Reproductive life in women with celiac disease; a nationwide, population-based matched cohort study.

STUDY QUESTION: How does celiac disease (CD) influence women's reproductive life, both prior to and after the diagnosis? SUMMARY ANSWER: Prior to the diagnosis of CD, an increased risk of adverse pregnancy outcomes was seen, whereas after the diagnosis, no influence on reproductive outcomes was found. WHAT IS KNOWN ALREADY: CD has been associated with several conditions influencing female reproduction and pregnancy outcomes including spontaneous abortion and stillbirth. STUDY DESIGN, SIZE, DURATION: A nationwide matched cohort study following 6319 women diagnosed with CD and 63166 comparison women and identifying reproductive events between the ages of 15 and 50 years. PARTICIPANTS/MATERIALS, SETTING, METHODS: Through linkage of several Danish national health registers, we identified all women diagnosed with CD between 1977 and 2016. We identified an age- and sex-matched comparison cohort and obtained data on reproductive outcomes for both cohorts. Adjusted stratified Cox and logistic regression models were used to estimate differences in reproductive outcomes between women with and without CD. MAIN RESULTS AND THE ROLE OF CHANCE: Comparing women with diagnosed CD with the non-CD women, the chance of pregnancy, live birth and risk of stillbirth, molar and ectopic pregnancy, spontaneous abortion and abortion due to foetal disease was the same. However, prior to being diagnosed, CD women had an excess risk of spontaneous abortion equal to 11 extra spontaneous abortions per 1000 pregnancies (adjusted odds ratio (OR) = 1.12, 95% CI: 1.03, 1.22) and 1.62 extra stillbirths per 1000 pregnancies (adjusted OR = 1.57, 95% CI: 1.05, 2.33) compared with the non-CD women. In the period 0-2 years prior to diagnosis fewer pregnancies occurred in the undiagnosed CD group, equal to 25 (95% CI: 20-31) fewer pregnancies per 1000 pregnancies compared to the non-CD group and in addition, fewer undiagnosed CD women initiated ART-treatment in this period, corresponding to 4.8 (95% CI: 0.9, 8.7) fewer per 1000 women compared to non-CD women. LIMITATIONS, REASONS FOR CAUTION: Validity of the diagnoses in the registers was not confirmed, but reporting to the registers is mandatory for all hospitals in Denmark. Not all spontaneous abortions will come to attention and be registered, whereas live- and stillbirths, ectopic and molar pregnancies and abortion due to foetal disease are unlikely not to be registered. We adjusted for several confounding factors but residual confounding cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS: These findings suggest that undiagnosed CD can affect female reproduction and the focus should be on early detection of CD in risk groups. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Health Research Fund of Central Denmark Region and The Hede Nielsens Foundation, Denmark. The authors report no conflicts of interest in this work.

The second Geneva Consensus: Recommendations for novel live TB vaccines.

Infection with Mycobacterium tuberculosis continues to be a major public health burden in most developing parts of the world and efforts to develop effective strategies for containing the disease remain a priority. It has long been evident that effective mass vaccination programmes are a cost effective and efficient approach to controlling communicable diseases in a public health setting and tuberculosis (TB) continues to be a major target. One approach with increasing acceptance is based upon on live mycobacterial vaccines, either as recombinant BCG or rationally attenuated M. tuberculosis, thus generating a new live TB vaccine. The Geneva Consensus published in March 2005 set out the opinion on priorities and requirements for developing live mycobacterial vaccines for Phase I trials. In the intervening period much progress has been made in both preclinical and clinical development of new TB vaccines and has provided the impetus for organising the second Geneva Consensus (held at WHO headquarters, April 2009) to discuss issues, including: i. Explore the regulatory requirements for live TB vaccines to enter Phase I trials, in particular those based on attenuated M. tuberculosis. Particular attention was paid to the characterisation and safety package likely to be required, including issues of attenuation, the presence of antibiotic resistance markers in live vaccines and the nature of any attenuated vaccine phenotype. ii. To identify the general criteria for further clinical development from Phase I through to Phase III. iii. Obtain a perspective of the regulatory landscape of developing countries where Phase II and III trials are to be held. iv. Review manufacturing considerations for live TB vaccines and relevance of the WHO and European Pharmacopeia guidelines and requirements for BCG vaccine. v. Consider requirements and associated issues related to the use of these new vaccines within an existing BCG vaccination programme.

