Hemagglutination Titers Research Articles

Evaluation of immunomodulatory effects of lamotrigine in BALB/c mice.

Modulation of the immune system has recently been shown to be involved in the pharmacological effects of old antiepileptic drugs and in the pathogenesis of epilepsy. Therefore, the most recent guidelines for immunotoxicological evaluation of drugs were consulted to investigate the immunomodulatory effects of lamotrigine, a newer antiepileptic drug, in BALB/c mice. These included the in vivo effects of lamotrigine on delayed-type hypersensitivity (DTH) response to sheep red blood cell (SRBC) antigens, hemagglutination titer assays and hematological changes. In vitro effects of lamotrigine on ConA-induced splenocyte proliferation and cytokine secretion were assessed. The results showed that lamotrigine treatment significantly increased the DTH response to SRBC in the mouse model of this study. This was accompanied by a significant increase in relative monocyte and neutrophil counts and in spleen cellularity. Lamotrigine significantly inhibited ConA-induced splenocyte proliferation in vitro and it significantly inhibited IL-2 and TNF-α secretion in ConA-stimulated splenocytes. In conclusion, the results demonstrated significant immunomodulatory effects of lamotrigine in BALB/c mice. These data could expand the understanding of lamotrigine-induced adverse reactions and its role in modulating the immune system in epilepsy.

Adaptation of velogenic Newcastle disease virus in Vero cells: velogenicity of virus unaltered after adaptation

In the present experiment, velogenic Newcastle disease virus (NDV) was adapted to Vero cells without the addition of exogenous trypsin. The cytopathic effect (CPE) in NDV-infected Vero cells was observed from passage 3 (P 3) onwards with concomitant increase in the haemagglutination (HA) and virus titres. The CPE was characterized by rounding of cells, their detachment from the surface and syncytia formation. The Vero-adapted NDV was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), which detected the amplification of haemagglutinin neuraminidase (HN) gene from virus-infected cells. The mean death time (MDT) of 58 hours upon inoculation to embryonated chicken eggs and the intravenous pathogenicity index (IVPI) of 2.12 following inoculation to SPF chicken suggested that Vero adapted virus retained the velogenicity even after ten passages. Furthermore, the nervous signs and haemorrhages in the pro-ventriculus of birds that died at day 5 post inoculation with PIO virus confirmed that the Vero-adapted NDV was a neurotropic viscerotropic velogenic strain.

Optimization of trypsins for influenza A/H1N1 virus replication in MDCK SI-6 cells, a novel MDCK cell line

A cell-based vaccine production method for influenza virus may be an effective and more rapid alternative to egg-based systems. For high-yield virus production, the effect of bovine, porcine, fungal, and recombinant trypsins on influenza A/H1N1 virus replication in MDCK SI-6 cells (SI-6 cells), a novel MDCK cell line developed by our research group, was examined. SI-6 cells infected with influenza A/H1N1 virus were incubated in the presence of four trypsin types at various concentrations, and virus yields in the culture medium were evaluated by a hemagglutination (HA) assay. Virus growth was most efficient in the presence of bovine and porcine trypsins. An analysis of the optimized concentration and definitive HA titer of each trypsin by Gaussian distribution revealed that comparable high virus yields (166.1 and 164.2 HAU/50μl) were obtained at the optimized concentrations of bovine (0.4μg/ml) and porcine (2.1μg/ml) trypsins, respectively, the yields of which were significantly higher than that of fungal and recombinant trypsins. We conclude that bovine and porcine trypsins are suitable for influenza A/H1N1 virus replication in SI-6 cells. This result complements our previous study and suggests the possible application of SI-6 cells to the development of cell-based influenza vaccines.

