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1.  Mother-to-Children Plasmodium falciparum Asymptomatic Malaria Transmission at Saint Camille Medical Centre in Ouagadougou, Burkina Faso 
Malaria Research and Treatment  2014;2014:390513.
Background. Malaria's prevalence during pregnancy varies widely in parts of sub-Saharan Africa, including Burkina Faso. The objective of this study was to evaluate the incidence of mother-to-child malaria transmission during childbirth at St. Camille Medical Centre in the city of Ouagadougou. Methods. Two hundred and thirty-eight (238) women and their newborns were included in the study. Women consenting to participate in this study responded to a questionnaire that identified their demographic characteristics. Asymptomatic malaria infection was assessed by rapid detection test Acon (Acon Malaria Pf, San Diego, USA) and by microscopic examination of Giemsa-stained thick and thin smears from peripheral, placental, and umbilical cord blood. Birth weights were recorded and the biological analyses of mothers and newborns' blood were also performed. Results. The utilization of long-lasting insecticidal nets (LLINs) and intermittent preventive treatment with sulfadoxine-pyrimethamine (SP) were 86.6% and 84.4%, respectively. The parasitic infection rates of 9.5%, 8.9%, and 2.8% were recorded, respectively, for the peripheral, placental, and umbilical cord blood. Placental infection was strongly associated with the presence of parasites in the maternal peripheral blood and a parasite density of >1000 parasites/µL. Conclusion. The prevalence of congenital malaria was reduced but was associated with a high rate of mother-to-child malaria transmission.
PMCID: PMC4259075  PMID: 25506464
2.  Antioxidant and Anti-Inflammatory Activities of Extracts from Cassia alata, Eleusine indica, Eremomastax speciosa, Carica papaya and Polyscias fulva Medicinal Plants Collected in Cameroon 
PLoS ONE  2014;9(8):e103999.
The vast majority of the population around the world has always used medicinal plants as first source of health care to fight infectious and non infectious diseases. Most of these medicinal plants may have scientific evidence to be considered in general practice.
The aim of this work was to investigate the antioxidant capacities and anti-inflammatory activities of ethanol extracts of leaves of Cassia alata, Eleusine indica, Carica papaya, Eremomastax speciosa and the stem bark of Polyscias fulva, collected in Cameroon.
Chemiluminescence was used to analyze the antioxidant activities of plant extracts against hydrogen peroxide or superoxide anion. Comet assays were used to analyze the protection against antioxidant-induced DNA damage induced in white blood cells after treating with hydrogen peroxide. Flow cytometry was used to measure γδ T cells proliferation and anti-inflammatory activity of γδ T cells and of immature dendritic cells (imDC) in the presence of different concentrations of plant extracts.
Ethanol extracts showed strong antioxidant properties against both hydrogen peroxide and superoxide anion. Cassia alata showed the highest antioxidant activity. The effect of plant extracts on γδ T cells and imDC was evidenced by the dose dependent reduction in TNF-α production in the presence of Cassia alata, Carica papaya, Eremomastax speciosa Eleusine indica, and Polyscias fulva. γδ T cells proliferation was affected to the greatest extent by Polyscias fulva.
These results clearly show the antioxidant capacity and anti-inflammatory activities of plant extracts collected in Cameroon. These properties of leaves and stem bark extracts may contribute to the value for these plants in traditional medicine and in general medical practice.
PMCID: PMC4121200  PMID: 25090613
3.  Evaluation of the nutritional status of infants from mothers tested positive to HIV/AIDS in the health district of Dschang, Cameroon 
Poor infant feeding practices are common in Africa, resulting in physical and intellectual developmental impairments. Good feeding practices are crucial, especially in the first year of growth. HIV/AIDS has worsened the clinical and nutritional status of both mothers and their children, exacerbating high rates of malnutrition. The aim of this study was to assess by participative approach, the nutritional status of infants from mothers tested positive to HIV in the health district of Dschang.
This is a cross sectional study with a period of recruitment of 2 years (2010-2012). Data Collection was done by the aim of a personal slip followed by training to strengthen the nutritional and hygienic capacity of targeted parents. Height and weight of infants were measured and body mass index (BMI) calculated.
