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Background: Dried bloodspots are easy to collect and to transport to assess various metabolites, such as amino acids. Dried bloodspots are routinely used for diagnosis and monitoring of some inherited metabolic diseases., Methods: Measurement of amino acids from dried blood spots by liquid chromatography-tandem mass spectrometry., Results: We describe a novel rapid method to measure underivatised urea cycle related amino acids. Application of this method enabled accurate monitoring of these amino acids to assess the efficacy of therapies in argininosuccinate lyase deficient mice and monitoring of these metabolites in patients with urea cycle defects., Conclusion: Measuring urea cycle related amino acids in urea cycle defects from dried blood spots is a reliable tool in animal research and will be of benefit in the clinic, facilitating optimisation of protein-restricted diet and preventing amino acid deprivation.

Group A Streptococcus (GAS) or Streptococcus pyogenes is responsible for an estimated 500,000 deaths worldwide each year. Protection against GAS infection is thought to be mediated by phagocytosis, enhanced by bacteria-specific antibody. There are no licenced GAS vaccines, despite many promising candidates in preclinical and early stage clinical development, the most advanced of which are based on the GAS M-protein. Vaccine progress has been hindered, in part, by the lack of a standardised functional assay suitable for vaccine evaluation. Current assays, developed over 50 years ago, rely on non-immune human whole blood as a source of neutrophils and complement. Variations in complement and neutrophil activity between donors result in variable data that is difficult to interpret. We have developed an opsonophagocytic killing assay (OPKA) for GAS that utilises dimethylformamide (DMF)-differentiated human promyelocytic leukemia cells (HL-60) as a source of neutrophils and baby rabbit complement, thus removing the major sources of variation in current assays. We have standardised the OPKA for several clinically relevant GAS strain types (emm1, emm6 and emm12) and have shown antibody-specific killing for each emm-type using M-protein specific rabbit antisera. Specificity was demonstrated by pre-incubation of the antisera with homologous M-protein antigens that blocked antibody-specific killing. Additional qualifications of the GAS OPKA, including the assessment of the accuracy, precision, linearity and the lower limit of quantification, were also performed. This GAS OPKA assay has the potential to provide a robust and reproducible platform to accelerate GAS vaccine development., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Neoehrlichiosis is an infectious disease caused by the tick-borne bacterium "Candidatus Neoehrlichia mikurensis". Splenectomy and rituximab therapies are risk factors for severe neoehrlichiosis. Our aim was to examine if neoehrlichiosis patients had low levels of natural IgM antibodies and/or were hypogammaglobulinemic, and if such deficiencies were associated with asplenia and vascular complications., Methods: Neoehrlichiosis patients (n = 9) and control subjects (n = 10) were investigated for serum levels of IgG, IgA, and IgM, and for levels of natural IgM antibodies to pneumococcal polysaccharides (6B, 14), and to the malondialdehyde acetaldehyde epitope of oxidized LDL. The multivariate method Projection to Latent Structures was used to analyze the data., Results: The levels of natural IgM antibodies of various specificities were decreased or not measurable in half of the studied patients with neoehrlichiosis. Only one patient and one control subject were hypogammaglobulinemic. An inverse relationship was noted between the levels of natural IgM antibodies and the development of deep vein thrombosis. Unexpectedly, no association was seen between having or not having a spleen and the levels of natural IgM antibody levels in the circulation., Conclusions: Neither hypogammaglobulinemia nor lack of natural IgM antibodies alone predisposes for severe neoehrlichiosis. The importance of the spleen in the immune defence against Ca. N. mikurensis probably lies in its capacity to generate or maintain specific antibodies.