In vitro and in vivo immunomodulatory potential of Pedicularis longiflora and Allium carolinianum in alloxan-induced diabetes in rats

Pedicularis longiflora Rudolph (Orobanchaceae) and Allium carolinianum Linn (Alliaceae) are two important medicinal plants found in trans-Himalayan Changthang. The immunomodulatory potential of these plants has not been explored. In the present study, we evaluated the in vitro and in vivo immunomodulatory potential of P. longiflora and A. carolinianum in alloxan-induced diabetes in Wistar rats. The ethanol extracts of the aerial parts of P. longiflora and whole plant parts of A. carolinianum were used for studying the in vitro immunomodulatory activity using lymphocyte stimulation and cytokine release assays. For the in vivo study, 5 groups of 6 rats per group, including alloxan-induced diabetic and plant extract-treated rats, were evaluated for cell-mediated immune (CMI) and humoral immune (HMI) responses in a 42-day experimental trial using doses of 500mg/kg b.wt. for P. longiflora and 250mg/kgbwt. for A. carolinianum. For P. longiflora, the median effective dose was found to be 500mg/kg. The in vitro lymphocyte stimulation index for P. longiflora was significantly higher (1.73±0.04, p<0.05) than that for A. carolinianum (1.27±0.06). However, the release of transforming growth factor-β1 (TGF-β1, 15.63±1.00, p<0.05) by P. longiflora was significantly lower than that by A. carolinianum (21.61±1.19), suggesting a better immune response by P. longiflora than by A. carolinianum. P. longiflora significantly increased the ear thickness (53.12%), inflammatory cellular infiltration (200.00±11.42), and total leukocyte count (7.44±0.02) compared to A. carolinianum (47.57%, 165.83±3.96, and 7.01±0.01, respectively). P. longiflora significantly reduced the percentage of leukocytes with depolarized mitochondria (3.24±0.16%) and apoptosis (1.81±0.07%), and induced a better CMI response than A. carolinianum. Significantly (p<0.05) higher hemagglutination titer (28.37±0.80) and IgG production (6.43±0.34mg/mL) were observed in the P. longiflora-treated group than in the A. carolinianum-treated group (23.93±0.58 and 6.23±0.37mg/mL). Plasma tumor necrosis factor-α (TNF-α) and TGF-β1 levels, and nuclear factor-κB (NF-κB) expression were significantly (p<0.05) lower in the P. longiflora-treated group than in the A. carolinianum-treated group. This may be due to better HMI responses produced by P. longiflora than by A. carolinianum. This is the first study to show that P. longiflora ethanol extract has more potent in vitro and in vivo immunomodulatory activities than A. carolinianum, especially in alloxan-induced diabetic rats. However, further research is needed to identify the different molecular mechanisms involved in mediating this immunomodulatory response.

The first external quality assessment of isolation and identification of influenza viruses in cell culture in the Asia Pacific region, 2016

BackgroundThe isolation and propagation of influenza viruses from clinical specimens are essential tools for comprehensive virologic surveillance. Influenza viruses must be amplified in cell culture for detailed antigenic analysis and for phenotypic assays assessing susceptibility to antiviral drugs or for other assays. ObjectivesTo conduct an external quality assessment (EQA) of proficiency for isolation and identification of influenza viruses using cell culture techniques among National Influenza Centres (NICs) in the World Health Organisation (WHO) South East Asia and Western Pacific Regions. Study designTwenty-one NICs performed routine influenza virus isolation and identification techniques on a proficiency testing panel comprising 16 samples, containing influenza A or B viruses and negative control samples. One sample was used exclusively to determine their capacity to measure hemagglutination titer and the other 15 samples were used for virus isolation and identification. ResultsAll NICs performed influenza virus isolation using Madin Darby canine kidney (MDCK) or MDCK-SIAT-1 cells. If virus growth was detected, the type, subtype and/or lineage of virus present in isolates was determined using immunofluorescence, RT-PCR and/or hemagglutination inhibition (HI) assays. Most participating laboratories could detect influenza virus growth and could identify virus amplified from EQA samples. However, some laboratories failed to isolate and identify viruses from EQA samples that contained lower titres of virus, highlighting issues regarding the sensitivity of influenza virus isolation methods between laboratories. ConclusionThis first round of EQA was successfully conducted by NICs in the Asia Pacific Region, revealing good proficiency in influenza virus isolation and identification.

