Recovery from mild Escherichia coli O157:H7 infection in young and aged C57BL/6 mice with intact flora estimated by fecal shedding, locomotor activity and grip strength.

Escherichia coli 0157:H7 is a food-borne pathogen that can cause severe complications in vulnerable populations. Mouse infection models of E. coli 0157:H7 are usually developed under severe animal suffering classification by depleting the normal flora, in which age plays a role. OBJECTIVE: To develop a refined method for longitudinal monitoring of E. coli 0157:H7 in young and old mice with intact flora. METHODS: We applied discriminant analysis and computed composite standardized scores from 19 variables obtained from physiological parameters, analysis of locomotor activity, grip strength measurement and fecal shedding in 16 aged and 16 young C57BL/6 mice after two mild oral challenges of E. coli 0157:H7. The resulting scores were validated in another experiment performed in 24 aged and 24 young mice including a group (8 aged and 8 young mice) treated with oxytetracycline. RESULTS: We show that our scores are significantly affected in the post-infection period and that can be used to measure and compare the recovery time after a treatment. The scores are most sensitive when separately developed in young and aged mice. CONCLUSIONS: We developed a method that minimizes the level of animal suffering and that can be applied in preclinical testing of new therapies.

Innovative design for a phase 1 trial with intra-patient dose escalation: The Crotoxin study.

INTRODUCTION: Crotoxin has a broad antitumor activity but has shown frequent neurotoxic toxicity. To induce tolerance and limit this toxicity, we propose a new design with intra-patient dose escalation. METHODS: A new Dose Limiting Toxicity definition was used. The concept of Target Ceiling Dose was introduced. RESULTS: Dose Limiting Toxicity was the inability to dose escalate twice. Target Ceiling Dose was the highest planned dose to be administered to a patient and could change for patients along time. Recommended Dose was defined similarly as in a (3 + 3) conventional design. CONCLUSION: This innovant design was used and the clinical trial is now closed for inclusions. Results will be presented later.

Interactions between Hepatitis C Virus and the Human Apolipoprotein H Acute Phase Protein: A Tool for a Sensitive Detection of the Virus.

The Hepatitis C virus (HCV) infection exhibits a high global prevalence frequently associated with hepatocellular carcinoma, taking years to develop. Despite the standardization of highly sensitive HCV quantitative RT-PCR (qRT-PCR) detection methods, false-negative diagnoses may be generated with current methods, mainly due to the presence of PCR inhibitors and/or low viral loads in the patient's sample. These false-negative diagnoses impact both public health systems, in developing countries, and an in lesser extent, in developed countries, including both the risk of virus transmission during organ transplantation and/or blood transfusion and the quality of the antiviral treatment monitoring. To adopt an appropriate therapeutic strategy to improve the patient's prognosis, it is urgent to increase the HCV detection sensitivity. Based upon previous studies on HBV, we worked on the capacity of the scavenger acute phase protein, Apolipoprotein H (ApoH) to interact with HCV. Using different approaches, including immunoassays, antibody-inhibition, oxidation, ultracentrifugation, electron microscopy and RT-PCR analyses, we demonstrated specific interactions between HCV particles and ApoH. Moreover, when using a two-step HCV detection process, including capture of HCV by ApoH-coated nanomagnetic beads and a home-made real-time HCV-RT-PCR, we confirmed the presence of HCV for all samples from a clinical collection of HCV-seropositive patients exhibiting an RT-PCR COBAS® TaqMan® HCV Test, v2.0 (COBAS)-positive result. In contrast, for HCV-seropositive patients with either low HCV-load as determined with COBAS or exhibiting HCV-negative COBAS results, the addition of the two-step ApoH-HCV-capture and HCV-detection process was able to increase the sensitivity of HCV detection or more interestingly, detect in a genotype sequence-independent manner, a high-proportion (44%) of HCV/RNA-positive among the COBAS HCV-negative patients. Thus, the immune interaction between ApoH and HCV could be used as a sample preparation tool to enrich and/or cleanse HCV patient's samples to enhance the detection sensitivity of HCV and therefore significantly reduce the numbers of false-negative HCV diagnosis results.

