Baseline malaria prevalence and care-seeking behaviours in rural Madagascar prior to a trial to expand malaria community case management to all ages.

BACKGROUND: Integrated community case management of malaria, pneumonia, and diarrhoea can reduce mortality in children under five years (CU5) in resource-poor countries. There is growing interest in expanding malaria community case management (mCCM) to older individuals, but limited empirical evidence exists to guide this expansion. As part of a two-year cluster-randomized trial of mCCM expansion to all ages in southeastern Madagascar, a cross-sectional survey was conducted to assess baseline malaria prevalence and healthcare-seeking behaviours. METHODS: Two enumeration areas (EAs) were randomly chosen from each catchment area of the 30 health facilities (HFs) in Farafangana district designated for the mCCM age expansion trial; 28 households were randomly selected from each EA for the survey. All household members were asked about recent illness and care-seeking, and malaria prevalence was assessed by rapid diagnostic test (RDT) among children < 15 years of age. Weighted population estimates and Rao-Scott chi-squared tests were used to examine illness, care-seeking, malaria case management, and malaria prevalence patterns. RESULTS: Illness in the two weeks prior to the survey was reported by 459 (6.7%) of 8050 respondents in 334 of 1458 households surveyed. Most individuals noting illness (375/459; 82.3%) reported fever. Of those reporting fever, 28.7% (112/375) sought care; this did not vary by participant age (p = 0.66). Most participants seeking care for fever visited public HFs (48/112, 46.8%), or community healthcare volunteers (CHVs) (40/112, 31.0%). Of those presenting with fever at HFs or to CHVs, 87.0% and 71.0%, respectively, reported being tested for malaria. RDT positivity among 3,316 tested children < 15 years was 25.4% (CI: 21.5-29.4%) and increased with age: 16.9% in CU5 versus 31.8% in 5-14-year-olds (p < 0.0001). Among RDT-positive individuals, 28.4% of CU5 and 18.5% of 5-14-year-olds reported fever in the two weeks prior to survey (p = 0.044). CONCLUSIONS: The higher prevalence of malaria among older individuals coupled with high rates of malaria testing for those who sought care at CHVs suggest that expanding mCCM to older individuals may substantially increase the number of infected individuals with improved access to care, which could have additional favorable effects on malaria transmission.

Combined evaluation of LC3B puncta and HMGB1 expression predicts residual risk of relapse after adjuvant chemotherapy in breast cancer.

In spite of adjuvant chemotherapy, a significant fraction of patients with localized breast cancer (BC) relapse after optimal treatment. We determined the occurrence of cytoplasmic MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3B)-positive puncta, as well as the presence of nuclear HMGB1 (high mobility group box 1) in cancer cells within surgical BC specimens by immunohistochemistry, first in a test cohort (152 patients) and then in a validation cohort of localized BC patients who all received adjuvant anthracycline-based chemotherapy (1646 patients). Cytoplasmic LC3B(+) puncta inversely correlated with the intensity of SQSTM1 staining, suggesting that a high percentage cells of LC3B(+) puncta reflects increased autophagic flux. After setting optimal thresholds in the test cohort, cytoplasmic LC3B(+) puncta and nuclear HMGB1 were scored as positive in 27.2% and 28.6% of the tumors, respectively, in the validation cohort, while 8.7% were considered as double positive. LC3B(+) puncta or HMGB1 expression alone did not constitute independent prognostic factors for metastasis-free survival (MFS) in multivariate analyses. However, the combined positivity for LC3B(+) puncta and nuclear HMGB1 constituted an independent prognostic factor significantly associated with prolonged MFS (hazard ratio: 0.49 95% confidence interval [0.26-0.89]; P = 0.02), and improved breast cancer specific survival (hazard ratio: 0.21 95% confidence interval [0.05-0.85]; P = 0.029). Subgroup analyses revealed that within patients with poor-prognosis BC, HMGB1(+) LC3B(+) double-positive tumors had a better prognosis than BC that lacked one or both of these markers. Altogether, these results suggest that the combined positivity for LC3B(+) puncta and nuclear HMGB1 is a positive predictor for longer BC survival.

