Improving the Th1 cellular efficacy of the lead Yersinia pestis rF1-V subunit vaccine using SA-4-1BBL as a novel adjuvant

The lead candidate plague subunit vaccine is the recombinant fusion protein rF1-V adjuvanted with alum. While alum generates Th₂ regulated robust humoral responses, immune protection against Yersinia pestis has been shown to also involve Th₁ driven cellular responses. Therefore, the rF1-V-based subunit vaccine may benefit from an adjuvant system that generates a mixed Th₁ and humoral immune response. We herein assessed the efficacy of a novel SA-4-1BBL costimulatory molecule as a Th₁ adjuvant to improve cellular responses generated by the rF1-V vaccine. SA-4-1BBL as a single adjuvant had better efficacy than alum in generating CD4⁺ and CD8⁺ T cells producing TNFα and IFNγ, signature cytokines for Th₁ responses. The combination of SA-4-1BBL with alum further increased this Th₁ response as compared with the individual adjuvants. Analysis of the humoral response revealed that SA-4-1BBL as a single adjuvant did not generate a significant Ab response against rF1-V, and SA-4-1BBL in combination with alum did not improve Ab titers. However, the combined adjuvants significantly increased the ratio of Th₁ regulated IgG_2c in C57BL/6 mice to the Th₂ regulated IgG₁. Finally, a single vaccination with rF1-V adjuvanted with SA-4-1BBL + alum had better protective efficacy than vaccines containing individual adjuvants. Taken together, these results demonstrate that SA-4-1BBL improves the protective efficacy of the alum adjuvanted lead rF1-V subunit vaccine by generating a more balanced Th₁ cellular and humoral immune response. As such, this adjuvant platform may prove efficacious not only for the rF1-V vaccine but also against other infections that require both cellular and humoral immune responses for protection.     

Circulating uncarboxylated matrix Gla protein,a marker of vitamin K status,as a risk factor of cardiovascular disease

Objectives

Vitamin K plays a pivotal role in the synthesis of Matrix Gla protein (MGP), a calcification inhibitor in vascular tissue. Vascular calcification has become an important predictor of cardiovascular disease. The aim of the current study was to examine the potential association of circulating desphospho-carboxylated and -uncarboxylated MGP (dp-cMGP and dp-ucMGP), reflecting vitamin K status, with the incidence of cardiovascular events and disease (CVD) in older individuals.

Study design

The study was conducted in 577 community-dwelling older men and women of the Longitudinal Aging Study Amsterdam (LASA), aged >55 year, who were free of cardiovascular disease at baseline. Multivariate Cox proportional hazards models were used to analyze the data.

Main outcome measures

Incidence of CVD.

Results

After a mean follow-up of 5.6 ± 1.2 year, we identified 40 incident cases of CVD. After adjustment for classical confounders and vitamin D status, we observed a more than 2-fold significantly higher risk of CVD for the highest tertile of dp-ucMGP with a HR of 2.69 (95% CI, 1.09–6.62) as compared with the lowest tertile. Plasma dp-cMGP was not associated with the risk of CVD.

Conclusions

Vitamin K insufficiency, as assessed by high plasma dp-ucMGP concentrations is associated with an increased risk for cardiovascular disease independent of classical risk factors and vitamin D status. Larger epidemiological studies on dp-ucMGP and CVD incidence are needed followed by clinical trials to test whether vitamin K-rich diets will lead to a decreased risk for cardiovascular events.     

Hemophagocytic lymphohistiocytosis and primary immune deficiency disorders

Hemophagocytic lymphohistiocytosis (HLH) is characterized by uncontrolled immune activation and is traditionally associated with inherited gene defects or acquired causes. In addition to abnormalities in cytotoxic granules and lysosomes, various primary immune deficiency disorders (PID) have been identified among patients suffering from HLH. Our purpose was twofold: to better characterize and detail the association between PID and HLH.     

Toxicological assessment of a particulate yeast (1,3/1,6)-beta-D-glucan in rats.

