Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells

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Infrared absorbance spectroscopy of aqueous proteins: Comparison of transmission and ATR data collection and analysis for secondary structure fitting

Attenuated total reflectance (ATR) infrared absorbance spectroscopy of proteins in aqueous solution is much easier to perform than transmission spectroscopy, where short path‐length cells need to be assembled reproducibly. However, the shape of the resulting ATR infrared spectrum varies with the refractive index of the sample and the instrument configuration. Refractive index in turn depends on the absorbance of the sample. In this work, it is shown that a room temperature triglycine sulfate detector and a ZnSe ATR unit can be used to collect reproducible spectra of proteins. A simple method for transforming the protein ATR spectrum into the shape of the transmission spectrum is also given, which proceeds by approximating a Kramers‐Krönig–determined refractive index of water as a sum of four linear components across the amide I and II regions. The light intensity at the crystal surface (with 45° incidence) and its rate of decay away from the surface is determined as a function of the wave number–dependent refractive index as well as the decay of the evanescent wave from the surface. The result is a single correction factor at each wave number. The spectra were normalized to a maximum of 1 between 1600 cm^?1 and 1700 cm^?1 and a self‐organizing map secondary structure fitting algorithm, SOMSpec, applied using the BioTools reference set. The resulting secondary structure estimates are encouraging for the future of ATR spectroscopy for biopharmaceutical characterization and quality control applications.     

Effect of pH, temperature and culture medium composition on the production of an extracellular cysteine proteinase by Micrococcus sp. INIA 528

Growth and proteinase production by Micrococcus sp. INIA 528 in a batch-operated laboratory fermentor were investigated, with trypticase soy broth as the basal medium for studies on optimum temperature, pH and medium composition. Maximum growth was recorded at 34°C and pH 715, whereas optimum temperature and pH for proteinase production were 31°C and pH 6.25. Maximum rate of enzyme production occurred during the late log and early stationary phases of growth. Addition of 5.0 g 1^-1 yeast extract, 1.0 g 1^-1 glucose, 1.0 g 1^-1 MgSO₄ or 1.0 g 1^-1 K₂HPO₄ to basal medium resulted in a lower enzyme yield, but supplementation of basal medium with 2.5 g 1^-1 (NH₄)₂SO₄ increased enzyme production by 45%. A high initial biomass added to fresh broth supplemented with 2.5 g 1^-1 (NH₄)₂SO₄ only increased enzyme activity by 19%, compared to the maximum enzyme activity achieved with the standard inoculum.     

The Vascular Endothelial Growth Factor Inhibitors Ranibizumab and Aflibercept Markedly Increase Expression of Atherosclerosis-Associated Inflammatory Mediators on Vascular Endothelial Cells

Introduction

Recent studies have suggested that the VEGF inhibitors, Ranibizumab and Aflibercept may be associated with an excess of cardiovascular events, potentially driven by increasing atheroma instability, leading to plaque rupture and clinical events. Inflammation plays a key role in the progression of atherosclerotic plaque and particularly conversion to an unstable phenotype. Here, we sought to assess the in vitro effects of these drugs on the expression of key inflammatory mediators on endothelial cells.

Methods

Human coronary artery endothelial cells were co-incubated for 16h with Ranibizumab (0.11nM) or Aflibercept (0.45nM), as determined by each drug’s peak serum concentration (Cmax). Expression at protein (ELISA) and gene (RT-PCR) level of inflammatory chemokines CCL2, CCL5 and CXC3L1 as well as gene expression for the cell adhesion molecules VCAM-1, ICAM-1 and the key NF-κb protein p65 was assessed. VEGF-A protein levels were also determined.

Results

Both drugs significantly increased chemokine, cell adhesion molecule (CAM) and p65 expression, while decreasing VEGF-A protein secretion. At equivalent Cmax concentrations, Aflibercept was significantly more pro-inflammatory than Ranibizumab. Reduction of secreted VEGF-A levels significantly attenuated inflammatory effects of both drugs, whereas blockade of the VEGF-A receptor or silencing of VEGF-A gene synthesis alone had no effect, suggesting that binding of drug to secreted VEGF-A is crucial in promoting inflammation. Finally, blockade of Toll-like receptor 4 significantly reduced inflammatory effects of both drugs.

Conclusion

We demonstrated here, for the first time, that both drugs have potent pro-inflammatory effects, mediated via activation of Toll-like receptor 4 on the endothelial cell surface by drug bound to VEGF-A. Further studies are required to investigate whether these effects are also seen in vivo.     

Costs of Dengue Control Activities and Hospitalizations in the Public Health Sector during an Epidemic Year in Urban Sri Lanka

Background

Reported as a public health problem since the 1960s in Sri Lanka, dengue has become a high priority disease for public health authorities. The Ministry of Health is responsible for controlling dengue and other disease outbreaks and associated health care. The involvement of large numbers of public health staff in dengue control activities year-round and the provision of free medical care to dengue patients at secondary care hospitals place a formidable financial burden on the public health sector.

Methods

We estimated the public sector costs of dengue control activities and the direct costs of hospitalizations in Colombo, the most heavily urbanized district in Sri Lanka, during the epidemic year of 2012 from the Ministry of Health’s perspective. The financial costs borne by public health agencies and hospitals are collected using cost extraction tools designed specifically for the study and analysed retrospectively using a combination of activity-based and gross costing approaches.

