to a CD4 cell count of <200 × 10⁶/liter in less than 9 years; P < 0.01) compared to that for 42 patients with slow progression (no AIDS and CD4 cell count of >200 × 10⁶/liter after at least 9 years from infection) or to that for 25 non-HIV-1-infected Swedish blood donors (P < 0.05). No differences were observed in the wild-type CCR5 sequences between the different groups of patients. For three analyzed patients, the 32-bp Δ32CCR5 gene deletions were identical. The antibody titers against gp41 and a V3_MN peptide in patients with the Δ32CCR5/CCR5 genotype were not significantly different from those in pair-matched CCR5/CCR5 controls. However, in 13 analyzed patients, a stronger serum neutralizing activity was associated with the Δ32CCR5/CCR5 genotype. Thus, a CCR5/CCR5 genotype correlates with a shortened AIDS-free HIV-1 infection period and possibly with a worse neutralizing activity, without an evident influence on the antibody response to two major antigenic regions of HIV-1 envelope.     

Characteristics and Help-Seeking Behaviors of Internet Gamblers Based on Most Problematic Mode of Gambling

Background

Previous studies of problem Internet gamblers have failed to distinguish whether their problem gambling relates to Internet or land-based gambling modes. Therefore, characteristics and help-seeking behaviors of people whose gambling problems relate specifically to Internet gambling are unknown, but could inform the optimal alignment of treatment and support services with the needs and preferences of problem gamblers.

Objective

This study aimed to compare (1) characteristics of problem Internet gamblers and problem land-based gamblers and (2) uptake of different types and modes of help between problem Internet gamblers and problem land-based gamblers. Hypothesis 1 was that problem Internet gamblers are less likely to seek help. Hypothesis 2 was that problem Internet gamblers are more likely to use online modes of help.

Methods

A sample of 620 respondents meeting criteria for problem gambling was drawn from an online survey of 4594 Australian gamblers. Respondents were recruited through advertisements on gambling and gambling help websites, Facebook, and Google. Measures consisted of gambling participation; proportion of gambling on the Internet; most problematic mode of gambling; help seeking from 11 different sources of formal help, informal help, and self-help for gambling problems; psychological distress (Kessler 6); problem gambling severity (Problem Gambling Severity Index, PGSI); and demographics.

Results

Problem Internet gamblers were significantly more likely than problem land-based gamblers to be male (χ² ₁=28.3, P<.001, φ=0.21), younger (t _616.33=4.62, P<.001, d=0.37), have lower psychological distress (χ² ₁=5.4, P=.02, φ=0.09), and experience problems with sports and race wagering (χ² ₄=228.5, P<.001, φ=0.61). Uptake of help was significantly lower among problem Internet compared to problem land-based gamblers (χ² ₁=6.9, P<.001, φ=0.11), including from face-to-face services, gambling helplines, online groups, self-exclusion from land-based venues, family or friends, and self-help strategies. Both problem Internet and problem land-based gamblers had similarly low use of online help. However, problem land-based gamblers (37.6%, 126/335) were significantly more likely to have sought land-based formal help compared to problem Internet gamblers (23.5%, 67/285; χ² ₁=14.3, P<.001, φ=0.15).

Conclusions

The findings suggest that more targeted and innovative efforts may be needed to increase use of gambling help by problem Internet gamblers. Alternatively, their lower PGSI and K6 scores suggest Internet problem gamblers may have less need for gambling-related help. This is the first known study to classify problem Internet gamblers as those whose problem gambling specifically relates to Internet gambling. Further research is needed to better understand why help-seeking rates are lower among Internet problem gamblers.     

Who Follows eHealth Interventions as Recommended? A Study of Participants' Personal Characteristics From the Experimental Arm of a Randomized Controlled Trial

Background

Computer-tailored eHealth interventions to improve health behavior have been demonstrated to be effective and cost-effective if they are used as recommended. However, different subgroups may use the Internet differently, which might also affect intervention use and effectiveness. To date, there is little research available depicting whether adherence to intervention recommendations differs according to personal characteristics.

Objective

The aim was to assess which personal characteristics are associated with using an eHealth intervention as recommended.

Methods

A randomized controlled trial was conducted among a sample of the adult Dutch population (N=1638) testing an intervention aimed at improving 5 healthy lifestyle behaviors: increasing fruit and vegetable consumption, increasing physical activity, reducing alcohol intake, and promoting smoking cessation. Participants were asked to participate in those specific online modules for which they did not meet the national guideline(s) for the respective behavior(s). Participants who started with fewer than the recommended number of modules of the intervention were defined as users who did not follow the intervention recommendation.