Patients with multiple myeloma and other B cell disorders respond poorly to pneumococcal vaccination. Vaccine responsiveness is commonly determined by measuring pneumococcal serotype-specific antibodies by enzyme-linked immunosorbent assay (ELISA), by a functional opsonophagocytosis assay (OPA), or by both assays. We compared the two methods in vaccinated elderly patients with multiple myeloma, Waldenstrom's macroglobulinemia, and monoclonal gammopathy of undetermined significance (MGUS). Postvaccination sera from 45 patients (n= 15 from each patient group) and 15 control subjects were analyzed by multiplexed OPA for pneumococcal serotypes 4, 6B, 14, and 23F, and the results were compared to IgG and IgM antibody titers measured by ELISA. While there were significant correlations between pneumococcal OPA and IgG titers for all serotypes among the control subjects (correlation coefficients [r] between 0.51 and 0.85), no significant correlations were seen for any of the investigated serotypes in the myeloma group (r= -0.18 to 0.21) or in the group with Waldenstrom's macroglobulinemia (borderline significant correlations for 2 of 4 serotypes). The MGUS group resembled the control group by having good agreement between the two test methods for 3 of 4 serotypes (r= 0.53 to 0.80). Pneumococcal postvaccination IgM titers were very low in the myeloma patients compared to the other groups and did not correlate with the OPA results. To summarize, our data indicate that ELISA measurements may overestimate antipneumococcal immunity in elderly subjects with B cell malignancies and that a functional antibody test should be used specifically for myeloma and Waldenstrom's macroglobulinemia patients., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background & Aims: Since 2009/10, a 10- and a 13-valent pneumococcal conjugate vaccine (PCV) are available, but only the 10-valent vaccine is now being used for the children in the Netherlands. As the vaccines differ in number of serotypes, antigen concentration, and carrier proteins this study was designed to directly compare quantity and quality of the antibody responses induced by PCV10 and PCV13 before and after the 11-month booster., Methods: Dutch infants (n = 132) were immunized with either PCV10 or PCV13 and DTaP-IPV-Hib-HepB at the age of 2, 3, 4 and 11 months. Blood samples were collected pre-booster and post-booster at one week and one month post-booster for quantitative and qualitative immunogenicity against 13 pneumococcal serotypes, as well as quantitative immunogenicity against diphtheria, tetanus, pertussis and Haemophilus influenzae type b. We compared immunogenicity induced by PCV13 and PCV10 for their ten shared serotypes., Results: One month post-booster, pneumococcal serotype-specific IgG geometric mean concentrations (GMCs) for the PCV13 group were higher compared with the PCV10 group for six serotypes, although avidity was lower. Serotype 19F showed the most distinct difference in IgG and, in contrast to other serotypes, its avidity was higher in the PCV13 group. One week post-booster, opsonophagocytosis for serotype 19F did not differ significantly between the PCV10- and the PCV13 group., Conclusion: Both PCV10 and PCV13 were immunogenic and induced a booster response. Compared to the PCV10 group, the PCV13 group showed higher levels for serotype 19F GMCs and avidity, pre- as well as post-booster, although opsonophagocytosis did not differ significantly between groups. In our study, avidity is not correlated to opsonophagocytotic activity (OPA) and correlations between IgG and OPA differ per serotype. Therefore, besides assays to determine IgG GMCs, assays to detect opsonophagocytotic activity, i.e., the actual killing of the pneumococcus, are important for PCV evaluation. How differences between the two vaccines relate to long-term protection requires further investigation., Trial Registration: www.trialregister.nl NTR3069.

Adequate access to effective and affordable vaccines is essential for the prevention of mortality due to infectious disease. Pneumonia--a consequence of Streptococcus pneumoniae infection--is the world's leading cause of death in children aged under 5 years. The development of a needle-free, thermostable pneumococcal-conjugate vaccine (PCV) could revolutionise the field by reducing cold-chain and delivery constraints. Skin patches have been used to deliver a range of vaccines, with some inducing significantly higher vaccine-specific immunogenicity than needle-injected controls in pre-clinical models, though they have yet to be used to deliver a PCV. We dry-coated a licensed PCV onto a microprojection-based patch (the Nanopatch) and delivered it to mouse skin. We analysed resulting anti-polysaccharide IgG responses. With and without adjuvant, anti-polysaccharide IgG titres induced by Nanopatch immunisation were significantly higher than dose-matched intramuscular controls. These improved responses were primarily obtained against pneumococcal serotypes 4 and 14. Importantly, capsule-specific IgG correlated with functionality in an opsonophagocytic killing assay. We demonstrate enhanced anti-PCV immunogenicity when delivered by Nanopatch over intramuscular injection. As the first study of a PCV delivered by a skin vaccination technology, this report indicates the potential for reduced costs and greater global distribution of such a vaccine., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The pneumococcus is a leading pathogen infecting