Utilization of RT-PCR and Restriction Enzyme Analysis in Detection and Differentiation of Pigeon Paramyxovirus-1 and Newcastle Disease Virus in Pigeons

Fifty-one pigeon houses from both commercial lofts and backyard of different ages and breeds were investigated for incrimination of pigeon paramyxovirus-1 (PPMV-1) and/ or Newcastle Disease virus (NDV) during 2013-2015. The results of clinical examination revealed nervous signs (wing paralysis, head tremors, torticollis, opisthotonos and leg paralysis), greenish diarrhea, and respiratory signs, with variable mortalities. The gross lesions included congestion and hemorrhage in brain; nephrosis and/ or nephritis in kidneys, ulcer in intestine and mucoid enteritis. Virus isolation via chicken embryo inoculation revealed embryo congestion and hemagglutination (HA) activity of harvested allantoic fluids were detected 41 samples (80.39%) with HA titers ranging from 26- 210. The hemagglutination inhibition (HI) results of the same isolates against PPMV-1 and NDV hyper immune sera which prepared in rabbits (1:5dilution) varied from 23 – 210 in 38 virus isolates. The allantoic fluids of 41 haemaglutinating virus isolates were subjected to Reverse transcriptase polymeraze chain reaction (RT-PCR), Thirty-eight isolates evidenced successful products for partial amplification of fusion protein gene of NDV and PPMV-1 at 356 bp using specific primers. Restriction fragment length polymorphism (RFLP) using a set of three enzymes; HhaI, MspI and MboI. revealed that 9 strains (mesogenic / lentogenic PPMV-1), 27(mesogenic / lentogenic NDV), one velogenic strain for PPMV-1 and the other was NDV. MDT value in ECE revealed 72 ± 0.0 and 62.4±5.89 indicating that both isolates could be mesogenic. It could be concluded that both NDV and PPMV-1 are co- circulating among pigeons and causing economical losses. Rt-PCR followed by RFLP can be useful rapid tools for detection and identification of both viruses. Beside, in the view of the failure of adopted prophylactic vaccination, both LaSota and Inactivated PPMV-1 vaccines must be included to overcome the impact of aforementioned disease problems in pigeons

Construction and comparison of different source neuraminidase candidate vaccine strains for human infection with Eurasian avian-like influenza H1N1 virus

Human infections with Eurasian avian-like swine influenza H1N1 viruses have been reported in China in past years. One case resulted in death and others were mild case. In 2016, the World Health Organization recommended the use of A/Hunan/42443/2015(H1N1) virus to construct the first candidate vaccine strain for Eurasian avian-like swine influenza H1N1 viruses. Previous reports showed that the neuraminidase of A/Puerto Rico/8/34(H1N1) might improve the viral yield of reassortant viruses. Therefore, we constructed two reassortant candidate vaccine viruses of A/Hunan/42443/2015(H1N1) by reverse genetic technology, with (6+2) and (7+1) gene constitution, respectively. The (6+2) virus had hemagglutinin and neuraminidase from A/Hunan/42443/2015, and the (7+1) one had hemagglutinin from A/Hunan/42443/2015, while all the other genes were from A/Puerto Rico/8/34. Our data revealed that although the neuraminidase of the (7+1) virus was from high yield A/Puerto Rico/8/34, the hemagglutination titer and the hemagglutinin protein content of the (7+1) virus was not higher than that of the (6+2) virus. Both of the (7+1) and (6+2) viruses reached a similar level to that of A/Puerto Rico/8/34 at the usual harvest time in vitro. Therefore, both reassortant viruses are potential candidate vaccine viruses, which could contribute to pandemic preparedness.

A nonsense mutation in TLR5 is associated with survival and reduced IL-10 and TNF-α levels in human melioidosis.

