Genotypically distinct strains of Leishmania major display diverse clinical and immunological patterns in BALB/c mice

Infection with Leishmania major species is endemic in many provinces of Iran. Isolates from four endemic areas located in north (Damghan), center (Kashan), west (Dehloran), and south (Shiraz) of country which showed major distinctive polymorphism by RAPD-PCR method were evaluated. Isolates were inoculated to different groups of BALB/c mice and their clinical and immunological status was compared. Lesion size, parasite burden and T cell phenotype in lymph node (LN), and cytokine secretion in the culture of LN mononuclear cells were determined. The results showed the lowest and highest lesion sizes in mice infected by Shiraz strain (3.02 ± 0.52 mm) and Kashan strain (5.20 ± 0.45), respectively, 8 weeks after inoculation. No significant difference was observed between other strains. The parasite burden was significantly lower in lymph node of mice infected with strain of Damghan (1.51 × 10⁷) than Kashan (3.60 × 10⁹) and Shiraz (7.08 × 10⁹) strains, 8 weeks post-infection. However, Dehloran strain showed intermediate load of viable parasites (1.51 × 10⁹) in LN, 8 weeks post-infection. High ratios of IFN-γ/IL-4 were shown in mice inoculated by strain of Dehloran (3.17) and Damghan (2.66), but not in mice infected by other strains, 8 weeks post-infection. The highest and lowest ratios of CD4⁺/CD8⁺ T cells were found in LN cells of mice infected with Kashan (1.82) and Dehloran (1.00) strains, respectively. Results indicate that the lowest and intermediate loads of parasites induced by Damghan and Dehloran strains along with higher ratio of IFN-γ/IL-4 produced by both strains in LN of inoculated mice suggest that these strains have the capacity to shift the immune responses to a predominant Th1 response after 8 weeks infection in BALB/c mice and might be the ideal strains for vaccine studies and development of candidate vaccine against leishmaniasis.     

Cost Effectiveness of Pegfilgrastim Versus Filgrastim After High-Dose Chemotherapy and Autologous Stem Cell Transplantation in Patients with Lymphoma and Myeloma

Background

Use of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) filgrastim accelerates neutrophil recovery following myelosuppressive chemotherapy. Since filgrastim requires multiple daily administrations, forms of rhG-CSF with a longer half life, including pegfilgrastim, have been developed. Pegfilgrastim is safe and effective in supporting neutrophil recovery and reducing febrile neutropenia after conventional chemotherapy. Pegfilgrastim has also been successfully used to support patients undergoing peripheral blood stem cell (PBSC) transplantation for haematological malignancies. To our knowledge, no cost-effectiveness analysis (CEA) of pegfilgrastim in this setting has been published yet.

Objective

We undertook a CEA to compare a single injection of pegfilgrastim versus repeated administrations of filgrastim in patients who had undergone PBSC transplantation for lymphoma or myeloma. The CEA was set in France and covered a period of 100 ± 10 days from transplant.

Methods

The CEA was designed as part of an open-label, multicentre, randomized phase II trial. Costs were assessed from the hospital’s point of view and are expressed in 2009 euros. Costs computation focused on inpatient, outpatient, and home care. Costs in the two arms of the study were compared using the Mann–Whitney test. When differences were statistically significant, multiple regression analyses were performed in order to identify cost drivers. Incremental cost-effectiveness ratios (ICER) were calculated for the major endpoints of the trial; i.e., duration of febrile neutropenia (absolute neutrophil count [ANC] <0.5 × 10⁹/L and temperature ≥38 °C), duration of neutropenia (ANC <1.0 × 10⁹/L and ANC <0.5 × 10⁹/L), duration of thrombopenia (platelets <50 × 10⁹/L and <20 × 10⁹/L), and days with a temperature ≥38 °C). Uncertainty around the ICER was captured by a probabilistic analysis using a non-parametric bootstrap method.