children and adults. Safe, effective vaccines exist, and they work by inducing antibodies to the polysaccharide capsule (unique for each serotype) that surrounds the cell; however, current vaccines are limited by the fact that only a few of the nearly 100 antigenically distinct serotypes are included in the formulations. Within the serotypes, serogroup 6 pneumococci are a frequent cause of serious disease and common colonizers of the nasopharynx in children. Serotype 6E was first reported in 2004 but was thought to be rare; however, we and others have detected serotype 6E among recent pneumococcal collections. Therefore, we analyzed a diverse data set of ∼1,000 serogroup 6 genomes, assessed the prevalence and distribution of serotype 6E, analyzed the genetic diversity among serogroup 6 pneumococci, and investigated whether pneumococcal conjugate vaccine-induced serotype 6A and 6B antibodies mediate the killing of serotype 6E pneumococci. We found that 43% of all genomes were of serotype 6E, and they were recovered worldwide from healthy children and patients of all ages with pneumococcal disease. Four genetic lineages, three of which were multidrug resistant, described ∼90% of the serotype 6E pneumococci. Serological assays demonstrated that vaccine-induced serotype 6B antibodies were able to elicit killing of serotype 6E pneumococci. We also revealed three major genetic clusters of serotype 6A capsular sequences, discovered a new hybrid 6C/6E serotype, and identified 44 examples of serotype switching. Therefore, while vaccines appear to offer protection against serotype 6E, genetic variants may reduce vaccine efficacy in the longer term because of the emergence of serotypes that can evade vaccine-induced immunity., (Copyright © 2015, van Tonder et al.)

Streptococcus pneumoniae serotype 1 (Sp1) constitutes an important cause of seasonal endemic meningitis in all age groups in the African meningitis belt. Despite a higher meningitis incidence, the Burkinabé population has an Sp1-specific antibody seroprevalence similar to that reported in the United Kingdom (UK). We aimed to establish whether the opsonophagocytic activity (OPA) of pneumococcal IgG naturally present in Burkina Faso differs from that seen in individuals in the UK and to compare the OPAs generated by natural and vaccine-induced immunity. Samples collected from pneumococcal vaccine-naive Burkinabé and UK subjects were matched for age (1 to 39 years) and anti-Sp1 IgG level, analyzed for OPA to 3 S. pneumoniae serotypes (1, 5, and 19A), and compared to postvaccine samples. Furthermore, the Burkinabé samples were assessed for IgG avidity and serotype-specific IgM concentrations. One hundred sixty-nine matched serum samples from both populations were selected. A greater proportion of Burkinabé subjects aged 1 to 19 years had functional Sp1 activity (OPA ≥ 8) compared to UK subjects (12% versus 2%, P < 0.001); however, the proportions were similar among adults (9%). The correlation between Sp1 IgG concentration and OPA was good (P < 0.001), but many individuals had nonfunctional IgG, which was not related to avidity. While the Sp1 IgM concentrations correlated with OPA, not all of the function in serum samples with low IgG could be attributed to IgM. Finally, vaccine-induced Sp1-specific IgG was more functional than equivalent amounts of naturally occurring IgG. In conclusion, despite a substantially higher pneumococcal meningitis incidence, no decreased functional immunity to Sp1 could be evidenced in the Burkinabé population compared to that in the population from the UK. Furthermore, the naturally induced antibodies were less functional than vaccine-induced antibodies., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Children with perinatally acquired HIV (paHIV) remain at an increased risk of pneumococcal infection despite highly active antiretroviral therapy (HAART). Beyond infancy, responses to pneumococcal conjugate vaccine (PCV) remain under-investigated. There are currently no published data on serological response to 13-valent PCV (PCV13) in the HIV-infected populations., Methods: We measured pneumococcal serotype-specific IgG in 48 paHIV-infected child patients (CP), 27 young adult healthy controls (AHC) and 30 child healthy controls (CHC). Opsonophagocytic assay (OPA) titres for three PCV13-exclusive serotypes were measured in a subset of children. Serotype-specific IgG was repeated 1 and 6 months following PCV13 vaccination of CP and AHC groups. OPA titres for four serotypes were measured at the 1-month time-point., Results: The majority of CP, CHC and AHC had serotype-specific IgG above 0.35  μg/ml at baseline, although OPA activity was undetectable for two of the three serotypes studied. Baseline IgG concentrations were significantly lower in CP than AHC for a proportion of serotypes and were strongly predictive of responses to vaccine. After adjusting for baseline, postvaccination IgG concentrations were comparable, although responses to some serotypes were impaired for CP. OPA correlated well with IgG after vaccination. Detectable HIV viral load was associated with significantly lower IgG concentration and OPA titre., Conclusion: Children with paHIV mount a robust serological response to PCV13 for most but not all vaccine serotypes. Viral load suppression with HAART and higher baseline IgG concentration are associated with higher postvaccination antibody levels. This has implications for HAART treatment and vaccination practices.

