A study of the protein antigens of listeria monocytogenes

No. ofwords in the main text of Thesis Cell wall components of L. monocytogenes and the sub-species of L. ivanovii were analysed by SDS-PAGE and Western blotting. Preliminary studies demonstrated the protein nature of blotted material. Difference's in the protein antigens of L. monocytogenes (serotypes 4a and 4b) and L. ivanovii (serotype 5) were established. Differences were not detected between proteins of serotypes 4a and 4b and serotype 7,Cell wall proteins of L. monocytogenes grown at 20°C and 37°C were defined. Differences of expression of proteins were noted, particularly with regard to a complex of 3 proteins of approximate molecular weight 29,000D expressed at 20°C but not at 37°C. This complex was found to be referable to bacterial flagellin and the results support the microscopic evidence for temperature dependent expression of flagella.The production of an anti-flagellin monoclonal antibody allowed analysis of flagellins from different serotypes. The monoclonal antibody detected the 29,000D flagellin of serotypes l/2b, 3b, 4a, 4b and 4d but not the flagellins of serotypes 3c and 5 which had a slightly lower molecular weight.A study of cross-reaction of L. monocytogenes proteins with proteins of other bacterial species was undertaken. Extensive cross-reaction was revealed between somatic antigens of L. monocytogenes and proteins of S_. epidermidis, S_. aureus, Str. fecalis and B_. subtilSerological studies revealed that convalescent serum from CBA mice recovering from a primary challenge of flagellate organisms (1 x LD50 organisms intraperitoneally or 100 x ID50 organisms by the oral route) did not elaborate antibody against L. monocytoger flagellin as determined by ELISA. In the same mice, antibody levels against L. monocytogenes cell wall proteins were low and inconsistent.Serum antibody levels in 14 field cases of ovine listerial encephalitis were measured by ELISA. Titres against both L^. monocytogenes cell wall protein and flagellin were of the same order as healthy control animals. Three out of 23 field cases had antilisterial antibody in the CSF. For two of these cases serum samples from the same sheep were also available. By Western blotting it was shown that the same spectrum of antibody was present in both serum and CSF.Sheep recovering from experimentally induced L. monocytogenes septicaemia did not have antibody levels, as measured by ELISA, significantly highter than normal uninfected controls.LD50 and ID50 experiments were conduced in CBA mice using flagellate and aflagellate organisms of the same serotype. No difference in pathogenicity could be determined by these methods. From these experiments it was possible to define the two syndromes of murine Listeriosis, of early septicaemic death and late neurological dysfunction. Nervous symptoms were recorded in 30% of mice which died, and the syndrome occurred following either intraperitoneal or intragastric challenge with both flagellate and aflagellate organisms.Repeated vaccination of CBA mice with adjuvanted flagella failed to protect them against a subsequent intraperitoneal challenge with flagellated organisms

Shakespeare and the English Sonnet: A History

https://digitalcommons.usu.edu/english_3315/1002/thumbnail.jp

Impact of multi-targeted antiretroviral treatment on gut T cell depletion and HIV reservoir seeding during acute HIV infection.

BackgroundLimited knowledge exists on early HIV events that may inform preventive and therapeutic strategies. This study aims to characterize the earliest immunologic and virologic HIV events following infection and investigates the usage of a novel therapeutic strategy.Methods and findingsWe prospectively screened 24,430 subjects in Bangkok and identified 40 AHI individuals. Thirty Thais were enrolled (8 Fiebig I, 5 Fiebig II, 15 Fiebig III, 2 Fiebig IV) of whom 15 completed 24 weeks of megaHAART (tenofovir/emtricitabine/efavirenz/raltegravir/maraviroc). Sigmoid biopsies were completed in 24/30 at baseline and 13/15 at week 24. At baseline, the median age was 29 years and 83% were MSM. Most were symptomatic (87%), and were infected with R5-tropic (77%) CRF01_AE (70%). Median CD4 was 406 cells/mm(3). HIV RNA was 5.5 log(10) copies/ml. Median total blood HIV DNA was higher in Fiebig III (550 copy/10(6) PBMC) vs. Fiebig I (8 copy/10(6) PBMC) (p = 0.01) while the median %CD4+CCR5+ gut T cells was lower in Fiebig III (19%) vs. Fiebig I (59%) (p = 0.0008). After 24 weeks of megaHAART, HIV RNA levels of <50 copies were achieved in 14/15 in blood and 13/13 in gut. Total blood HIV DNA at week 0 predicted reservoir size at week 24 (p<0.001). Total HIV DNA declined significantly and was undetectable in 3 of 15 in blood and 3 of 7 in gut. Frequency of CD4+CCR5+ gut T cells increased from 41% at baseline to 64% at week 24 (p>0.050); subjects with less than 40% at baseline had a significant increase in CD4+CCR5+ T cells from baseline to week 24 (14% vs. 71%, p = 0.02).ConclusionsGut T cell depletion and HIV reservoir seeding increases with progression of AHI. MegaHAART was associated with immune restoration and reduced reservoir size. Our findings could inform research on strategies to achieve HIV drug-free remission