BackgroundMelioidosis, caused by the flagellated bacterium Burkholderia pseudomallei, is a life-threatening and increasingly recognized emerging disease. Toll-like receptor (TLR) 5 is a germline-encoded pattern recognition receptor to bacterial flagellin. We evaluated the association of a nonsense TLR5 genetic variant that truncates the receptor with clinical outcomes and with immune responses in melioidosis.Methodology/Principal findingsWe genotyped TLR5 c.1174C>T in 194 acute melioidosis patients in Thailand. Twenty-six (13%) were genotype CT or TT. In univariable analysis, carriage of the c.1174C>T variant was associated with lower 28-day mortality (odds ratio (OR) 0.21, 95% confidence interval (CI) 0.05–0.94, P = 0.04) and with lower 90-day mortality (OR 0.25, 95% CI 0.07–086, P = 0.03). In multivariable analysis adjusting for age, sex, diabetes and renal disease, the adjusted OR for 28-day mortality in carriers of the variant was 0.24 (95% CI 0.05–1.08, P = 0.06); and the adjusted OR for 90-day mortality was 0.27 (95% CI 0.08–0.97, P = 0.04). c.1174C>T was associated with a lower rate of bacteremia (P = 0.04) and reduced plasma levels of IL-10 (P = 0.049) and TNF-α (P < 0.0001). We did not find an association between c.1174C>T and IFN-γ ELISPOT (T-cell) responses (P = 0.49), indirect haemagglutination titers or IgG antibodies to bacterial flagellin during acute melioidosis (P = 0.30 and 0.1, respectively).Conclusions/SignificanceThis study independently confirms the association of TLR5 c.1174C>T with protection against death in melioidosis, identifies lower bacteremia, IL-10 and TNF-α production in carriers of the variant with melioidosis, but does not demonstrate an association of the variant with acute T-cell IFN-γ response, indirect haemagglutination antibody titer, or anti-flagellin IgG antibodies.

Comparative efficacy of cryoprotectant in the lyophilizationof pigeon paramyxovirus-1 vaccine

Preparation of live attenuated freeze dried Pigeon Paramyxo Virus-1 (PPMV-1) vaccine selection of proper cryoprotectant which could preserve and protect the virus against injury due to reduction of temperature at the time of lyophilization, was very necessary. PPMV-1 locally isolated lentogenic strain was selected as seed virus which was properly attenuated by serially passaging in 9 to 10 days old embryonated specific pathogen free (SPF) fowl eggs and produced master seed virus and working seed virus. Five per cent lactalbumin hydrolysate with 10% sucrose (LAS) and 1% polyvinyl pyrrolidone (PVP) were used as two different cryoprotectant for lyophilization of working seed virus. After lyophilization value of haemagglutination (HA) titer and embryo infective dose fifty per ml (EID50/ml) were changed and concluded that both LAS and PVP could be used as suitable cryoprotectant where LAS was better than PVP which was first time done in the whole world.

Induction of IL21 in Peripheral T Follicular Helper Cells Is an Indicator of Influenza Vaccine Response in a Previously Vaccinated HIV-Infected Pediatric Cohort.

HIV-infected patients of all ages frequently underperform in response to seasonal influenza vaccination, despite virologic control of HIV. The molecular mechanisms governing this impairment, as well as predictive biomarkers for responsiveness, remain unknown. This study was performed in samples obtained prevaccination (T0) from HIV-infected children who received the 2012-2013 seasonal influenza vaccine. Response status was determined based on established criterion for hemagglutination inhibition titer; participants with a hemagglutination titer ≥1:40 plus a ≥4-fold increase over T0 at 3 wk postvaccination were designated as responders. All children had a history of prior influenza vaccinations. At T0, the frequencies of CD4 T cell subsets, including peripheral T follicular helper (pTfh) cells, which provide help to B cells for developing into Ab-secreting cells, were similar between responders and nonresponders. However, in response to in vitro stimulation with influenza A/California/7/2009 (H1N1) Ag, differential gene expression related to pTfh cell function was observed by Fluidigm high-density RT-PCR between responders and nonresponders. In responders, H1N1 stimulation at T0 also resulted in CXCR5 induction (mRNA and protein) in CD4 T cells and IL21 gene induction in pTfh cells that were strongly associated with H1N1-specific B cell responses postvaccination. In contrast, CD4 T cells of nonresponders exhibited increased expression of IL2 and STAT5 genes, which are known to antagonize peripheral Tfh cell function. These results suggest that the quality of pTfh cells at the time of immunization is important for influenza vaccine responses and provide a rationale for targeted, ex vivo Ag-driven molecular profiling of purified immune cells to detect predictive biomarkers of the vaccine response.