Results

151 patients were enrolled at ten French centres from October 2008 to September 2009. The mean total cost in the pegfilgrastim arm of the study (n = 74) was €25,024 (SD 9,945). That in the filgrastim arm (n = 76) was €28,700 (SD 20,597). Pegfilgrastim strictly dominated filgrastim for days of febrile neutropenia avoided, days of neutropenia (ANC <1.0 × 10⁹/L) avoided, days of thrombopenia (platelets <20 × 10⁹/L) avoided, and days with temperature ≥38 °C) avoided. Pegfilgrastim was less costly and less effective than filgrastim for the number of days with ANC <0.5 × 10⁹/L avoided and the number of days with platelets <50.0 × 10⁹/L avoided. Taking uncertainty into account, the probabilities that pegfilgrastim strictly dominated filgrastim were 67 % for febrile neutropenia, 86 % for neutropenia (ANC <1.0 × 10⁹/L), 59 % for thrombopenia (platelets <20 × 10⁹/L), 86 % for temperature ≥38 °C, 32 % for neutropenia (ANC <0.5 × 10⁹/L), and 43 % for thrombopenia (platelets <50 × 10⁹/L). Conversely, the probability that filgrastim strictly dominated pegfilgrastim for neutropenia (ANC <0.5 × 10⁹/L) is 5 %.

Conclusion

This study found no evidence that the use of pegfilgrastim is associated with greater cost in lymphoma and myeloma patients after high-dose chemotherapy and PBSC transplantation.     

Inhibition of Glutathione Reductase Activity from Baker’s Yeast (Saccharomyces cerevisiae) By Copper(II) Oxide Nanoparticles and Copper(II) Chloride

In this study, glutathione reductase (GR) from baker’s yeast (Saccharomyces cerevisiae) was exposed to 0, 25, 50, 100, 250 and 500 mg/L copper(II) oxide nanoparticles (CuO NPs) and copper(II) chloride (CuCl₂). Changes in GR% activity upon exposure to 25, 50, 100, 250 and 500 mg/L CuO NPs and CuCl₂ were found to be + 0.3, − 3.4, − 8.1, − 25.7 and − 37.4 and − 60.7, − 72.7, − 77.8, − 85.3 and − 90.6, respectively. The 50% inhibition concentration (IC50) was 625 ppm (78.6 × 10⁻⁴ M) for CuO NPs and 21 ppm (1.56 × 10⁻⁴ M) for CuCl₂. Moreover, CuO NPs and CuCl₂ inhibited GR competitively and noncompetitively, respectively.

     

Development of a high-dose vaccine formulation for prevention of megalocytivirus infection in rock bream (Oplegnathus fasciatus)

A formalin-inactivated red sea bream iridovirus (RSIV) vaccine was prepared using the culture supernatant of a persistently infected Pagrus major fin cell line (PI-PMF) with IVS-1 strain (RSIV subtype II Meglaocytivirus). Rock bream (Oplegnathus fasciatus) were injected with a high-dose, ultracentrifuged megalocytivirus vaccine (Ultra HSCMV, 7.0 × 10¹⁰ copies/mL), a high-dose supernatant of cultured megalocytivirus vaccine (HSCMV, 1.0 × 10¹⁰ copies/mL), a supernatant of cultured megalocytivirus vaccine (SCMV, 1.0 × 10⁹ copies/mL), and a low-dose of cultured megalocytivirus vaccine (LSCMV, 1.0 × 10⁸ copies/mL). The vaccine efficacies for the various vaccine formulations were determined done following injection challenge with IVS-1 (1.0 × 10⁴ copies/0.1 mL/fish), and the four different vaccines exhibited cumulative mortalities of 10.0 ± 0.0%, 48.3 ± 7.6%, 75.0 ± 5.0%, and 100.0 ± 0.0%, respectively. Additionally, the dose-dependent vaccine efficacy was also confirmed using two different cohabitation methods that included challenges G (general) and I (individual). When squalene + aluminum hydroxide (SqAl) was used as an adjuvant for the HSCMV or SCMV vaccine, cumulative mortalities of 30.0 ± 5.0% and 48.3 ± 7.6%, respectively, were obtained; moreover, these two adjuvants exhibited the highest efficacy in this study. The observed difference in survival post-challenge for the different vaccine concentrations was not reflected in the differences in neutralizing antibody titers. It was found that the water temperature during immune induction plays a less important a role than the water temperature during the challenge test, in which lowering the water temperature from 25 °C to 21 °C during a challenge improved the level of protection from cumulative mortalities from 35% to 10%. This study demonstrated that protection against mortality using inactivated vaccines against RSIVD in rock bream, which are known to be the most susceptible species to RSIV infection, is dependent upon antigen dose and temperature during the challenge.     