Significant difference (p ≤ 0.05) was noticed in height-for-age z-score (HAZ) of girls aged between 1 to 2 years compared to 1-year old girls as well as to boys of all ages, defining them as stunted. Furthermore, the weight-for-age z-score (WAZ) results indicate that both girls and boys of all age are in moderate state of malnutrition. The results of BMI thinness classified according to gender and age groups, indicates that most infants (68/130, 52.3%) showed grade 2 thinness predominantly in 2-years old both boys and girls. However, no participants fall within the normal category for age and sex, as well as overweight and obesity categories.
Undernutrition exists among infants from mothers tested positive to HIV residing in Dschang, as most of the infants are underweight, and malnourished.
PMCID: PMC4231317  PMID: 25400858
Nutritional status; HIV/AIDS; infants; Body Mass Index
5.  HS1,2 Ig Enhancer Alleles Association to AIDS Progression in a Pediatric Cohort Infected with a Monophyletic HIV-Strain 
BioMed Research International  2014;2014:637523.
Alteration in the humoral immune response has been observed during HIV infection. The polymorphisms of enhancer HS1,2, member of the 3′ regulatory region of the Ig heavy chain cluster, may play a role in the variation of the humoral response leading to pathological conditions. To assess the role of the HS1,2 polymorphic variants in the progression of AIDS, the HS1,2-A allelic frequencies were investigated in a cohort of HIV infected pediatric subjects from a nosocomial outbreak with a monophyletic strain of HIV. From a total group of 418 HIV infected children in the outbreak cohort, 42 nonprogressors and 31 progressors without bias due to antiretroviral therapy were evaluated. HS1,2 allele ∗1 has been associated with nonprogressors (allelic frequency: 51.19% versus 33.87% in progressors, OR 0.5, and P = 0.0437), while allele ∗2 has been associated with progression (allelic frequency: 48.39% versus 30.95% in nonprogressors, OR 2.1, and P = 0.0393). Further, only subjects carrying allele ∗2 in absence of allele ∗1, either in homozygous condition for allele ∗2 [nonprogressors 2/42 (4.76%), Progressors 7/31 (22.58%), OR 5.8, and P = 0.0315] or in combination with other allelic variants [nonprogressors 7/42 (16.67%), Progressors 13/31 (41.93%), OR 3.61, and P = 0.0321], have been associated with HIV progression to AIDS. In conclusion, while the HS1,2 allele ∗1 has a protective effect on HIV progression when present, allele ∗2 is associated with progression toward AIDS when allele ∗1 is absent.
PMCID: PMC4055013  PMID: 25009819
6.  Population-Based Monitoring of Emerging HIV-1 Drug Resistance on Antiretroviral Therapy and Associated Factors in a Sentinel Site in Cameroon: Low Levels of Resistance but Poor Programmatic Performance 
PLoS ONE  2013;8(8):e72680.
Scale-up of antiretroviral therapy (ART) in resource-limited settings has drastically reduced HIV-related morbidity and mortality. However, challenges in long-term ART, adherence and HIV drug resistance (HIVDR) itself, require monitoring to limit HIVDR emergence among ART-experienced populations, in order to ensure regimen efficacy.
A longitudinal study was conducted from 2009–2011 in a cohort of 141 HIV-infected adult patients (aged >21) at the national social insurance centre hospital in Yaounde, Cameroon. As per-WHO HIVDR protocol, HIV-1 protease-reverse transcriptase genotyping was performed at baseline and at endpoint (12 months) on first-line ART using ViroSeq™ Genotyping kit.
At baseline, a prevalence of 3.6% (5/139) HIVDR was observed [protease inhibitors M46I (1/5), G73A (1/5), L90LM (1/5); nucleoside reverse transcriptase inhibitors: M184V (1/5), T215F (1/5); non-nucleoside reverse transcriptase inhibitors: K103N (1/5), Y181Y/C (2/5), M230ML (1/5)]. At endpoint, 54.0% (76) patients were followed-up, 9.2% (13) died, and 3.5% (5) transferred, 38.5% (47) lost to follow-up (LTFU). 69.7% (53/76) of those followed-up had viremia <40 copies/ml and 90.8% (69/76) <1000 copies/ml. 4/7 patients with viremia ≥1000 copies/ml harbored HIVDR (prevalence: 5.3%; 4/76), with M184V/I (4/4) and K103K/N (3/4) being the most prevalent mutations. LTFU was favored by costs for consultation/laboratory tests, drug shortages, workload (physician/patient ratio: 1/180) and community disengagement.