Clinical impact of the NKp30/B7-H6 axis in high-risk neuroblastoma patients.

The immunosurveillance mechanisms governing high-risk neuroblastoma (HR-NB), a major pediatric malignancy, have been elusive. We identify a potential role for natural killer (NK) cells, in particular the interaction between the NK receptor NKp30 and its ligand, B7-H6, in the metastatic progression and survival of HR-NB after myeloablative multimodal chemotherapy and stem cell transplantation. NB cells expressing the NKp30 ligand B7-H6 stimulated NK cells in an NKp30-dependent manner. Serum concentration of soluble B7-H6 correlated with the down-regulation of NKp30, bone marrow metastases, and chemoresistance, and soluble B7-H6 contained in the serum of HR-NB patients inhibited NK cell functions in vitro. The expression of distinct NKp30 isoforms affecting the polarization of NK cell functions correlated with 10-year event-free survival in three independent cohorts of HR-NB in remission from metastases after induction chemotherapy (n = 196, P < 0.001), adding prognostic value to known risk factors such as N-Myc amplification and age >18 months. We conclude that the interaction between NKp30 and B7-H6 may contribute to the fate of NB patients and that both the expression of NKp30 isoforms on circulating NK cells and the concentration of soluble B7-H6 in the serum may be clinically useful as biomarkers for risk stratification.

Antitumor immunity triggered by melphalan is potentiated by melanoma cell surface-associated calreticulin.

Systemic chemotherapy generally has been considered immunosuppressive, but it has become evident that certain chemotherapeutic drugs elicit immunogenic danger signals in dying cancer cells that can incite protective antitumor immunity. In this study, we investigated whether locoregionally applied therapies, such as melphalan, used in limb perfusion for melanoma (Mel-ILP) produce related immunogenic effects. In human melanoma biopsies, Mel-ILP treatment upregulated IL1B, IL8, and IL6 associated with their release in patients' locoregional sera. Although induction of apoptosis in melanoma cells by melphalan in vitro did not elicit threshold levels of endoplasmic reticulum and reactive oxygen species stress associated with danger signals, such as induction of cell-surface calreticulin, prophylactic immunization and T-cell depletion experiments showed that melphalan administration in vivo could stimulate a CD8(+) T cell-dependent protective antitumor response. Interestingly, the vaccination effect was potentiated in combination with exogenous calreticulin, but not tumor necrosis factor, a cytokine often combined with Mel-ILP. Our results illustrate how melphalan triggers inflammatory cell death that can be leveraged by immunomodulators such as the danger signal calreticulin.

Viral envelope is a major determinant of enhanced fitness of a multidrug-resistant HIV-1 variant.

Multidrug-resistant (MDR) HIV-1 viruses are thought to be less pathogenic than wild-type viruses because of the fitness costs of drug-resistance mutations. However, we identified an individual infected with MDR virus associated with rapid disease progression referred to as MDR-1. To study the contribution of virologic factors to rapid disease progression, we constructed molecular clones that demonstrated high replication fitness and cytopathicity. To dissect determinants of enhanced fitness of a cytopathic clone, pMDR-1c, we divided its genome into 2 parts: the envelope (gp160) and the remaining backbone genome, and constructed mutual chimeric viruses with a reference, wild-type virus clone, pNL4-3. The growth competition assay indicated that pMDR-1c has high fitness (1.62), although its envelope confers remarkably enhanced fitness (2.29) and its backbone confers reduced fitness (0.56) as compared with pNL4-3. We also performed a similar study with a less cytopathic pMDR-5a, a molecular clone derived from another subject MDR-5, infected with MDR HIV-1, and associated with slower clinical progression. The results indicated that pMDR-5a has reduced fitness (0.82), although its envelope confers enhanced fitness (1.64) and its backbone confers reduced fitness (0.49), a fitness pattern compatible with envelope-mediated fitness compensation. These results suggest that the viral envelope may be a major determinant of the enhanced fitness of the MDR HIV-1 variant isolated from a patient with rapid disease progression. Furthermore, we speculate that compensation conferred by envelope may be a mechanism by which MDR HIV-1 maintains overall fitness despite the presence of changes in pol, which reduce replication capacity.

