Apoptotic vesicles crossprime CD8 T cells and protect against tuberculosis

CD8 T lymphocytes are important effectors in protective immunity against Mycobacterium tuberculosis. We recently characterized the detour pathway of CD8 T cell activation in tuberculosis mediated by apoptotic vesicles from infected cells that transport mycobacterial antigens to dendritic cells (DCs). Here we demonstrate that apoptotic vesicles from mycobacteria-infected macrophages stimulate CD8 T cells in vivo. Homing of DCs to draining lymph nodes was critically required for effective crosspriming. Subsequent fate of vesicle-associated antigens in recipient DCs was characterized by endosomal mechanisms predominating over proteasomal processing. In addition, vesicle processing depended on the presence of saposins to disintegrate apoptotic membranes. Apoptotic vesicles displayed potent adjuvant activity by stimulating through Toll-like receptors (TLR). Ultimately, vaccination with vesicles from infected cells induced protection against M. tuberculosis infection. Taken together, we propose the detour pathway to represent a genuine immunological mechanism mediating crosspriming of CD8 T cells in vivo and protection against tuberculosis.     

Sulfolipid deficiency does not affect the virulence of Mycobacterium tuberculosis H37Rv in mice and guinea pigs

Lipids that are found only in the cell envelope of pathogenic mycobacteria, such as those containing multiple methyl-branched fatty acids, have long been thought to play a role in pathogenesis. Among these complex lipids, sulfolipids have been the most extensively studied over the last 50 years. The numerous biological effects exhibited by purified sulfolipids on phagocytic cells led to the idea that these molecules are probably important virulence factors facilitating the intracellular survival of Mycobacterium tuberculosis. However, definitive evidence to support this concept has been lacking. The recent construction of an isogenic sulfolipid-deficient mutant of M. tuberculosis H37Rv (Sirakova et al., J. Biol. Chem. 276:16833-16839, 2001) has for the first time provided the opportunity to directly assess the contribution of these complex lipids to pathogenesis. In the present study, we show that against all expectations, sulfolipid deficiency does not significantly affect the replication, persistence, and pathogenicity of M. tuberculosis H37Rv in mice and guinea pigs or in cultured macrophages.     

Genetic information and life insurance: a 'real' risk?

Public concern about genetic discrimination, particularly access to insurance following genetic testing, has been reported in the literature. This paper aims to separate myths from realities regarding genetic discrimination in life insurance and to underline the positive aspects of allowing insurers access to relevant genetic information for underwriting purposes. We present a review of the literature pertinent to discrimination in life insurance and a comparative analysis of industries guidelines. There are few reported cases in the literature of validated genetic discrimination. However, the benefits to be gained by allowing insurers access to relevant genetic data could justify fostering a more active role in the use of genetic information by insurance companies.     

Methylation profiling in individuals with uniparental disomy identifies novel differentially methylated regions on chromosome 15

