We performed a genome-wide siRNA screen to identify host factors that regulated pathogen load in human macrophages infected with a virulent strain of Mycobacterium tuberculosis. Iterative rounds of confirmation, followed by validation, identified 275 such molecules that were all found to functionally associate with each other through a dense network of interactions. This network then yielded to a molecular description of the host cell functional modules that were both engaged and perturbed by the pathogen. Importantly, a subscreen against a panel of field isolates revealed that the molecular composition of the host interface varied with both genotype and the phenotypic properties of the pathogen. An analysis of these differences, however, permitted identification of those host factors that were invariantly involved, regardless of the diversification in adaptive mechanisms employed by the pathogen. Interestingly, these factors were found to predominantly function through the regulation of autophagy.

Staphylococcal enterotoxins (SE) can cause toxin-mediated disease, and those that function as superantigens are implicated in the pathogenesis of allergic diseases. The prevalence of 19 enterotoxin genes was determined by PCR in clinical S. aureus strains derived from wounds (108) and blood (99). We performed spa typing and multilocus sequence typing (MLST) to determine clonal origin, and for selected strains staphylococcal enterotoxin B (SEB) production was measured by enzyme-linked immunosorbent assay. Strains carried a median of five SE genes. For most SE genes, the prevalence rates among methicillinresistant and methicillin-sensitive S. aureus isolates, as well as wound-and blood-derived isolates, did not differ. At least one SE gene was detected in all except two S. aureus isolates (>99%). Complete egc clusters were found in only 11% of S. aureus isolates, whereas the combination of sed, sej, and ser was detected in 24% of clinical strains. S. aureus strains exhibited distinct combinations of SE genes, even if their pulsed-field gel electrophoresis and MLST patterns demonstrated clonality. USA300 strains also showed considerable variability in SE content, although they contained a lower number of SE genes (mean, 3). By contrast, SE content was unchanged in five pairs of serial isolates. SEB production by individual strains varied up to 200-fold, and even up to 15-fold in a pair of serial isolates. In conclusion, our results illustrate the genetic diversity of S. aureus strains with respect to enterotoxin genes and suggest that horizontal transfer of mobile genetic elements encoding virulence genes occurs frequently.As a commensal, Staphylococcus aureus colonizes the nasal mucosa of 20 to 40% of humans (54), and as a pathogen it causes pyogenic diseases and toxin-mediated diseases (38). S. aureus produces many different virulence factors, including enterotoxins (SEs), which can cause defined toxic shock syndromes (4). The characterization of some of these toxins led to the discovery of superantigens (41), which bind to major histocompatibility complex class II molecules and V␤ chains of T-cell receptors, resulting in the activation of large numbers of T cells (20 to 30%) and massive cytokine production (10, 18). These superantigen-induced "cytokine storms" are responsible for the toxic effects seen in staphylococcal entertoxin B (SEB)-and toxic shock syndrome toxin (TSST)-associated shock syndromes in S. aureus infections (13, 40, 47). To date, 19 SEs have been identified based on sequence homologies, and studies have reported enterotoxin genes in up to 80% of all S. aureus strains (4, 21). Although many new enterotoxins have been identified, i.e., seg ser and seu (33,37,44,49), their precise functions have not been characterized yet. The majority of experimental work with SEs is still done with SEB, toxic shock syndrome toxin 1, and SEA (27, 31), because these toxins are commercially available. Most SEs are located on mobile elements in bacterial genomes such as plasmids or pathogenicity islands and can thu...

T-cell stimulating activity of Staphylococcal enterotoxin B (SEB) is an important factor in the pathogenesis of certain staphylococcal diseases including SEB mediated shock. SEB is one of the most potent superantigens known and treatment of SEB induced shock remains a challenge. We generated and characterized murine monoclonal antibodies (mAbs) to SEB in mice. We tested mAbs neutralize mitogenic effects of SEB in vitro and in vivo with T-cell proliferation assays and 2 murine models for SEB induced lethal shock (SEBILS). Epitope mapping suggests that all these mAbs recognize conformational epitopes that are destroyed by deleting the C terminus of the protein. Further site-directed mutagenesis identified potential residues involved in binding to SEB that differ between Methicillin resistant and sensitive Staphylococcus aureus strains. Only mAb 20B1 was effective as a monotherapy in treating SEBILS in HLA DR3 transgenic mice, which exhibit enhanced sensitivity to SEB. It is noteworthy that mAbs, 14G8 and 6D3 were not protective when given alone in the HLA DR3 mice but their efficacy of protection could be greatly enhanced when mAbs were co-administered simultaneously. Our data suggest combinations of defined mAbs may constitute a better treatment strategy and provide a new insight for the development of passive immunotherapy. The Staphylococcal enterotoxins (SEs)2 comprise a family of distinct toxins (A-E) all of which are excreted by various strains of Staphylococcus aureus (S. aureus) (1). Staphylococcal enterotoxin B (SEB) is a well characterized 28 kDa protein that is related to SEC1-3 on the basis of sequence homology (1, 2). SEB is a superantigen that triggers cytokine production and T-cell proliferation by cross-linking MHC class II molecules on antigen presenting cells and T-cell receptors (TCR) (2-5). In humans, SEB can trigger toxic shock, profound hypotension and multi-organ failure. SEB is the major enterotoxin associated with non-menstrual toxic shock syndrome and accounts for the majority of intoxications that are not caused by toxic shock syndrome toxin 1 (TSST-1). In addition, some reports indicate that SEB induces an IgE response and thereby might contribute to the pathogenesis of asthma, chronic rhinitis, and dermatitis (6 -9). SEB is considered a select agent. The quantities needed to produce a desired effect are much lower than with synthetic chemicals. Also SEB can be easily produced in large quantities (10).Currently there are no therapies available for treating enterotoxin-induced shock, but clinical data suggests that immunoglobulins can alleviate disease (11). Moreover, passive administration of pooled human immunoglobulin, as well as murine and chicken antibodies (Abs) can protect against SEB induced lethal shock (SEBILS) in murine and primate animal models as well as against SEB triggered release of cytokines by SEB stimulated T-cells (12, 13). The efficacy of humoral immunity in protection against SEB was established by demonstrating an inverse relationship between susceptibility and an...

