A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Background

Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects.

Methods

A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 – 10⁹ copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.

Results

Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242–292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes.

Conclusion

Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials.     

Glucocorticoid receptor gene polymorphisms and disease activity during pregnancy and the postpartum period in rheumatoid arthritis

Introduction

The mechanism underlying the spontaneous improvement of rheumatoid arthritis (RA) during pregnancy and the subsequent postpartum flare is incompletely understood, and the disease course varies widely between pregnant RA patients. In pregnancy, total and free levels of cortisol increase gradually, followed by a postpartum decrease to prepregnancy values. The glucocorticoid receptor (GR) polymorphisms BclI and N363S are associated with relatively increased glucocorticoid (GC) sensitivity, whereas the 9β and ER22/23EK polymorphisms of the GR gene are associated with a relatively decreased GC sensitivity. We examined the relation between the presence of these GR polymorphisms and level of disease activity and disease course of RA during pregnancy and postpartum.

Methods

We studied 147 participants of the PARA study (Pregnancy-Induced Amelioration of Rheumatoid Arthritis study), a prospective study investigating the natural improvement during pregnancy and the postpartum flare in women with RA. Patients were visited, preferably before pregnancy, at each trimester and at three postpartum time points. On all occasions, disease activity was scored by using DAS28. All patients were genotyped for the GR polymorphisms BclI, N363S, 9β, and ER22/23EK and divided in groups harboring either polymorphisms conferring increased GC sensitivity (BclI and N363S; GC-S patients) or polymorphisms conferring decreased GC sensitivity (9β or 9β + ER22/23EK; GC-I patients). Data were analyzed by using a mixed linear model, comparing GC-S patients with GC-I patients with respect to improvement during pregnancy and the postpartum flare. The cumulative disease activity was calculated by using time-integrated values (area under the curve, AUC) of DAS28 in GC-I patients versus GC-S patients. Separate analyses were performed according to the state of GC use.

Results

GC-S patients treated with GC had a significantly lower AUC of DAS28 in the postpartum period than did GC-I patients. This difference was not observed in patients who were not treated with GCs. During pregnancy, GC-S and GC-I patients had comparable levels of disease activity and course of disease.

Conclusions

Differences in relative GC sensitivity, as determined by GR polymorphisms, are associated with the level of disease activity in the postpartum period in GC-treated patients, but they do not seem to influence the course of the disease per se.     

Gene Expression Analysis Reveals Inhibition of Radiation-Induced TGFβ-Signaling by Hyperbaric Oxygen Therapy in Mouse Salivary Glands

A side effect of radiation therapy in the head and neck region is injury to surrounding healthy tissues such as irreversible impaired function of the salivary glands. Hyperbaric oxygen therapy (HBOT) is clinically used to treat radiation-induced damage but its mechanism of action is largely unknown. In this study, we investigated the molecular pathways that are affected by HBOT in mouse salivary glands two weeks after radiation therapy by microarray analysis. Interestingly, HBOT led to significant attenuation of the radiation-induced expression of a set of genes and upstream regulators that are involved in processes such as fibrosis and tissue regeneration. Our data suggest that the TGFβ-pathway, which is involved in radiation-induced fibrosis and chronic loss of function after radiation therapy, is affected by HBOT. On the longer term, HBOT reduced the expression of the fibrosis-associated factor α-smooth muscle actin in irradiated salivary glands. This study highlights the potential of HBOT to inhibit the TGFβ-pathway in irradiated salivary glands and to restrain consequential radiation induced tissue injury.     

Impact of Anti-Peroxynitrite-Damaged-Thymidine- Monophosphate Antibodies on Disease Activity in Patients with Systemic Lupus Erythematosus

Present study probes the role of peroxynitrite (ONOO^-)-modified thymidine-5′-monophosphate (TMP) in SLE patients with different disease activity scores according to the SLE Disease Activity Index (SLEDAI). Serum analysis showed significant increased number of subjects positive for anti-ONOO^--TMP-protein antibodies in SLE patients with different SLEDAI scores. Interestingly, the levels of these antibodies were significantly higher among SLE patients, whose SLEDAI scores were ≥20. In addition, a significant correlation was observed between the levels of anti-ONOO^--TMP-protein antibodies and the SLEDAI score (r = 0.595, p < 0.0001). In short, this study shows a positive association between anti-ONOO^--TMP-protein antibodies and SLEDAI. The stronger response observed in patients with higher SLEDAI scores suggests that anti-ONOO^--TMP-protein antibodies may be useful in evaluating the progression of SLE and in elucidating the mechanisms of disease pathogenesis.     

The Biology of Gregarina fernandoi n. sp. in the Cockroach Pysnoscelus surinamensis with Observations on its Development and Cytochemical Nature

SYNOPSIS. Gregarina fernandoi n. sp. is a eugregarine. Its structure, development and life history in the gut of the cockroach Pycnoscelus surinamensis are described. It is named as a new species on the basis of its size, nuclear structure, structure and form of the gametocyst and oocyst. Observations were made on the different stages of the parasite and related to the pH of the gut. In the ceca, pH 4.5–5, the parasite was in its early stages of development, in the midgut, pH 6.5–7, it was in syzygy and in the rectum, pH 7.5–8, gametocysts were found.