Leukocyte Telomere Length in Alzheimer's Disease Patients with a Different Rate of Progression.

BACKGROUND: Age and short leukocyte telomeres have been associated with a higher risk of Alzheimer's disease (AD). Inflammation is involved in AD and it is suggested that anti-inflammatory interleukin-10 (IL-10) may partly antagonize these processes. OBJECTIVE: The aim is to correlate telomere length (TL) in peripheral blood mononuclear cells (PBMC) from patients with AD to disease progression rate. Moreover, we evaluated whether TL was associated with IL-10 production by unstimulated or amyloid-ß (Aß)-stimulated PBMC. METHODS: We enrolled 31 late-onset AD and 20 age-matched healthy elderly (HE). After a two-year follow-up period, patients were retrospectively evaluated as slow-progressing (ADS) (Mini Mental State Examination (MMSE) decline over the two years of follow-up ≤3 points) or fast progressing AD (ADF) (MMSE decline ≥5 points). TL was measured by flow cytometry and in vitro IL-10 production by enzyme-linked immunosorbent assay. RESULTS: TL (mean±SD) for HE, ADS, and ADF was 2.3±0.1, 2.0±0.1, and 2.5±0.1 Kb, respectively. ADS showed a shorter TL compared to HE (p = 0.034) and to ADF (p = 0.005). MMSE decline correlated with TL in AD (R2 = 0.284; p = 0.008). We found a significant difference in IL-10 production between unstimulated and Aß-stimulated PBMC from ADS (40.7±13.7 versus 59.0±27.0; p = 0.004) but not from ADF (39.7±14.4 versus 42.2±22.4). HE showed a trend toward significance (47.1±25.4 versus 55.3±27.9; p = 0.10). CONCLUSION: PBMC from ADF may be characterized by an impaired response induced by Aß and by a reduced proliferative response responsible for the longer telomeres. TL might be a contributing factor in predicting the rate of AD progression.

Interleukin-10 Production in Response to Amyloid-ß Differs between Slow and Fast Decliners in Patients with Alzheimer's Disease.

We investigated IL-10 and IL-6 production in amyloid-ß (Aß) stimulated peripheral blood mononuclear cells (PBMCs) in twenty Alzheimer's disease (AD) patients with slow progression, eleven with fast progression, and twenty age-matched controls. Promoter polymorphisms in IL-10 (position -592, -819, -1082), IL-6 (-174), transforming growth factor-ß1 (TGF-ß1) (-10, -25), interferon-γ (IFN-γ) (-874), and tumor necrosis factor-α (TNF-α) (-308) genes were analyzed. IL-10 production after Aß stimulation was high in PBMCs from slow decliners and almost completely abrogated in fast decliners. Association between AA IFN-γ low-producing genotype and fast progression was demonstrated. Investigations in a larger sample will clarify these findings.

Treatment of periodontal disease results in improvements in endothelial dysfunction and reduction of the carotid intima-media thickness.

Several cohort studies reported a relation of cardiovascular events and periodontal disease. In particular, Porphyromonas gingivalis is associated with the development of atherosclerotic plaques. We verified in a longitudinal study whether inflammation biomarkers, endothelial adhesion molecules, leukocyte activation markers, and intima-media thickness could be beneficially modified by periodontal treatment alone. Thirty-five otherwise healthy individuals affected by mild to moderate parodontopathy were enrolled in the study. Echo-Doppler cardiography of the carotid artery, fluorescence-activated cell sorting analyses on lymphocytes and monocytes, and plasma inflammatory indices were evaluated at baseline and at multiple time points after the periodontal treatment. Results showed that inflammation biomarkers were abnormally increased at baseline. Periodontal treatment resulted in a significant reduction of the total oral bacterial load that was associated with a significant amelioration of inflammation biomarkers and of adhesion and activation proteins. Notably, intima-media thickness was significantly diminished after treatment. Inflammatory alterations associated with the genesis of atherosclerotic plaques are detected in otherwise healthy individuals affected by parodontopathy and are positively influenced by periodontal treatment. Reduction of oral bacterial load results in a modification of an anatomical parameter directly responsible for atherosclerosis. These results shed light on the pathogenesis of atherosclerosis and could have practical implications for public health.

