Vaccination with endosomal unknown epitopes produces therapeutic response in rheumatoid arthritis patients and modulates adjuvant arthritis of rats

BACKGROUND: Our previous results showed that intrasynovial Rifamycin SV caused the lysis of synoviocites and freed the autoantigens which in turn stimulated the immunoregulatory rather than autoreactive T cell response in rheumatoid patients. Here, we hypothesize that disruption in vitro of peripheral blood mononuclear cells, by freeze/thawing or by lytic action of Rifamycin SV, would induce the release of endosomal pathogenic autoantigens from APCs present in the circulation, which could then be isolated from degrading enzymes by ultrafiltration. METHODS: The preparation of the ultrafiltrates are based on the rupture of PBMCs (5 7 10(6) cells/mL) by the addition of Rifamycin SV in culture (250 \u3bcg/mL), which causes the lysis of 90 % of the cells in 3 h, or by three cycles of freeze/thawing of the PBMC, from -80 \ub0C to room temperature. The lysate and the fragmented cells were then centrifuged and ultrafiltered by passage through a filtration device with a cut-off of 10 kDa. Also the synovial fluid was subjected to ultrafiltration. RESULTS AND CONCLUSIONS: At clinical monitoring of the 30th day, 22/58 (38 %) patients subcutaneously treated with the autologous ultrafiltrate prepared by the freeze/thawing of PBMCs reached an ACR20. Comparable results were obtained with the other two ultrafiltrates. Cell cultures The addition of ultrafiltrates to rheumatoid PBMCs cultures led to the upregulation of a marker for T-regulatory cells, and downregulation of a cell proliferation marker; changes that together have the meaning of a global immunomodulatory response and that only a specific antigen (ultrafiltrate UF-f/t) might induce in the rheumatoid patient, probably by activating pre-existing protective network. Experimental arthritis All the ultrafiltrates except that prepared by Rifamycin SV were able to modulate the adjuvant arthritis in rats. In particular, longlasting synovial fluid induced a significant reduction of the severity of subsequent arthritis (p < 0.01) while SF from recent RA effusion (5-10 days after a previous complete extraction) and knee osteoarthrosis were ineffective. It is reasonable to assume there are at least two unknown endosomal immunoactive epitopes; one developing its immunotherapeutic property in RA, and the other, related to the molecule of HSP60, reduces the severity of oncoming arthritis. Both epitopes are present in humans, have a molecular weight of 6410 kDa and do not appear to be bystander antigens. Please see Additional file 1 for the abstract in Italian

Imported dengue fever: a 16-years retrospective analysis in Milan (Italy) and a brief review of the European literature

Dengue Fever (DF), transmitted by Aedes mosquitoes, is the most common arthropod-borne infection, it is almost ubiquitous in tropical and subtropical areas with an estimate of 360 million infections per year. A competent vector (A. albopictus) is present in most of Southern Europe and is endemic in Italy. We conducted a 16-year retrospective study of probable/confirmed dengue fever observed at the Department of Infectious Diseases of Luigi Sacco Hospital in Milan, Italy. Overall 122 patients were included in the study, 106 with probable and 16 with proven diagnosis of dengue fever. Most patients (91%) were Italian, with a median age of 35 years (IQR 29-46 years) and similar gender distribution, travelling for tourism (80%). Asia (mainly South East Asia and Indian Subcontinent) was the most frequent travel destination (55%), followed by Central America and the Caribbeans (22%). August-September was the peak season of presentation (42.6%). The majority of our diagnoses were based on serology alone. The most common signs and symptoms were fever (99,2%), maculopapular rash (50,8%), headache (50,8%), arthralgias (50,8%) and myalgias (46,7%). Leukopenia (77%), thrombocytopenia (81%) and altered LDH, AST and ALT (respectively 60,6%, 54,1% and 45,9%) were the most common laboratory test's abnormalities. No cases of severe DF were recorded. Our epidemiological and clinical findings are largely in accordance with most recent studies about imported DF in Europe. Although very similar in presentation to other arthropod-borne illnesses, some clinical features may help in differentiating DF from other causes of fever in the returning traveler

Comparative evaluation of the new xTAG GPP multiplex assay in the laboratory diagnosis of acute gastroenteritis. Clinical assessment and potential application from a multicentre Italian study.

Objective: Gastroenteritis caused by a single pathogen or multiple pathogens remains a major diagnostic challenge for the laboratory. The treatment of diarrhoea is based on microbiological results. Diagnosis is achieved using different laboratory techniques that have variable sensitivity and specificity. xTAG GPP is a new multiplex PCR assay that simultaneously detects 15 different pathogens responsible for diarrhoea. The results of the first multicentre study in Italy to evaluate the potential clinical application of the GPP assay in the laboratory diagnosis of diarrhoea are reported here. Methods: Faeces specimens (N = 664) from hospitalized patients were tested with the GPP assay using a Luminex 200 instrument. All specimens were run using comparator methods following a routine algorithm: culture for bacteria, enzyme immunoassay and PCR for viruses, and microscopy for parasites. Results: Of the samples tested with the GPP, 53.61% (356/664) gave positive results, as compared to 45.33% by routine testing. Of the positive specimens, 34.55% showed the presence of genomic DNA from multiple pathogens. The Luminex method showed an increase in the percentage of positivity of 8.28%. Conclusions: The GPP assay can be considered a helpful tool for the detection of gastrointestinal pathogens, with a hands-on time of 5 h; it provides accurate data for the clinical management of hospitalized patients and for epidemiological surveillance

