Cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus

To understand the role of cytokines during rotavirus infection, we assessed the kinetics of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) (proinflammatory), IL-12 (Th1 inducer), gamma interferon (IFN-gamma) (Th1), IL-4 and IL-10 (Th2), and transforming growth factor beta (Th3) cytokine responses by enzyme-linked immunosorbent assay in serum and intestinal contents of neonatal gnotobiotic pigs and IL-12, IFN-gamma, IL-4, and IL-10 cytokine-secreting cell (CSC) responses of mononuclear cells from ileum, spleen, and blood by ELISPOT. Pigs received the virulent Wa P1A[8]G1 strain of human rotavirus (HRV) (VirHRV), attenuated Wa HRV (AttHRV), or mock (controls). The TNF-alpha levels peaked earlier and remained elevated in serum of the VirHRV group but peaked later in the AttHRV group. In serum, IL-6 was significantly elevated at postinoculation day (PID) 1 in the VirHRV group and at PID 3 in both HRV groups. The IL-12 was detected in serum of all pigs including controls with significantly elevated peaks in both HRV-infected groups, indicating a role for IL-12 in the induction of immune responses to rotavirus infection. Only low and transient IFN-gamma responses occurred in serum and intestinal contents of the AttHRV-infected pigs, compared to significantly higher and prolonged IFN-gamma responses in the VirHRV-infected pigs. This observation coincides with the diarrhea and viremia induced by VirHRV. The number of IFN-gamma-secreting cells was significantly higher in the ileum of the VirHRV group than in that of the controls. The number of IL-4 CSCs was significantly higher in ileum of both HRV groups than in that of the controls. Significantly higher levels of IL-10 in the serum occurred early in the VirHRV group, compared to lower levels in the AttHRV group. However, the number of IL-10 CSCs was significantly higher later in ileum and spleen of the AttHRV than in the VirHRV group, suggesting a delayed initiation of a Th2 response induced by AttHRV. A significantly higher percentage of pigs had IFN-gamma and IL-10 responses in serum after VirHRV infection than after AttHRV infection or in controls. These data indicate a balanced Th1/Th2 response during rotavirus infection, with higher cytokine levels early after infection with VirHRV compared to that with AttHRV. Mapping the kinetics and patterns of cytokine responses after rotavirus infection has important implications for induction of protective immunity by HRV vaccines. Higher protection rates may be associated with more balanced Th1- and Th2-type responses, but induction of higher earlier IFN-gamma (Th1) and proinflammatory cytokines triggered by VirHRV may also play an important role in the higher intestinal immunoglobulin A responses and protection rates induced by VirHRV.     

Vitamin A Deficiency Impairs Adaptive B and T Cell Responses to a Prototype Monovalent Attenuated Human Rotavirus Vaccine and Virulent Human Rotavirus Challenge in a Gnotobiotic Piglet Model

Rotaviruses (RV) are a major cause of gastroenteritis in children. Widespread vitamin A deficiency is associated with reduced efficacy of vaccines and higher incidence of diarrheal infections in children in developing countries. We established a vitamin A deficient (VAD) gnotobiotic piglet model that mimics subclinical vitamin A deficiency in children to study its effects on an oral human rotavirus (HRV) vaccine and virulent HRV challenge. Piglets derived from VAD and vitamin A sufficient (VAS) sows were orally vaccinated with attenuated HRV or mock, with/without supplemental vitamin A and challenged with virulent HRV. Unvaccinated VAD control piglets had significantly lower hepatic vitamin A, higher severity and duration of diarrhea and HRV fecal shedding post-challenge as compared to VAS control pigs. Reduced protection coincided with significantly higher innate (IFNα) cytokine and CD8 T cell frequencies in the blood and intestinal tissues, higher pro-inflammatory (IL12) and 2-3 fold lower anti-inflammatory (IL10) cytokines, in VAD compared to VAS control pigs. Vaccinated VAD pigs had higher diarrhea severity scores compared to vaccinated VAS pigs, which coincided with lower serum IgA HRV antibody titers and significantly lower intestinal IgA antibody secreting cells post-challenge in the former groups suggesting lower anamnestic responses. A trend for higher serum HRV IgG antibodies was observed in VAD vs VAS vaccinated groups post-challenge. The vaccinated VAD (non-vitamin A supplemented) pigs had significantly higher serum IL12 (PID2) and IFNγ (PID6) compared to vaccinated VAS groups suggesting higher Th1 responses in VAD conditions. Furthermore, regulatory T-cell responses were compromised in VAD pigs. Supplemental vitamin A in VAD pigs did not fully restore the dysregulated immune responses to AttHRV vaccine or moderate virulent HRV diarrhea. Our findings suggest that that VAD in children in developing countries may partially contribute to more severe rotavirus infection and lower HRV vaccine efficacy.     