Low levels of HIVDR at baseline and at endpoint suggest a probable effectiveness of ART regimens used in Cameroon. However the possible high rate of HIVDR among LTFUs limited the strengths of our findings. Evaluating HIVDR among LTFU, improving adherence, task shifting, subsidizing/harmonizing costs for routine follow-up, are urgent measures to ensure an improved success of the country ART performance.
PMCID: PMC3753336  PMID: 23991142
7.  Immunoinformatic Docking Approach for the Analysis of KIR3DL1/HLA-B Interaction 
BioMed Research International  2013;2013:283805.
KIR3DL1 is among the most interesting receptors studied, within the killer immunoglobulin receptor (KIR) family. Human leukocyte antigen (HLA) class I Bw4 epitope inhibits strongly Natural Killer (NK) cell's activity through interaction with KIR3DL1 receptor, while Bw6 generally does not. This interaction has been indicated to play an important role in the immune control of different viral infectious diseases. However, the structural interaction between the KIR3DL1 receptor and different HLA-B alleles has been scarcely studied. To understand the complexity of KIR3DL1-HLA-B interaction, HLA-B alleles carrying Bw4/Bw6 epitope and KIR3DL1∗001 allele in presence of different peptides has been evaluated by using a structural immunoinformatic approach. Different energy minimization force fields (ff) have been tested and NOVA ff enables the successful prediction of ligand-receptor interaction. HLA-B alleles carrying Bw4 epitope present the highest capability of interaction with KIR3DL1∗001 compared to the HLA-B alleles presenting Bw6. The presence of the epitope Bw4 determines a conformational change which leads to a stronger interaction between nonpolymorphic arginine at position 79 of HLA-B and KIR3DL1∗001 136–142 loop. The data shed new light on the modalities of KIR3DL1 interaction with HLA-B alleles essential for the modulation of NK immune-mediated response.
PMCID: PMC3747338  PMID: 23984333
8.  Declining trends in early warning indicators for HIV drug resistance in Cameroon from 2008–2010: lessons and challenges for low-resource settings 
BMC Public Health  2013;13:308.
Rapid scale-up of antiretroviral therapy (ART) and limited access to genotyping assays in low-resource settings (LRS) are inevitably accompanied by an increasing risk of HIV drug resistance (HIVDR). The current study aims to evaluate early warning indicators (EWI) as an efficient strategy to limit the development and spread of preventable HIVDR in these settings, in order to sustain the performance of national antiretroviral therapy (ART) rollout programmes.
Surveys were conducted in 2008, 2009 and 2010 within 10 Cameroonian ART clinics, based on five HIVDR EWIs: (1) Good prescribing practices; (2) Patient lost to follow-up; (3) Patient retention on first line ART; (4) On-time drug pick-up; (5) Continuous drug supply. Analysis was performed as per the World Health Organisation (WHO) protocol.
An overall decreasing performance of the national ART programme was observed from 2008 to 2010: EWI1 (100% to 70%); EWI2 (40% to 20%); EWI3 (70% to 0%); EWI4 (0% throughout); EWI5 (90% to 40%). Thus, prescribing practices (EWI1) were in conformity with national guidelines, while patient adherence (EWI2, EWI3, and EWI4) and drug supply (EWI5) were lower overtime; with a heavy workload (median ratio ≈1/64 staff/patients) and community disengagement observed all over the study sites.
In order to limit risks of HIVDR emergence in poor settings like Cameroon, continuous drug supply, community empowerment to support adherence, and probably a reduction in workload by task shifting, are the potential urgent measures to be undertaken. Such evidence-based interventions, rapidly generated and less costly, would be relevant in limiting the spread of preventable HIVDR and in sustaining the performance of ART programmes in LRS.
PMCID: PMC3627634  PMID: 23565992
Early warning indicator; HIV drug resistance; Surveillance and prevention; Cameroon
9.  Molecular Epidemiology of HIV Type 1 CRF02_AG in Cameroon and African Patients Living in Italy 
AIDS Research and Human Retroviruses  2011;27(11):1173-1182.
