CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.

Transcriptomics-based analysis using RNA-Seq of the coconut (Cocos nucifera) leaf in response to yellow decline phytoplasma infection

Invasive phytoplasmas wreak havoc on coconut palms worldwide, leading to high loss of income, food insecurity and extreme poverty of farmers in producing countries. Phytoplasmas as strictly biotrophic insect-transmitted bacterial pathogens instigate distinct changes in developmental processes and defence responses of the infected plants and manipulate plants to their own advantage; however, little is known about the cellular and molecular mechanisms underlying host–phytoplasma interactions. Further, phytoplasma-mediated transcriptional alterations in coconut palm genes have not yet been identified. This study evaluated the whole transcriptome profiles of naturally infected leaves of Cocos nucifera ecotype Malayan Red Dwarf in response to yellow decline phytoplasma from group 16SrXIV, using RNA-Seq technique. Transcriptomics-based analysis reported here identified genes involved in coconut innate immunity. The number of down-regulated genes in response to phytoplasma infection exceeded the number of genes up-regulated. Of the 39,873 differentially expressed unigenes, 21,860 unigenes were suppressed and 18,013 were induced following infection. Comparative analysis revealed that genes associated with defence signalling against biotic stimuli were significantly overexpressed in phytoplasma-infected leaves versus healthy coconut leaves. Genes involving cell rescue and defence, cellular transport, oxidative stress, hormone stimulus and metabolism, photosynthesis reduction, transcription and biosynthesis of secondary metabolites were differentially represented. Our transcriptome analysis unveiled a core set of genes associated with defence of coconut in response to phytoplasma attack, although several novel defence response candidate genes with unknown function have also been identified. This study constitutes valuable sequence resource for uncovering the resistance genes and/or susceptibility genes which can be used as genetic tools in disease resistance breeding.

Being diagnosed with a sexually transmitted infection (STI): sources of support for young women

BACKGROUND: Sexually transmitted infections (STIs) are prevalent throughout the world with the rate of these infections increasing on a daily basis. STI acquisition has the ability to cause personal adversity and elicit feelings of stigma and shame. AIM: The aim of this paper is to report on the sources of support young women who acquired STIs drew on to overcome their associated adversity.

METHODS: This study utilised a feminist qualitative methodology.

FINDINGS: Findings revealed that the women drew on both personal and anonymous sources of support.

CONCLUSION: Nurses and other health-care professionals need to be equipped with knowledge and strategies to promote personal wellbeing and minimise the adversity felt among people having acquired these infections.

Foot-and-mouth disease in red deer - experimental infection and test methods performance

Summary: The aim of this study was to evaluate a number of foot-and-mouth disease (FMD) test methods for use in red deer. Ten animals were intranasally inoculated with the FMD virus (FMDV) O UKG 11/2001, monitored for clinical signs, and samples taken regularly (blood, serum, oral swabs, nasal swabs, probang samples and lesion swabs, if present) over a 4-week period. Only one animal, deer 1103, developed clinical signs (lesions under the tongue and at the coronary band of the right hind hoof). It tested positive by 3D and IRES real-time reverse transcription polymerase chain reaction (rRT-PCR) in various swabs, lesion materials and serum. In a non-structural protein (NSP) in-house ELISA (NSP-ELISA-IH), one commercial ELISA (NSP-ELISA-PR) and a commercial antibody NSP pen side test, only deer 1103 showed positive results from day post-inoculation (dpi) 14 onwards. Two other NSP-ELISAs detected anti-NSP serum antibodies with lower sensitivity. It also showed rising antibody levels in the virus neutralization test (VNT), the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR at dpi 9, and in another two commercial SPO-ELISAs at dpi 12 (SPO-ELISA-IV) and dpi 19 (SPO-ELISA-IZ), respectively. Six of the red deer that had been rRT-PCR and antibody negative were re-inoculated intramuscularly with the same O-serotype FMDV at dpi 14. None of these animals became rRT-PCR or NSP-ELISA positive, but all six animals became positive in the VNT, the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR. Two other commercial SPO-ELISAs were less sensitive or failed to detect animals as positive. The rRT-PCRs and the four most sensitive commercial ELISAs that had been used for the experimentally inoculated deer were further evaluated for diagnostic specificity (DSP) using 950 serum samples and 200 nasal swabs from non-infected animals. DSPs were 100% for the rRT-PCRs and between 99.8 and 100% for the ELISAs.