Persistence of Lactobacillus reuteri DSM17938 in the Human Intestinal Tract: Response to Consecutive and Alternate-Day Supplementation

Background: Probiotics may enhance gastrointestinal health and immune function. The efficacy of different probiotic dosing strategies on colonization and persistence of probiotics is undefined.

Objective: The authors assessed colonization and persistence of Lactobacillus reuteri (L. reuteri) DSM17938 (BioGaia AB, Stockholm, Sweden) after daily or alternate-day dosing.

Methods: Volunteers ate pudding with L. reuteri (10⁹ CFU) daily (n = 9) or on alternate days (n = 9) over 7 days. Fecal samples were collected on dosing days (D1–7) and after dosing ended (D13–15 and D20–22) and were analyzed for the presence of L. reuteri. Results are reported in 3-day increments (D2–4, D5–7, D13–15, and D20–22).

Results: L. reuteri count rose in response to daily supplementation ([mean ± SD] D2–4: 4 × 10⁴ ± 2 × 10⁴ CFU, p < 0.01; D5–7: 10 × 10⁴ ± 9 × 10⁴ CFU, p < 0.01) and alternate-day supplementation (D2–4: 21 × 10⁴ ± 20 × 10⁴ CFU, p < 0.01; D5–7: 11 × 10⁴ ± 15 × 10⁴ CFU, p = 0.06) and fell in both groups 1 week after dosing ended (p < 0.01). Total volunteers with detectable L. reuteri 1 and 2 weeks after dosing ended was similar in response to daily feeding (4/9 and 2/9, respectively) and alternate-day feeding (3/9 and 2/9, respectively). L. reuteri count was higher D2–4 in response to alternate-day vs daily feeding (p < 0.05) but similar thereafter.

Conclusions: Alternate-day probiotic intake achieves equivalent colonization to daily intake, but colonization declines rapidly once dosing stops. It is possible that, initially, responsiveness to probiotics may differ between individuals, but those differences do not persist with longer consumption.     

Effect of Agrochemical Use on the Drinking Water Quality of Agogo,a Tomato Growing Community in Ashanti Akim,Ghana

The effect of agrochemical use in agricultural activities on the drinking water quality of ground and surface water within Agogo, a prominent tomato growing area in the Ashanti region of Ghana was assessed by monitoring physicochemical parameters, trace metals and microbial quality of two water sources. Levels of contamination were greater in surface water than groundwater. Trace metal levels (mg/L) were 1.40, 0.12, 0.08 and 0.18 in surface water and 0.08, 0.10, 0.05 and 0.08 in groundwater for Fe, Pb, Zn and Cd, respectively. Lead and Cd in surface and groundwater exceeded USEPA maximum acceptable levels (MCLs) for drinking water. Bacterial indicator numbers (geometric means/100 mL) in surface water varied from 9.35 × 10⁵ to 1.57 × 10¹¹ for total coliforms, 4.15 × 10⁴ to 2.10 × 10⁷ for faecal coliforms and 2.80 × 10 to 3.25 × 10² for enterococci, but none was found in groundwater.     

Evolutionary analysis of human respiratory syncytial virus collected in Myanmar between 2015 and 2018

