BACKGROUND:We have previously reported that an early initiation of highly active antiretroviral therapy (HAART) in HIV-1 vertically infected children enhanced the function of memory B-cells gained ...during childhood routine vaccinations. On the other hand, a significant waning of immunity was observed for patients with a late treatment. In this follow-up study, we report data from a sample of patients in our cohort including late-treated patients being revaccinated with routine childhood vaccines.
METHODS:The levels of serum antibodies and cellular immunity were measured by antigen-specific enzyme-linked immunosorbent assay and B-cell ELISpot. Moreover, flow cytometry on the frequencies of mature-activated (CD10−CD21−) and double-negative (CD27–IgD–) B-cells as hallmarks of immune activation and immune senescence, respectively, was performed for all patients.
RESULTS:Reduced protective humoral immunity and cellular immunity to routine childhood vaccines was observed in late-treated patients. Moreover, we found that timing of HAART related with the frequencies of mature activated and double negative.
CONCLUSIONS:Altogether the data presented in this follow-up study reenforce the importance for an early start of HAART in HIV-1 vertically infected individuals and suggest that timing of HAART is a fundamental factor to take into account for vaccination design in this population.
Pseudomonas savastanoi pv. savastanoi is the causal agent of olive knot disease. The strains isolated from oleander and ash belong to the pathovars nerii and fraxini, respectively. When artificially ...inoculated, pv. savastanoi causes disease also on ash, and pv. nerii attacks also olive and ash. Surprisingly nothing is known yet about their distribution in nature on these hosts and if spontaneous cross-infections occur. On the other hand sanitary certification programs for olive plants, also including P. savastanoi, were launched in many countries. The aim of this work was to develop several PCR-based tools for the rapid, simultaneous, differential and quantitative detection of these P. savastanoi pathovars, in multiplex and in planta.
Specific PCR primers and probes for the pathovars savastanoi, nerii and fraxini of P. savastanoi were designed to be used in End Point and Real-Time PCR, both with SYBR Green or TaqMan chemistries. The specificity of all these assays was 100%, as assessed by testing forty-four P. savastanoi strains, belonging to the three pathovars and having different geographical origins. For comparison strains from the pathovars phaseolicola and glycinea of P. savastanoi and bacterial epiphytes from P. savastanoi host plants were also assayed, and all of them tested always negative. The analytical detection limits were about 5 - 0.5 pg of pure genomic DNA and about 102 genome equivalents per reaction. Similar analytical thresholds were achieved in Multiplex Real-Time PCR experiments, even on artificially inoculated olive plants.
Here for the first time a complex of PCR-based assays were developed for the simultaneous discrimination and detection of P. savastanoi pv. savastanoi, pv. nerii and pv. fraxini. These tests were shown to be highly reliable, pathovar-specific, sensitive, rapid and able to quantify these pathogens, both in multiplex reactions and in vivo. Compared with the other methods already available for P. savastanoi, the identification procedures here reported provide a versatile tool both for epidemiological and ecological studies on these pathovars, and for diagnostic procedures monitoring the asymptomatic presence of P. savastanoi on olive and oleander propagation materials.
Thromboxane-Dependent CD40 Ligand Release in Type 2 Diabetes Mellitus
Francesca Santilli, Giovanni Davì, Agostino Consoli, Francesco Cipollone, Andrea Mezzetti, Angela Falco, Tea Taraborelli, ...Eleonora Devangelio, Giovanni Ciabattoni, Stefania Basili, Carlo Patrono
we attempted to characterize the platelet contribution to soluble CD40 ligand (sCD40L) in type 2 diabetes (T2DM). Urinary 8-iso-prostaglandin (PG)F2αand 11-dehydro-thromboxane (TX)B2, plasma CD40L, and C-reactive protein (CRP) were measured in 114 diabetic patients and 114 control patients. A 17-day aspirin study was performed in 18 T2DM patients and 6 healthy volunteers. Twenty poorly controlled patients were re-evaluated after improved metabolic control. Diabetic patients showed higher 8-iso-PGF2α, 11-dehydro-TXB2, sCD40L, and CRP than control patients, with 11-dehydro-TXB2and 8-iso-PGF2αpredicting sCD40L. Diabetic patients and control patients showed significantly reduced sCD40L after seven days of aspirin. Improved metabolic control led to a reduction in sCD40L and urinary metabolites. Abnormal sCD40L release occurs during TXA2-dependent platelet activation in T2DM.