Evaluation of efficacy of Vero cell-adapted, thermostable Newcastle disease vaccine in broilers

The main theme of this project was to develop a Vero cell-adapted, thermostable NDV I-2 vaccine and evaluate its efficacy against challenge infection. For this purpose, serial passages of virus were done in Vero cell line up to 13 times and after each passage samples were subjected to heat treatment at 56°C for 40 min. After 13 passages, the virus was completely adapted on Vero cell line and cytopathic effects were observed, including syncytial formation, rounding, degeneration, and detachment of cells. Hemagglutination and infectivity titers showed that the virus was thermostable after each passages in Vero cell line. One-day-old broiler chicks (Group 1) were vaccinated orally with thermostable NDV I-2 vaccine. A commercially available thermolabile NDV LaSota was used in Group 2 used as positive control. NDV I-2 vaccine produced maximum % inhibition migration at d 6 (50%) as compared with LaSota ND vaccine (i.e., 32%). On encounter with virulent NDV I-2, 100% safety was accomplished in group 1 and 60% in case of group 2. All the birds in the control negative group had died. This study led to the conclusion that thermostable Vero cell adapted I-2 strain vaccine resulted in better immunization in broiler birds than obtained by the use of commercially available thermolabile vaccines.

Anti-inflammatory and immuno-modulatory studies on LC-MS characterised methanol extract of Gentiana kurroo Royle

BackgroundIn ayurvedic traditional medicine Gentiana kurroo Royle (family; Gentianaceae) is used to treat several metabolic diseases. This plant is rich in various compounds belonging to flavonoids and glycosides. Till now little work has been carried out on immunomodulatory and anti-inflammatory potential of this plant. This study confirms the presence of bioactive compounds and evaluates the anti-inflammatory and immunomodulatory effect of this plant.MethodsTo carry out this work, the methanol extract was investigated in different doses using in vivo and in vitro models. In vivo study involved haemagglutination titre and DTH methods, and in vitro study was done using splenocyte proliferation assay and LPS stimulated macrophage culture. TNF-α, IL-6 and NO were assayed using ELISA kit methods, while NF-κB was evaluated by western blotting. LC-ESI-MS/MS was used for the characterization of the methanol extract.ResultsThe results showed suppression of both humoral and cell mediated immunity in vivo. This effect was also observed by inhibition of B and T cell proliferation in splenocyte proliferation assay. TNF-α, IL-6 and NO concentrations were also less in extract treated macrophage cultures. The NF-κB expression was also lowered in treated macrophages as compared to untreated macrophages. All these observations were found to be dose dependent. LC-MS characterization of this extract showed the presence of known compounds which are glycosides, alkaloids and flavonoids in nature.ConclusionThe methanol extract of this plant was found to be rich in glycoside, alkaloid and flavonoid compounds. These compounds are probably responsible for the suppression of immune response and anti-inflammatory activity. The extract as such and identified bioactive compounds can be useful for the treatment of inflammatory disorders.

Investigating immunocompetence in Aseel, Kadaknath and White Leghorn chicken

The study aimed to investigate immunocompetence in Aseel, Kadaknath and IWH line of White Leghorn chicken maintained at this institute. Five to six weeks aged pedigreed 75, 55 and 100 chicks progenies of 8, 8 and 20 sires of these respective genotypes were immunized against 1% (v/v) SRBC suspension. Five dpi sera were analyzed through haemagglutination (HA) test to assess humoral immune response. Serum lysozyme and immunoglobulin-G (IgG) concentrations were assessed through agarose lysoplate and single radial immunodiffusion assay, respectively. The data were analyzed by least squares analysis of variance. The estimated corresponding means of HA titre, serum lysozyme and serum IgG concentrations were 10.84±0.18, 21.28±0.78 μg/ml and 24.23±2.05 μg/μl in Aseel, 11.62±0.21, 16.91±0.93 μg/ml and 15.70±2.44 μg/μl in Kadaknath, and 11.94±0.15, 9.42±0.68 μg/mland 10.27±1.79 μg/μlin IWH line of White Leghorn chicken. The estimates significantly varied among the three genotypes. The HA titre mean was higher in IWH line of WLH followed by Kadaknath and Aseel chicken; whereas serum lysozyme and serum IgG concentration means were higher in Aseel followed by Kadaknath and IWH line. Sire within genotype or sire and sex of the birds had nonsignificant effect on the traits. This information could be important while selecting birds for improvement in general immune responsiveness and the high immunocompetence in Aseel and Kadaknath might be exploited by their selective introgression in high productive but less immunocompetent chicken germplasms.