The mucosae-associated epithelial chemokine (MEC/CCL28) modulates immunity in HIV infection.

BACKGROUND: CCL28 (MEC) binds to CCR3 and CCR10 and recruits IgA-secreting plasma cells (IgA-ASC) in the mucosal lamina propria (MLP). Mucosal HIV-specific IgA are detected in HIV-infection and exposure. The CCL28 circuit was analyzed in HIV-infected and-exposed individuals and in HIV-unexposed controls; the effect of CCL28 administration on gastrointestinal MLP IgA-ASC was verified in a mouse model. METHODOLOGY/FINDINGS: CCL28 was augmented in breast milk (BM) plasma and saliva of HIV-infected and -exposed individuals; CCR3+ and CCR10+ B lymphocytes were increased in these same individuals. Additionally: 1) CCL28 concentration in BM was associated with longer survival in HIV vertically-infected children; and 2) gastro-intestinal mucosal IgA-ASC were significantly increased in VSV-immunized mice receiving CCL28. CONCLUSIONS: CCL28 mediates mucosal immunity in HIV exposure and infection. CCL28-including constructs should be considered in mucosal vaccines to prevent HIV infection of the gastro-intestinal MLP via modulation of IgA-ASC.

Dengue-virus-infected dendritic cells trigger vascular leakage through metalloproteinase overproduction.

Dengue virus (DV) is an important re-emerging arthropod-borne virus of global significance. The defining characteristic of DV infection-associated pathology is haemorrhagic fever, which often leads to a fatal shock-like syndrome (DHF/DSS) owing to an increase in vascular endothelial permeability. Here, we show, in a viral dose-dependent manner, that DV-infected immature dendritic cells overproduce soluble gelatinolytic matrix metalloproteinase (MMP)-9-and to a lesser extent MMP-2-which enhances endothelial permeability, but which are reduced by specific inhibitors and a neutralizing anti-MMP-9 antibody. This permeability was associated with a loss of expression of the platelet endothelial adhesion molecule 1 (PECAM-1) and vascular endothelium (VE)-cadherin cell adhesion molecules and redistribution of F-actin fibres. These in vitro observations were confirmed in an in vivo vascular-leakage mouse model. These results provide a molecular basis for DHF/DSS that could be a basis for a general model of haemorrhagic fever-inducing viruses, and identify a new therapeutic approach for the treatment of viral-induced vascular leakage by specifically targeting gelatinolytic metalloproteases.

Immunization with gp120-depleted whole killed HIV immunogen and a second-generation CpG DNA elicits strong HIV-specific responses in mice.

HIV-1 Immunogen is a gp120-depleted whole killed virus vaccine candidate formulated with Incomplete Freund's Adjuvant (HIV-IFA). We evaluated in a mouse model the immunogenicity of HIV-IFA by itself and when combined with HYB2055, an immunomodulatory oligonucleotide consisting of a novel DNA structure and synthetic CpR immunostimulatory motif, as an adjuvant. C57/BL6 mice were immunized with HIV-IFA alone or combined with HYB2055. Mice treated with HYB2055 or with PBS were used as controls. Compared to HIV-IFA alone, immunization with HIV-IFA and HYB2055 combination elicited strong production of HIV- and p24-specific IFNgamma, RANTES, MIP 1alpha, and MIP 1beta, as well as high titers of HIV- and p24-specific antibodies. Inclusion of HYB2055 also reduced levels of IL-5 produced by HIV-IFA alone. HYB2055 enhances the immunogenicity of HIV-IFA and shifts responses towards a type 1 cytokine profile. The immune enhancing effects of HYB2055 adjuvant were dose-dependent. These findings warrant clinical evaluation of the HIV-1 immunogen/HYB2055 candidate as a therapeutic vaccine for HIV-1 infected patients.

Immunomodulation induced by tucaresol in HIV infection: results of a 16 week pilot Phase I/II trial.

