Influenza Virus Surveillance in Pakistan during 2008-2011

Background

There is little information about influenza among the Pakistani population. In order to assess the trends of Influenza-like-Illness (ILI) and to monitor the predominant circulating strains of influenza viruses, a country-wide lab-based surveillance system for ILI and Severe Acute Respiratory Illness (SARI) with weekly sampling and reporting was established in 2008. This system was necessary for early detection of emerging novel influenza subtypes and timely response for influenza prevention and control.

Methods

Five sentinel sites at tertiary care hospitals across Pakistan collected epidemiological data and respiratory samples from Influenza-like illness (ILI) and severe acute respiratory illness (SARI) cases from January 2008 to December 2011. Samples were typed and sub-typed by Real-Time RT-PCR assay.

Results

A total of 6258 specimens were analyzed; influenza virus was detected in 1489 (24%) samples, including 1066 (72%) Influenza type A and 423 (28%) influenza type B viruses. Amongst influenza A viruses, 25 (2%) were seasonal A/H1N1, 169 (16%) were A/H3N2 and 872 (82 %) were A(H1N1)pdm09. Influenza B virus circulation was detected throughout the year along with few cases of seasonal A/H1N1 virus during late winter and spring. Influenza A/H3N2 virus circulation was mainly observed during summer months (August-October).

Conclusions

The findings of this study emphasize the need for continuous and comprehensive influenza surveillance. Prospective data from multiple years is needed to predict seasonal trends for vaccine development and to further fortify pandemic preparedness.     

Hypothalamic S-Nitrosylation Contributes to the Counter-Regulatory Response Impairment following Recurrent Hypoglycemia

Aims

Hypoglycemia is a severe side effect of intensive insulin therapy. Recurrent hypoglycemia (RH) impairs the counter-regulatory response (CRR) which restores euglycemia. During hypoglycemia, ventromedial hypothalamus (VMH) production of nitric oxide (NO) and activation of its receptor soluble guanylyl cyclase (sGC) are critical for the CRR. Hypoglycemia also increases brain reactive oxygen species (ROS) production. NO production in the presence of ROS causes protein S-nitrosylation. S-nitrosylation of sGC impairs its function and induces desensitization to NO. We hypothesized that during hypoglycemia, the interaction between NO and ROS increases VMH sGC S-nitrosylation levels and impairs the CRR to subsequent episodes of hypoglycemia. VMH ROS production and S-nitrosylation were quantified following three consecutive daily episodes of insulin-hypoglycemia (RH model). The CRR was evaluated in rats in response to acute insulin-induced hypoglycemia or via hypoglycemic-hyperinsulinemic clamps. Pretreatment with the anti-oxidant N-acetyl-cysteine (NAC) was used to prevent increased VMH S-nitrosylation.

Results

Acute insulin-hypoglycemia increased VMH ROS levels by 49±6.3%. RH increased VMH sGC S-nitrosylation. Increasing VMH S-nitrosylation with intracerebroventricular injection of the nitrosylating agent S-nitroso-L-cysteine (CSNO) was associated with decreased glucagon secretion during hypoglycemic clamp. Finally, in RH rats pre-treated with NAC (0.5% in drinking water for 9 days) hypoglycemia-induced VMH ROS production was prevented and glucagon and epinephrine production was not blunted in response to subsequent insulin-hypoglycemia.

Conclusion

These data suggest that NAC may be clinically useful in preventing impaired CRR in patients undergoing intensive-insulin therapy.     

Treatment of nitric oxide supplemented with nitrogen and sulfur regulates photosynthetic performance and stomatal behavior in mustard under salt stress

Nitric oxide (NO) is a hormone that connects numerous reactions in plant cells under normal and environmental stress conditions. The application of 100 µM NO as sodium nitroprusside (SNP; NO donor) applied individually or in combination with N or S in different combinations (i.e. 100 mg N or S kg⁻¹ soil applied at the time of sowing [100 N + 100S]_0d or with split, 50 mg N or S kg⁻¹ soil at the time of sowing and similar dose at 20 d after sowing [50 N + 50S]_0d + [50 N + 50S]_20d) was tested to alleviate salt stress in mustard (Brassica juncea L.). Application of 100 µM NO plus split application of N and S more significantly promoted stomatal behavior, photosynthetic and growth performance in the absence of salt stress and maximally alleviated effects of salt stress through increased N- and S-use efficiency, proline and antioxidant system. The combined application of N and S at the time of sowing was lesser effective in promoting photosynthesis and growth under salt or no salt stress conditions in presence or absence of NO. The study suggests that salt stress effects on the photosynthetic performance are mitigated more efficiently when NO was applied together with the split application of N and S given at two stages, and the photosynthetic activity was promoted under salt stress through increased N and S assimilation and antioxidant system. This strategy may be adopted in agricultural system for overcoming salt stress effects on performance of mustard.     