The maternal and paternal genomes possess distinct epigenetic marks that distinguish them at imprinted loci. In order to identify imprinted loci, we used a novel method, taking advantage of the fact that uniparental disomy (UPD) provides a system that allows the two parental chromosomes to be studied independently. We profiled the paternal and maternal methylation on chromosome 15 using immunoprecipitation of methylated DNA and hybridization to tiling oligonucleotide arrays. Comparison of six individuals with maternal versus paternal UPD15 revealed 12 differentially methylated regions (DMRs). Putative DMRs were validated by bisulfite sequencing, confirming the presence of parent-of-origin-specific methylation marks. We detected DMRs associated with known imprinted genes within the Prader-Willi/Angelman syndrome region, such as SNRPN and MAGEL2, validating this as a method of detecting imprinted loci. Of the 12 DMRs identified, eight were novel, some of which are associated with genes not previously thought to be imprinted. These include a site within intron 2 of IGF1R at 15q26.3, a gene that plays a fundamental role in growth, and an intergenic site upstream of GABRG3 that lies within a previously defined candidate region conferring an increased maternal risk of psychosis. These data provide a map of parent-of-origin-specific epigenetic modifications on chromosome 15, identifying DNA elements that may play a functional role in the imprinting process. Application of this methodology to other chromosomes for which UPD has been reported will allow the systematic identification of imprinted sites throughout the genome.Imprinting is a phenomenon in which the expression status of a gene is dependent on the sex of the parent from which it is inherited. Imprinted genes generally exhibit monoallelic expression accompanied by parent-of-origin-specific epigenetic marks such as differential DNA methylation and histone modifications that distinguish the maternal and paternal genomes at these loci (Reik and Walter 2001; Dindot et al. 2009). More than 60 imprinted genes have been identified in humans (http://www.geneimprint.com/), and their clustered nature suggests that many are regulated by regional control mechanisms.To date, the discovery of imprinted sites in both mouse and human has largely been driven through the use of phenotype-based approaches. The vast majority of loci subject to parent-of-origin effects were first recognized through the observation that maternal and paternal transmission of the same genetic mutation results in different phenotypes (Nicholls et al. 1989). For example, the identification of imprinted gene clusters in 15q11-q13 associated with Prader-Willi/Angelman syndrome, 11p11.5 associated with Beckwith-Wiedemann syndrome, and imprinted loci at 14q32, 6q24, and 20q13.2 were all catalyzed by the initial observation that genetic disease occurred specifically in patients with either uniparental disomy (UPD) or deletions of these regions of specific parental origin. In combination with chromosomal engineering techniques that can systematically generate defined aneuploidies, this notion has been applied to screen the mouse genome for imprinting with great success, resulting in the identification of more than 130 murine imprinted genes (Williamson et al. 2009). However, because this methodology relies on the recognition of overt phenotypic differences between individuals to detect imprinting, it is likely to miss imprinting that may cause subtle phenotype differences or those that manifest in ways that are not easily recognized by typical methods of phenotypic characterization. Further, imprinted genes will also be missed or masked by phenotypes that are lethal.In order to circumvent this limitation, a variety of genomic techniques have been developed to identify parent-of-origin effects. Several previous studies have attempted to detect imprinting based on the differential expression of parental alleles at imprinted loci. Studies using subtractive cDNA hybridization (Kaneko-Ishino et al. 1995) and high-throughput cDNA sequencing in hybrid mouse strains (Wang et al. 2008) have been used to detect imprinted expression with some success. However, these approaches are limited in that they can only assay the subset of genes expressed in the tissue(s) under investigation, and for some genes, imprinted expression is only observed in specific tissues or at certain developmental stages (Deltour et al. 1995; Rougeulle et al. 1997; Zhou et al. 2006). Furthermore, sequencing-based approaches are only able to assay allelic bias in genes containing transcribed polymorphisms (Daelemans et al. 2010).Alternative approaches to detect imprinting have used the fact that the maternal and paternal genomes have differential epigenetic marks at most imprinted loci. This approach has the advantage over expression-based methods, in that these differential methylation marks are generally conserved, even in tissues that lack imprinted expression (Dockery et al. 2009). The presence of overlapping euchromatin and heterochromatin marks has been used to highlight imprinted domains in human (Wen et al. 2008), and restriction landmark genome scanning (Hayashizaki et al. 1994) and methylation-sensitive representational difference analysis (Kelsey et al. 1999; Smith et al. 2003) have been applied as methods to detect differentially methylated regions in the mouse genome. However, the reliance of these latter techniques on restriction enzyme digestion means that they can only assay a small subset of CpGs that overlap the enzyme recognition site, and if used in outbred genomes, are liable to artifacts generated by the presence of single nucleotide variants that alter restriction patterns.Because one of the key features of imprinted genes is the presence of parent-of-origin-specific methylation, we hypothesized that the systematic comparison of DNA methylation patterns in maternal versus paternal chromosomes should represent an optimal method for the detection of imprinted loci. Based on this hypothesis, we have taken advantage of the fact that uniparental disomy provides a unique system that allows the separate study of chromosomes derived from a single parent and combined this with a methodology in which the methylation of entire chromosomes can be analyzed in an unbiased fashion. By analyzing methylation patterns in cases of maternal UPD15 (matUPD15) and paternal UPD15 (patUPD15) using immunoprecipitation of methylated DNA and high-density tiling arrays with complete coverage of human chromosome 15, we generated separate methylation profiles of the maternally and paternally derived alleles. Comparison of the two parental epigenotypes identifies numerous loci on chromosome 15 that show parent-of-origin-specific methylation differences, defining a set of DNA elements that are likely responsible for the establishment and/or maintenance of imprinting on this chromosome. We identify novel imprinted loci both within and outside of the known PWS/AS imprinted domain, suggesting candidate loci that may exert parent-of-origin effects in several human phenotypes.     