The goals of this study were to characterize the platelet contribution to soluble CD40 ligand (sCD40L), to correlate its formation with the extent of oxidative stress and platelet activation, and to investigate the effects of improved metabolic control and low-dose aspirin on these processes.
Inflammation, oxidative stress, and platelet activation are involved in the pathogenesis of type 2 diabetes (T2DM) and its complications. The CD40-CD40L interactions result in inflammatory and pro-thrombotic responses.
Urinary 8-iso-prostaglandin (PG)F2αand 11-dehydro-thromboxane (TX)B2, in vivo markers of oxidative stress and platelet activation, respectively, plasma CD40L, and C-reactive protein (CRP) were measured in 114 T2DM patients and 114 control patients. A randomized, parallel group, 17-day study of aspirin (30, 100, or 325 mg/day) was performed in 18 T2DM patients. A similar study was performed in six healthy volunteers (aspirin, 100 mg/day). Twenty poorly controlled T2DM patients were studied before and after improved metabolic control.
Compared with control patients, diabetic patients showed significantly higher levels of 8-iso-PGF2α, 11-dehydro-TXB2, sCD40L, and CRP. On multiple regression analysis, 11-dehydro-TXB2and 8-iso-PGF2αexcretion rates predicted sCD40L levels. Soluble CD40L linearly correlated with 11-dehydro-TXB2(rho = 0.67, p < 0.0001), and both were reduced after one week of aspirin (p < 0.0026), with slow recovery over 10 days after aspirin withdrawal. Improved metabolic control was associated with a reduction in sCD40L, 8-iso-PGF2α, and 11-dehydro-TXB2.
This study provides several lines of evidence for the dependence of sCD40L release on TXA2-dependent platelet activation in T2DM and provides novel mechanistic insight into the amplification loops of persistent platelet activation in this setting.
Insulin Resistance as a Determinant of Platelet Activation in Obese Women
Stefania Basili, Giovanni Pacini, Maria Teresa Guagnano, Maria Rosaria Manigrasso, Francesca Santilli, Caterina Pettinella, ...Giovanni Ciabattoni, Carlo Patrono, Giovanni Davì
Insulin resistance, per se, might contribute to increased platelet activation observed in obesity, independently of underlying inflammation. Forty obese women had significantly higher 11-dehydro-thromboxane B2(11-dehydro-TXB2) excretion, an index of platelet activation, C-reactive protein, and CD40 ligand levels and lower SI, an index of insulin sensitivity and adiponectin, than healthy controls. Waist-to-hip ratio and SIpredicted 11-dehydro-TXB2, independently of adiponectin, inflammatory, and lipid patterns. Improved insulin sensitivity achieved with either successful weight loss or pioglitazone treatment significantly decreased thromboxane biosynthesis in obese women. Thus, insulin resistance is a major determinant of platelet activation in female obesity.
We tested the hypothesis that insulin resistance, per se, contributes to increased platelet activation in obesity, independently of underlying inflammation.
Obesity, insulin resistance, and atherosclerosis are closely linked phenomena associated with low-grade inflammation. Obesity is associated with persistent platelet activation in otherwise healthy women.
We performed a cross-sectional study in 40 obese and 20 non-obese healthy women using urinary thromboxane metabolite excretion as a non-invasive index of platelet activation. An index of insulin sensitivity, SI, and plasma adiponectin, C-reactive protein (CRP), and CD40 ligand (CD40L) levels were measured.
Obese women had significantly (p < 0.0001) higher 11-dehydro-thromboxane B2(11-dehydro-TXB2) excretion (median 718 vs. 211 pg/mg creatinine), CRP (1.13 vs. 0.48 mg/l), and CD40L levels (4.45 vs. 0.90 ng/ml) than controls. Obese women had lower SI(median 2.51 vs. 5.0 104min−1/μU/ml, p < 0.002) and adiponectin (6.3 vs. 10 μg/ml, p < 0.01) than control subjects. On multiple regression analysis, waist-to-hip ratio (β = 0.27, p < 0.05) and SI(β = −0.72, p < 0.04) predicted 11-dehydro-TXB2excretion rate, independently of adiponectin, CRP, CD40L, and lipid patterns. In order to investigate the cause-effect relationship of these associations, we examined the effects of a 12-week weight loss program or a 3-week pioglitazone treatment on urinary 11-dehydro-TXB2in 10 women with impaired SIand visceral obesity. Successful weight loss (0.6 kg loss/week) achieved in 5 subjects was associated with increased SI(+92%) and decreased CD40L (−27%), CRP (−37%), and 11-dehydro-TXB2(−53%) (p < 0.05). Consistently, improvement of insulin sensitivity achieved with pioglitazone significantly decreased urinary 11-dehydro-TXB2excretion (−43%, p < 0.05) without changes in body weight.