Cationic microparticles consisting of poly(lactide-co-glycolide) and polyethylenimine as carriers systems for parental DNA vaccination.

Cationic microparticles for DNA adsorption were formulated by blending poly(lactide-co-glycolide) (PLGA) (50:50), with different cationic agents, either PEI 25 kDa (polyethylenimine) or CTAB (cetyl-trimethyl-ammonium-bromide). The aim was to create adjuvant delivery systems increasing the efficiency of DNA vaccines. Microparticles formulated with 10% PEI exhibited a highly positive zeta-potential, small particle sizes, in contrast to particles prepared with CTAB, which revealed highly aggregated structures in scanning electron micrographs. PEI 10% microparticles efficiently adsorbed DNA and protected DNA from enzymatic degradation. Microparticles with up to 10% PEI did not affect membrane integrity whereas CTAB particles showed higher LDH release. Transfection efficiencies were assessed using a luciferase reporter gene assay compared to naked DNA and PEI/DNA polyplexes. DNA adsorbed onto microspheres with 10% or 50% PEI generally had higher transfection efficiencies than CTAB but reached lower expression levels than PEI/DNA polyplexes alone. This documented the intact release of DNA. The mechanism of gene delivery to non-phagocytic cells was studied via covalent fluorescence labeling of both the DNA and PEI by confocal microscopy and suggested uptake of DNA. Immunization of mice was performed using plasmids encoding immunodominant antigens of Listeria monocytogenes adsorbed onto RG 502 H+PEI 10% microparticles. The efficiency was tested by intravenous challenge with an otherwise lethal dose of L. monocytogenes. PLGA+PEI microspheres can be used as adjuvant delivery systems for DNA but further optimization is necessary to exploit their full potential.

Limited mycobacterial infection of the liver as a consequence of its microanatomical structure causing restriction of mycobacterial growth to professional phagocytes.

Among sites of extrapulmonary growth of Mycobacterium tuberculosis, the liver is the least infected. Our data suggest that this is due to the complete restriction of mycobacterial growth to liver macrophages. Unlike in organs more persistently seeded by M. tuberculosis, in the liver the bacteria do not infect cell types other than professional phagocytes.

Contribution of MHC class I-dependent immune mechanisms induced by attenuated recombinant Salmonella typhimurium secreting superoxide dismutase to protection against murine listeriosis.

A recombinant (r)Salmonella typhimurium aroA strain secreting the naturally non-secreted superoxide dismutase (SOD) of Listeria monocytogenes controls murine listeriosis dependent on 'transporter associated with antigen processing' (TAP)-mediated immune mechanisms. TAP1-deficient mice (devoid of most CD8 T cells) vaccinated with this rSalmonella SODs strain succumbed to lethal L. monocytogenes challenge, whereas C57BL/6 mice were protected by this vaccine. Moreover, vaccination of H-2I-Abeta-deficient mice (lacking major histocompatibility class (MHC) II molecules and thus devoid of mature CD4 TCR-alphabeta cells), of TAP1-deficient as well as of beta2microglobulin-deficient mice (devoid of conventional CD8 T cells) with a sublethal dose of L. monocytogenes and subsequent challenge with rSalmonella control or SODs strain revealed contribution of both MHC class I- and MHC class II-dependent immune mechanisms to the control of secondary Salmonella infection. Finally, the clearance of rSalmonella SODs bacteria was achieved in TAP1-deficient animals vaccinated with L. monocytogenes. Our data suggest a role of TAP-dependent mechanisms in priming of protective immunity by rSalmonella micro-organisms secreting SOD.

Recombinant attenuated bacteria for the delivery of subunit vaccines.