Presence of new mecA and mph(C) variants conferring antibiotic resistance in Staphylococcus spp. isolated from the skin of horses before and after clinic admission

Because of the frequency of multiple antibiotic resistance, Staphylococcus species often represent a challenge in incisional infections of horses undergoing colic surgery. To investigate the evolution of antibiotic resistance patterns before and after preventative peri- and postoperative penicillin treatment, staphylococci were isolated from skin and wound samples at different times during hospitalization. Most staphylococci were normal skin commensals and belonged to the common coagulase-negative group. In some cases they turned out to be opportunistic pathogens present in wound infections. MICs were determined for 12 antibiotics, and antibiotic resistance genes were detected by microarray. At hospital admission, horses harbored staphylococci that were susceptible to antibiotics or resistant to one group of drugs, mainly due to the presence of new variants of the methicillin and macrolide resistance genes mecA and mph(C), respectively. After 3 days, the percentage of Staphylococcus isolates displaying antibiotic resistance, as well as the number of resistance genes per isolate, increased moderately in hospitalized horses without surgery or penicillin treatment but dramatically in hospitalized horses after colic surgery as well as penicillin treatment. Staphylococcus species displaying multiple resistance were found to harbor mainly genes conferring resistance to beta-lactams (mecA and blaZ), aminoglycosides [str and aac(6')-Ie-aph(2')-Ia], and trimethoprim [dfr(A) and dfr(D)]. Additional genes conferring resistance to macrolides [mph(C), erm(C), and erm(B)], tetracycline [tet(K) and tet(M)], chloramphenicol [cat(pC221) and cat(pC223)], and streptothricin (sat4) appeared in several strains. Hospitalization and preventive penicillin use were shown to act as selection agents for multidrug-resistant commensal staphylococcal flora.     

Unusual association of a unique CAG interruption in 5′ of DM1 CTG repeats with intergenerational contractions and low somatic mosaicism

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder associated with high variability of symptoms and anticipation. DM1 is caused by an unstable CTG repeat expansion that usually increases in successive generations and tissues. DM1 family pedigrees have shown that ～90% and 10% of transmissions result in expansions and contractions of the CTG repeat, respectively. To date, the mechanisms of CTG repeat contraction remain poorly documented in DM1. In this report, we identified two new DM1 families with apparent contractions and no worsening of DM1 symptoms in two and three successive maternal transmissions. A new and unique CAG interruption was found in 5′ of the CTG expansion in one family, whereas multiple 5′ CCG interruptions were detected in the second family. We showed that these interruptions are associated with maternal intergenerational contractions and low somatic mosaicism in blood. By specific triplet‐prime PCR, we observed that CTG repeat changes (contractions/expansions) occur preferentially in 3′ of the interruptions for both families.     

Rapid Push vs Pump-Infused Subcutaneous Immunoglobulin Treatment: a Randomized Crossover Study of Quality of Life in Primary Immunodeficiency Patients

Purpose

Subcutaneous immunoglobulin replacement therapy (IgRT) may be administered once a week with a pump or every other day with a syringe (rapid push). The objective of the study was to compare the impact of pump and rapid push infusions on patient’s life quality index (LQI).

Methods

This study was a randomized, crossover, multicenter, non-inferiority trial conducted in adults with primary immunodeficiency (PID) accustomed to weekly infusions at home by pump. Patients used pump or rapid push for 3 months each according to the randomized sequence. Main criterion was PID-LQI factor I (treatment interference). Non-inferiority ratio was set at 90%.

Results

Thirty patients entered the study; 28 completed the two periods. IgRT exposure was similar during each period. At the end of each period, mean LQI factor 1 was 87.0 (IC95% [80.3; 94.3]) and 77.80 (IC95% [71.5; 84.7]) for pump and rapid push, respectively. There was a slightly larger effect of rapid push on treatment interference than with pump so that the primary endpoint could not be met. No difference was found on other LQI components, satisfaction (TSQM), or quality of life (SF36v2). Eight patients declared to prefer rapid push while 19 others preferred pump. Of rapid push infusions, 67.2% led to local reactions vs 71.8% of pump infusions (p?=?0.11) illustrating its good tolerance. Rapid push and pump infusions achieved similar trough IgG levels with similar incidence of infections. Rapid push saved 70% of administration cost when compared to pump.