Background: Efficacy of the 13-valent pneumococcal conjugate vaccine (PCV13) was inferred before licensure from an aggregate correlate of protection established for the seven-valent vaccine (PCV7). We did a postlicensure assessment of serotype-specific vaccine effectiveness and immunogenicity in England, Wales, and Northern Ireland to derive the correlates of protection for individual serotypes., Methods: We assessed vaccine effectiveness against invasive pneumococcal disease using the indirect cohort method. We measured serotype-specific IgG concentration in infants after they were given two priming doses of PCV7 (n=126) or PCV13 (n=237) and opsonophagocytic antibody titre from a subset of these infants (n=100). We derived correlates of protection by relating percentage protection to a threshold antibody concentration achieved by an equivalent percentage of infants. We used multivariable logistic regression to estimate vaccine effectiveness and reverse cumulative distribution curves to estimate correlates of protection., Findings: For the 706 cases of invasive pneumococcal disease included in the study, PCV13 vaccine effectiveness after two doses before age 12 months or one dose from 12 months was 75% (95% CI 58-84). Vaccine effectiveness was 90% (34-98) for the PCV7 serotypes and 73% (55-84) for the six additional serotypes included in PCV13. Protection was shown for four of the six additional PCV13 serotypes (vaccine effectiveness for serotype 3 was not significant and no cases of serotype 5 infection occurred during the observation period). The vaccine effectiveness for PCV13 and PCV7 was lower than predicted by the aggregate correlate of protection of 0·35 μg/mL used during licensing. Calculated serotype-specific correlates of protection were higher than 0·35 μg/mL for serotypes 1, 3, 7F, 19A, 19F, and lower than 0·35 μg/mL for serotypes 6A, 6B, 18C, and 23F. Opsonophagocytic antibody titres of 1 in 8 or higher did not predict protection., Interpretation: PCV13 provides significant protection for most of the vaccine serotypes. Although use of the aggregate correlate of protection of 0·35 μg/mL has enabled the licensing of effective new PCVs, serotype-specific correlates of protection vary widely. The relation between IgG concentration after priming and long-term protection needs to be better understood., Funding: Public Health England and UK Department of Health Research and Development Directorate., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Protection against disease or colonization from serotypes related to those in pneumococcal conjugate vaccines (i.e. cross-protection) vary by serotype; the basis for this variation is not understood. The 13-valent pneumococcal conjugate vaccine (PCV13) replaced 7-valent conjugate (PCV7) in the USA in 2010 allowing assessment of PCV7 and PCV13 immunogenicity and functional cross-protection in vitro., Methods: Post-primary, pre-booster and post-booster sera from American Indian children receiving exclusively PCV7 or PCV13 were collected. IgG was measured by ELISA for 13 vaccine serotypes; functional antibody was assessed by opsonophagocytic killing assays for serotypes 6A/B/C and 19A/F., Results: Post-primary IgG geometric mean concentrations (GMC) for serotypes 4 and 9V were lower in PCV13 recipients while 19F GMCs were higher. Only 19F differences persisted after receipt of the booster dose. Functional antibody activity was higher among PCV13 recipients for 6A, 6C, 19A and 19F (p<0.04), and among PCV7 recipients for 6B (p = 0.01). Following PCV7, functional antibodies to 6A but not 19A were observed. High levels of 6C functional activity were seen after PCV13 but not PCV7., Conclusions: Functional antibody activity against 6A/B/C and 19A/F suggest that PCV13 is likely to control the 19A disease and 6C disease remaining despite widespread use of PCV7.