Experimental foot-and-mouth disease virus infection in white tailed deer

White tailed deer (Odocoileus virginianus) were inoculated with foot-and-mouth disease virus (FMDV) O UKG 11/2001 and monitored for the development of clinical signs, histopathological changes and levels of virus replication. All FMDV-infected deer developed clinical signs starting at 2 days post inoculation and characterized by an increase in body temperature, increased salivation and lesions in the mouth and on the feet. Virus spread to various tissues was determined by quantifying the amount of FMDV RNA using quantitative reverse transcriptase polymerase chain reaction. Virus or viral antigen was also detected in tissues using traditional isolation techniques, enzyme linked immunosorbent assay and immunohistochemistry. Deer-to-cattle transmission of the virus was observed in this experimental setting; however, inoculated deer were not found to become carriers of FMDV.

Epitope-specific CD4+, but not CD8+, T-cell responses induced by recombinant influenza A viruses protect against Mycobacterium tuberculosis infection

Tuberculosis remains a global health problem, in part due to failure of the currently available vaccine, BCG, to protect adults against pulmonary forms of the disease. We explored the impact of pulmonary delivery of recombinant influenza A viruses (rIAVs) on the induction of Mycobacterium tuberculosis (M. tuberculosis)-specific CD4(+) and CD8(+) T-cell responses and the resultant protection against M. tuberculosis infection in C57BL/6 mice. Intranasal infection with rIAVs expressing a CD4(+) T-cell epitope from the Ag85B protein (PR8.p25) or CD8(+) T-cell epitope from the TB10.4 protein (PR8.TB10.4) generated strong T-cell responses to the M. tuberculosis-specific epitopes in the lung that persisted long after the rIAVs were cleared. Infection with PR8.p25 conferred protection against subsequent M. tuberculosis challenge in the lung, and this was associated with increased levels of poly-functional CD4(+) T cells at the time of challenge. By contrast, infection with PR8.TB10.4 did not induce protection despite the presence of IFN-γ-producing M. tuberculosis-specific CD8(+) T cells in the lung at the time of challenge and during infection. Therefore, the induction of pulmonary M. tuberculosis epitope-specific CD4(+), but not CD8(+) T cells, is essential for protection against acute M. tuberculosis infection in the lung.

Properties of HIV-1 associated cholesterol in addition to raft formation are important for virus infection

The overall HIV-1 membrane lipid contents resemble lipid rafts, and we have previously demonstrated that raft-promoting properties of virus-associated cholesterol (with modifications in either the 3β-OH group or AB rings) are important for HIV-1 infectivity. As cholesterol is present in both rafts and non-rafts domains of HIV-1 membrane, we question whether the interpretation of rafts property of virus-associated cholesterol being an absolute requirement for HIV-1 function is too simplistic. The carbon side chain of cholesterol is the third component of cholesterol that can affect the fluidity of membrane depending on its context within the lipid membrane bilayers. In this work, we have used synthetic cholesterol analogues that have different lengths of carbon side chain for our investigation. In contrast to our previous report, we have found that cholesterol side chain analogues that lack in vitro defined raft promoting-property is able to support HIV-1 replication. More specifically, cholesterol analogues with side chains of intermediate length have greater capacity to support HIV-1 infection, suggesting HIV-1 is able to maintain function using cholesterol variants that promote a range of non-rafts- to rafts-properties. Our data demonstrate cholesterol properties other than raft-promoting function also contribute to the infectivity of HIV-1.