Ethanol extract and chromatographic fractions of Tamarindus indica stem bark inhibits Newcastle disease virus replication

Context: The plethora of ethnomedicinal applications of Tamarindus indica Linn. (Leguminosae), tamarind, includes treatment of human and livestock ailments; preparations are recognized antipyretics in fevers, laxatives and carminatives. African folklore has various applications of tamarind. However, in Nyasaland, domestic fowl are fed with preparations for prophylactic properties.Objectives: The objective of this study is to evaluate the antiviral properties of T. indica extract.Materials and methods: Tamarindus indica stem bark was extracted through ethanol maceration over 24 h, and the crude extract was fractionated by gravity-propelled column chromatography. Newcastle disease virus (NDV) inhibitory activity of extract and fractions were evaluated in vivo using 10-d-old embryonated chicken egg (ECE) as the medium for virus cultivation and antivirus assay. About 240 ECE were grouped into eight (three controls and five experimental) and, 200 μL of the extract and fractions respectively inoculated into NDV pre-infected eggs and incubated at 37 °C. Allantoic fluid was harvested 5 d post-virus infection and assayed for haemagglutination (HA).Results: Anti-NDV assessment showed 62.5 mg/mL of crude extract and fractions: TiA, TiC and TiD to yield a HA titre of 1:128 each, while TiB showed 1:64 HA titre. At 125 mg/mL, a titre of 1:16 was recorded against TiB and TiD and, 1:8 against TiA. Similarly, crude extract and TiC, each recorded 1:4 HA titre. However, the minimum concentrations of extract and fraction for virus inactivation were 0.24 mg/mL and 0.49 mg/mL, respectively.Conclusion: The antiviral activity shown by T. indica portends novel antiviral drugs and, perhaps, as scaffold for new drugs.

Assessment of Immunomodulatory Activity of the Biofield Energy Treated Novel Herbomineral Formulation After Oral Administration in Female &amp;lt;i&amp;gt;Sprague Dawley&amp;lt;/i&amp;gt; Rats

The use of an herbomineral formulation in the healthcare sector for different chronic diseases is gaining popularity due to its fewer side-effects, high safety profile, and cost effectiveness. A new proprietary herbomineral formulation was formulated, consisting of four essential ingredients viz. herbal (ashwagandha root extract) and minerals (zinc chloride, magnesium gluconate, and sodium selenate). The objective of this study was to evaluate the immunomodulatory effect of the Biofield Energy Treated test formulation on female Sprague Dawley (SD) rats. The test formulation was divided into two parts; one was represented as control, while the other part was treated with the Biofield Energy Healing Treatment remotely by renowned Biofield Energy Healers (The Trivedi Effect®) and defined as the Biofield Energy Treated formulation. The effect of the test formulation was monitored using humoral immune response, delayed type hypersensitivity, hematology, biochemistry, relative organ weight, and histopathology. The primary hemagglutination (HA) antibody titre level was significantly (p≤0.05) increased by 185.88% in the Biofield Energy Treated group (G3); while 41.18% increased in the untreated test formulation group (G4) as compared to the disease control group (G2). The secondary HA titre was significantly increased by 540% (p≤0.001) and 166.7% in the G3 and G4 groups, respectively as compared to the G2 group. The secondary HA titre was also increased significantly (p≤0.05) by 193% in the Biofield Energy Treated group per se at day -15 (G6), as compared to the G2 group. The paw volume data showed an alteration in the G3 and G6 groups as compared to the G2 group. The platelet count was significantly increased by 11.16% and 8.69% in the G3 and G4 groups, respectively as compared to the G2 group. The magnesium level was increased by 9.31% in the G3 group as compared to the G2 group. The uric acid was significantly reduced by 23.88% in the G3 group; while it was increased by 17.47% in the G4 group as compared to the G2 group. Since, the relative organ weight and histopathological findings did not produce any signs of toxicity as compared to the normal control group (G1). These data suggested that the Biofield Energy Treated herbomineral formulation was found to be safe and showed better immunomodulatory response as compared to the untreated formulation. Thus, it is assumed that the Biofield Energy Treated herbomineral formulation could be better and more useful to boost-up the immune system in healthy human and patients.