To evaluate how the 7-valent pneumococcal vaccine (PCV7) programme and the very high vaccination coverage reached for over 4 years affected the prevalence of Streptoccoccus pneumoniae serotypes in the paediatric population and to evaluate demographic, behavioural and risk factors for carriage in the post-vaccination era, a cross-sectional study on nasopharyngeal carriage was performed. Six hundred sixty-nine children under the age of 5, representative of the open population, were enrolled by cluster sampling. High sensitive techniques for detection of multi-serotype carriage, i.e. broth enrichment and real-time PCR and sequential PCRs for detection and typing, respectively, were used. Of the 669 enrolled children, 97.8% were compliant with the recommended PCV7 vaccination schedule. Post-stratification adjustment for age was applied considering the Ligurian population as standard population. Age-weighted carriage rate was 50.1% and 78% of carriers were colonized by more than one serotype. The prevalence of carriage increased with age from 22% in the first year of life, to 48.6% in the second year of life and to 60% in the 25-59 month age group. Age-weighted prevalence of any of the PCV7, PCV10 or PCV13 serotypes was 10.3%, 20.3% and 27.5%, respectively. PCV7 serotypes were mainly represented by serotype 4 that was carried since the 3rd year of life and was responsible for invasive pneumococcal disease (IPD) and non-IPD in adults, but not in children confirming the high vaccine effectiveness. Among the serotypes included in recently available vaccines, serotypes 5 and 19A showed a higher prevalence, being carried by 15.2% and 8.8% of the population, respectively. A multivariate analysis showed that age, the presence of child siblings at home and day care attendance covariates were strongly associated with S. pneumoniae carriage. In conclusion, over 7 years of vaccination with PCV7 and very high coverage in the last 4 years has led to low carriage prevalence in the first year of life rapidly increasing in the following years and high prevalence of non-PCV7 serotypes carriage., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A laboratory-based surveillance study was conducted from January 2007 to May 2010 in San Martino Tertiary Referral Hospital in Genoa, Italy in which the molecular epidemiology of multidrug-resistant Acinetobacter baumannii was investigated in the five intensive care units (ICUs). A total of 53 A. baumannii strains were isolated from patients admitted to ICUs (69.8%) and to other epidemiologically linked hospital wards (30.2%) and were genotyped by repetitive extragenic palindromic polymerase chain reaction (REP-PCR), multilocus sequence typing (MLST) and adeB sequence typing. REP-PCR fingerprinting analysis, MLST and adeB typing results were well correlated and allowed us to classify strains causing epidemic events into three major epidemic clones: A (REP-I/ST4, adeB-STII genotype) isolated for the first time in May 2007, B (REP-IV/ST95, adeB-STI genotype) from November 2007 to May 2009 and C (REP-VII/ST118, adeB-STII genotype) from July 2008 to May 2010. MLST results demonstrated that epidemic clones A and C were related as they were members of the widespread clonal complex CC92. The genetic determinants of carbapenem resistance were investigated and resistance associated with the presence of the bla(OxA-58-like) gene with ISAba2 and ISAba3 elements flanking it in clone A, and with the bla(OxA-23-like) gene flanked by ISAba1 in clones B and C. A molecular approach allowed the prompt introduction of infection control measures and the evaluation of data in a global epidemiological context., (Copyright © 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Introduction: Following the observation that 1 or 2 pandemic peak due to the circulation ofAHINlv had occurred in most countries and in most World Health Organization (WHO) Regions, WHO declared on August 10"h, 2010 that the world was moving into the post-pandemic period, whose surveillance presents considerable interest both from epidemiological and clinical point of view. We described the epidemiological picture emerged from syndromic and virological surveillance during the post-pandemic season in Liguria, Italy., Materials and Methods: An Emergency Department Syndrome surveillance system, based on data collected at "San Martino" and IRCCS "G. Gaslini" Liguria Regional Reference University Hospitals for adults and children is active since July 2007. Monitored syndromes include "Influenza-Like Illness" (ILl) and "Low Respiratory Tract Infections" (LRTI). The Ligurian Regional Reference laboratory for Influenza virological surveillance and diagnosis offers rapid detection of influenza viruses by real-time and block RT-PCR, viral culture and genetic characterization by entire sequence analysis of haemagglutinin- and neuraminidase-coding regions in accordance with the international standards established by the global laboratory network., Results and Discussion: The integration of syndromic surveillance system and laboratory surveillance for rapid detection and characterization of the disease responsible agent represented a specific and sensitive tool for influenza surveillance. The post-pandemic season was characterized by early onset and by the heaviest impacts for ILI and LRTI among the recent epidemic seasons. In contrast to the picture observed during the pandemic season, the 2010/11 winter was characterized by the intensive circulation of pandemic AH1N1v coupled with sustained activity due to influenza B and Respiratory Syncytial Virus (RSV). Antigenic and molecular characterization of influenza strains confirmed the good matching between circulating and 2010/11 vaccine viruses.