HIV-1 CRF02_AG accounts for >50% of infected individuals in Cameroon. CRF02_AG prevalence has been increasing both in Africa and Europe, particularly in Italy because of migrations from the sub-Saharan region. This study investigated the molecular epidemiology of CRF02_AG in Cameroon by employing Bayesian phylodynamics and analyzed the relationship between HIV-1 CRF02_AG isolates circulating in Italy and those prevalent in Africa to understand the link between the two epidemics. Among 291 Cameroonian reverse transcriptase sequences analyzed, about 70% clustered within three distinct clades, two of which shared a most recent common ancestor, all related to sequences from Western Africa. The major Cameroonian clades emerged during the mid-1970s and slowly spread during the next 30 years. Little or no geographic structure was detected within these clades. One of the major driving forces of the epidemic was likely the high accessibility between locations in Southern Cameroon contributing to the mobility of the population. The remaining Cameroonian sequences and the new strains isolated from Italian patients were interspersed mainly within West and Central African sequences in the tree, indicating a continuous exchange of CRF02_AG viral strains between Cameroon and other African countries, as well as multiple independent introductions in the Italian population. The evaluation of the spread of CRF02_AG may provide significant insight about the future dynamics of the Italian and European epidemic.
PMCID: PMC3206741  PMID: 21453131
10.  B-Pred, a structure based B-cell epitopes prediction server 
The ability to predict immunogenic regions in selected proteins by in-silico methods has broad implications, such as allowing a quick selection of potential reagents to be used as diagnostics, vaccines, immunotherapeutics, or research tools in several branches of biological and biotechnological research. However, the prediction of antibody target sites in proteins using computational methodologies has proven to be a highly challenging task, which is likely due to the somewhat elusive nature of B-cell epitopes. This paper proposes a web-based platform for scoring potential immunological reagents based on the structures or 3D models of the proteins of interest. The method scores a protein’s peptides set, which is derived from a sliding window, based on the average solvent exposure, with a filter on the average local model quality for each peptide. The platform was validated on a custom-assembled database of 1336 experimentally determined epitopes from 106 proteins for which a reliable 3D model could be obtained through standard modeling techniques. Despite showing poor sensitivity, this method can achieve a specificity of 0.70 and a positive predictive value of 0.29 by combining these two simple parameters. These values are slightly higher than those obtained with other established sequence-based or structure-based methods that have been evaluated using the same epitopes dataset. This method is implemented in a web server called B-Pred, which is accessible at The server contains a number of original features that allow users to perform personalized reagent searches by manipulating the sliding window’s width and sliding step, changing the exposure and model quality thresholds, and running sequential queries with different parameters. The B-Pred server should assist experimentalists in the rational selection of epitope antigens for a wide range of applications.
PMCID: PMC3413014  PMID: 22888263
B-cell epitopes; immunoinformatics; bioinformatics; web server; epitope prediction
11.  Early Warning Indicators for HIV Drug Resistance in Cameroon during the Year 2010 
PLoS ONE  2012;7(5):e36777.
Rapid scale-up of antiretroviral therapy (ART) in resource-limited settings is accompanied with an increasing risk of HIV drug resistance (HIVDR), which in turn could compromise the performance of national ART rollout programme. In order to sustain the effectiveness of ART in a resource-limited country like Cameroon, HIVDR early warning indicators (EWI) may provide relevant corrective measures to support the control and therapeutic management of AIDS.
A retrospective study was conducted in 2010 among 40 ART sites (12 Approved Treatment Centers and 28 Management Units) distributed over the 10 regions of Cameroon. Five standardized EWIs were selected for the evaluation using data from January through December, among which: (1) Good ARV prescribing practices: target = 100%; (2) Patient lost to follow-up: target ≤20%; (3) Patient retention on first line ART: target ≥70%; (4) On-time drug pick-up: target ≥90%; (5) ARV drug supply continuity: target = 100%. Analysis was performed using a Data Quality Assessment tool, following WHO protocol.