Using attenuated intracellular bacteria as carriers, we have developed two different approaches for the delivery of subunit vaccines encoding heterologous antigens. The first system is based on the direct secretion of the heterologous antigens in Gram-negative bacteria via the hemolysin secretion system of Escherichia coli into either phagosome or cytosol of infected cells. The second approach is based on the transport of eukaryotic antigen expression vectors by intracellular bacteria like Listeria and Salmonella into the host cell and here, preferably, into the cytosolic compartment. After release of the plasmid DNA from the bacteria, the plasmid-encoded antigens can be expressed directly by the host cell. Finally, we combined both types of subunit vaccines in one live vector - we equipped Salmonella strains with a phagosomal escape function by utilization of the hemolysin secretion system and used this recombinant vaccine strain for the delivery of a eukaryotic antigen expression vector into the cytosol of macrophages.

Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG.

A recombinant (r-) Salmonella typhimurium aroA vaccine that secretes the naturally secreted protein of Mycobacterium bovis strain BCG, Ag85B, by means of the HlyB/HlyD/TolC export machinery (termed p30 in the following) was constructed. In contrast to r-S. typhimurium control, oral vaccination of mice with the r-S. typhimurium p30 construct induced partial protection against an intravenous challenge with the intracellular pathogen Mycobacterium tuberculosis, resulting in similar vaccine efficacy comparable to that of the systemically administered attenuated M. bovis BCG strain. The immune response induced by r-S. typhimurium p30 was accompanied by augmented interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) levels produced by restimulated splenocytes. These data suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated r-S. typhimurium as carrier is capable of inducing an immune response against mycobacterial antigens.

Secretion of different listeriolysin cognates by recombinant attenuated Salmonella typhimurium: superior efficacy of haemolytic over non-haemolytic constructs after oral vaccination.

Viable antigen (Ag) delivery systems expressing defined pathogen-derived proteins represent powerful candidates for future vaccination strategies. Here, recombinant (r)Salmonella typhimurium aroA strains secreting listeriolysin (Hly) of Listeria monocytogenes in haemolytic or non-haemolytic form were constructed to direct these carriers into cytosolic or phagosomal host cell compartments, respectively. Oral and intravenous (i.v.) vaccination of mice with either construct induced 'transporter associated with antigen processing'-dependent protection against the intracellular bacterial pathogen L. monocytogenes. Comparison of oral immunization with both rSalmonella constructs revealed superior vaccine efficacy of the haemolytic rS. typhimurium Hlys construct as compared to the non-haemolytic rSalmonella Hlys(492) strain. In contrast, efficacy of i.v. vaccination with either rSalmonella strain did not significantly differ. Therefore, rSalmonella strains secreting biologically active Hly represent valuable delivery systems for heterologous rAg or DNA which should be exploited for future mucosal vaccination strategies.

Effective DNA vaccination against listeriosis by prime/boost inoculation with the gene gun.

Protective immunity against Listeria monocytogenes strongly depends on CD8+ T lymphocytes, and both IFN-gamma secretion and target cell killing are considered relevant to protection. We analyzed whether we could induce a protective type 1 immune response by DNA vaccination with the gene gun using plasmids encoding for two immunodominant listerial Ags, listeriolysin and p60. To induce a Th1 response, we 1) coprecipitated a plasmid encoding for GM-CSF, 2) employed a prime/boost vaccination schedule with a 45-day interval, and 3) coinjected oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. DNA immunization of BALB/c mice with plasmids encoding for listeriolysin (pChly) and p60 (pCiap) efficiently induced MHC class I-restricted, Ag-specific CD8+ T cells that produced IFN-gamma. Coinjection of CpG-ODN significantly increased the frequency of specific IFN-gamma-secreting T cells. Although pChly induced specific CD8+ T cells expressing CTL activity, it failed to stimulate CD4+ T cells. Only pCiap induced significant CD4+ T cell and humoral responses, which were predominantly of Th2 type. Vaccination with either plasmid induced protective immunity against listerial challenge, and coinjection of CpG ODN improved vaccine efficacy in some situations. This study demonstrates the feasibility of gene gun administration of plasmid DNA for inducing immunity against an intracellular pathogen for which protection primarily depends on type 1 CD8+ T cells.