Results

The fewer modules recommended to participants, the better participants adhered to the intervention modules. Following the intervention recommendation increased when participants were older (χ² ₁=39.8, P<.001), female (χ² ₁=15.8, P<.001), unemployed (χ² ₁=7.9, P=.003), ill (χ² ₁=4.5, P=.02), or in a relationship (χ² ₁=7.8, P=.003). No significant relevant differences were found between groups with different levels of education, incomes, or quality of life.

Conclusion

Our findings indicate that eHealth interventions were used differently by subgroups. The more frequent as-recommended intervention use by unemployed, older, and ill participants may be an indication that these eHealth interventions are attractive to people with a greater need for health care information. Further research is necessary to make intervention use more attractive for people with unhealthy lifestyle patterns.     

IL12Rβ1ΔTM Is a Secreted Product of il12rb1 That Promotes Control of Extrapulmonary Tuberculosis

IL12RB1 is a human gene that is important for resistance to Mycobacterium tuberculosis infection. IL12RB1 is expressed by multiple leukocyte lineages, and encodes a type I transmembrane protein (IL12Rβ1) that associates with IL12p40 and promotes the development of host-protective T_H1cells. Recently, we observed that il12rb1—the mouse homolog of IL12RB1—is alternatively spliced by leukocytes to produce a second isoform (IL12Rβ1ΔTM) that has biological properties distinct from IL12Rβ1. Although the expression of IL12Rβ1ΔTM is elicited by M. tuberculosis in vivo, and its overexpression enhances IL12p40 responsiveness in vitro, the contribution of IL12Rβ1ΔTM to controlling M. tuberculosis infection has not been tested. Here, we demonstrate that IL12Rβ1ΔTM represents a secreted product of il12rb1 that, when absent from mice, compromises their ability to control M. tuberculosis infection in extrapulmonary organs. Furthermore, elevated M. tuberculosis burdens in IL12Rβ1ΔTM-deficient animals are associated with decreased lymph node cellularity and a decline in T_H1 development. Collectively, these data support a model wherein IL12Rβ1ΔTM is a secreted product of il12rb1 that promotes resistance to M. tuberculosis infection by potentiating T_H cells response to IL-12.     

Cardiovascular effects of the intracerebroventricular injection of adrenomedullin: roles of the peripheral vasopressin and central cholinergic systems

Our objective was to investigate in conscious Sprague-Dawley (6-8 weeks, 250-300 g) female rats (N = 7 in each group) the effects of intracerebroventricularly (icv) injected adrenomedullin (ADM) on blood pressure and heart rate (HR), and to determine if ADM and calcitonin gene-related peptide (CGRP) receptors, peripheral V₁ receptors or the central cholinergic system play roles in these cardiovascular effects. Blood pressure and HR were observed before and for 30 min following drug injections. The following results were obtained: 1) icv ADM (750 ng/10 µL) caused an increase in both blood pressure and HR (ΔMAP = 11.8 ± 2.3 mmHg and ΔHR = 39.7 ± 4.8 bpm). 2) Pretreatment with a CGRP receptor antagonist (CGRP_8-37) and ADM receptor antagonist (ADM_22-52) blocked the effect of central ADM on blood pressure and HR. 3) The nicotinic receptor antagonist mecamylamine (25 µg/10 µL, icv) and the muscarinic receptor antagonist atropine (5 µg/10 µL, icv) prevented the stimulating effect of ADM on blood pressure. The effect of ADM on HR was blocked only by atropine (5 µg/10 µL, icv). 4) The V₁ receptor antagonist [β-mercapto-β-β-cyclopentamethylenepropionyl¹, O-me-Tyr²,Arg⁸]-vasopressin (V2255; 10 µg/kg), that was applied intravenously, prevented the effect of ADM on blood pressure and HR. This is the first study reporting the role of specific ADM and CGRP receptors, especially the role of nicotinic and muscarinic central cholinergic receptors and the role of peripheral V₁ receptors in the increasing effects of icv ADM on blood pressure and HR.     