A randomized open-label study of 3- versus 5-drug combination antiretroviral therapy in newly HIV-1-infected individuals.

BACKGROUND: To understand whether combination antiretroviral therapy (cART) has been optimized, we asked whether 3-drug protease inhibitor (PI)-based cART intensified with raltegravir and maraviroc and initiated during early infection would improve outcomes when compared with similarly applied 3-drug PI-based cART. METHODS: Forty newly HIV-1-infected patients were randomized 1:2 to receive 3-drug (N = 14) or 5-drug (N = 26) therapy. The primary end point was the percent of subjects with undetectable plasma viremia using standard reverse transcriptase-polymerase chain reaction and the single copy assay after 48 weeks. Secondary end points included levels of cell-associated HIV-1 DNA and RNA and levels of infectious virus in resting CD4 T cells at week 96 and quantitative and qualitative immunologic responses. RESULTS: At 48 weeks, 34 subjects remained on study and are included in the as-treated analysis. Three of 11 (27.3%) in the 3-drug arm and 9 of 21 (42.9%) in the 5-drug arm had plasma HIV-1 RNA levels below detection by both standard reverse transcriptase-polymerase chain reaction and single copy assay (P = 0.46, Fisher exact test). No significant differences in absolute levels of proviral DNA or changes in cell-associated RNA were seen during 96 weeks of therapy. Mean levels of infectious HIV-1 in resting CD4 T cells at week 96 in 7 subjects treated with 3-drugs and 13 with 5-drugs were 0.67 and 0.71 infectious units per million, respectively (P = 0.81). No differences were seen in quantitative or qualitative immunologic determinations including markers of immune activation. CONCLUSIONS: Intensified 5-drug cART initiated during early infection fails to significantly further impact virologic or immunologic responses beyond those achieved with standard 3-drug PI-based cART.

Analysis of NKp30/NCR3 isoforms in untreated HIV-1-infected patients from the ANRS SEROCO cohort.

Natural killer (NK) cells play a prominent role at the intersection between innate and cognate immunity, thus influencing the development of multiple pathological conditions including HIV-1-induced AIDS. Not only NK cells directly kill HIV-1-infected cells, but also control the maturation and/or elimination of dendritic cells (DCs). These functions are regulated by the delicate balance between activating and inhibiting receptors expressed at the NK-cell surface. Among the former, NKp30 has raised significant interest since the alternative splicing of its intracellular domain leads to differential effector functions, dictating the prognosis of patients bearing gastrointestinal sarcoma, and B7-H6 has recently been identified as its main ligand. Since NKp30 is downregulated in CD56-/CD16+ NK cells expanded in viremic, chronically infected HIV-1+ patients, we decided to investigate the predictive value of NKp30 splice variants for spontaneous disease progression in 89 therapy-naïve HIV-1-infected individuals enrolled in an historical cohort of patients followed since diagnosis (ANRS SEROCO cohort). We found no difference in the representation of NK-cell subsets (CD56bright, CD56dim, CD56neg) in HIV-1-infected patients as compared with healthy subjects. NKp30 downregulation was detected in CD56dim and CD56neg NK-cell subsets, yet this did not convey any prognostic value. None of the NKp30 isoforms did affect disease progression, as measured in terms of time-to-loss of circulating CD4+ T cells, time-to-AIDS-defining events and overall survival. NKp30 isoforms do not seem to play a major role in the outcome of HIV-1 infection, but the heterogeneity of the immuno-virological status of patients at enrollment could have to be taken into account.