The PTEX component EXP2 is critical for establishing a patent malaria infection in mice

Export of most malaria proteins into the erythrocyte cytosol requires the Plasmodium Translocon of Exported proteins (PTEX) and a cleavable Plasmodium Export Element (PEXEL). In contrast, the contribution of PTEX in the liver stages and export of liver stage proteins is unknown. Here, using the FLP/FRT conditional mutatagenesis system, we generate transgenic P. berghei parasites deficient in EXP2, the putative pore-forming component of PTEX. Our data reveal that EXP2 is important for parasite growth in the liver and critical for parasite transition to the blood, with parasites impaired in their ability to generate a patent blood-stage infection. Surprisingly, whilst parasites expressing a functional PTEX machinery can efficiently export a PEXEL-bearing GFP reporter into the erythrocyte cytosol during a blood stage infection, this same reporter aggregates in large accumulations within the confines of the parasitophorous vacuole membrane during hepatocyte growth. Notably HSP101, the putative molecular motor of PTEX, could not be detected during the early liver stages of infection, which may explain why direct protein translocation of this soluble PEXEL-bearing reporter or indeed native PEXEL proteins into the hepatocyte cytosol has not been observed. This suggests that PTEX function may not be conserved between the blood and liver stages of malaria infection. This article is protected by copyright. All rights reserved.

Novel targeting of PEGylated liposomes for codelivery of TGF-β1 siRNA and four antitubercular drugs to human macrophages for the treatment of mycobacterial infection: a quantitative proteomic study

Tuberculosis (TB) is still a major public health issue in developing countries, and its chemotherapy is compromised by poor drug compliance and severe side effects. This study aimed to synthesize and characterize new multimodal PEGylated liposomes encapsulated with clinically commonly used anti-TB drugs with linkage to small interfering RNA (siRNA) against transforming growth factor-β1 (TGF-β1). The novel NP-siRNA liposomes could target THP-1-derived human macrophages that were the host cells of mycobacterium infection. The biological effects of the NP-siRNA liposomes were evaluated on cell cycle distribution, apoptosis, autophagy, and the gene silencing efficiency of TGF-β1 siRNA in human macrophages. We also explored the proteomic responses to the newly synthesized NP-siRNA liposomes using the stable isotope labeling with amino acids in cell culture approach. The results showed that the multifunctional PEGylated liposomes were successfully synthesized and chemically characterized with a mean size of 265.1 nm. The novel NP-siRNA liposomes functionalized with the anti-TB drugs and TGF-β1 siRNA were endocytosed efficiently by human macrophages as visualized by transmission electron microscopy and scanning electron microscopy. Furthermore, the liposomes showed a low cytotoxicity toward human macrophages. There was no significant effect on cell cycle distribution and apoptosis in THP-1-derived macrophages after drug exposure at concentrations ranging from 2.5 to 62.5 μg/mL. Notably, there was a 6.4-fold increase in the autophagy of human macrophages when treated with the NP-siRNA liposomes at 62.5 μg/mL. In addition, the TGF-β1 and nuclear factor-κB expression levels were downregulated by the NP-siRNA liposomes in THP-1-derived macrophages. The Ingenuity Pathway Analysis data showed that there were over 40 signaling pathways involved in the proteomic responses to NP-siRNA liposome exposure in human macrophages, with 160 proteins mapped. The top five canonical signaling pathways were eukaryotic initiation factor 2 signaling, actin cytoskeleton signaling, remodeling of epithelial adherens junctions, epithelial adherens junction signaling, and Rho GDP-dissociation inhibitor signaling pathways. Collectively, the novel synthetic targeting liposomes represent a promising delivery system for anti-TB drugs to human macrophages with good selectivity and minimal cytotoxicity.

Dietary protein intake may reduce hospitalisation due to infection in Māori of advanced age: LiLACS NZ

OBJECTIVE: To investigate factors related to hospital admission for infection, specifically examining nutrient intakes of Māori in advanced age (80+ years). METHOD: Face-to-face interviews with 200 Māori (85 men) to obtain demographic, social and health information. Diagnoses were validated against medical records. Detailed nutritional assessment using the 24-hour multiple-pass recall method was collected on two separate days. FOODfiles was used to analyse nutrient intake. National Health Index (NHI) numbers were matched to hospitalisations over a two-year period (12 months prior and 12 months following dietary assessment). Selected International Classification of Disease (ICD) codes were used to identify admissions related to infection. RESULTS: A total of 18% of participants were hospitalised due to infection, most commonly lower respiratory tract infection. Controlling for age, gender, NZ deprivation index, diabetes, CVD and chronic lung disease, a lower energy-adjusted protein intake was independently associated with hospitalisation due to infection: OR (95%CI) 1.14 (1.00-1.29), p=0.046. CONCLUSIONS: Protein intake may have a protective effect on the nutrition-related morbidity of older Māori. Improving dietary protein intake is a simple strategy for dietary modification aiming to decrease the risk of infections that lead to hospitalisation and other morbidities.

