Inhibition of IL-10 production by maternal antibodies against Group B Streptococcus GAPDH confers immunity to offspring by favoring neutrophil recruitment

Group B Streptococcus (GBS) is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10) by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab')(2) fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10(-/-)) mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a structurally conserved enzyme that is metabolically essential for bacterial growth in media containing glucose as the sole carbon source (i.e., the blood), this protein constitutes a powerful candidate for the development of a human vaccine against this pathogen.     

Relationship between the magnitude of the inbreeding coefficient and milk traits in Holstein and Jersey dairy bull semen used in Brazil

Artificial insemination has been used to improve production in Brazilian dairy cattle; however, this can lead to problems due to increased inbreeding. To evaluate the effect of the magnitude of inbreeding coefficients on predicted transmitting abilities (PTAs) for milk traits of Holstein and Jersey breeds, data on 392 Holstein and 92 Jersey sires used in Brazil were tabulated. The second-degree polynomial equations and points of maximum or minimal response were estimated to establish the regression equation of the variables as a function of the inbreeding coefficients. The mean inbreeding coefficient of the Holstein bulls was 5.10%; this did not significantly affect the PTA for percent milk fat, protein percentage and protein (P = 0.479, 0.058 and 0.087, respectively). However, the PTAs for milk yield and fat decreased significantly after reaching inbreeding coefficients of 6.43 (P = 0.034) and 5.75 (P = 0.007), respectively. The mean inbreeding coefficient of Jersey bulls was 6.45%; the PTAs for milk yield, fat and protein, in pounds, decreased significantly after reaching inbreeding coefficients of 15.04, 9.83 and 12.82% (P < 0.001, P = 0.002, and P = 0.001, respectively). The linear regression was only significant for fat and protein percentages in the Jersey breed (P = 0.002 and P = 0.005, respectively). The PTAs of Holstein sires were more affected by smaller magnitudes of inbreeding coefficients than those of Jersey sires. It is necessary to monitor the inbreeding coefficients of sires used for artificial insemination in breeding schemes in Brazil, since the low genetic variability of the available sires may lead to reduced production.     

Studies on the peptidase activity of transthyretin (TTR)

Transthyretin (TTR) is a plasma protein transporter of thyroxine (T₄) and retinol and also has peptidase activity. In order to characterize TTR peptidase activity we used fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLRSSK-Q-EDDnp and from two portion-mixing libraries as substrates. Most of the susceptible FRET peptides were cleaved at more than one peptide bond, without particular substrate specificity. The more relevant observation was that the peptides containing E or D were cleaved at only one peptide bond and TTR was competitively inhibited by glutathione analog peptide γ-E-A-G-OH that contains two free carboxyl groups. The dependence on ionic interactions of TTR hydrolytic activity was confirmed by the large inhibitory effects of salt and ionic surfactants. TTR was not inhibited by any usual peptidase inhibitors, except by ortho-phenanthroline and EDTA. The mechanism of TTR catalysis was explored by the pH-profile of TTR hydrolytic activity in different temperatures and by proton inventory. The obtained pK and heat of ionization values suggest that a carboxylate and an ammonium group, possibly from a lysine side chain are involved. These results support the recently proposed inducible metalloprotease mechanism for TTR based on its 3D structure in presence of Zn²⁺ and a series of point mutations [Liz et al., Biochem. J 443 (2012) 769–778].     

Diagnosis of Streptococcus pneumoniae meningitis by polymerase chain reaction amplification of the gene for pneumolysin

Diagnosis of bacterial meningitis has long been based on classical methods of Gram stain, serological tests, and culture of cerebrospinal fluid (CSF). The performance of these methods, especially culture and direct smear is thwarted by failure to detect bacteria following administration of antimicrobial agents and reluctance to performance lumbar punctures at admission. Indeed, patients with meningitis frequently receive antibiotics orally or by injection before the diagnosis is suspected or established Thus an alternative method has become necessary to help clinicians and epidemiologists to management and control of bacterial meningitis. We evaluate the application of a polymerase chain reaction-based (PCR) assay for amplification of pneumolysin gene (ply) to diagnosis of Streptococcus pneumoniae meningitis. The PCR assay sensitivity for CSF was 96% (95% confidence interval, CI, 90-99%) compared to a sensitivity of 59% for culture (95% CI 49-69%), 66% for Gram stain (95% CI 56-74%), and 78% for latex agglutination test (95% CI 69-86%); PCR specificity was 100% (95% CI 83-100%). PCR results were available within 4 h of the start of the assay. This molecular approach proved to be reliable and useful to identify this bacterium compared with other classical laboratory methods for identification of bacterial meningitis pathogens.     

Escalating Plasmodium falciparum antifolate drug resistance mutations in Macha, rural Zambia

Background

Artemisinin and its derivatives have been used for falciparum malaria treatment in China since late 1970s. Monotherapy and uncontrolled use of artemisinin drugs were common practices for a long period of time. In vitro tests showed that the susceptibility of Plasmodium falciparum to artemisinins was declining in China. A concern was raised about the resistance to artemisinins of falciparum malaria in the country. It has been reported that in vitro artemisinin resistance was associated with the S769N mutation in the PfATPase6 gene. The main purpose of this study was to investigate whether that mutation has occurred in field isolates from China.