Two Novel Functions of Hyaluronidase from Streptococcus agalactiae Are Enhanced Intracellular Survival and Inhibition of Proinflammatory Cytokine Expression

Streptococcus agalactiae is the causative agent of septicemia and meningitis in fish. Previous studies have shown that hyaluronidase (Hyl) is an important virulence factor in many Gram-positive bacteria. To investigate the role of S. agalactiae Hyl during interaction with macrophages, we inactivated the gene encoding extracellular hyaluronidase, hylB, in a clinical Hyl⁺ isolate. The isogenic hylb mutant (Δhylb) displayed reduced survival in macrophages compared to the wild type and stimulated a significantly higher release of proinflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), than the wild type in macrophages as well as in mice. Furthermore, only Hyl⁺ strains could grow utilizing hyaluronic acid (HA) as the sole carbon source, suggesting that Hyl permits the organism to utilize host HA as an energy source. Fifty percent lethal dose (LD₅₀) determinations in zebrafish demonstrated that the hylb mutant was highly attenuated relative to the wild-type strain. Experimental infection of BALB/c mice revealed that bacterial loads in the blood, spleen, and brain at 16 h postinfection were significantly reduced in the ΔhylB mutant compared to those in wild-type-infected mice. In conclusion, hyaluronidase has a strong influence on the intracellular survival of S. agalactiae and proinflammatory cytokine expression, suggesting that it plays a key role in S. agalactiae pathogenicity.     

Contribution of the SitABCD, MntH, and FeoB metal transporters to the virulence of avian pathogenic Escherichia coli O78 strain chi7122

The roles of SitABCD, MntH, and FeoB metal transporters in the virulence of avian pathogenic Escherichia coli (APEC) O78 strain χ7122 were assessed using isogenic mutants in chicken infection models. In a single-strain infection model, compared to χ7122, the Δsit strain demonstrated reduced colonization of the lungs, liver, and spleen. Complementation of the Δsit strain restored virulence. In a coinfection model, compared to the virulent APEC strain, the Δsit strain demonstrated mean 50-fold, 126-fold, and 25-fold decreases in colonization of the lungs, liver, and spleen, respectively. A ΔmntH Δsit strain was further attenuated, demonstrating reduced persistence in blood and mean 1,400-fold, 954-fold, and 83-fold reduced colonization in the lungs, liver, and spleen, respectively. In coinfections, the ΔfeoB Δsit strain demonstrated reduced persistence in blood but increased colonization of the liver. The ΔmntH, ΔfeoB, and ΔfeoB ΔmntH strains were as virulent as the wild type in either of the infection models. Strains were also tested for sensitivity to oxidative stress-generating agents. The ΔmntH Δsit strain was the most sensitive strain and was significantly more sensitive than the other strains to hydrogen peroxide, plumbagin, and paraquat. sit sequences were highly associated with APEC and human extraintestinal pathogenic E. coli compared to commensal isolates and diarrheagenic E. coli. Comparative genomic analyses also demonstrated that sit sequences are carried on conjugative plasmids or associated with phage elements and were likely acquired by distinct genetic events among pathogenic E. coli and Shigella sp. strains. Overall, the results demonstrate that SitABCD contributes to virulence and, together with MntH, to increased resistance to oxidative stress.     

Is there any association between leptin levels and bone mineral density in haemophiliac men?

Introduction

Conflicting data exist regarding the role of leptin in bone metabolism. The purpose of the present study was to investigate serum leptin concentrations in male patients with haemophilia A and B, a disease known to be associated with low bone mass.

Material and methods

Eighty-one male patients, aged 45.4 ±15 years, were screened. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA) in lumbar spine (LS), femoral neck (FN) and total hip (TH).

Results

Low bone mass was diagnosed in 20 patients (24.7%). Serum leptin concentrations were strongly associated with body weight (r _s = 0.457, p = 0.0001) and body mass index (BMI) (r _s = 0.491, p = 0.0001). In unadjusted analysis leptin was inversely associated with BMD in LS (r _s = –0.255, p = 0.023), but not in FN and TH (r _s = –0.205, p = 0.068 and r _s = –0.191, p = 0.090, respectively). However, after adjusting for BMI and body weight, leptin was inversely associated with BMD in FN (F _1,76 = 7.727, p = 0.007, β = –0.371, ΔR ² = 0.089) and TH (F _1,76 = 4.533, p = 0.036, β = –0.290, ΔR ² = 0.054), but not in LS (F _1,75 = 2.076, p = 0.154, β = –0.202, ΔR ² = 0.026). No association was found between age, presence of HBV, HCV or HIV infection or alkaline phosphatase and leptin levels.

Conclusions

Our study showed a negative association between circulating leptin levels and bone mass in males, independently of body weight and BMI.