Novel integrase inhibitors for HIV.

IMPORTANCE OF THE FIELD: Integrase inhibitors are the newest class of antiretroviral agents developed to treat HIV-1 infection. Raltegravir (RAL), the only integrase inhibitor (INI) currently approved for the treatment of HIV-infected patients, has proven to be a potent and well-tolerated antiretroviral (ARV) agent. It is currently approved and used for the treatment of both ARV-experienced and ARV-naive patients. Nevertheless, the relatively low genetic barrier for resistance of RAL encourages the search for new INIs with different mechanisms of actions and resistance profiles. AREAS COVERED IN THIS REVIEW: Here we review the data available about INI that are currently being tested in clinical trials or are in preclinical development: elvitegravir (EVG), S/GSK1349572, S/GSK1265744 and LEDGINs. We focus on their clinical efficacy, pharmacokinetic, safety and resistance profiles. WHAT THE READER WILL GAIN: Up-to-date overview on the currently available, clinically relevant INIs and promising preclinical inhibitors at all phases of development. TAKE HOME MESSAGE: Integrase inhibitors represent the newest therapeutic class available to treat HIV-1 infection. There are a variety of compounds either available in the clinic (RAL), advancing to Phase III trials (EVG), or in earlier phases of development. Taken together, this class offers new treatment options for the HIV-infected individual.

Natural polymorphisms of human immunodeficiency virus type 1 integrase and inherent susceptibilities to a panel of integrase inhibitors.

We evaluated the human immunodeficiency virus type 1 (HIV-1) integrase coding region of the pol gene for the presence of natural polymorphisms in patients during early infection (AHI) and with triple-class drug-resistant HIV-1 (MDR). We analyzed selected recombinant viruses containing patient-derived HIV-1 integrase for susceptibility to a panel of strand transfer integrase inhibitors (InSTI). A pretreatment sequence analysis of the integrase coding region was performed for 112 patients identified during acute or early infection and 15 patients with triple-class resistance. A phenotypic analysis was done on 10 recombinant viruses derived from nine patients against a panel of six diverse InSTI. Few of the polymorphisms associated with in vitro InSTI resistance were identified in the samples from newly infected individuals or those patients with MDR HIV-1. We identified polymorphisms V72I, L74I, T97A, V151I, M154I/L, E157Q, V165I, V201I, I203M, T206S, and S230N. V72I was the most common, seen in 63 (56.3%) of the AHI samples. E157Q was the only naturally occurring mutation thought to contribute to resistance to elvitegravir, raltegravir, and L-870,810. None of the patient-derived viruses demonstrated any significant decrease in susceptibility to the drugs tested. In summary, the integrase coding region contains as much natural variation as that seen in protease, but mutations associated with high-level resistance to existing InSTI are rarely, if ever, present in integrase naïve patients, especially those being used clinically. Most of the highly prevalent polymorphisms have little effect on InSTI susceptibility in the absence of specific primary mutations. Baseline testing for integrase susceptibility in InSTI-naïve patients is not currently warranted.

Drug-susceptible HIV-1 infection despite intermittent fixed-dose combination tenofovir/emtricitabine as prophylaxis is associated with low-level viremia, delayed seroconversion, and an attenuated clinical course.