Metagenomic study of the viruses of African straw-coloured fruit bats: detection of a chiropteran poxvirus and isolation of a novel adenovirus

Viral emergence as a result of zoonotic transmission constitutes a continuous public health threat. Emerging viruses such as SARS coronavirus, hantaviruses and henipaviruses have wildlife reservoirs. Characterising the viruses of candidate reservoir species in geographical hot spots for viral emergence is a sensible approach to develop tools to predict, prevent, or contain emergence events. Here, we explore the viruses of Eidolon helvum, an Old World fruit bat species widely distributed in Africa that lives in close proximity to humans. We identified a great abundance and diversity of novel herpes and papillomaviruses, described the isolation of a novel adenovirus, and detected, for the first time, sequences of a chiropteran poxvirus closely related with Molluscum contagiosum. In sum, E. helvum display a wide variety of mammalian viruses, some of them genetically similar to known human pathogens, highlighting the possibility of zoonotic transmission

Phase I Safety and Immunogenicity Evaluation of MVA-CMDR, a Multigenic, Recombinant Modified Vaccinia Ankara-HIV-1 Vaccine Candidate

We conducted a Phase I randomized, dose-escalation, route-comparison trial of MVA-CMDR, a candidate HIV-1 vaccine based on a recombinant modified vaccinia Ankara viral vector expressing HIV-1 genes env/gag/pol. The HIV sequences were derived from circulating recombinant form CRF01_AE, which predominates in Thailand. The objective was to evaluate safety and immunogenicity of MVA-CMDR in human volunteers in the US and Thailand.MVA-CMDR or placebo was administered intra-muscularly (IM; 10(7) or 10(8) pfu) or intradermally (ID; 10(6) or 10(7) pfu) at months 0, 1 and 3, to 48 healthy volunteers at low risk for HIV-1 infection. Twelve volunteers in each dosage group were randomized to receive MVA-CMDR or placebo (10∶2). Volunteers were actively monitored for local and systemic reactogenicity and adverse events post vaccination. Cellular immunogenicity was assessed by a validated IFNγ Elispot assay, an intracellular cytokine staining assay, lymphocyte proliferation and a (51)Cr-release assay. Humoral immunogenicity was assessed by ADCC for gp120 and binding antibody ELISAs for gp120 and p24. MVA-CMDR was safe and well tolerated with no vaccine related serious adverse events. Cell-mediated immune responses were: (i) moderate in magnitude (median IFNγ Elispot of 78 SFC/10(6) PBMC at 10(8) pfu IM), but high in response rate (70% (51)Cr-release positive; 90% Elispot positive; 100% ICS positive, at 10(8) pfu IM); (ii) predominantly HIV Env-specific CD4(+) T cells, with a high proliferative capacity and durable for at least 6 months (100% LPA response rate by the IM route); (iv) dose- and route-dependent with 10(8) pfu IM being the most immunogenic treatment. Binding antibodies against gp120 and p24 were detectable in all vaccination groups with ADCC capacity detectable at the highest dose (40% positive at 10(8) pfu IM).MVA-CMDR delivered both intramuscularly and intradermally was safe, well-tolerated and elicited durable cell-mediated and humoral immune responses.ClinicalTrials.gov NCT00376090