Insulin resistance is a major determinant of platelet activation in female obesity.
We evaluated severe acute respiratory syndrome coronavirus 2 RNA clearance in 22 children. The estimation of positivity at day 14 was 52% for nasopharyngeal swab and 31% for stool samples. These data ...underline the significance of nasopharyngeal and stoolsample for detecting infected children. Additional studies are needed for transmissibility.
To investigate early events possibly related to the development of diabetic angiopathy, we examined whether 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha) formation, a marker of in vivo oxidant ...stress, is altered in different stages of type 1 diabetes (T1DM) and whether it correlates with the rate of thromboxane (TX) A2 biosynthesis, a marker of in vivo platelet activation. We also investigated the relationship between inflammatory markers and F2-isoprostane formation in this setting.
A cross-sectional study was performed in 23 insulin-treated patients aged <18 years with new-onset T1DM (<or=6 weeks, group A), matched for age and gender with 23 patients with stable disease (>1 year, group B). Urinary 8-iso-PGF2alpha and 11-dehydro-TXB2 were measured in all patients and in age- and gender-matched controls. Circulating interleukin-6 (IL-6), tumor necrosis factor-alpha, and C-reactive protein were also determined as markers of the inflammatory response. Fifteen of the 23 children in group A were reexamined after 12 months. Compared with either controls or group B, diabetic children in group A showed significantly higher levels of 8-iso-PGF2alpha, 11-dehydro-TXB2, IL-6, tumor necrosis factor-alpha, and C-reactive protein. Statistically significant correlations between IL-6 and both 8-iso-PGF2alpha (r=0.63, P<0.001) and 11-dehydro-TXB2 (r=0.51, P<0.01) were observed. The 15 patients reexamined after 1 year showed a significant reduction in lipid peroxidation and platelet activation (P<0.02 and P<0.001, respectively), consistent with reduced levels of IL-6 and tumor necrosis factor-alpha.
These results demonstrate that enhanced lipid peroxidation and platelet activation represent early events in T1DM that are possibly related to an acute inflammatory response. These noninvasive indexes may help in further examining T1DM pathophysiology and monitoring pharmacological interventions to interfere with disease development and progression.
The methylenetetrahydrofolate reductase (MTHFR) 677 C→T polymorphism may be associated with elevated total homocysteine (tHcy) levels, an independent risk factor for cardiovascular disease. It was ...the study objective to evaluate
in vivo
lipid peroxidation and platelet activation in carriers of the MTHFR 677 C→T polymorphism and in non-carriers, in relation to tHcy and folate levels. A cross-sectional comparison of urinary 8-iso-prostaglandin (PG)F2α and 11-dehydro-thromboxane (TX)B2 (markers of
in vivo
lipid peroxidation and platelet activation, respectively) was performed in 100 carriers and 100 non-carriers of the polymorphism. A methionine-loading test and folic acid supplementation were performed to investigate the causal relationship of the observed associations. Urinary 8-iso-PGF2α and 11-dehydro-TXB2 were higher in carriers with hyperhomocysteinaemia than in those without hyperhomocysteinaemia (p
Children with perinatally acquired HIV (PHIV) have special vaccination needs, as they make suboptimal immune responses. Here, we evaluated safety and immunogenicity of 2 doses of 4-component group B ...meningococcal vaccine in antiretroviral therapy-treated children with PHIV and healthy controls (HCs). Assessments included the standard human serum bactericidal antibody (hSBA) assay and measurement of IgG titers against capsular group B Neisseria meningitidis antigens (fHbp, NHBA, NadA). The B cell compartment and vaccine-induced antigen-specific (fHbp+) B cells were investigated by flow cytometry, and gene expression was investigated by multiplexed real-time PCR. A good safety and immunogenicity profile was shown in both groups; however, PHIV demonstrated a reduced immunogenicity compared with HCs. Additionally, PHIV showed a reduced frequency of fHbp+ and an altered B cell subset distribution, with higher fHbp+ frequency in activated memory and tissue-like memory B cells. Gene expression analyses on these cells revealed distinct mechanisms between PHIV and HC seroconverters. Overall, these data suggest that PHIV presents a diverse immune signature following vaccination. The impact of such perturbation on long-term maintenance of vaccine-induced immunity should be further evaluated in vulnerable populations, such as people with PHIV.