Avian influenza infection dynamics under variable climatic conditions, viral prevalence is rainfall driven in waterfowl from temperate, south-east Australia

Understanding Avian Influenza Virus (AIV) infection dynamics in wildlife is crucial because of possible virus spill over to livestock and humans. Studies from the northern hemisphere have suggested several ecological and environmental drivers of AIV prevalence in wild birds. To determine if the same drivers apply in the southern hemisphere, where more irregular environmental conditions prevail, we investigated AIV prevalence in ducks in relation to biotic and abiotic factors in south-eastern Australia. We sampled duck faeces for AIV and tested for an effect of bird numbers, rainfall anomaly, temperature anomaly and long-term ENSO (El-Niño Southern Oscillation) patterns on AIV prevalence. We demonstrate a positive long term effect of ENSO-related rainfall on AIV prevalence. We also found a more immediate response to rainfall where AIV prevalence was positively related to rainfall in the preceding 3-7 months. Additionally, for one duck species we found a positive relationship between their numbers and AIV prevalence, while prevalence was negatively or not affected by duck numbers in the remaining four species studied. In Australia largely non-seasonal rainfall patterns determine breeding opportunities and thereby influence bird numbers. Based on our findings we suggest that rainfall influences age structures within populations, producing an influx of immunologically naïve juveniles within the population, which may subsequently affect AIV infection dynamics. Our study suggests that drivers of AIV dynamics in the northern hemisphere do not have the same influence at our south-east Australian field site in the southern hemisphere due to more erratic climatological conditions.

Nematicidal activity of Solanum nigrum and S. sisymbriifolium extracts against the root-lesion nematode Pratylenchus goodeyi and its effects on infection and gene expression

The control of Pratylenchus goodeyi a common nematode parasite of banana crop in Madeira Island can benefit from searching for natural nematicides through plants extracts. With this aim we submitted Solanum nigrum and S. sisymbriifolium dried plants to a sequential extraction in the solvent sequence of dichloromethane, acetone, ethanol and water, and to na aqueous extraction of the fresh and dried plants. Analyses with the extracts at several concentrations were used to assess mobility and mortality on P. goodeyi. Results showed that the water extract and aqueous extracts from both plants at a concentration of 10 mg/mL affected nematode mobility and caused mortality but the acetone extract from S. nigrum was the most efficient, causing 100% mortality whereas dichloromethane had no effect on P. goodeyi. Determination of the lipophilic and phenolic compounds present in the two most effective Solanum extracts (acetone and water) and in dichloromethane extract revealed that some of these compounds had nematicidal activity. S. nigrum acetone extract (10 mg/mL) was used to find out the nematicidal potential following the effect at gene expression level and nematode behaviour. Genes coding for calreticulin and beta-1,4- endoglucanase related to parasitism and translocon-associated protein putatively connected to stress were obtained and its relative expression assessed in nematodes exposed to the extract. Results revealed that expression of Pg-CRT decreased showing to influence the infection, Pg-ENG remained steady and Pg-TRAPδ was induced over time exposure. Biological assays showed that P. goodeyi mobility and ability to infect the banana roots were affected as a decrease in the number of nematodes that reached the roots was obtained with the increased exposure time to the extract being implicated in the infection success. The information obtained from this thesis showed that S. nigrum has potential to be used for the development of a new control strategy against plant-parasitic nematodes.