Characterization of NMCR-2, a new non-mobile colistin resistance enzyme: implications for an MCR-8 ancestor

MCR-4 and MCR-8 are two recently identified members of an ongoing MCR family of colistin resistance. Although that aquatic reservoir for MCR-4 is proposed, the origin and mechanism of MCR-8 is poorly understood. Here we report a previously unrecognized non-mobile colistin resistance enzyme, termed NMCR-2, originating from the plant pathogen Kosakonia pseudosacchari. NMCR-2 (551aa) gives 67.3% identity to MCR-8 (565aa). NMCR-2 is placed as a progenitor/ancestor for MCR-8 in phylogeny of MCR members. Genetic study reveals that nmcr-2 is comparable to mcr-8 in the ability of producing phenotypic colistin resistance. Biochemical analyses determine that these two enzymes catalyse the transfer of PEA from the donor PE lipid substrate to the recipient lipid A molecule by a putative ‘ping-pong’ trade-off. Further experiment of protein engineering demonstrates that the two motifs (TM region and catalytic domain) of NMCR-2 are functionally exchangeable with that of MCR-8, rather than MCR-1. Physiological impacts of nmcr-2 and/or mcr-8 are detected in Escherichia coli, featuring with fitness cost. Evidently, the action and mechanism of NMCR-2 is analogous to that of MCR-8. Therefore, our finding underlines that NMCR-2 might be a possible progenitor of MCR-8.     

Cardiac myocytes' dynamic contractile behavior differs depending on heart segment

Cardiac myocytes originating from different parts of the heart exhibit varying morphology and ultrastructure. However, the difference in their dynamic behavior is unclear. We examined the contraction of cardiac myocytes originating from the apex, ventricle, and atrium, and found that their dynamic behavior, such as amplitude and frequency of contraction, differs depending on the heart segment of origin. Using video microscopy and high‐precision image correlation, we found that: (1) apex myocytes exhibited the highest contraction rate (～17 beats/min); (2) ventricular myocytes exhibited the highest contraction amplitude (～5.2 micron); and (3) as myocyte contraction synchronized, their frequency did not change significantly, but the amplitude of contraction increased in apex and ventricular myocytes. In addition, as myocyte cultures mature they formed contractile filaments, further emphasizing the difference in myocyte dynamics is persistent. These results suggest that the dynamic behavior (in addition to static properties) of myocytes is dependent on their segment of origin. Biotechnol. Bioeng. 2013; 110: 628–636. © 2012 Wiley Periodicals, Inc.     

Viremia and nasal and rectal shedding of rotavirus in gnotobiotic pigs inoculated with Wa human rotavirus

Respiratory symptoms with rotavirus shedding in nasopharyngeal secretions have been reported in children with and without gastrointestinal symptoms (Zheng et al., 1991, J. Med. Virol. 34:29-37). To investigate if attenuated and virulent human rotavirus (HRV) strains cause upper respiratory tract infections or viremia in gnotobiotic pigs, we inoculated them with attenuated or virulent HRV intranasally, intravenously, or orally or via feeding tube (gavage) and assayed virus shedding. After oral or intranasal inoculation with attenuated HRV, the pigs remained asymptomatic, but 79 to 95% shed virus nasally and 5 to 17% shed virus rectally. After inoculation by gavage, no pigs shed virus nasally or rectally, but all pigs seroconverted with antibodies to HRV. No viremia was detected through postinoculation day 10. Controls inoculated intranasally with nonreplicating rotavirus-like particles or mock inoculated did not shed virus. In contrast, 100% of pigs inoculated with virulent HRV (oral, intranasal, or gavage) developed diarrhea, shed virus nasally and rectally, and had viremia. The infectivity of sera from the viremic virulent HRV-inoculated pigs was confirmed by inoculating gnotobiotic pigs orally with pooled HRV-positive serum. Serum-inoculated pigs developed diarrhea and fecal and nasal virus shedding and seroconverted with serum and intestinal HRV antibodies. Pigs inoculated intravenously with serum or intestinal contents from the viremic virulent HRV-inoculated pigs developed diarrhea, virus shedding, and viremia, similar to the orally inoculated pigs. This study provides new evidence that virulent HRV causes transient viremia and upper respiratory tract infection in addition to gastrointestinal infection in gnotobiotic pigs, confirming previous reports of rotavirus antigenemia (Blutt et al., Lancet 362:1445-1449, 2003). Our data also suggest that intestinal infection might be initiated from the basolateral side of the epithelial cells via viremia. Additionally, virus shedding patterns indicate a different pathogenesis for attenuated versus virulent HRV.     

Crystal structure of the Bacillus anthracis nucleoside diphosphate kinase and its characterization reveals an enzyme adapted to perform under stress conditions