We studied genetic variation in the second hypervariable region (HVR) of the G gene of human respiratory syncytial virus (HRSV) from 1701 nasal swab samples collected from outpatients with acute respiratory infections at two general hospitals in the cities Yangon and Pyinmana in Myanmar from 2015 to 2018. HRSV genotypes were characterized using phylogenetic trees constructed using the maximum likelihood method. Time-scale phylogenetic tree analyses were performed using the Bayesian Markov chain Monte Carlo method. In total, 244 (14.3%) samples were HRSV-positive and were classified as HRSV-A (n = 84, 34.4%), HRSV-B (n = 158, 64.8%), and co-detection of HRSV-A/HRSV-B (n = 2, 0.8%). HRSV epidemics occurred seasonally between July (1.9%, 15/785) and August (10.5%, 108/1028), with peak infections in September (35.8%, 149/416) and October (58.2%, 89/153). HRSV infection rate was higher in children ≥1 year of age than in those <1 year of age (70.5% vs. 29.5%). The most common HRSV symptoms in children were cough (80%–90%) and rhinorrhea (70%–100%). The predominant genotypes were ON1for HRSV-A (78%) and BA9 for HRSV-B (64%). Time to the most recent common ancestor was 2014 (95% highest posterior density [HPD], 2012–2015) for HRSV-A ON1 and 2009 (95% HPD, 2004–2012) for HRSV-B BA9. The mean evolutionary rate (substitutions/site/year) for HRSV-B (2.12 × 10⁻², 95% HPD, 8.53 × 10⁻³–3.63 × 10⁻²) was slightly higher than that for HRSV-A (1.39 × 10⁻², 95% HPD, 6.03 × 10⁻³–2.12 × 10⁻²). The estimated effective population size (diversity) for HRSV-A increased from 2015 to 2016 and declined in mid-2018, whereas HRSV-B diversity was constant in 2015 and 2016 and increased in mid-2017. In conclusion, the dominant HRSV-A and HRSV-B genotypes in Myanmar were ON1 and BA9, respectively, between 2015 and 2018. HRSV-B evolved slightly faster than HRSV-A and exhibited unique phylogenetic characteristics.     

Immunogenicity and protective efficacy of clade 2.3.2.1c and clade 2.3.4.4c H5Nx avian influenza antigen bank vaccines in mice,Korea

• The immunogenicity and protective efficacy of inactivated clade 2.3.2.1c (rgKA435) and clade 2.3.4.4c (rgES2) H5Nx vaccines, which are representatives of an avian influenza antigen bank in Korea, were examined in mice. Mice were vaccinated twice and then challenged with homologous virus. Hemagglutinin inhibition and serum neutralizing antibody titers in the rgES2-vaccinated group were higher (4.4 ± 1.7 and 10.8 ± 2.3 log₂, respectively) than those in the rgKA435-vaccinated group (2.8 ± 1.1 and 2.5 ± 0.9 log₂, respectively). rgES2 conferred 100% protection, with no morbidity, no severe body weight loss, and no virus replication in any of the tissues tested. By contrast, 80% of mice in the rgKA435 group survived. One mouse in this group died at 10 dpi. Virus titers in the lung and turbinate were 10^2.5–3.5 TCID₅₀/0.1 ml at 3–7 dpi and 10^1.5 TCID₅₀/0.1 ml at 3–5 dpi, respectively. In particular, the viral titer in the turbinate from the rgKA435 group at 3 dpi was significantly lower than that in the equivalent control group (p < 0.05). The data suggest that both of these antigen bank vaccines are promising candidates for further evaluation in humans.

     

Beneficial effects of polyphenol-rich olive oil in patients with early atherosclerosis

Purpose

Diets rich in plant-derived polyphenols such as olive oil (OO) and/or catechins such as epigallocatechin 3-gallate (EGCG) have been shown to reduce the incidence of cardiovascular diseases, potentially by improving endothelial function, an important surrogate for atherosclerosis. The possible augmentation of endothelial function with the combined efforts of OO and EGCG is intriguing, yet unknown.

Methods

Eighty-two patients with early atherosclerosis (presence of endothelial dysfunction) were enrolled in this double-blind, randomized trial with 52 completing the study. The aim of the study was to compare the effect of a daily intake of 30 ml simple OO, with 30 ml of EGCG-supplemented OO, on endothelial function as well as on inflammation and oxidative stress after a period of 4 months. Endothelial function was assessed noninvasively via peripheral arterial tonometry (Endo-PAT^®).

Results

After 4 months, when OO and EGCG-supplemented OO groups were combined, OO significantly improved endothelial function (RHI, 1.59 ± 0.25–1.75 ± 0.45; p < 0.05). However, there were no significant differences in results between the two olive oil groups. Interestingly, with OO supplementation there was a significant reduction in inflammatory parameters: sICAM (196 to 183 ng/mL, p = < 0.001); white blood cells (WBCs) (6.0 × 10⁹/L–5.8 × 10⁹/L, p < 0.05); monocytes (0.48 × 10⁹/L to 0.44 × 10⁹/L, p = 0.05); lymphocytes (1.85 × 10⁹/L to 1.6 × 10⁹/L, p = 0.01); and platelets (242–229 × 10⁹/L, p = 0.047).

