A certain number of studies were carried out to address the question of how dysbiosis could affect the onset and development of rheumatoid arthritis (RA), but little is known about the reciprocal influence between microbiota composition and immunosuppressive drugs, and how this interaction may have an impact on the clinical outcome. The aim of this study was to characterize the intestinal microbiota in a groups of RA patients treatment-naïve, under methotrexate, and/or etanercept (ETN). Correlations between the gut microbiota composition and validated immunological and clinical parameters of disease activity were also evaluated. In the current study, a 16S analysis was employed to explore the gut microbiota of 42 patients affected by RA and 10 healthy controls. Disease activity score on 28 joints (DAS-28), erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, anti-cyclic citrullinated peptides, and dietary and smoking habits were assessed. The composition of the gut microbiota in RA patients free of therapy is characterized by several abnormalities compared to healthy controls. Gut dysbiosis in RA patients is associated with different serological and clinical parameters; in particular, the phylum of Euryarchaeota was directly correlated to DAS and emerged as an independent risk factor. Patients under treatment with ETN present a partial restoration of a beneficial microbiota. The results of our study confirm that gut dysbiosis is a hallmark of the disease, and shows, for the first time, that the anti-tumor necrosis factor alpha (TNF-α) ETN is able to modify microbial communities, at least partially restoring a beneficial microbiota.

Background: The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. Results: Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. Conclusion: In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology.

This report describes a new flow cytometry technique to measure phagocytic activity and discriminate simultaneously between internalized and membranebound particles. Fluorescein‐conjugated heat‐killed Candida albicans (F‐Ca) are opsonized with purified antibodies or normal human serum and used as targets for human polymorphonuclear granulocytes (PMN). The procedure is based on the observation that F‐Ca lose their green fluorescence and acquire red fluorescence upon incubation with ethidium bromide (EB) through the resonance energy‐transfer phenomenon occurring between the two fluorochromes. PMN are incubated with opsonized F‐Ca particles for 20 min at 37°C or, as a control, at 4°C and in the presence of cytochalasin B, an inhibitor of the phagocytic process that does not affect membrane binding of F‐Ca. EB is added, and green and red fluorescence associated with PMN is evaluated using a mercury‐lamp‐powered instrument. Because EB does not penetrate intact cell membranes, internalized particles are not affected by EB and remain green, whereas membrane‐bound particles assume an intense red stain. By means of contour plot analysis, the number of PMN containing and/or binding F‐Ca particles can be readily assessed. The method described here allows precise quantitative analysis of the phagocytic process on the part of human PMN in a single, one‐step assay that does not require sophisticated instrumentation or reagents and should prove to become a test suitable for clinical purposes.

Infections which complicate rheumatic diseases such as Rheumatoid Arthritis (RA) and Spondyloarthropathy (SpA) (Psoriatic Arthritis [PA] and Ankylosing Spondylitis [AS]), may cause significant morbidity and mortality. However, among the studies on the incidence rate (IR) of infections in such patients, very few have involved controls and the results have been controversial, probably due to methodological difficulties.

In higher vertebrates, mucosal sites at the border between the internal and external environments, directly interact with bacteria, viruses, and fungi. Through co-evolution, hosts developed mechanisms of tolerance or ignorance toward some infectious agents, because hosts established “gain of function” interactions with symbiotic bacteria. Indeed, some bacteria assist hosts in different functions, among which are digestion of complex carbohydrates, and absorption and supply of vitamins. There is no doubt that microbiota modulate innate and acquired immune responses starting at birth. However, variations in quality and quantity of bacterial species interfere with the equilibrium between inflammation and tolerance. In fact, correlations between gut bacteria composition and the severity of inflammation were first described for inflammatory bowel diseases and later extended to other pathologies. The genetic background, environmental factors (e.g., stress or smoking), and diet can induce strong changes in the resident bacteria which can expose the intestinal epithelium to a variety of different metabolites, many of which have unknown functions and consequences. In addition, alterations in gut permeability may allow pathogens entry, thereby triggering infection and/or chronic inflammation. In this context, a local event occurring at a mucosal site may be the triggering cause of an autoimmune reaction that eventually involves distant sites or organs. Recently, several studies attributed a pathogenic role to altered oral microbiota in rheumatoid arthritis (RA) and to gut dysbiosis in spondyloarthritis (SpA). There is also growing evidence that different drugs, such as antibiotics and immunosuppressants, can influence and be influenced by the diversity and composition of microbiota in RA and SpA patients. Hence, in complex disorders such RA and SpA, not only the genetic background, gender, and immunologic context of the individual are relevant, but also the history of infections and the structure of the microbial community at mucosal sites should be considered. Here the role of the microbiota and infections in the initiation and progression of chronic arthritis is discussed, as well as how these factors can influence a patient’s response to synthetic and biologic immunosuppressive therapy.