Nucleoside diphosphate kinases (Ndks) play an important role in a plethora of regulatory and metabolic functions. Inhibition of the B. anthracis Ndk mRNA results in the formation of nonviable aberrant spores. We report the characterization and crystal structure of the enzyme from B. anthracis nucleoside diphosphate kinase (BaNdk), the first from sporulating bacteria. The enzyme, although from a mesophilic source, is active at extremes of pH (3.5–10.5), temperature (10–95°C) and ionic strength (0.25–4.0M NaCl). It exists as a hexamer that is composed of two SDS‐stable trimers interacting in a back‐to‐back association; mutational analysis confirms that the enzyme is a histidine kinase. The high‐resolution crystal structure reported here reveals an unanticipated change in the conformation of residues between 43 and 63 that also regulates substrate entry in other Ndks. A comparative structural analysis involving Ndks from seven mesophiles and three thermophiles has resulted in the delineation of the structure into relatively rigid and flexible regions. The analysis suggests that the larger number of intramolecular hydrogen bonds and to a lesser extent ionic interactions in BaNdk contributes to its high thermostability. Mutational analysis and Molecular Dynamics simulations were used to probe the role of a highly conserved Gly19 (present at the oligomeric interface in most of the Ndks). The results suggest that the mutation leads to a rigidification of those residues that facilitate substrate entry and consequently leads to a large reduction in the kinase activity. Overall, the enzyme characterization helps to understand its apparent adaptation to perform under stress conditions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Coral recruitment and early benthic community development on several materials used in the construction of artificial reefs and breakwaters

Artificial reefs are increasingly being promoted as a means to mitigate impacts from human activities in coastal urban areas. Coastal defense structures such as breakwaters are becoming recognized as large-scale artificial reefs that support abundant and diverse marine communities and play important roles in coastal ecology and management. However, there is limited understanding of how substrate materials used to construct artificial reefs or breakwaters can influence the development of habitat-forming benthic organisms. To assess the influence of substrata on coral recruitment and overall benthic community development, we deployed standard-size tiles of materials used in the construction of breakwaters and artificial reefs (concrete, gabbro, granite, and sandstone), along with terra-cotta for comparative purposes, at two breakwaters (DDD, PRT) and two natural reef sites (NR1, NR2) in Dubai, United Arab Emirates, for one year. Kruskal-Wallis ANOVA with post-hoc Mann-Whitney U-tests were used to examine differences in coral recruitment among sites and materials. Coral recruitment was highest at the DDD (4.9 ± 0.5 recruits 100 cm^− 2), while recruitment was low and did not differ among other sites (PRT: 0.1 ± 0.04, NR1:0.3 ± 0.1, NR2: 0.1 ± 0.03 recruits 100 cm^− 2). There were significant differences in coral recruitment among materials at DDD, where gabbro had higher recruit densities than concrete and sandstone; sandstone also contained less coral recruits than terra-cotta. Variability associated with low coral recruit densities precluded significant differences among materials at other sites. Overall benthic community structure differed more as a result of differences among sites than among substrate materials. Higher community dissimilarity was observed among sites than among material in SIMPER analysis, and significant differences were only observed among sites in ANOSIM. Univariate comparison of the benthos correlated with community differences in NMS ordination also showed significant differences among sites but not material. Overall, these results indicate that site-specific differences in recruitment patterns are more important in determining early benthic community structure and coral recruitment than are differences among substrate material. However, where coral recruitment is high, these results suggest that gabbro should be used preferentially over concrete or sandstone where it is feasible, but that granite may be a suitable alternative where it is the dominant stone. Coral recruitment on terra-cotta was comparable to all materials but sandstone, supporting its continued use in recruitment studies. These results also indicate that using stone amenable to coral recruitment is unlikely to influence the wider benthic community.     

Gender Disparities in the Presentation,Management and Outcomes of Acute Coronary Syndrome Patients: Data from the 2nd Gulf Registry of Acute Coronary Events (Gulf RACE-2)

Background

Gender-related differences in mortality of acute coronary syndrome (ACS) have been reported. The extent and causes of these differences in the Middle-East are poorly understood. We studied to what extent difference in outcome, specifically 1-year mortality are attributable to demographic, baseline clinical differences at presentation, and management differences between female and male patients.

Methodology/Principal Findings

Baseline characteristics, treatment patterns, and 1-year mortality of 7390 ACS patients in 65 hospitals in 6 Arabian Gulf countries were evaluated during 2008–2009, as part of the 2nd Gulf Registry of Acute Coronary Events (Gulf RACE-2). Women were older (61.3±11.8 vs. 55.6±12.4; P<0.001), more overweight (BMI: 28.1±6.6 vs. 26.7±5.1; P<0.001), and more likely to have a history of hypertension, hyperlipidemia or diabetes. Fewer women than men received angiotensin-converting enzyme inhibitors (ACE), aspirin, clopidogrel, beta blockers or statins at discharge. They also underwent fewer invasive procedures including angiography (27.0% vs. 34.0%; P<0.001), percutaneous coronary intervention (PCI) (10.5% vs. 15.6%; P<0.001) and reperfusion therapy (6.9% vs. 20.2%; P<0.001) than men. Women were at higher unadjusted risk for in-hospital death (6.8% vs. 4.0%, P<0.001) and heart failure (HF) (18% vs. 11.8%, P<0.001). Both 1-month and 1-year mortality rates were higher in women than men (11% vs. 7.4% and 17.3% vs. 11.4%, respectively, P<0.001). Both baseline and management differences contributed to a worse outcome in women. Together these variables explained almost all mortality disparities.

Conclusions/Significance

Differences between genders in mortality appeared to be largely explained by differences in prognostic variables and management patterns. However, the origin of the latter differences need further study.