Prevalence of helicobacter pylori infection in advanced gastric carcinoma

There is substantial evidence that infection with Helicobacter pylori plays a role in the development of gastric cancer and that it is rarely found in gastric biopsy of atrophic gastritis and gastric cancer. On advanced gastric tumors, the bacteria can be lost from the stomach. Aims - To analyze the hypothesis that the prevalence of H.pylori in operated advanced gastric carcinomas and adjacent non-tumor tissues is high, comparing intestinal and diffuse tumors according to Lauren’s classifi cation. Methods - A prospective controlled study enrolled 56 patients from “Hospital Universitário”, Federal University of Rio Grande do Norte, Natal, RN, Brazil, with advanced gastric cancer, treated from February 2000 to March 2003. Immediately after partial gastrectomy, the resected stomach was opened and several mucosal biopsy samples were taken from the gastric tumor and from the adjacent mucosa within 4 cm distance from the tumor margin. Tissue sections were stained with hematoxylin and eosin. Lauren‘s classifi cation for gastric cancer was used, to analyse the prevalence of H. pylori in intestinal or diffuse carcinomas assessed by the urease rapid test, IgG by ELISA and Giemsa staining. H. pylori infected patients were treated with omeprazole, clarithromycin and amoxicillin for 7 days. Follow-up endoscopy and serology were performed 6 months after treatment to determine successful eradication of H. pylori in non-tumor tissue. Thereafter, follow-up endoscopies were scheduled annually. Chi-square and MacNemar tests with 0.05 signifi cance were used. Results - Thirty-four tumors (60.7%) were intestinal-type and 22 (39.3%) diffuse type carcinomas. In adjacent non-tumor gastric mucosa, chronic gastritis were found in 53 cases (94.6%) and atrophic mucosa in 36 patients (64.3%). All the patients with atrophic mucosa were H. pylori positive. When examined by Giemsa and urease test, H. pylori positive rate in tumor tissue of intestinal type carcinomas was higher than that in diffuse carcinomas. In tumor tissues, 34 (60.7%) H. pylori-positive in gastric carcinomas were detected by Giemsa method. H. pylori was observed in 30 of 56 cases (53.5%) in tissues 4 cm adjacent to tumors. This difference was not signifi cant. Eradication of H. pylori in non-tumor tissue of gastric remnant led to a complete negativity on the 12th postoperative month. Conclusions - The data confi rmed the hypothesis of a high prevalence of H. pylori in tumor tissue of gastric advanced carcinomas and in adjacent non-tumor mucosa of operated stomachs. The presence of H. pylori was predominant in the intestinal-type carcinoma

Surgical Site Infection in a University Hospital in Northeast Brazil

We examined prevention of surgical site infection (SSI) in a tertiary teaching hospital in northeast Brazil, from January 1994 to December 2003. The survey included 5,742 patients subjected to thoracic, urologic, vascular and general surgery. The criteria for diagnosing SSI were those of the Centers for Disease Control, USA, and the variables of the National Nosocomial Infection Surveillance risk index were used. Data analysis revealed that anesthetic risk scores, wound class and duration of surgery were significantly associated with SSI. A total of 296 SSIs were detected among the 5,742 patients (5.1%). The overall incidence of SSI was 8.8% in 1994; it decreased to 3.3% in 2003. In conclusion, the use of educational strategies, based on guidelines for SSI prevention reduced SSI incidence. Appropriate management of preoperative, intraoperative, and postoperative incision care, and a surveillance system based on international criteria, were useful in reducing SSI rates in our hospital

The effect of phospholipase A2 from Crotalus durissus collilineatus on Leishmania (Leishmania) amazonensis infection

In this study, the effect of phospholipase A2 (PLA2) derived from Crotalus durissus collilineatus was evaluated in vitro and in vivo on experimental cutaneous leishmaniasis. The promastigote and amastigote forms treated with PLA2 presented increased growth rate. In vivo studies showed that PLA2-treated Leishmania (Leishmania) amazonensis promastigotes increased the size of lesions in BALB/c mice, and histopathological analysis showed numerous necrotic regions presenting a higher density of polymorphonuclear, mononuclear, and amastigote cells. Additionally, infected macrophages treated with PLA2 were able to generate prostaglandin E2 (PGE2). Cytokine quantification showed that the supernatant from infected macrophages presented moderate and high amounts of IL-2 and IL-10, respectively. However, in PLA2-treated infected macrophages, suppression of IL-2 levels occurred, but not of IL-10 levels. Observation also revealed that both the supernatant and lysate of L. (L.) amazonensis promastigotes exhibited PLA2 activity, which, in the presence of dexamethasone, showed no reduction in their activities; while glucocorticoid maintained the ability of promastigote forms to infect macrophages, which presented values similar to controls. In conclusion, the results indicate that PLA2 may be a progression factor for cutaneous leishmaniasis, since the PLA2 effect suppressed IL-2 levels and generated PGE2, an inflammatory lipid mediator.