OBJECTIVE: Immune reconstitution in highly active antiretroviral therapy (HAART)-treated individuals is incomplete and immunomodulatory compounds are needed to improve the outcome of HIV therapy. In a Phase I/II clinical trial performed on HIV-positive patients we analysed the safety and immunomodulating effects of tucaresol, a novel compound that has previously been described tn enhance cell-mediated immune responses. PATIENTS AND METHODS: Sixteen weeks pulse dose escalation protocol. Four groups of HIV-positive patients were enrolled: group A (n=6): HAART, CD4+ 300-500 cells/microl, HIV RNA <80 copies/ml; group B (n=6): HAART-naive, CD4+ <500 cells/microl, HIV RNA >10 000 copies/ml; group C (n=3): HAART-naive, CD4+ >500 cells/microl, HIV RNA <10000 copies/ml; and group D (n=6): HAART, CD4+ <200cells/microl, HIV RNA <80 copies/ml. Tucaresol was added to HAART in group A and D patients; group B patients started tucaresol with HAART, group C patients received tucaresol alone. Clinical and immunological analyses were performed at different time points. RESULTS: Tucaresol-related serious adverse events were observed in the first week of therapy in 2/21 patients who were viraemic when commencing treatment, but did not occur in patients on stable HAART. Tucaresol did not affect HIV viraemia whereas increases in CD4+ percentages, mainly supported by naive CD4+ cells, were observed. CD8+/28-/45RA+ cells and HIV-specific CD8+ IFNgamma- and perforin-producing cells improved whereas IL-10 mRNA diminished in tucaresol-treated patients. The effects were greater with 25 mg given every other day for 1 week. CONCLUSION: In HAART-receiving patients with proper virus suppression, tucaresol was not associated with serious adverse events and resulted in qualitative and quantitative stimulation of HIV-specific cytotoxic T lymphocyte activity and generation of naive T cells. These data may support further exploration of tucaresol use in reconstitution of immune system parameters in HIV patients with proper virus suppression while on HAART.

Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: 18-month follow-up of the HIVNET 012 randomised trial.

BACKGROUND: In 1999, we reported safety and efficacy data for short-course nevirapine from a Ugandan perinatal HIV-1 prevention trial when 496 babies were followed up to age 14-16 weeks. Safety and efficacy data are now presented for all babies followed up to 18 months of age. METHODS: From November, 1997, to April, 1999, HIV-1 infected pregnant women in Kampala, Uganda, were randomly assigned nevirapine (200 mg at labour onset and 2mg/kg for babies within 72 h of birth; regimen A) or zidovudine (600 mg orally at labour onset and 300 mg every 3 h until delivery, and 4 mg/kg orally twice daily for babies for 7 days, regimenB). Infant HIV-1 testing was done at birth, age 6-8 and 14-16 weeks, and age 12 months by HIV-1 RNA PCR, and by HIV-1 antibody at 18 months. HIV-1 transmission and HIV-1-free survival were assessed using Kaplan-Meier analysis. We recorded adverse experiences through 6-8 weeks postpartum for mothers, and 18 months for babies. Efficacy analyses were by intention to treat. FINDINGS: We enrolled 645 mothers to the study: 313 were assigned regimen A, 313 regimen B, and 19 placebo. Eight mothers were lost to follow-up before delivery. 99% of babies were breastfed (median duration 9 months). Estimated risks of HIV-1 transmission in the zidovudine and nevirapine groups were 10.3% and 8.1% at birth (p=0.35); 20.0% and 11.8% by age 6-8 weeks (p=0.0063); 22.1% and 13.5% by age 14-16 weeks (p=0.0064); and 25.8% and 15.7% by age 18 months (p=0.0023). Nevirapine was associated with a 41% (95% CI 16-59) reduction in relative risk of transmission through to age 18 months. Both regimens were well-tolerated with few serious side-effects. INTERPRETATION: Intrapartum/neonatal nevirapine significantly lowered HIV-1 transmission risk in a breastfeeding population in Uganda compared with a short intrapartum/neonatal zidovudine regimen. The absolute 8.2% reduction in transmission at 6-8 weeks was sustained at age 18 months (10.1% [95% CI 3.5-16.6]). This simple, inexpensive, well-tolerated regimen has the potential to significantly decrease HIV-1 perinatal transmission in less-developed countries.