Resistenza di Pseudomonas aeruginosa a ciprofloxacina e levofloxacina : 1998-2002

The aim of this study was to evaluate resistance rates to ciprofloxacin and levofloxacin of Pseudomonas aeruginosa (n=1917 strains) isolated at Laboratory of Microbiology at L. Sacco Teaching Hospital in Milan, Italy in the period between January 1998 and October 2002. Twenty-five percent of tested strains were isolated from sputum, 18% from bronchial lavage, 10% from urine, 9% from ear, 4.5% from blood and 26% from other materials. Ciprofloxacin-resistant strains were 121/ 411 (29%) in 1998, 158/ 526 (30%) in 1999, 136/ 400 (34%) in 2000, 129/390 (33%) in 2001 and 53/ 190 (28%) in 2002. Resistance rates for levofloxacin were 132/472 (28%) in 1999, 104/ 400 (26%) in 2000, 101/ 390 (26 %) in 2001 and 47/ 190 (25%) in 2002. Our data highlight overall stability in resistance to ciprofloxacin and levofloxacin with minor variations for ciprofloxacin. Moreover, in contrast with international worldwide studies, resistance rates to ciprofloxacin remained higher than those related to levofloxacin throughout the period studied

Favorable therapeutic response with an antiretroviral salvage regimen in an HIV-1-positive subject infected with a CRF11-cpx virus

HIV drug resistance still represents a crucial problem in antiretroviral therapy. We report a case of a naive patient, harboring a CRF11-cpx virus, which showed drug resistance mutations in the reverse transcriptase. Drug resistance genotyping test was performed for pol (protease, reverse transcriptase, and integrase) and V3 regions. The initial clinical parameters results showed a 4 logs level of HIV-RNA (12,090 cp/mL) and a very low CD4+ cell count (35 cells/\ub5L). We designed an initial HAART regimen including 3TC+ABC+DRV/r. The virus resulted highly resistant to all NRTIs and NNRTIs except to ABC, TDF, and EFV and susceptible to all PIs and INIs. A salvage regimen including RAL+DRV/r was started. Ten months after, the immuno-virological status shows CD4+ 142/\ub5L and HIV-RNA <37 cp/mL. Our results demonstrate the effectiveness of a treatment combination that includes RAL+DRV/r in a patient infected with a complex X4-tropic CRF11-cpx virus

Chagas disease knocks on our door : A cross-sectional study among Latin American immigrants in Milan, Italy

Objectives: We aimed to assess the prevalence and risk factors for Chagas disease (CD) in Latin American immigrants and to evaluate the accuracy of diagnostic tests. Moreover, we offered to all positive subjects a complete free-of-charge clinical/instrumental evaluation as well as benznidazole treatment in order to stage the disease and verify drug tolerability. Methods: A cross-sectional survey of CD among Latin Americans living in Milan and its metropolitan area was conducted between July 2013 and July 2014. Blood samples were tested for serologic evidence of CD together with a questionnaire covering demographic and clinical-epidemiological information. Results: Forty-eight (9.6%) of the 501 tested subjects were conclusively diagnosed as having CD. The highest prevalence of CD was among those from Bolivia (43/169, 25.4%) and El Salvador (4/68, 5.9%). Older age (adjusted odds ratio (aOR)] 1.05, p =0.004), a Bolivian origin (aOR 8.80; p =0.003), being born in the department of Santa Cruz (aOR 3.72, p =0.047), having lived in mud houses (aOR 2.68; p =0.019), and having an affected relative (aOR 12.77, p =0.001) were independently associated with CD. The ARCHITECT Chagas test showed the highest sensitivity (100%) and specificity (99.8%). Twenty-nine of the subjects with CD (60.4%) underwent disease staging, 10 of whom (35.7%) showed cardiac and/or digestive involvement. Benznidazole treatment was associated with high frequency of adverse reactions (19/27, 70.4%) and permanent discontinuation (8/27, 29.6%). Conclusions: CD is highly prevalent among Bolivians and Salvadorans living in Milan. Regions with a large Latin American immigrant population should implement programmes of active detection and treatment

Salinomycin and other ionophores as a new class of antimalarial drugs with transmission-blocking activity

The drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages of Plasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin on Plasmodium falciparum asexual and sexual erythrocytic stages and on the development of the Plasmodium berghei and P. falciparum mosquito stages is reported here. Gametocytogenesis of the P. falciparum strain 3D7 was induced in vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads

Sharing a common understanding in biosafety and biosecurity

Effective response to CBRN risk depends on cooperation and co-ordination between all levels of government, response organizations and international partners. An effective strategy to deal with the CBRN threat requires a very high level of co-operation and co-ordination among many different authorities within and among countries. Lack of harmonised national preparedness and fragmentation of responsibilities within the regional or international anti-trafficking network may be easily exploited by non-state actors to traffic and develop CBRN weapons. The creation of CBRN Centres of Excellence (CoE) aims at improving national CBRN policies in the targeted third countries by defining comprehensive tailored assistance packages in the areas of export control, CBRN illicit trafficking, redirection of scientists, emergency planning, bio-safety and security, crisis response and a culture of safety and security. This process will be developed with the help of EU Member States in a coherent and effective combination of national and regional capabilities