. FIRST MOLECULAR DETECTION OF LEISHMANIA INFANTUM IN SERGENTOMYIA MINUTA (DIPTERA, PSYCHODIDAE) IN ALENTEJO, SOUTHERN PORTUGAL

One Sergentomyia minuta female was positive for L. infantum infection and another for human blood as a meal source

Expansion of Inefficient HIV-Specific CD8 T Cells during Acute Infection

ABSTRACT Attrition within the CD4 + T cell compartment, high viremia, and a cytokine storm characterize the early days after HIV infection. When the first emerging HIV-specific CD8 + T cell responses gain control over viral replication it is incomplete, and clearance of HIV infection is not achieved even in the rare cases of individuals who spontaneously control viral replication to nearly immeasurably low levels. Thus, despite their partial ability to control viremia, HIV-specific CD8 + T cell responses are insufficient to clear HIV infection. Studying individuals in the first few days of acute HIV infection, we detected the emergence of a unique population of CD38 + CD27 − CD8 + T cells characterized by the low expression of the CD8 receptor (CD8 dim ). Interestingly, while high frequencies of HIV-specific CD8 + T cell responses occur within the CD38 + CD27 − CD8 dim T cell population, the minority populations of CD8 bright T cells are significantly more effective in inhibiting HIV replication. Furthermore, the frequency of CD8 dim T cells directly correlates with viral load and clinical predictors of more rapid disease progression. We found that a canonical burst of proliferative cytokines coincides with the emergence of CD8 dim T cells, and the size of this population inversely correlates with the acute loss of CD4 + T cells. These data indicate, for the first time, that early CD4 + T cell loss coincides with the expansion of a functionally impaired HIV-specific CD8 dim T cell population less efficient in controlling HIV viremia. IMPORTANCE A distinct population of activated CD8 + T cells appears during acute HIV infection with diminished capacity to inhibit HIV replication and is predictive of viral set point, offering the first immunologic evidence of CD8 + T cell dysfunction during acute infection

Biomarker Driven Antifungal Stewardship (BioDriveAFS) in acute leukaemia—a multi-centre randomised controlled trial to assess clinical and cost effectiveness: a study protocol for a randomised controlled trial

BACKGROUND: Acute leukaemias (AL) are life-threatening blood cancers that can be potentially cured with treatment involving myelosuppressive, multiagent, intensive chemotherapy (IC). However, such treatment is associated with a risk of serious infection, in particular invasive fungal infection (IFI) associated with prolonged neutropenia. Current practice guidelines recommend primary antifungal (AF) prophylaxis to be administered to high-risk patients to reduce IFI incidence. AFs are also used empirically to manage prolonged neutropenic fever. Current strategies lead to substantial overuse of AFs. Galactomannan (GM) and β-D-glucan (BG) biomarkers are also used to diagnose IFI. Combining both biomarkers may enhance the predictability of IFI compared to administering each test alone. Currently, no large-scale randomised controlled trial (RCT) has directly compared a biomarker-based diagnostic screening strategy without AF prophylaxis to AF prophylaxis (without systematic biomarker testing). METHODS: BioDriveAFS is a multicentre, parallel, two-arm RCT of 404 participants from UK NHS Haematology departments. Participants will be allocated on a 1:1 basis to receive either a biomarker-based antifungal stewardship (AFS) strategy, or a prophylactic AF strategy, which includes existing standard of care (SoC). The co-primary outcomes will be AF exposure in the 12-month post randomisation and the patient-reported EQ-5D-5L measured at 12-month post randomisation. Secondary outcomes will include total AF exposure, probable/proven IFI, survival (all-cause mortality and IFI mortality), IFI treatment outcome, AF-associated adverse effects/events/complications, resource use, episodes of neutropenic fever requiring hospital admission or outpatient management, AF resistance in fungi (non-invasive and invasive) and a Desirability of Outcome Ranking. The trial will have an internal pilot phase during the first 9 months. A mixed methods process evaluation will be integrated in parallel to the internal pilot phase and full trial, aiming to robustly assess how the intervention is delivered. Cost-effectiveness analysis will also be performed. DISCUSSION: The BioDriveAFS trial aims to further the knowledge of strategies that will safely optimise AF use through comparison of the clinical and cost-effectiveness of a biomarker-led diagnostic strategy versus prophylactic AF to prevent and manage IFI within acute leukaemia. The evidence generated from the study will help inform global clinical practice and approaches within antifungal stewardship. TRIAL REGISTRATION: ISRCTN11633399. Registered 24/06/2022