The number of sites attaining the required performance are: 90% (36/40) for EWI1, 20% (8/40) for EWI2; 20% (8/40) for EWI3; 0% (0/37) for EWI4; and 45% (17/38) for EWI 5. ARV prescribing practices were in conformity with the national guidelines in almost all the sites, whereas patient adherence to ART (EWI2, EWI3, and EWI4) was very low. A high rate of patients was lost-to-follow-up and others failing first line ART before 12 months of initiation. Discontinuity in drug supply observed in about half of the sites may negatively impact ARV prescription and patient adherence. These poor ART performances may also be due to low number of trained staff and community disengagement.
The poor performance of the national ART programme, due to patient non-adherence and drug stock outs, requires corrective measures to limit risks of HIVDR emergence in Cameroon.
PMCID: PMC3355154  PMID: 22615810
12.  Validation of a single-platform, volumetric, flow cytometry for CD4 T cell count monitoring in therapeutic mobile unit 
A mobile health unit may be useful to follow up adult and pediatric patients on antiretroviral treatment and living in remote areas devoid of laboratory facilities. The study evaluated the use of the simplified, robust, single-plateform, volumetric, pan-leucogating Auto40 flow cytometer (Apogee Flow Systems Ltd, Hemel Hempstead, UK) for CD4 T cell numeration in a mobile unit, compared against a reference flow cytometry method.
The therapeutic mobile unit of the Laboratoire National de Santé Hygiène Mobile, Yaoundé, Cameroon, was equipped with the Auto40. A FACSCalibur flow cytometer (Becton Dickinson Immuno-cytometry System, San Jose, CA, USA) was used as reference method. EDTA-blood samples from volunteers were first subjected to CD4 T cell count in the mobile unit, and an aliquot was sent within 4 hours to Centre International de Référence Chantal Biya, Yaoundé, for FACSCalibur assay.
Two HIV screening campaigns with the mobile unit were organised in December 2009 and January 2010. The campaign in the suburb of Yaoundé which was 20 km from the reference laboratory included 188 volunteers comprising 93 children less than 5 years old. The campaign in Ambang Bikok (53 km far from Yaoundé) included 69 adult volunteers. In Yaoundé suburb, mean ± standard deviation (SD) CD4 T cell count was 996 ± 874 cells/μl by Auto40, and 989 ± 883 cells/μl by FACSCalibur; in Ambang Bikok, mean ± SD CD4 T cell count was 1041 ± 317 cells/μl by Auto40, and 1032 ± 294 cells/μl by FACSCalibur. Results by Auto40 and FACSCalibur were highly correlated in Yaoundé (r2 = 0.982) as in Ambang Bikok (r2 = 0.921). Bland-Altman analysis showed a close agreement between Auto40 and FACSCalibur results expressed in absolute count as in percentage in Yaoundé and Ambang Bikok. When pooling the 257 CD4 T cell count measurements, the Auto40 yielded a mean difference of +7.6 CD4 T cells/μl higher than by reference flow cytometry; and the sensitivity and specificity of Auto40 in enumerating absolute CD4 T cell counts of less than 200 cells/μl were 87% and 99%, respectively, and in enumerating absolute CD4 T cell counts of less than 350 cells/μl were 87% and 98%, respectively. The intrarun and interun precisions of the Auto40 assay assessed in the mobile unit were 5.5% and 7.9%, respectively.
The Auto40 flow cytometer installed in a therapeutic mobile unit and operated far from its reference laboratory gave a perfect correlation with the reference method, and could be useful in carrying out immunological monitoring of HIV-infected patients living in areas without access to laboratory facilities.