BACKGROUND: Continued high rates of HIV-1 transmission have fueled interest in the use of antiretrovirals to prevent infection. Attenuated infection with failure of tenofovir as prophylaxis has been reported in animal models. Here, we report a case of HIV-1 infection despite intermittent use of fixed-dose combination tenofovir and emtricitabine (FTC). METHODS: The patient was treated with tenofovir DF/FTC for reported repeated high-risk sexual exposures. After seroconversion, he was subjected to routine laboratory testing, CCR5 and HLA genotyping, and biopsy of gastrointestinal (GI) tissue. Resistance testing was performed both as bulk sequencing of plasma and cloning and sequencing of virus derived from plasma, peripheral blood mononuclear cells, and GI tissue. RESULTS: In this patient with no readily identifiable modifying host factors, acute HIV-1 infection with tenofovir DF/FTC-susceptible HIV-1 was associated with an attenuated clinical course, very low postseroconversion HIV-1 RNA levels, slow kinetics of seroconversion, and relative sparing of mucosal CD4+ T cells in the GI tract. CONCLUSIONS: Despite the failure of tenofovir DF/FTC as prophylaxis, selection for drug-resistant transmission did not occur and the blunting of postinfection levels of viremia likely reduced the probability of subsequent forward transmissions during the acute phase. These results support continued investigations of the use of antiretrovirals as a means to reduce HIV-1 transmission.

Homeostatic cytokines and expansion of regulatory T cells accompany thymic impairment in children with Down syndrome.

Down syndrome (DS), the most common chromosomal abnormality in humans, is characterized by precocious immunologic aging that results, among other things, in alterations of B and T lymphocyte subsets and natural killer cells, defective phagocytosis, and chemotaxis of polymorphonuclear leukocytes. We studied 30 children affected by DS, compared them to 29 healthy controls, and evaluated the functionality of the thymus (by measuring the amount of lymphocytes that express the signal-joint T cell receptor rearrangement excision circles [sj-TREC+]), the plasma levels of interleukin (IL)-7 and IL-15, the proliferative T cell response to these cytokines, the expression of the alpha chain of the IL-7 receptor (CD127), the extrathymic differentiation of T lymphocytes, and the presence of natural regulatory T cells (Tregs) in peripheral blood. We found that DS children had a significantly lower number of sj-TREC+ lymphocytes, the levels of which were strongly correlated with age. We found higher plasma levels of IL-7 and IL-15 than in healthy controls, and a higher proliferative T cell response to IL-15. DS children also showed a lower percentage of CD4(+) cells and profound alterations of T cell differentiation, along with increased amount of Tregs and of cells expressing markers of apoptosis. We can thus hypothesize that the precocious thymic involution occurring in DS is mirrored by a high production of IL-7 and IL-15, which is crucial for cell survival and proliferation. The complex alterations present in the periphery are likely the result of a compensatory mechanism: the overproduction of homeostatic cytokines could be a reaction to the impaired intrathymic production of T lymphocytes and/or to the expansion of Treg in the periphery, and could be required to allow the survival of T cells.

Mitochondrial alterations and tendency to apoptosis in peripheral blood cells from children with Down syndrome.

Different types of cells from subjects with Down syndrome (DS) have an increased susceptibility to cell death. We have studied apoptosis and mitochondrial (mt) membrane potential (DeltaPsi(m)) in peripheral blood mononuclear cells (PBMC) from DS children and age-matched healthy donors after in vitro treatment with apoptogenic molecules, along with mtDNA content. We found that PBMC from DS and healthy controls had a similar tendency to undergo apoptosis and a similar amount of mtDNA. However, in cells from DS subjects, mitochondria showed a higher loss of DeltaPsi(m), underlying the presence of an increasing susceptibility of these organelles to damaging agents.

Characterization of cells with different mitochondrial membrane potential during apoptosis.

