YopP-expressing variant of Y. pestis activates a potent innate immune response affording cross-protection against yersiniosis and tularemia [corrected].

Plague, initiated by Yersinia pestis infection, is a rapidly progressing disease with a high mortality rate if not quickly treated. The existence of antibiotic-resistant Y. pestis strains emphasizes the need for the development of novel countermeasures against plague. We previously reported the generation of a recombinant Y. pestis strain (Kim53ΔJ+P) that over-expresses Y. enterocolitica YopP. When this strain was administered subcutaneously to mice, it elicited a fast and effective protective immune response in models of bubonic, pneumonic and septicemic plague. In the present study, we further characterized the immune response induced by the Kim53ΔJ+P recombinant strain. Using a panel of mouse strains defective in specific immune functions, we observed the induction of a prompt protective innate immune response that was interferon-γ dependent. Moreover, inoculation of mice with Y. pestis Kim53ΔJ+P elicited a rapid protective response against secondary infection by other bacterial pathogens, including the enteropathogen Y. enterocolitica and the respiratory pathogen Francisella tularensis. Thus, the development of new therapies to enhance the innate immune response may provide an initial critical delay in disease progression following the exposure to highly virulent bacterial pathogens, extending the time window for successful treatment.

Frequency Analyses Can Be Improved by a Modified t-test in Sample-based Preclinical Efficacy Studies.

Sample-based preclinical drug efficacy studies compare frequency (proportion) or incidences of successes within respective samples of test and control groups. The word success in principle refers to a protected (e.g., due to vaccination), recovered, or surviving animal, depending on the particular experiment. We introduce here a modified t-test for two independent groups, aimed at statistical analysis of the difference between frequencies of successes in sample based preclinical studies. The test is applicable whenever the study is based on repeating replicate experiments, as required by certain procedures such as validation. Such experiments are based on constant drug dose and performed under identical conditions and protocol. The proposed test combines the computational rules of t-test for two independent groups and analysis of variance. In the initial steps, incidences are transformed to proportions, and variance between proportions in samples of the j(th) group (s(p(j))(2)), is then transformed into theoretical weighted variance within the i(th) repetition (sample) of the j(th) group (s(i,j)(2)). The variance of proportions in samples of the size of the whole group (SE(j)(2)) is then calculated. The t-statistic is computed according to the rules of t-test for two independent groups. Significance is calculated using (N(1) - 1) + (N(2) - 1) degrees of freedom, where N(j) denotes the total number of animals in the j(th) group. The proposed model offers an important advantage over incidence or proportion distribution models, such as chi-square or normal approximation of binomial distribution, respectively, because it considers variance between replicate experiments. It moreover offers important flexibility by limiting the requirement for identical sample sizes only to samples within the control or test group. A difference between groups in sample sizes, number of samples, or both, preventing application of block designs or the standard formats of t-test, may still exist. Theoretical considerations and working examples are provided. LAY ABSTRACT: Sample based preclinical drug efficacy studies compare frequency (proportion) or incidences of successful results (e.g., protected, recovered, or surviving animals, depending on the particular experiment) within respective samples of the test and the control groups. Certain procedures, such as validation, require replicate experiments that are identical in all controllable factors, such as drug dose, sample sizes within each group, general experimental conditions, etc. Still, the control sample size is not required to be identical to that of the respective test sample size. In such cases, t-test or block designs are not applicable for statistical analyses. Moreover, incidence or frequency distribution models, such as chi-square or normal approximation of binomial distribution, respectively, which are performed on pooled data of the examined groups, ignore variance between experiments and thereby result in impaired validity of the statistical inference. We propose here a modified t-test that limits the requirement for identical sample sizes to only within each group. This aim is achieved by combining the computational rules of t-test together with analysis of variance. The proposed t-test allows the incorporating of variance between experiments into frequency or incidence assessments. We recommend using the proposed modified t-test as a complementary test to incidence distribution models.

A Lecinoxoid, an oxidized phospholipid small molecule, constrains CNS autoimmune disease.

Oxidized phospholipids (Ox-PLs) are generated in abundance at sites of inflammation. Recent studies have indicated that Ox-PLs may also exhibit anti-inflammatory activities. In this study, we investigated the beneficial effect of VB-201, a pure synthetic Ox-PL analog that we synthesized, on the development of a central nervous system (CNS) autoimmune inflammatory disease, in vivo. Oral administration of VB-201 ameliorated the severity of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide MOG35-55, and restrained the encephalogenicity of MOG35-55-specific T-cells. Our data presents a novel prospect for the role of Ox-PL analogs in CNS inflammatory diseases.

The search for early markers of plague: evidence for accumulation of soluble Yersinia pestis LcrV in bubonic and pneumonic mouse models of disease.

Markers of the early stages of plague, a rapidly progressing deadly disease, are crucial for enabling the onset of an effective treatment. Here, we show that V-antigen protein (LcrV) is accumulated in the serum of Yersinia pestis-infected mice before bacterial colonization of the spleen and dissemination to blood, in a model of bubonic plague. LcrV accumulation is detected earlier than that of F1 capsular antigen, an established marker of disease. In a mouse model of pneumonic plague, LcrV can be determined in the bronchoalveolar lavage fluid somewhat later than F1, but before dissemination of Y. pestis to the blood. Thus, determination of soluble LcrV is suggested as a potential useful tool for monitoring disease progression in both bubonic and pneumonic plague. Moreover, it may be of particular advantage in cases of infections with F1 nonproducing strains.

Yersinia pestis endowed with increased cytotoxicity is avirulent in a bubonic plague model and induces rapid protection against pneumonic plague.