Conclusions

Since IgRT is a lifelong treatment in PID patients, individualization of treatment is of paramount importance. Rapid push is a new administration method in the physician’s armamentarium which is preferred by some patients and is cost-effective.

ClinicalTrials.gov Identifier

NCT02180763

Clinical Implications

Self-administration of small volumes of immunoglobulins at home, every other day, using a syringe (rapid push) is a cost-effective alternative to administration of larger volumes by pump once a week.

Capsule Summary

This study compared subcutaneous infusions of immunoglobulins either weekly via a pump or every other day via a syringe (rapid push). Rapid push is preferred by some patients and is cost-effective, therefore completing a physician’s armamentarium.

     

Comparison of cardiac output as assessed by transesophageal echo-Doppler and transpulmonary thermodilution in patients undergoing thoracic surgery

Study ObjectiveTo evaluate the accuracy of cardiac index (CI) as measured by echo-transesophageal Doppler monitoring (echo-TDM) with CI measured by the transpulmonary thermodilution technique.DesignProspective, observational study.SettingUniversity hospital.Patients16 patients scheduled for elective lung cancer resection.InterventionsPatients underwent two-lung ventilation (TLV) and one-lung ventilation (OLV).Measurements and Main ResultsCI measurements were analyzed using Bland-Altman plots. Absolute values of CI as measured by both devices were highly correlated (r² ranging from 0.72 to 0.77), as were relative changes in CI after the start of OLV (r² = 0.48, P = 0.006). Before, during, and after OLV, TDM-CI biases were 0.46 ± 0.28 L/min/m², 0.25 ± 0.18 L/min/m², and 0.35 ± 0.29 L/min/m², respectively. Limits of agreement remained stable throughout the three measurement periods (range ?1.08 to 0.21 L/min/m²). The mean percentage error of CI measurements was 21.9% compared with the thermodilution technique. Although no adverse events were reported, 11% of measurement sets were incomplete due to poor signal detection.ConclusionsEcho-TDM is a safe technique, allowing continuous semi-invasive assessment of hemodynamic changes in most patients undergoing open-chest surgery. Doppler-derived CI values showed significant biases and moderate clinical agreement with transpulmonary thermodilution during TLV and OLV.     

Pentraxin 3 in plasma and vaginal fluid in women with preterm delivery

OBJECTIVE To investigate the role of pentraxin 3 (PTX3), an acute-phase protein produced by cells of innate immunity in response to inflammatory signals, in spontaneous preterm delivery (PTD). DESIGN Cohort study. SETTING Department of Obstetrics and Gynecology of the University of Milano-Bicocca. POPULATION Forty-six pregnant women with preterm rupture of membranes (n=33) or preterm labour with intact membranes (n=13) delivering at <34 weeks of gestation and 34 women with uncomplicated pregnancies (control group). METHODS We compared plasma and vaginal PTX3 levels between study group and controls, and in women with versus women without clinical or histologic evidence of intrauterine infection using statistical analysis. MAIN OUTCOME MEASURES Peak PTX3 concentration. RESULTS Peak PTX3 concentration in plasma samples of study group was significantly higher than that in controls (1175, 0-9630 versus 650, 0-1450 pg/ml; P=0.0003) but not in vaginal swabs (1660, 0-6604 versus 457, 0-4649 pg/ml; P=0.386). PTX3 levels in plasma were significantly higher in women with placenta vasculopathy compared with that in women with no placental lesions (2910, 0-9630 versus 636, 0-5692 pg/ml; P=0.04). Peak plasma and vaginal PTX3 concentrations were not significantly different in women with versus women without intrauterine infection (1168, 0-7110 versus 845, 0-9630 pg/ml, P=0.34 and 1975, 471-6604 versus 1919, 0-4150 pg/ml, P=0.38, respectively). CONCLUSIONS Spontaneous PTD is associated with a significant increase of maternal plasma concentrations of PTX3. PTX3 seems to be a marker of placenta vasculopathy rather than intrauterine infection.