Herein, we show that γδT, CD8(+) αβT lymphocytes and natural killer (NK) cells display a different sensitivity to survival signals delivered via NKG2D surface receptor. All the three effector cell populations activate Akt1/PKBalpha through the engagement of this molecule. Upon binding to leukemic cells expressing NKG2D ligands (NKG2DL), including chronic lymphocytic leukemias treated with transretinoic acid, most γδT (>60%) and half CD8(+) αβT cells (about 50%) received a survival signal, at variance with the majority of NK cells (>80%) that underwent apoptosis by day 5. Interestingly, oligomerization of NKG2D in γδT or CD8(+) αβT cells, led to a significant rise in nuclear/cytoplasmic ratio of both NF-kBp52 and RelB, the two NF-kB subunits mainly involved in the transcription of antiapoptotic proteins of the Bcl family. Indeed, the ratio between the antiapoptotic protein Bcl-2 or Bcl-x(L) and the proapoptotic protein Bax raised in γδT or CD8(+) αβT cells following NKG2D engagement by specific monoclonal antibodies or by NKG2DL expressing leukemic cells. Conversely, nuclear translocation of NF-kBp52 or RelB did not increase, nor the Bcl-2/Bax or the Bcl-x(L) /Bax ratios changed significantly, in NK cells upon oligomerizaton of NKG2D. Of note, transcripts for α5 importin, responsible for nuclear translocation of NF-kBp52/Rel B heterodimer, are significantly higher in γδT and CD8(+) αβT cells than in NK cells. These biochemical data may explain, at least in part, why γδT and CD8(+) αβT cells are cytolytic effector cells more resistant to target-induced apoptosis than NK cells., (Copyright © 2010 UICC.)

MF59 is already known to enhance the breadth of antibody response to mismatched influenza seasonal and avian strains. However, little is known on the effect of MF59 on immunogenicity of influenza vaccines when "apparent" good matching between circulating and vaccine strains exists. To this end, we compared the immune response elicited by MF59-adjuvanted or non-adjuvanted subunit vaccine, containing A/California/7/04(H3N2) strain, against circulating viruses isolated between 2004/2005 and 2006/2007 seasons, belonging to different clades. The advantage offered by MF59 in terms of higher immunogenicity, expressed as higher post-vaccination HI titres, is observable also against viruses showing antigenic and molecular pattern undistinguishable from vaccine strain, but it became even more evident as the antigenic and molecular distance between vaccine and circulating strains grew. These data show that seasonal influenza vaccine adjuvanted with MF59 can offer a stronger benefit as compared to non-adjuvanted vaccine in protecting against a broader range of virus strains circulating during the influenza season., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Introduction: Rubella is generally a mild rush fever disease when acquired in childhood, but when infection occurs during the first months of pregnancy, high risk of trans-placental transmission to the foetus and of congenital anomalies exists. In November 2003, a National Plan for measles and congenital rubella elimination was approved in Italy. The aim was to reduce and maintain Congenital Rubella Syndrome incidence lower than 1 case per 100,000 live births/year by 2007. Since June 2006, Liguria Administrative Region recognized U.O. Hygiene, "San Martino" University Hospital, Genoa, as regional reference laboratory for diagnosis of rubella infection during pregnancy and post-partum., Methods: Twenty-one-month virological-surveillance results between April 2007 and December 2008 were reported in terms of demographic data, risk factors, access reasons, clinical picture, vaccination, previous rubella disease, laboratory results of pregnant women and newborns., Results and Conclusion: Since the beginning of surveillance, 65 pregnant women with suspected virus infection and 18 newborns with suspected congenital rubella were followed up. The results of laboratory surveillance highlighted (i) the importance of an early screening, (ii) the suboptimal specificity of chemiluminescent assays, that often yield false positive IgM results and (iii) the fundamental role of second-level laboratory to confirm the serological diagnosis and to detect the virus by molecular techniques.