BACKGROUND: Until now, the simultaneous analysis of several parameters during apoptosis, including DNA content and mitochondrial membrane potential (DeltaPsi), has not been possible because of the spectral characteristics of the commonly used dyes. Using polychromatic flow cytometry based upon multiple laser and UV lamp excitation, we have characterized cells with different DeltaPsi during apoptosis. METHODS: U937 cells were treated with the flavonoid quercetin (Qu) and stained with JC-1 to detect DeltaPsi, propidium iodide (PI) for cell viability, Hoechst 33342 for DNA content, Annexin V conjugated with Alexa Fluor-647 for detection of phosphatidilserine (PS) exposure, marker of early apoptosis, or Mitotracker Deep Red for the determination of mitochondrial mass. RESULTS: Treatment with Qu provoked the onset of three cell populations with different DeltaPsi: (1) healthy cells, with normal DeltaPsi, DNA content and physical parameters, high mitochondrial mass, PI- and Annexin V-negative; (2) cells with intermediate DeltaPsi and normal DNA content, but with physical parameters typical of apoptotic cells and low mitochondrial mass; most of them were PI+ and Annexin V+; (3) cells with collapsed DeltaPsi that had low mitochondrial mass and were Annexin-V+, PI+; half of them showed diminished DNA content. Similar results, i.e. the presence of cells with intermediate DeltaPsi, were observed in other models of apoptosis. CONCLUSIONS: During Qu-induced apoptosis, loss of DeltaPsi, PS exposure, and decrease of mitochondrial mass are early events that precede permeability to PI and loss of DNA. Populations of cells with different DeltaPsi, as revealed by flow cytometry after JC-1 staining, differed also for other parameters associated to apoptosis. Thus, the simultaneous analysis of several parameters by polychromatic flow cytometry permits a better identification of many stages of cell death, and, more in general, allows to evaluate the eventual heterogenic sensibility of the population under study to a given compound.

Changes in mitochondrial RNA production in cells treated with nucleoside analogues.

BACKGROUND: To investigate mitochondrial (mt) toxicity of antiretroviral drugs further, we developed a novel real-time PCR-based assay for the quantification of mtRNA. We analysed the effects of stavudine (d4T), didanosine (ddl) and zidovudine (AZT) on the production of mtRNAs in different human cell lines and compared the production with the amount of mtDNA present in the same cells. MATERIALS AND METHODS: HUT78, CEM and U937 cells were exposed to different nucleoside reverse transcriptase inhibitors (NRTIs) for 7 days. Thereafter, nucleic acids were isolated and Taqman-based real-time PCR was used to quantify mtDNA and three different mtRNAs (ND1, CYTB and ND6 gene products). RESULTS: Different amounts of mtRNAs exist in different cell lines. When mtRNA was measured in cells exposed to an NRTI, a marked decrease was observed in cells treated with d4T, but not with ddl or AZT. Changes in mtRNA production did not always correspond to modifications in mtDNA content: 1 microM d4T significantly changed mtRNA but not mtDNA content. CONCLUSIONS: d4T, but not ddl or AZT, significantly alters mtRNA quantity and quality. The method we have developed can reveal changes that are not observed by measuring mtDNA content only, and can be used for ex vivo studies on drug toxicity.

Direct analysis of thymic function in children with Down's syndrome.

BACKGROUND: Down's syndrome (DS) is characterized by several immunological defects, especially regarding T cell compartment. DS is considered the best example of accelerated ageing in humans. Direct observations of the thymus have shown that in DS this organ undergoes severe histological and morphological changes. However, no data on its capacity to generate T cells are present in the literature. Here, using a new technology based upon real time PCR, we have investigated the capacity of the thymus to produce and release newly generated T lymphocytes (the so called "recent thymic emigrants", RTE) in children with DS. METHODS: We studied 8 children affected by DS, aged 2-7 years, compared with 8 age- and sex-matched healthy controls. Flow cytometry was used to determine different lymphocytes subsets. Real time PCR with the Taqman system was used to quantify the amount of RTE, i.e. peripheral blood lymphocytes that express the T cell receptor rearrangement excision circles (TREC). RESULTS: In comparison with control children, those with DS had a significant lower number of TREC+ peripheral blood cells. Moreover, in DS children but not in controls, a strong negative correlation between age and the levels of TREC+ cells was found. CONCLUSIONS: The direct measure of thymic output indicates that the impairment of the organ results in a reduced production of newly generated T cells. This observation could suggest that cytokines able to modulate thymic function, such as interleukins, could be useful to improve the functionality of the organ and to treat the immunodeficiency present in DS subjects.