NO and CO differentially activate soluble guanylyl cyclase via a heme pivot-bend mechanism

Diatomic ligand discrimination by soluble guanylyl cyclase (sGC) is paramount to cardiovascular homeostasis and neuronal signaling. Nitric oxide (NO) stimulates sGC activity 200-fold compared with only four-fold by carbon monoxide (CO). The molecular details of ligand discrimination and differential response to NO and CO are not well understood. These ligands are sensed by the heme domain of sGC, which belongs to the heme nitric oxide oxygen (H-NOX) domain family, also evolutionarily conserved in prokaryotes. Here we report crystal structures of the free, NO-bound, and CO-bound H-NOX domains of a cyanobacterial homolog. These structures and complementary mutational analysis in sGC reveal a molecular ruler mechanism that allows sGC to favor NO over CO while excluding oxygen, concomitant to signaling that exploits differential heme pivoting and heme bending. The heme thereby serves as a flexing wedge, allowing the N-terminal subdomain of H-NOX to shift concurrent with the transition of the six- to five-coordinated NO-bound state upon sGC activation. This transition can be modulated by mutations at sGC residues 74 and 145 and corresponding residues in the cyanobacterial H-NOX homolog.     

Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes

To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks.     

Molecular Epidemiology of Influenza A(H1N1)pdm09 Viruses from Pakistan in 2009-2010

Background

In early 2009, a novel influenza A(H1N1) virus that emerged in Mexico and United States rapidly disseminated worldwide. The spread of this virus caused considerable morbidity with over 18000 recorded deaths. The new virus was found to be a reassortant containing gene segments from human, avian and swine influenza viruses.

Methods/Results

The first case of human infection with A(H1N1)pdm09 in Pakistan was detected on 18^th June 2009. Since then, 262 laboratory-confirmed cases have been detected during various outbreaks with 29 deaths (as of 31^st August 2010). The peak of the epidemic was observed in December with over 51% of total respiratory cases positive for influenza. Representative isolates from Pakistan viruses were sequenced and analyzed antigenically. Sequence analysis of genes coding for surface glycoproteins HA and NA showed high degree of high levels of sequence identity with corresponding genes of regional viruses circulating South East Asia. All tested viruses were sensitive to Oseltamivir in the Neuraminidase Inhibition assays.

Conclusions

Influenza A(H1N1)pdm09 viruses from Pakistan form a homogenous group of viruses. Their HA genes belong to clade 7 and show antigenic profile similar to the vaccine strain A/California/07/2009. These isolates do not show any amino acid changes indicative of high pathogenicity and virulence. It is imperative to continue monitoring of these viruses for identification of potential variants of high virulence or drug resistance.     

Obesity and postprandial lipemia in adolescents: risk factors for cardiovascular disease

In the last 50 years, obesity has become a global epidemic and is one of the main public health problems in many parts of the world. Adolescence is a critical period regarding weight control. The factors determining obesity include a complex group of interrelated biological, behavioral and environmental factors which reinforce each other. In children and adolescents, obesity is associated with premature cardiovascular diseases, diabetes mellitus type 2, acanthosis nigricans, respiratory and skeletal muscle problems, as well as psychological problems. The clinical manifestations of cardiovascular disease begin in middle age. Nevertheless, studies indicate that the atherosclerotic process begins to develop during childhood. Postprandial hyperlipemia is a physiological process that occurs several times a day after the complete absorption of a diet including lipids and has been suggested as a risk factor for coronary heart disease (CHD). New study areas include the effects of different fatty acids, lipid sources (endogenous and exogenous), and the effect ingesting alcoholic beverages during meals. Given the evidence that postprandial lipidemia is an independent risk factor for CHD, it is vital to establish normative values for children and adolescents such that more effective and efficient preventive and therapeutic measures can be adopted.     

Soluble signalling factors derived from differentiated cartilage tissue affect chondrogenic differentiation of rat adult marrow stromal cells.

BACKGROUND: Chondral defects show lack of proper regeneration whereas osteochondral lesions display limited regeneration capacity. Latter is probably due to immigration of chondroprogenitor cells from the subchondral bone. Known chondroprogenitor cells for cartilage tissues are multi-potent adult marrow stromal or mesenchymal stem cells (MSCs). In vitro chondrogenic differentiation of these precursor cells usually require cues from growth and signalling factors provided in vivo by surrounding tissues and cells. We hypothesise that signalling factors secreted by differentiated cartilage tissue can initiate and maintain chondrogenic differentiation status of MSCs. METHODS: To study such paracrine communication between allogenic rat articular cartilage and rat MSCs embedded in alginate beads a novel coculture system without addition of external growth factors has been established. RESULTS: Impact of cartilage on differentiating MSCs was observed at two different time points. Firstly, sustained expression of Sox9 was observed at an early stage which indicated induction of chondrogenic differentiation. Secondly, late stage repression of collagen X indicated pre-hypertrophic arrest of differentiation. In the culture supernatant we have identified vascular endothelial growth factor alpha (VEGF-164 alpha), matrix metalloproteinase (MMP) -13 and tissue inhibitors of MMPs (TIMP-1 and TIMP-2) which could be traced back either to the cartilage explant or to the MSCs under the influence of cartilage. CONCLUSION: The identified factors might be involved in regulation of collagen X gene and protein expression and therefore, may have an impact on the control and regulation of MSCs differentiation.