Conclusions

Improvement in endothelial dysfunction in patients with early atherosclerosis in association with significant reduction in leukocytes may suggest an important role of early cellular inflammatory mediators on endothelial function. The current study supports one potential mechanism for the role of olive oil, independent of EGCG, modestly supplemented to a healthy cardiovascular diet.     

Pathogenesis of Brucella ovis in pregnant mice and protection induced by the candidate vaccine strain B. Ovis ΔabcBA

Ovine brucellosis caused by Brucella ovis is a major cause of reproductive failure in sheep. This study aimed to evaluate transplacental infection and pathogenicity of B. ovis wild type strain ATCC 25,840 (WT B. ovis) and the candidate vaccine strain B. ovis ΔabcBA in pregnant mice. A total of 40 BALB/c mice were equally divided into 4 groups: (i) non immunized and uninfected control mice (3/10 mice became pregnant); (ii) non immunized and challenged with WT B. ovis (5/10 pregnant); (iii) inoculated only with B. ovis ΔabcBA (6/10 pregnant); (iv) immunized with B. ovis ΔabcBA and challenged with WT B. ovis (5/10 pregnant). Female mice bred, and five days after visualization of the vaginal plug, they were inoculated intraperitoneally (ip) with 100 µL of sterile PBS, 100 µL of 1 × 10⁶ CFU of B. ovis ΔabcBA, or 100 µL of 1 × 10⁶ CFU of B. ovis WT, according to each group. At the 17th day of gestation, samples of spleen, liver, uterus, placenta, fetus and mammary gland were obtained for bacteriology, histopathology and immunohistochemistry. Non immunized mice challenged with B. ovis WT developed necrotizing placentitis as well as microgranulomas in the liver and spleen. These findings support the notion that B. ovis infection in pregnant mice induces lesions that are similar to those caused by B. abortus in the same animal model. B. ovis ΔabcBA was not recovered from any of the sampled organs, and it did not cause any gross or microscopic lesions, indicating that it is a safe and attenuated strain in this experimental model. In addition, B. ovis ΔabcBA was induced protective immunity as demonstrated by decreased numbers of B. ovis WT in the liver, uterus and fetuses of immunized mice after the challenge with B. ovis WT.     

Cistanches herba induces testis cytotoxicity in male mice

We investigated the effects of Cistanches herba (CH) on the male reproductive system in mice, assessing CREM gene expression and spermatogenesis. Our results demonstrate that CH treatment lead to a significant decrease in sperm count dose-dependently, 298.3 ± 48.9 vs. 296.6 ± 102.4 (250 mg/kg), 236.7 ± 75.1 (500 mg/kg), 223.0 ± 48.7 × 10⁶ (1000 mg/kg), respectively. Additionally, serum testosterone levels decreased following CH treatment to as low as ~57% compared with the vehicle-treated group. CREM gene expression was also down-regulated following CH treatment and histological examination of the testicular seminiferous tubules showed severe damage on CH treatment. These results suggest that CH induces cytotoxicity in the male reproductive system, through the inhibition of spermatogenesis, testicular damage, and limited hormonal function.     

Genome characterization,prevalence and tissue distribution of astrovirus,hepevirus and norovirus among wild and laboratory rats (Rattus norvegicus) and mice (Mus musculus) in Hungary

Rodents including rats are reservoir of several pathogens capable of affecting human health. In this study, faecal and different organ specimens from free-living Norway rats (Rattus norvegicus) (N = 18) and faecal samples from laboratory rodents (rats N = 21 and mice N = 20) collected from different geographic areas in Hungary between 2017 and 2020 were investigated by viral metagenomics and conventional RT-PCR methods. The complete genome of three different RNA viruses, rat astrovirus, rat norovirus and rat hepevirus were characterized and analysed in detail. Rat norovirus was detected in faecal (17.6%, 3/17) and kidney (7.1%, 1/14) samples; rat astrovirus in faecal (23.5%, 4/17) and spleen (13.3%, 2/15) samples, and rat hepevirus in 43% to 67% the faecal, liver, kidney, lung, heart, muscle, brain and blood samples from Norway rats, respectively. Rat norovirus was also identifiable in 5% (1/21) of laboratory rats and rat astrovirus in 40% (8/20) of faecal samples from laboratory mice. Co-infections were found in 28% (5/18) wild Norway rats. The highest RNA viral load of astrovirus (1.81 × 10⁸ copy/g) and norovirus (3.49 × 10⁷ copy/g) were measured in faecal samples; while the highest RNA viral load of hepevirus (1.16 × 10⁹ copy/g) was found in liver samples of Norway rats, respectively. This study confirms the wide geographic distribution and high prevalence of astrovirus, norovirus and hepevirus among wild rats in Hungary with confirmation of different organ involvement of as well as the detection of norovirus and astrovirus in laboratory rats and mice, respectively. This finding further strengthens the role of rodents in the spread of viral pathogens especially infecting human.     