A Double-Blind Randomized Phase I Clinical Trial Targeting ALVAC-HIV Vaccine to Human Dendritic Cells

BACKGROUND: We conducted a novel pilot study comparing different delivery routes of ALVAC-HIV (vCP205), a canarypox vaccine containing HIV gene inserts: env, gag and pol. We explored the concept that direct ex vivo targeting of human dendritic cells (DC) would enhance the immune response compared to either conventional intramuscular or intradermal injections of the vaccine alone. METHODOLOGY/PRINCIPAL FINDINGS: Healthy HIV-1 uninfected volunteers were administered ALVAC-HIV or placebo by intramuscular injection (i.m.), intradermal injection (i.d.) or subcutaneous injection (s.q.) of autologous ex vivo transfected DC at months 0, 1, 3 and 6. All vaccine delivery routes were well tolerated. Binding antibodies were observed to both the ALVAC vector and HIV-1 gp160 proteins. Modest cellular responses were observed in 2/7 individuals in the DC arm and 1/8 in the i.m. arm as determined by IFN-γ ELISPOT. Proliferative responses were most frequent in the DC arm where 4/7 individuals had measurable responses to multiple HIV-1 antigens. Loading DC after maturation resulted in lower gene expression, but overall better responses to both HIV-1 and control antigens, and were associated with better IL-2, TNF-α and IFN-γ production. CONCLUSIONS/SIGNIFICANCE: ALVAC-HIV delivered i.m., i.d. or s.q. with autologous ex vivo transfected DC proved to be safe. The DC arm was most immunogenic. Proliferative immune responses were readily detected with only modest cytotoxic CD8 T cell responses. Loading mature DC with the live viral vaccine induced stronger immune responses than loading immature DC, despite increased transgene expression with the latter approach. Volunteers who received the autologous vaccine loaded mature DC developed a broader and durable immune response compared to those vaccinated by conventional routes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00013572

Resources and Geographic Access to Care for Severe Pediatric Pneumonia in Four Resource-limited Settings

Rationale: Pneumonia is the leading cause of death in children worldwide. Identifying and appropriately managing severe pneumonia in a timely manner improves outcomes. Little is known about the readiness of healthcare facilities to manage severe pediatric pneumonia in low-resource settings. Objectives: As part of the HAPIN (Household Air Pollution Intervention Network) trial, we sought to identify healthcare facilities that were adequately resourced to manage severe pediatric pneumonia in Jalapa, Guatemala (J-GUA); Puno, Peru (P-PER); Kayonza, Rwanda (K-RWA); and Tamil Nadu, India (T-IND). We conducted a facility-based survey of available infrastructure, staff, equipment, and medical consumables. Facilities were georeferenced, and a road network analysis was performed. Measurements and Main Results: Of the 350 healthcare facilities surveyed, 13% had adequate resources to manage severe pneumonia, 37% had pulse oximeters, and 44% had supplemental oxygen. Mean (±SD) travel time to an adequately resourced facility was 41 ± 19 minutes in J-GUA, 99 ± 64 minutes in P-PER, 40 ± 19 minutes in K-RWA, and 31 ± 19 minutes in T-IND. Expanding pulse oximetry coverage to all facilities reduced travel time by 44% in J-GUA, 29% in P-PER, 29% in K-RWA, and 11% in T-IND (all P < 0.001). Conclusions: Most healthcare facilities in low-resource settings of the HAPIN study area were inadequately resourced to care for severe pediatric pneumonia. Early identification of cases and timely referral is paramount. The provision of pulse oximeters to all health facilities may be an effective approach to identify cases earlier and refer them for care and in a timely manner