It is thought that human natural killer (NK) lymphocytes should not damage self-tissues due to the inhibiting signal initiated by the engagement of one or another inhibitory receptor superfamily (IRS) members with self-human histocompatibility antigen (HLA)-I. During viral infection, the low expression of self-HLA-I on infected-cells leads to a reduction of the inhibiting signal and thus NK cells kill self-cells (missing self-hypothesis). Here, we have analyzed human NK cell interaction with self-cells as antigen-presenting cells (APC) or stromal cells isolated from bone marrow or skin. Despite the expression of high levels of HLA-I, APC and stromal cells are killed by interleukin (IL)-2-activated NK cells upon lymphocyte function antigen (LFA)1-(intracellular adhesion molecule) (ICAM)1 interaction. The natural cytotoxicity receptors NKp30 and NKp46 are responsible for the delivery of lethal hit to APC, whereas NKG2D-activating receptor, the ligand of the major histocompatibility complex (MHC)-related molecule MICA, and the UL16-binding protein are involved in stromal cell killing. These events are dependent on the activation of phosphoinositol 3-kinase and consequent release of perforins and granzymes. Both bone marrow stromal cells and skin fibroblasts inhibit T cell proliferation to alloantigen or triggering through CD3/T cell receptor complex. Importantly, NK cells can revert this veto effect. Altogether, these findings support the notion that NK cells can recognize self-cells possibly affecting both APC function and interaction between lymphocytes and microenvironment leading to autoreactivity.

Background and Objectives: Mesenchymal stem cells (MSC) have been proposed as a way to treat graft-versus-host disease based of their immunosuppressive effect. We analyzed whether regulatory T cells can be generated in co-cultures of peripheral blood mononuclear cells (PBMC) and MSC., Design and Methods: MSC were obtained from the bone marrow of four healthy donors and nine patients with acute leukemia in complete remission following chemotherapy. Short-term (4 days) co-cultures of MSC and autologous or allogeneic PBMC were set up, the lymphocytes harvested and their regulatory activity assessed., Results: Lymphocytes harvested from MSC-PBMC co-cultures strongly inhibit (up to 95%) mixed lymphocyte reaction (MLR), recall to alloantigen, and CD3- or PHA-induced lymphocyte proliferation. These lymphocytes, termed regulatory cells (Regc), were all CD45+CD2+ with variable proportions of CD25+ cells (range 40-75% n=10) and a minor fraction expressed CTLA4 (2-4%, n=10) or glucocorticoid-induced tumor necrosis factcor receptor-related gene (0.5-4% n=10). Both CD4+ and CD8+ Regc purified from MSC-PBMC co-cultures strongly inhibited lymphocyte proliferation at a 1:100 Regc:responder cell ratio. CD4+ Regc expressed high levels of forkhead box P3 (Foxp3) mRNA while CD8+ Regc did not. The effectiveness of Regc, whether CD4+ or CD8+, was 100-fold higher than that of CD4+CD25+high regulatory T cells. Regc were also generated from highly purified CD25- PBMC or CD4+ or CD8+ T cell subsets. Soluble factors, such as interleukin-10, transforming growth factor-b and prostaglandin E2 did not appear to be involved in the generation of Regc or in the Regc-mediated immunosuppressive effect. Furthermore, cyclosporin A did not affect Regc generation or the immunosuppression induced by Regc., Interpretation and Conclusions: These findings indicate that powerful regulatory CD4+ or CD8+ lymphocytes are generated in co-cultures of PBMC with MSC. This strongly suggests that these regulatory cells may amplify the reported MSC-mediated immunosuppressive effect.