Rapid detection of the New Delhi metallo-β-lactamase (NDM) gene by recombinase polymerase amplification

New Delhi metallo-β-lactamase (NDM) is a series of enzyme conferring resistance to β-lactam antibiotics including the carbapenems. The bla_NDM gene has been reported in a variety of Gram-negative bacilli, especially in the Enterobacteriaceae and Acinetobacter spp., which is deeply disconcerting for public health worldwide. In this study, recombinase polymerase amplification assays using a basic detection (Basic-RPA) and a real-time fluorescent detection (Exo-RPA) were established for detecting bla_NDM gene. The RPA reactions were performed at 39 °C and finished within 20 min. Using different copy numbers of pMD18T-NDM plasmid DNA as templates, we identified the detection limit of Basic-RPA assay (1.85 × 10³ copies/μL), conventional PCR assay (1.85 × 10⁴ copies/μL), Exo-RPA assay (1.85 × 10² copies/μL) and real-time PCR assay (1.85 × 10² copies/μL). Both Basic-RPA and Exo-RPA assays were highly specific for detecting bla_NDM, as there were no cross-reactions with the strains without bla_NDM gene. Examination of 62 clinical samples by RPA assays and PCR assays showed the same results, suggesting that RPA assays are reliable in clinical diagnosis. The amplification time of RPA is much shorter than that of other molecular techniques, it is easy to implement and has the potential for clinical application.     

Genotoxicity of Fenpropathrin and Fenitrothion on Root Tip Cells of <Emphasis Type="Italic">Vicia faba</Emphasis>

The genotoxicity of fenpropathrin and fenitrothion on root tip cells of Vicia faba was studied. The symptoms were investigated about the mitotic index, the micronucleus frequency and chromosomal aberration frequency of root tip cells of Vicia faba which were induced by different concentrations of fenpropathrin and fenitrothion (1 × 10⁻¹⁰–1 × 10⁻² g L⁻¹). Results showed that fenpropathrin and fenitrothion could induce the micronucleus of root tip cells of Vicia faba. It occurred in a dose-dependent manner. Peaks were observed at 1 × 10 ⁻⁶ g L⁻¹ fenpropathrin and 1 × 10⁻⁴ g L⁻¹ fenitrothion, and micronucleus frequency reached 14.587 ± 1.511‰ and 14.164 ± 1.623‰, respectively. From 1 × 10⁻¹⁰ g L⁻¹ to 1 × 10 ⁻⁶ g L⁻¹ fenpropathrin and 1 × 10⁻⁴ g L⁻¹ fenitrothion, the micronucleus frequency increased with the increase of the concentrations, but beyond this range, the micronucleus frequency decreased with the further increase of the concentrations. A similar trend was observed for mitotic index. Moreover, fenpropathrin and fenitrothion could induce various types of chromosome aberration, such as lagging chromosomes, chromosome fragment, chromosome bridge, multipolar, nuclear buds, karyorrhexis, etc.     