Temporal Regulation of fim Genes in Uropathogenic Escherichia coli during Infection of the Murine Urinary Tract.

Uropathogenic Escherichia coli (UPEC) adhere to cells in the human urinary tract via type 1 pili that undergo phase variation where a 314-bp fimS DNA element flips between Phase-ON and Phase-OFF orientations through two site-specific recombinases, FimB and FimE. Three fim-lux operon transcriptional fusions were created and moved into the clinical UPEC isolate NU149 to determine their temporal regulation in UPEC growing in the urinary tract. Within murine urinary tracts, the UPEC strains demonstrated elevated transcription of fimA and fimB early in the infection, but lower transcription by the fifth day in murine kidneys. In contrast, fimE transcription was much lower than either fimA or fimB early, increased markedly at 24 h after inoculation, and then dropped five days after inoculation. Positioning of fimS was primarily in the Phase-ON position over the time span in UPEC infected bladders, whereas in UPEC infected murine kidneys the Phase-OFF orientation was favored by the fifth day after inoculation. Hemagglutination titers with guinea pig erythrocytes remained constant in UPEC growing in infected murine bladders but fell substantially in UPEC infected kidneys over time. Our results show temporal in vivo regulation of fim gene expression in different environmental niches when UPEC infects the murine urinary tract.

Antithrombocytopenic and immunomodulatory potential of metabolically characterized aqueous extract of Carica papaya leaves

Context: Carica papaya Linn. (Caricaceae) leaf (CPL) juice has long been traditionally used in ethnomedicine for dengue fever. Objective: The study examines the effects of standardized CPL aqueous extract (SCPLE) on platelet count, extramedullary haematopoiesis (EMH), and immunomodulation in cyclophosphamide (CP)-induced animal model of thrombocytopenia. Materials and methods: The extract was analyzed for myricetin, caffeic acid, trans-ferulic acid, and kaempferol using HPTLC for standardization followed by UPLC-qTOF/MS fingerprinting for metabolite signature. The effects of SCPLE (50 and 150 mg/kg p.o.) on proliferative response of platelet count and total leucocyte count (TLC) were observed up to 14 days in Wistar rat. However, delayed-type hypersensitivity (DTH), haemagglutination titre (HT), and in vivo carbon clearance were examined as immunomodulatory parameters in albino mice at 150 mg/kg p.o. against CP. Results: The quantitative HPTLC estimation of SCPLE showed the presence of myricetin, caffeic acid, trans-ferulic acid, and kaempferol up to 280.16 ± 5.99, 370.18 ± 6.27, 1110.86 ± 2.97, and 160.53 ± 2.48 (μg/g), respectively. Twenty-four metabolites were identified using UPLC-qTOF/MS. Oral administration of SCPLE (150 mg/kg) in thrombocytopenic rats exhibited significant (p < 0.01) increase in thrombocytes (1014.83 × 103 cells/mm3), DTH response (0.16 ± 0.004), and phagocytic index (63.15% increase) as compared to CP-induced thrombocytopenia group. Histopathological studies showed minimal fibrosis in spleen histology. Discussion and conclusions: Results suggest CPL can mediate the release of platelets providing the means for the treatment and prevention of dengue.