Therapeutic HIV immunization is intended to induce new HIV-specific cellular immune responses and to reduce viral load, possibly permitting extended periods without antiretroviral drugs. A multigene, ...multi-subtype A, B, C HIV-DNA vaccine (HIVIS) has been used in clinical trials in both children and adults with the aim of improving and broadening the infected individuals' immune responses. Despite the different country locations, different regimens and the necessary variations in assays performed, this is, to our knowledge, the first attempt to compare children's and adults' responses to a particular HIV vaccine. Ten vertically HIV-infected children aged 4-16 years were immunized during antiretroviral therapy (ART). Another ten children were blindly recruited as controls. Both groups continued their antiretroviral treatment during and after vaccinations. Twelve chronically HIV-infected adults were vaccinated, followed by repeated structured therapy interruptions (STI) of their antiretroviral treatment. The adult group included four controls, receiving placebo vaccinations. The HIV-DNA vaccine was generally well tolerated, and no serious adverse events were registered in any group. In the HIV-infected children, an increased specific immune response to Gag and RT proteins was detected by antigen-specific lymphoproliferation. Moreover, the frequency of HIV-specific CD8+ T-cell lymphocytes releasing perforin was significantly higher in the vaccinees than the controls. In the HIV-infected adults, increased CD8+ T-cell responses to Gag, RT and viral protease peptides were detected. No augmentation of HIV-specific lymphoproliferative responses were detected in adults after vaccination. In conclusion, the HIV-DNA vaccine can elicit new HIV-specific cellular immune responses, particularly to Gag antigens, in both HIV-infected children and adults. Vaccinated children mounted transient new HIV-specific immune responses, including both CD4+ T-cell lymphoproliferation and late CD8+ T-cell responses. In the adult cohort, primarily CD8+ T-cell responses related to MHC class I alleles were noted. However, no clinical benefits with respect to viral load reduction were ascribable to the vaccinations alone. No severe adverse effects related to the vaccine were found in either cohort, and no virological failures or drug resistances were detected.
Abstract The PEDVAC study is the first trial designed to analyze safety and immunogenicity of a therapeutic vaccination with a multiclade multigene HIV DNA vaccine (HIVIS) in infected children. ...Twenty HIV-1 vertically infected children (6–16 years of age), on stable antiretroviral treatment for at least 6 months with HIV-1 RNA < 50 copies/ml and stable CD4 counts (>400 cells/mm3 or 25%) over 12 months of follow-up, were recruited into the study. Enrolled patients have been randomized into two arms: a control group of 10 children who continued previous antiretroviral treatment (HAART) (arm A) and a group of 10 children immunized intramuscularly with the HIVIS DNA vaccine in addition to previous HAART (arm B). Immunizations took place at week 0, 4, 12 and the boosting dose is planned at week 36. The 10 children in the vaccine group have received the first 3 priming doses of the HIVIS vaccine. Safety data showed good tolerance to the vaccination schedule. Mild cutaneous self-limeted reactions consisted of local irritation, usually itching or erythema +/− swelling at the injection site, were reported. No severe systemic adverse events have been observed. No vaccinated children had a decrease of CD4 T-cell counts from baseline. None experienced virological failure. Analysis of cellular immune responses was scheduled at week 0, 4, 12, 16, 20, 40, 60, 72 and 96 by standard lymphoproliferation assay, intracellular cytokine staining and cell-ELISA, a miniaturized assay to measure antigen-induced IFNγ secretion. Evaluation of these results is in progress and will provide key information on the status and changes of antigen specific immunity during HIV DNA immunization.
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