Rational design of medium supplementation strategy for improved influenza viruses production based on analyzing nutritional requirements of MDCK Cells

Influenza vaccine production using cell culture technology has become popular nowadays. However, to meet the ever increasing demand of influenza vaccine, it is prerequisite to improve the yield of influenza virus in cells. To achieve this, in the present study, the nutritional requirements of MDCK cells in the virus production process were analyzed and a nutrient-feeding strategy was developed accordingly. Based on the consumption rates and corresponding concentration optimization, glucose and fast metabolized amino acids were supplemented into the maintaining medium at the time of infection. Compared with the non-supplemented culture, the average cell specific death rate during 0–48 h post-infection was 0.013 h⁻¹, which was 40.91% lower in the nutrient-supplemented culture. Total virus titer, HA antigen protein concentration and cell-specific virus yield were (1.88 ± 0.23) × 10³ HA units/50 μL, 11.70 ± 0.22 μg/mL and (10.06 ± 1.16) × 10³ virions/cell, respectively, which were 84.04 ± 22.50%, 31.46 ± 2.87% and 86.64 ± 25.81% higher than those in the control, respectively. These data showed that the appropriate supplementation of nutrients during virus production process could reduce cell death, and improve cell-specific virus yield and total influenza virus output. This study laid foundation for the development of cell culture technology for influenza vaccine production.     

Semi-perfusion cultures of suspension MDCK cells enable high cell concentrations and efficient influenza A virus production

Control and prevention of rapid influenza spread among humans depend on the availability of efficient and safe seasonal and pandemic vaccines, made primarily from inactivated influenza virus particles. Current influenza virus production processes rely heavily on embryonated chicken eggs or on cell culture as substrate for virus propagation. Today’s efforts towards process intensification in animal cell culture could innovate viral vaccine manufacturing using high-yield suspension cells in high cell density perfusion processes. In this work, we present a MDCK cell line adapted to grow as single cell suspension with a doubling time of less than 20 h, achieving cell concentrations over 1 × 10⁷ cells/mL in batch mode. Influenza A virus titer obtained in batch infections were 3.6 log₁₀(HAU/100 µL) for total- and 10⁹ virions/mL for infectious virus particles (TCID₅₀), respectively. In semi-perfusion mode concentrations up to 6 × 10⁷ cells/mL, accumulated virus titer of 4.5 log₁₀(HAU/100 µL) and infectious titer of almost 10¹⁰ virions/mL (TCID₅₀) were possible. This exceeds results reported previously for cell culture-based influenza virus propagation by far and suggests perfusion cultures as the preferred method in viral vaccine manufacturing.     

Genetic Markers Associated With Plasma Protein C Level in African Americans: The Atherosclerosis Risk in Communities (ARIC) Study

Protein C is an endogenous anticoagulant protein with anti‐inflammatory properties. Single‐nucleotide polymorphisms (SNPs) affect the levels of circulating protein C in European Americans. We performed a genome‐wide association (GWA) scan of plasma protein C concentration with approximately 2.5 million SNPs in 2,701 African Americans in the Atherosclerosis Risk in Communities Study. Seventy‐nine SNPs from the 20q11 and 2q14 regions reached the genome‐wide significance threshold of 5 × 10^‐8. A missense variant rs867186 in the PROCR gene at 20q11 is known to affect protein C levels in individuals of European descent and showed the strongest signal (P = 9.84 × 10^‐65) in African Americans. The minor allele of this SNP was associated with higher protein C levels (β = 0.49 μg/ml; 10% variance explained). In the 2q14 region, the top SNPs were near or within the PROC gene: rs7580658 (β = 0.15 μg/ml; 2% variance explained, P = 1.7 × 10^‐12) and rs1799808 (β = 0.15 μg/ml; 2% variance explained, P = 2.03 × 10^‐12). These two SNPs were in strong linkage disequilibrium (LD) with another SNP rs1158867 that resides in a biochemically functional site and in weak to strong LD with the top PROC variants previously reported in individuals of European descent. In addition, two variants outside the PROC region were significantly and independently associated with protein C levels: rs4321325 in CYP27C1 and rs13419716 in MYO7B. In summary, this first GWA study for plasma protein C levels in African Americans confirms the associations of SNPs in the PROC and PROCR regions with circulating levels of protein C across ethnic populations and identifies new candidates for protein C regulation.     