Oil-in-water emulsion formulated with eucalyptus leaves extract inhibit influenza virus binding and replication in vitro.

Throughout human history, the human-beings have been used different types of plants as antimicrobial agents in fight against infectious diseases. Influenza virus is one of the most common causes of respiratory infection and transmitted through direct contact with flu infected individuals and contaminated substances or droplets. In the current study, both oil-in-water and water-in-oil emulsions with hydroalcoholic extract of eucalyptus leaves (OLHE) were developed and their antiviral efficiency was evaluated. To doing so, Madin-Darbey Canine Kidney (MDCK) cells were treated with effective minimal cytotoxic concentration of the formulated emulsions. The treated cells were then infected with 50% cell culture infectious dose (100 CCID50) of the A/H1N1 virus (the swine flu). The viral titers were measured by hemagglutination (HA) and cell culture infectious dose 50% (CCID50) assays. Also, to check the virus binding inhibition via the formulated extract, the viruses were incubated with the formulated extracts. Our study showed that the oil-in-water emulsions formulated with 2% eucalyptus leaves extract inhibited virus replication completely when the cells were infected by 100 CCID50 and decreased HA titer up to four fold. Therefore, this formulation, may hold promising application to prevent influenza virus transmission through direct contact among children and passengers.

Validation of the Immunomodultory Potential of Amarkand Species

Amarkand plants are routinely used by Indian tribes for health and longevity. Taxonomically, Amarkand includes species from genera Eulophia and Dioscorea. Comparative immunomodulatory potential of Amarkand extracts were examined through biochemical, hematological, serological and histopathological analysis. A significant effect on the modulation of immune reactivity was observed in bulbils of Dioscorea bulbifera and tubers of Eulophia ochreata. A significant increase was also observed in relative organ weights, hematological parameters and total protein by E. ochreata (500 mg/kg) treatment compared to the negative control group. While aminotransferase enzyme levels have shown a significant decrease by the treatment of E. ochreata and D. bulbifera extracts, D. bulbifera (300 mg/kg) showed greater (61.7±3.62%) neutrophil adhesion and a significant increase in delayed type hypersensitivity response. E. ochreata treatment showed increased haemagglutination titre compared to negative control, thereby providing evidence that D. bulbifera and E. ochreata possessed immunomodulatory activity. Histopathology of rat organs treated with D. bulbifera and E. ochreata, showed normal cytoarchitecture and less neutrophil infiltration as compared to the negative control group, suggesting no evidence of pathological signs. Our findings demonstrated that E. ochreata and D. bulbifera have immunomodulatory potential and provide the basis for an alternative approach to investigate new traditional remedies.

Viral type characterization and clinical aspects of canine parvovirus in naturally infected dogs in São Paulo State, Brazil

ABSTRACT: Since the first isolation of canine parvovirus type 2 (CPV-2) in late 70’s new virus types as CPV-2a and CPV-2b have been emerged and becoming prevalent in natural canine population and more recently, a third subtype was identified , CPV-2c. The main purpose of this study was to detect and characterize canine parvovirus currently present in Central-West region of São Paulo state, in Brazil. Fecal samples were collected of vaccinated and non-vaccinated dogs, clinically suspected of having CPV infection brought to the Infectious Diseases Service, Veterinary Hospital of FMVZ-UNESP. All samples (n=30) were screening for canine parvovirus through hemagglutination test and those resulting as positive (n=20) were submitted to PCR and the products were subsequently sequenced for subtype characterization. Results were tested for association with age, hematological values, viral hemagglutination titers in the feces, vaccination status and survival. Leukopenia was found in all animals, death occurred in 30% of unvaccinated dogs and in 42% of vaccinated ones. In a total of 20 positive sequenced samples, 18 were classified as CPV-2b, one as CPV-2c, and one as CPV-2a, being CPV2a and CPV2c detected in unvaccinated puppies. Compared to the reference samples amino acid change at position 426 in those circling virus was identified. The study results demonstrate the predominance of CPV-2b and the presence of CPV-2a and CPV-2c in naturally infected, vaccinated and unvaccinated dogs in in São Paulo region.