Results

The total cost of dengue control and reported hospitalizations was estimated at US$3.45 million (US$1.50 per capita) in Colombo district in 2012. Personnel costs accounted for the largest shares of the total costs of dengue control activities (79%) and hospitalizations (46%). The results indicated a per capita cost of US$0.42 for dengue control activities. The average costs per hospitalization ranged between US$216–609 for pediatric cases and between US$196–866 for adult cases according to disease severity and treatment setting.

Conclusions

This analysis is a first attempt to assess the economic burden of dengue response in the public health sector in Sri Lanka. Country-specific evidence is needed for setting public health priorities and deciding about the deployment of existing or new technologies. Our results suggest that dengue poses a major economic burden on the public health sector in Sri Lanka.     

Laboratory-Enhanced Dengue Sentinel Surveillance in Colombo District,Sri Lanka: 2012-2014

Introduction

Dengue has emerged as a significant public health problem in Sri Lanka. Historically surveillance was passive, with mandatory dengue notifications based on clinical diagnosis with only limited laboratory confirmation. To obtain more accurate data on the disease burden of dengue, we set up a laboratory-based enhanced sentinel surveillance system in Colombo District. Here we describe the study design and report our findings of enhanced surveillance in the years 2012–2014.

Methods

Three outpatient clinics and three government hospitals in Colombo District that covered most of the Colombo metropolitan area were selected for the sentinel surveillance system. Up to 60 patients per week presenting with an undifferentiated fever were enrolled. Acute blood samples from each patient were tested by dengue specific PCR, NS1 ELISA and IgM ELISA. A sub-set of samples was sent to Duke-NUS Singapore for quality assurance, virus isolation and serotyping. Trained medical research assistants used a standardized case report form to record clinical and epidemiological data. Clinical diagnoses by the clinicians-in-charge were recorded for hospitalized cases.

Results

Of 3,127 febrile cases, 43.6% were PCR and/or NS1 positive for dengue. A high proportion of lab confirmed dengue was observed from inpatients (IPD) (53.9%) compared to outpatient (clinics in hospitals and general practice) (7.6%). Dengue hemorrhagic fever (DHF) was diagnosed in 11% of patients at the time of first contact, and the median day of illness at time of presentation to the sentinel sites was 4. Dengue serotype 1 was responsible for 85% of the cases and serotype 4 for 15%. The sensitivity and specificity of the clinicians’ presumptive diagnosis of dengue was 84% and 34%, respectively.

Conclusion

DENV-1, and to a lesser degree DENV-4, infection were responsible for a high proportion of febrile illnesses in Colombo in the years 2012 to 2014. Clinicians’ diagnoses were associated with high sensitivity, but laboratory confirmation is required to enhance specificity.     

Determination of arginine-vasopressin in small volumes of rat plasma

A radioimmunoassay to determine arginine-vasopressin concentration in rat plasma is described. This method was a modification from a previously described technique from which the incubation volume was reduced. It allows us to use only 300 microliter of plasma, 1/4 of the original volume, without any impairment to the sensitivity. The intra- and inter-assay coefficients of variation are 11% and 13% respectively. The accuracy ranges between 90 and 99%. To investigate the influence of sampling blood conditions on arginine-vasopressin levels, five groups of Sprague-Dawley rats are analyzed: Group I, decapitation; Group II, ether anaesthesia and cardiac puncture; Group III, ether anaesthesia and decapitation; Group IV, ketamine anaesthesia and cardiac puncture; Group V, ketamine anaesthesia and decapitation. Arginine-vasopressin levels in Group V (2.2 +/- 0.8 pg/ml) are significantly lower than those from the other four groups and similar to those obtained by other authors using a chronic catheter.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes'milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 × 10² Enterobacteriaceae/ml, 3.9 × 10² coliforms/ml and 2.0 × 10² faecal coliforms/ml, whereas the respective mean counts were 6.2 × 10³/ml, 5.4 × 10³/ml and 1.3 × 10³/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

Effect of atrial natriuretic peptide on arterial pressure and renal function in cirrhotic rats with ascites

Glomerular filtration rate, urine volume, sodium excretion and mean arterial pressure were measured in 10 rats with Cl4C induced cirrhosis presenting sodium retention and ascites, and in 10 control rats before and during the iv administration of the 28 aminoacid rat alpha-Atrial Natriuretic Peptide (alpha-ANP) (a bolus of 1 microgram followed by a constant infusion of 33 ng/min). alpha-ANP induced a similar increase in glomerular filtration rate and filtered sodium load in both groups of rats. In contrast, the increase in urine volume and sodium excretion produced by alpha-ANP was significantly lower in cirrhotic rats (from 13.8 +/- 1.9 to 37.9 +/- 9.1 microliters/min., and from 0.5 +/- 0.1 to 3.3 +/- 1.0 microEq/min) than in control animals (from 14.6 +/- 1.3 to 102.5 +/- 17.7 microliters/min., p less than 0.005; and from 1.0 +/- 0.3 to 14.1 +/- 3.2 microEq/min., p less than 0.001). The results indicate that in rats with experimental cirrhosis and ascites there are blunted diuretic and natriuretic responses to alpha-ANP, probably as a consequence of the exaggerated tubular sodium reabsorption present in these animals.