Gammadelta T lymphocytes are thought to be involved in multiple sclerosis (MS) pathogenesis. In this work, we discuss the characteristics of these cells and possible implications in the pathogenesis of MS, focusing on the mechanism(s) underlying extravasation and tissue localization. Phenotype and transendothelial migration of gammadelta T cells from healthy donors and patients with relapsing-remitting MS were studied. In MS patients the V delta 2 T cell subset, expressing NKRP1A/CD161 adhesion molecule, is expanded and capable of transendothelial migration. V delta 1/V delta 2 subsets use distinct signal transduction pathways: V delta 1 cells lack NKRP1A and express PECAM-1/CD31, which drives transmigration, while V delta 2 cells are PECAM-1 negative and use NKRP1A. V delta 2 migration is coupled with CAMKII, whereas V delta 1 depend on PI-3K. NKRP1A and PECAM-1 selectively activate the two pathways: indeed, oligomerization of NKRP1A on V delta 2 T cells leads to CAMKII activation, occupancy of PECAM-1 on V delta 1 cells triggers the PI-3K-dependent Akt/PKB pathway. Moreover, V delta 2 T cells are CXCR3(bright)CXCR4(dull), while V delta 1 are mostly CXCR4(+). V delta 1 and V delta 2 cells transmigrate in response to IP-10/CXCL10 and SDF-1/CXCL12 according to the expression of their specific receptors. In a fraction of V delta 1 T cells coexpressing CXCR3 and CXCR4, the homeostatic chemokine 6Ckine/SLC/CCL21 is more effective. IP-10/CXCL10 or 6Ckine/SLC/CCL21 and SDF-1/CXCL12-induced transmigration is coupled to PI-3K/Akt/PKB, but only CXCR3 is capable of inducing CAMKII activation. We suggest that both subsets of gammadelta T lymphocytes may migrate to the site of lesion in MS using two different signaling pathways to extravasate and responding to different chemokines.

Liposome encapsulation of anticancer agents results in reduced side effects of the entrapped drug and improved therapeutic efficacy. The external surface of the lipidic envelope can be coupled with antibodies directed against tumor-associated antigens. The resulting immunoliposomes allow to increase the therapeutic index of cytotoxic drugs while minimizing their systemic toxicity. In this regard, the disialoganglioside GD2 is a very promising tumor-associated antigen since it is expressed at high intensity on human neuroblastoma cells, but is detected only in normal cerebellum and peripheral nerves. Immunoliposomes can be used as vectors to deliver antisense oligonucleotides to cancer cells with the aim to modulate oncogene expression. Furthermore, antisense oligonucleotides have attracted much interest because of their ability to stimulate immune responses. Here, we will describe a novel experimental therapeutic approach for neuroblastoma based on anti-GD2 liposomal c-myb-selective antisense oligonucleotides.

Neuroectodermal tumors are highly malignant and increasingly common tumors. Because the cure rate of these neoplasias by conventional treatment is very low, new therapeutic approaches are needed. Entrapping high concentrations of cytotoxic drugs and/or oligonucleotides within stabilized liposomal formulations represents an emerging modality of antitumor treatment. Here, we tested the in vitro and in vivo antitumor effects of a novel antisense oligodeoxynucleotide (asODN) liposomal formulation, the coated cationic liposomes (CCL), by targeting the c-myc and the c-myb oncogenes on melanoma and neuroblastoma, respectively, through the use of a monoclonal antibody against the disialoganglioside GD2, selectively expressed by neuroectoderma-derived tumors. Our methods produced GD2-targeted liposomes that stably entrapped 90 percent of added asODNs. These liposomes showed selective binding for GD2-positive tumor cells in vitro. Neuroblastoma cells treated with free myb-as or nontargeted CCL-myb-as showed the same level of c-myb protein expression as control cells. In contrast, c-myb protein expression of cells treated with aGD2-CCL-myb-as was inhibited by approximately 70 percent. Melanoma and neuroblastoma cell proliferation was inhibited to a greater extent by GD2-targeted liposomes containing c-myc or c-myb asODNs than by nontargeted liposomes or free asODNs. Mice bearing established subcutaneous human melanoma xenografts treated with aGD2-CCL-myc-as exhibited significantly reduced tumor growth and increased survival. The mechanism for the antitumor effects appears to be downregulation of the expression of the c-myc protein, induction of p53, and inhibition of Bcl-2 proteins, leading to extensive tumor cell apoptosis. In contrast, the increased life span obtained in a neuroblastoma pseudometastatic mouse model with the liposomal c-myb asODNs seems to be due to a synergistic mechanism: specific targeting to neuroblastoma cancer cells, downmodulation of c-myb protein expression, and stimulation of the innate immune system. These results suggest that inhibition of c-myc or c-myb proto-oncogenes by GD2-targeted antisense therapy could provide an effective approach for the treatment of neuroectodermal tumors in an adjuvant setting.