This study investigates the toxicity of WGP 3-6, a yeast-derived beta-glucan ingredient, during single-dose acute and sub-chronic toxicity studies in rats. For the acute study, Fisher-344 rats were administered WGP 3-6 via gavage at a dose of 2000 mg/kg body weight, and any evidence of toxicity was monitored over a 14-day period. WGP 3-6 was well tolerated, indicating that the LD(50) value is greater than 2000 mg/kg body weight. For the sub-chronic study, Fisher-344 rats (10/sex/group) were randomly allocated to receive daily gavage treatment with WGP 3-6 at doses of 0, 2, 33.3, or 100 mg/kg body weight. Control and high-dose satellite recovery groups of each sex also were included. Full toxicological monitoring and endpoint investigations were performed throughout and upon completion of the study. No negative effects on animal weights or food consumption attributable to WGP 3-6 were evident at any dose. In addition, no mortality, clinical pathology, functional/behavioral, microscopic, or gross observations indicating toxicity were observed. Sporadic changes in some biochemical and hematological parameters were observed; however, since the effects were within the physiological ranges in historical controls, were not dose-responsive, or were not observed in both sexes, they were determined to be of no toxicological significance. In conclusion, no adverse or toxic effects were observed after subchronic oral administration of 2, 33.3, or 100mg/kg body weight/day of WGP 3-6 in Fisher-344 rats, and therefore, a no observed adverse effect level (NOAEL) of 100 mg/kg body weight/day, the highest dose tested, was determined.     

Selective impairment of drug-metabolizing enzymes in pig liver during subchronic dietary exposure to aflatoxin B1.

Consequences of subchronic exposure to aflatoxin B1 (AFB1) on liver monooxygenase and transferase enzymes were compared in control pigs and pigs given 385, 867 or 1,807 microg AFB1/kg of feed for 4 weeks. Animals exposed to the highest dose of toxin developed clinical signs of aflatoxicosis, like liver fibrosis, hepatic dysfunction and decreased weight gain. This group had significantly lower levels of liver cytochrome P450, ethoxyresorufin O-deethylase (EROD) activity, testosterone metabolism, P450 1A and P450 3A protein expression. By comparison, mild degenerative hepatic changes, no hepatic dysfunction but a similar pattern of liver P450 enzymes activity without changes in P450 3A expression were observed in pigs exposed to 867 microg AFB1/kg of feed. Benzphetamine and aminopyrine N-demethylase activities were increased in pigs exposed to 867 or 1,807microg AFB1/kg of feed. Pigs exposed to 385 microg AFB1/kg of feed had low levels of EROD activity and all other biotransformation and clinical parameters remained at control levels. Aniline hydroxylase activity, P450 2C protein expression, UDP-glucuronosyl and glutathione S-transferase activities were unaffected at all doses of AFB1. In conclusion, P450 1A and P450 3A appear to be specific targets of AFB1 even if pig did not display clinical sign of liver toxicosis.     

A 90-day ad libitum administration toxicity study of oligoglucosamine in F344 rats.

A 90-day ad libitum administration toxicity study of oligoglucosamine (OG) was carried out using F344 rats of both sexes. The animals were divided into four groups of 20 animals each, 10 of each sex, and fed a diet containing 0, 0.04, 0.2 or 1.0 (w/w)% OG. During the administration period, no animals of either sex died or exhibited abnormal signs in the 0.04% OG and 0.2% OG groups. In the 1% OG group, in both sexes, erythema and swelling of the snout and forelimbs and loss of fur in the forelimbs were observed. On macroscopic observation, emaciation, swelling of the snout, auricles and forelimbs and alopecia of the forelimbs were also observed in 2-3 males of the 1% OG group. It was suggested that these topical abnormalities might be due to dermal responses to OG adhering to the skin and fur, which are easily soiled with saliva during grooming. In the animals of the 1% OG group, food consumption decreased, resulting in body weight gain being suppressed. This was found concomitantly with the abnormal findings mentioned above. Thus, feeding difficulties due to the topical lesions on the snout and forelimbs were thought to affect body weight. In hematology, platelet count, lymphocyte count and differential neutrophil count increased in males of the 1% OG group. These changes might be related to the dermal inflammation. Abnormalities in urinalysis and blood chemistry, as well as a small thymus, small spleen, dark spots or areas on the glandular stomach mucosa, pale Harderian glands and small testes in histopathology, were also observed in males in the 1% OG group. Whether or not all these changes were related only to the malnutrition remains to be elucidated. From these results, OG gave rise to no adverse effects in rats up to the dose level of 0.2 (w/w)%. Thus, the no observed adverse effect level was determined to be 0.2 (w/w)% for rats of either sex (124.0mg/kg/day in males, 142.0mg/kg/day in females).     

662 A/G gene variation in human tumor necrosis factor receptor superfamily,member 9 (TNFRSF9)

The aim of this paper is to report a new coding variance of the TNFRSF9 gene, a candidate for auto-immune diseases. We found the variation in two families with type 2 diabetes mellitus by D-HPLC mutation screening method and confirmed our results by direct sequencing and PCR-RFLP. Although without changing the amino acid coding, the variance may have an effect on codon usage and play a role in disease development, such as type 2 diabetes mellitus. However, we cannot define the role of this variance because the frequency of the minor allele is low in the Chinese population and no homozygote of the variance was found. More research in multiple populations will be necessary to define the role of this variance.