Methods

Plasmodium falciparum field isolates were collected in 2006–2007 from Hainan and Yunnan provinces, China. A nested PCR-sequencing assay was developed to analyse the genotype of the PfATPase6 S769N polymorphism in the P. falciparum field isolates.

Results

The genotyping results of six samples could not be obtained due to failure of PCR amplification, but no S769N mutation was detected in any of the 95 samples successfully analysed.

Conclusion

The results indicate that the S769N mutation in the PfATPase6 gene is not present in China, suggesting that artemisinin resistance has not yet developed, but the situation needs to be watched very attentively.     

Passion fruit flowers: Kunitz trypsin inhibitors and cystatin differentially accumulate in developing buds and floral tissues

In order to better understand the physiological functions of protease inhibitors (PIs) the PI activity in buds and flower organs of passion fruit (Passiflora edulis Sims) was investigated. Trypsin and papain inhibitory activities were analyzed in soluble protein extracts from buds at different developmental stages and floral tissues in anthesis. These analyses identified high levels of inhibitory activity against both types of enzymes at all bud stages. Intriguingly, the inhibitory activity against both proteases differed remarkably in some floral tissues. While all organs tested were very effective against trypsin, only sepal and petal tissues exhibited strong inhibitory activity against papain. The sexual reproductive tissues (ovary, stigma-style and stamen) showed either significantly lower activity against papain or practically none. Gelatin–SDS–PAGE assay established that various trypsin inhibitors (TIs) homogenously accumulated in developing buds, although some were differentially present in floral organs. The N-terminal sequence analysis of purified inhibitors from stamen demonstrated they had homology to the Kunitz family of serine PIs. Western-blot analysis established presence of a ∼60 kDa cystatin, whose levels progressively increased during bud development. A positive correlation between this protein and strong papain inhibitory activity was observed in buds and floral tissues, except for the stigma-style. Differences in temporal and spatial accumulation of both types of PIs in passion fruit flowers are thus discussed in light of their potential roles in defense and development.     

Ovarian follicular dynamics during the estrous cycle in buffalo (Bubalus bubalis)

The growth, selection, regression and ovulation of ovarian follicles was ultrasonically monitored in 30 Murrah buffalo throughout a spontaneous estrous cycle during the breeding season (autumn). Examinations revealed that follicular growth during the estrous cycle occurs in waves; the buffalo showed 1-wave (3.3%, n = 1), 2-wave (63.3%, n = 19) or 3-wave (33.3%, n = 10) follicular growth. The first wave began at 1.00, 1.16 +/-0.50 and 1.10 +/- 0.32 d in buffalo with 1, 2 and 3 waves, respectively (ovulation = Day 0). The second wave appeared at 10.83 +/- 1.09 and 9.30 +/- 1.25 d (P < 0.01) for the 2 and 3 wave cycle animals, respectively. The third wave started at 16.80 +/- 1.22 d. Structural persistence of the first dominant follicle was longer in the 2- than 3-wave cycles (20.67 +/- 1.18 vs 17.90 +/- 3.47 d ; P < 0.05). The duration of the growth and static phases of the first dominant follicle differed between the 2 and 3 wave cycles (P < 0.05), whereas there were no differences in linear growth rates (cm/d). Two and three wave cycles differed (P < 0.05) with respect to the maximum diameter of both the first dominant follicle (1.51 +/- 0.24 vs 1.33 +/- 0.18 cm) and the ovulatory follicles (1.55 +/- 0.16 vs 1.34 +/- 0.13 cm). No relationship was found between dominant follicle development and the presence of either a CL or a previous dominant follicle in either ovary. Two and three wave cycles also differed with respect to the mean length of intervals between ovulation (22.27 +/- 0.89 vs 24.50 +/- 1.88 d; P < 0.01) and the mean length of luteal phases (10.40 +/- 2.11 vs 12.66 +/- 2.91 d; P < 0.05). These results demonstrate that buffalo have estrous cycles with 1, 2 or 3 follicular waves; that 2-wave cycles are the most common; and that the number of waves in a cycle is associated with the luteal phase and with estrous cycle length.     

Variations of Mn,Fe and S concentrations in sediment pore waters of Ria Formosa at different time scales

Intertidal sediments of Ria Formosa have been surveyed at different time scales. Pore water of short sediment cores was collected bimonthly at five stations, from May 1993 to June 1994, around low-tide. One location was studied intensively every 1.5 hour, during a 6-hour air-exposure period, and during 20 minutes (1, 5, 10 and 20 minutes) immediately after tidal water had inundated the sampling site. Determinations of vertical profiles of pH, Eh, water content, O₂, total dissolved Mn (Mn_diss), total dissolved Fe (Fe_diss) and inorganic sulphur [(HS^–)_t] were carried out in all samples. The results obtained indicate that daily variations of Mn_diss, Fe_diss and (HS^–)_t concentrations appear to be superimposed to the seasonal ones. Probably, the renewal of the pore water at periods of tidal inundation causes advective transport of manganese and exchange of iron, between the pore water and the solid fraction of the sediment that masks seasonal fluctuations induced by temperature and organic matter input variations.