An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica Oratio8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10(7)-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD(50) of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.

Neutralization of Yersinia pestis-mediated macrophage cytotoxicity by anti-LcrV antibodies and its correlation with protective immunity in a mouse model of bubonic plague.

Plague is a life-threatening disease caused by Yersinia pestis, for which effective-licensed vaccines and reliable predictors of in vivo immunity are lacking. V antigen (LcrV) is a major Y. pestis virulence factor that mediates translocation of the cytotoxic Yersinia protein effectors (Yops). It is a well-established protective antigen and a part of currently tested plague subunit vaccines. We have developed a highly sensitive in vitro macrophage cytotoxicity neutralization assay which is mediated by anti-LcrV antibodies; and studied the potential use of these neutralizing antibodies as an in vitro correlate of plague immunity in mice. The assay is based on a Y. pestis strain with enhanced cytotoxicity to macrophages in which endogenous yopJ was replaced by the more effectively translocated yopP of Y. enterocolitica O:8. Mice passively immunized with rabbit anti-LcrV IgG or actively immunized with recombinant LcrV were protected against lethal doses of a virulent Y. pestis strain, in a mouse model of bubonic plague. This protection significantly correlated with the in vitro neutralizing activity of the antisera but not with their corresponding ELISA titers. In actively immunized mice, a cutoff value for serum neutralizing activity, above which survival was assured with high degree of confidence, could be established for different vaccination regimes. The impact of overall findings on the potential use of serum neutralizing activity as a correlate of protective immunity is discussed.

Effects of oleic acid and macrophage recruitment on cholesterol efflux in cell culture and in vivo.

BACKGROUND AND AIM: Monounsaturated fatty acids in diets are beneficial for the plasma lipoprotein profile, but studies in cell culture point out that they may also be detrimental by inhibiting cholesterol efflux to apo AI. METHODS AND RESULTS: In the present study we used mouse peritoneal macrophages, loaded with cholesterol and upregulated by cyclic AMP or by LXR/RXR ligands and compared the effect of oleic acid on cholesterol efflux to 3 different acceptors. Inhibition of cholesterol efflux by oleic acid ranged from 10 to 25% with HDL or 2.5% mouse serum, while efflux to phosphatidyl choline vesicles was not affected. Previously we reported that the LXR ligand, TO901317, retarded cholesterol removal in vivo from a modified LDL depot in muscle. This could have resulted from inhibition by unsaturated fatty acids or from reduction in macrophage recruitment due to the anti-inflammatory action of LXR. CONCLUSIONS: Our current findings, of retardation of cholesterol clearance from the depot in the presence of low macrophage recruitment, support the latter possibility.

Lower macrophage recruitment and atherosclerosis resistance in FVB mice.

The aim of this study was to compare some aspects of cholesterol accretion and cholesterol efflux in cellular components of the aortic wall derived from mice resistant or susceptible to atherosclerosis, FVB or C57BL, respectively. Cholesterol efflux, from cholesterol loaded smooth muscle cells or elicited macrophages, to apo A-I or HDL was similar in the two strains under basal conditions, and after cAMP or LXR upregulation. Recruitment of peritoneal macrophages, 3 days after thioglycollate injection, was 65% lower in FVB than in C57BL mice, commensurate with a 40% reduction in MCP-1 in peritoneal lavage. In additional three atherosclerosis resistant strains, NZB, A/J and 129(SvJ), macrophage recruitment was reduced to a similar extent despite high MCP-1 levels. Since impaired macrophage recruitment in CCR2(-/-) or MCP-1(-/-) C57BL mice was reported to reduce atherosclerosis, it seems plausible that in some mouse strains reduction in macrophage mobilization could contribute to atherosclerosis resistance.

LXR activation and cholesterol efflux from a lipoprotein depot in vivo.

Activation of LXR in cultured cells results in enhancement of cholesterol efflux to apo Al. To study cholesterol efflux, in vivo cationized LDL was injected into the rectus femoris muscle of mice to create a lipoprotein depot. LXR ligand TO901317, 10 mg/kg, was given by gavage for 8 days, starting 4 days after injection of the lipoprotein. The rate of cholesterol efflux from the depot was compared in treated and control mice. Administration of the ligand resulted in a 70% increase in plasma cholesterol and 40% in phospholipids, but HDL-cholesterol and HDL-phospholipids increased by 43% and 24% only. Efflux of the injected cholesterol from the lipoprotein depot of treated mice was not enhanced but even somewhat delayed. This impairment was unexpected and its cause could be multifactorial. A plausible explanation seems that induced hypercholesterolemia, and a decrease in HDL-cholesterol to total cholesterol ratio, delayed the clearance.

In CCR2-/- mice monocyte recruitment and egress of LDL cholesterol in vivo is impaired.

Recruitment of macrophages plays an important role in initiation of atheroma, but their involvement in cholesterol clearance during regression is unknown. We developed a mouse model to quantitate cholesterol clearance from a depot of cationized LDL injected into a leg muscle, which evokes a sterile inflammatory reaction. In the CCR2(-/-) mice, cholesterol clearance was significantly slower than in C57BL controls because of decrease in cholesteryl ester (CE) hydrolysis, which is mandatory prior to cholesterol efflux. In CCR2(-/-) mice, macrophage recruitment to the injected site, identified by immunohistochemistry, was markedly delayed. CE hydrolysis was also significantly reduced in thioglycollate elicited peritoneal exudate cells of CCR2(-/-) mice, related to paucity of macrophages in the cell differential. The present study provides definite evidence that recruitment of macrophages is required for LDL cholesterol clearance, which plays a prominent role in regression of an atheroma.