Staphylococcus aureus can cause devastating and life-threatening infections. With the increase in multidrug resistant strains, novel therapies are needed. Limited success with active and passive immunization strategies have been attributed to S. aureus immune evasion. Here, we report on a monoclonal antibody, 514G3, that circumvents a key S. aureus evasion mechanism by targeting the cell wall moiety Protein A (SpA). SpA tightly binds most subclasses of immunoglobulins via their Fc region, neutralizing effector function. The organism can thus shield itself with a protective coat of serum antibodies and render humoral immunity ineffective. The present antibody reactivity was derived from an individual with natural anti-SpA antibody titers. The monoclonal antibody is of an IgG3 subclass, which differs critically from other immunoglobulin subclasses since its Fc is not bound by SpA. Moreover, it targets a unique epitope on SpA that allows it to bind in the presence of serum antibodies. Consequently, the antibody opsonizes S. aureus and maintains effector function to enable natural immune mediated clearance. The data presented here provide evidence that 514G3 antibody is able to successfully rescue mice from S. aureus mediated bacteremia.

mAb 20B1, an SEB-neutralizing mAb, is effective against MRSA infection. mAb 20B1 protects against lethal sepsis and reduces skin tissue invasion and deep-abscess formation. The mAb penetrates well into the abscess and binds to SEB. It affects the outcome of S. aureus infection by modulating the host's proinflammatory immune response.

Exploring gender-specific metabolic differences in biofluids provides a basic understanding of the physiological and metabolic phenotype of healthy subjects. Many reports have shown gender-specific metabolome profiles in the urine and serum of healthy subjects; however, limited studies focusing on exhaled human breath are available in the literature. In this study, we profiled the exhaled breath (~450 mL) volatile organic compounds (VOCs) of 47 healthy volunteers (age: 19-47; 23 male (M) and 24 female (F)) using a multidimensional gas chromatography and mass spectrometry and employed chemometric analysis to identify gender-specific VOCs. Eleven exhaled breath VOCs were identified from both uni and multivariate analysis from a training set (M = 15, F = 15) that could differentiate the genders within a healthy population. A partial least-squares discriminate analysis (PLS-DA) model built using these putative markers showed high accuracy in predicting (area under the receiver operating characteristic curve >0.9) a hold out/test sample set (n = 17). The outcomes of this report open up new avenues to undertake larger studies to elucidate the association of exhaled breath metabolites with gender-specific disease phenotypes and pharmacokinetics in the future.

Background: Staphylococcal enterotoxin B is a potent superantigen that causes lethal toxic shock syndrome. Results: Ternary and binary complex of SEB with SEB specific mAbs identified three distinct epitopes. Conclusion: Two different mechanisms illustrate how cocktails of mAbs enhance neutralization efficacy. Significance: SEB neutralization via combination of mAbs is superior to monotherapy and can include non-neutralizing mAbs.

Remitting Seronegative Symmetrical Synovitis with Pitting Edema (RS3PE) is a rare clinical entity that is easily missed due to lack of knowledge. It was formerly considered as a subset of rheumatoid arthritis (RA), but is now regarded as a distinct disease/syndrome. The diagnosis of RS3PE is not easy, as it is always hindered by the lack of definite diagnostic criteria and presence of other much common rheumatological disorders that mimic it. We report a series of seven cases that attended our clinic in the last year, which highlight the salient features of the disease. The disease was found to have a heterogeneous presentation. Immunogenetic, clinical, laboratory, radiological, and possible etiological factors and associations with the neoplasm are described, as also other peculiar presentations. Finally, a comparison with other common rheumatological disorders is made to alert the clinician about this rare, but easily treatable disease.