Protective immunity against Megalocytivirus infection in rock bream (Oplegnathus fasciatus) following CpG ODN administration

Rock bream iridovirus (RBIV) disease in rock bream (Oplegnathus fasciatus) remains an unsolved problem in Korea aquaculture farms. CpG ODNs are known as immunostimulant, can improve the innate immune system of fish providing resistance to diseases. In this study, we evaluated the potential of CpG ODNs to induce anti-viral status protecting rock bream from different RBIV infection conditions. We found that, when administered into rock bream, CpG ODN 1668 induces better antiviral immune responses compared to other 5 CpG ODNs (2216, 1826, 2133, 2395 and 1720). All CpG ODN 1668 administered fish (1/5 µg) at 2 days before infection (1.1 × 10⁷) held at 26 °C died even though mortality was delayed from 8 days (1 µg) and 4 days (5 µg). Similarly, CpG ODN 1668 administered (5 µg) at 2 days before infection (1.2 × 10⁶) held at 23/20 °C had 100% mortality; the mortality was delayed from 9 days (23 °C) and 11 days (20 °C). Moreover, when CpG ODN 1668 administered (1/5/10 µg) at 2/4/7 days before infection or virus concentration was decreased to 1.1 × 10⁴ and held at 20 °C had mortality rates of 20/60/30% (2 days), 30/40/60% (4 days) and 60/60/20% (7 days), respectively, for the respective administration dose, through 100 dpi. To investigate the development of a protective immune response, survivors were re-infected with RBIV (1.1 × 10⁷) at 100 and 400 dpi, respectively. While 100% of the previously unexposed fish died, 100% of the previously infected fish survived. The high survival rate of fish following re-challenge with RBIV indicates that protective immunity was established in the surviving rock bream. Our results showed the possibility of developing preventive measures against RBIV using CpG ODN 1668 by reducing RBIV replication speed (i.e. water temperature of 20 °C and infection dose of 1.1 × 10⁴).     

Pervious and Impervious Urban Stormwater Runoff in a Rapidly Urbanizing Region: Occurrence of Fluoranthene and Pyrene

Stormwater runoff in a rapidly urbanizing region was analyzed for organic contamination to compare impervious and pervious surfaces. Total petroleum hydrocarbons (TPH) ranged between 5 and 277 mg/L, with impervious surfaces showing, on average, greater TPH concentrations. Pyrene and fluoranthene were identified in all impervious stormwater samples. Sediments from receiving waters also contained pyrene and fluoranthene. Runoff samples had concentrations in the range of 11–191 μg/L. Sediment samples ranged from 2.3 × 10¹–1.3 × 10⁴ μg/kg. Results from this study are useful for identifying major urban contaminants and understanding the role of pervious surfaces in filtering urban contaminants.     

Comparative Toxicity of Nine Metals to Two Malaysian Aquatic Dipterian Larvae with Reference to Temperature Variation

A study was conducted to determine the suitability of using selected aquatic dipterian larvae for biomonitoring bioassays. The organisms included a member of the biting midge family that was identified as Culicoides furens and a member of the non-biting midge family, identified as Chironomus plumosus. Median lethal toxicity tests were conducted to observe the variation between metal sensitivities between the two larval forms and how variations in temperature could affect the experimental setup. Nine heavy metals were used in the study. It was observed that the 96 h LC₅₀ (in mg/L) for the different metals was found to be Zn-16.21 (18.55 ± 13.87); Cr-0.96 (1.08 ± 0.84); Ag-4.22 (6.87 ± 1.57); Ni-0.42 (0.59 ± 0.25); Hg-0.42 (0.59 ± 0.25); Pb-16.21 (18.31 ± 14.11); Cu-42.24 (45.18 ± 39.30); Mn-4.22 (7.19 ± 1.25); Cd-0.42 (0.59 ± 0.25) for the Chironomus plumosus and Zn-4.22 (6.56 ± 1.88); Cr-0.42 (0.54 ± 0.30); Ag-0.42 (0.54 ± 0.30); Ni-0.42 (0.54 ± 0.30); Hg-0.04 (0.07 ± 0.01); Pb-0.42 (0.54 ± 0.30); Cu-42.24 (45.18 ± 39.30); Mn-4.22 (6.56 ± 1.88); Cd-0.42 (0.54 ± 0.30) in the case of the Culicoides furens. With temperature as a variable the LC₅₀ values were observed to increase from 2.51 mg/L at 10°C to 4.22 ppm at 30°C and to reduce slightly to 3.72 mg/L at 35°C as seen in the case of Zn. It was also observed that at 40°C thermal toxicity and chemical toxicity overlapped as 100% mortality was observed in the controls. This trend was observed in all metals for both C. plumosus and C. furens. Thus indicating temperature played an important role in determining LC₅₀ values of toxicants.