PMCID: PMC3293735  PMID: 22309994
Flow cytometry; CD4 T cell count; HIV; Resource-limited settings; Therapeutic mobile unit
13.  Reference Values of Lymphocyte Subsets in Healthy, HIV-Negative Children in Cameroon▿ 
Lymphocyte subset reference values used to monitor infectious diseases, including HIV/AIDS, tuberculosis, malaria, or other immunological disorders in healthy children in Cameroon, are lacking. Values for Caucasian cohorts are already being utilized for clinical decisions but could be inappropriate for African populations. We report here the immunological profile for children aged from birth through 6 years in Cameroon and also compare our values to data from other African and Caucasian populations. In a cohort of 352 healthy children, aged 0 to 6 years, the relative and absolute numbers of T-cell subsets, B cells, and NK lymphocytes were determined from peripheral blood collected in EDTA tubes. Samples were stained with BD Multitest reagents in Trucount tubes and analyzed by using CellQuest-Pro and FlowJo software. We evaluated about 23 different lymphocyte subsets in which the absolute number and percentage values differed significantly (P < 0.05) with age and peaked between 6 and 12 months. B-cell values were higher compared to reported values from developed countries. Differences in activated and differentiated T cells were observed in subjects between 1 and 6 years of age. The absolute CD8+ T-cell count and the CD4+/CD8+ ratio seem to depend on gender. Normal lymphocyte subsets values among children from Cameroon differ from reported values in Caucasian and some African populations. The differences observed could be due to genetic and environmental factors coupled with the methodology used. These values could be used as initial national reference guidelines as more data are assembled.
PMCID: PMC3122514  PMID: 21411603
14.  HMGB1 and Cord Blood: Its Role as Immuno-Adjuvant Factor in Innate Immunity 
PLoS ONE  2011;6(8):e23766.
In newborn the innate immune system provides essential protection during primary infections before the generation of an appropriate adaptive immune response that is initially not fully operative. Innate immune response is evoked and perpetuated by molecules derived from microorganisms or by the damage/death of host cells. These are collectively known as damage-associated molecular-pattern (DAMP) molecules. High-mobility group box 1 protein (HMGB1) or amphoterin, which previously was considered to be only a nuclear factor, has been recently identified as a DAMP molecule. When it is actively secreted by inflammatory cells or passively released from necrotic cells, HMGB1 mediates the response to infection, injury and inflammation, inducing dendritic cells maturation and T helper-1-cell responses. To characterize the role of HMGB1 in the innate and immature defense mechanisms in newborns, human cord blood (CB) mononuclear cells, in comparison to adult peripheral blood (PB) mononuclear cells, have been analyzed for its expression. By flow cytometry and western blot analysis, we observed that in CB and PB cells: i) HMGB1 is expressed on cell surface membranes of myeloid dendritic cell precursors, mostly, and lymphocytes (gamma/delta and CD4+ T cells) to a lesser extent; ii) different pro-inflammatory stimuli or molecules that mimic infection increased cell surface expression of HMGB1 as well as its secretion into extracellular environment; iii) the treatment with synthetic molecules such as aminobisphosphonates (ABs), identified to be γδ T cell antigens, triggered up-regulation of HMGB1 expression on mononuclear cells, as well γδ T lymphocytes, inducing its secretion. The modulation of its secretion and the HMGB1-mediated migration of monocytes indicated HMGB1 as regulator of immune response in an immature system, like CB, through engagement of γδ T lymphocytes and myeloid dendritic cell precursors, essential components of innate immunity. In addition, the increased HMGB1 expression/secretion triggered by ABs, previously characterized for their immuno-modulating and immune-adjuvant capabilities, indicated that immunomodulation might represent a new therapeutical approach for neonatal and adult pathologies.
PMCID: PMC3161821  PMID: 21915243
15.  Different Transcriptional Profiles of Human Monocyte-Derived Dendritic Cells Infected with Distinct Strains of Mycobacterium tuberculosis and Mycobacterium bovis Bacillus Calmette-Guérin 
In order to analyze dendritic cells (DCs) activation following infection with different mycobacterial strains, we studied the expression profiles of 165 genes of human monocyte-derived DCs infected with H37Rv, a virulent Mycobacterium tuberculosis (MTB) laboratory strain, CMT97, a clinical MTB isolate, Mycobacterium bovis bacillus Calmette-Guérin (BCG), Aventis Pasteur, and BCG Japan, both employed as vaccine against tuberculosis. The analysis of the gene expression reveals that, despite a set of genes similarly modulated, DCs response resulted strain dependent. In particular, H37Rv significantly upregulated EBI3 expression compared with BCG Japan, while it was the only strain that failed to release a significant IL-10 amount. Of note, BCG Japan showed a marked increase in CCR7 and TNF-α expression regarding both MTB strains and it resulted the only strain failing in exponential intracellular growth. Our results suggest that DCs display the ability to elicit a tailored strain-specific immune response.
PMCID: PMC3062957  PMID: 21436989
16.  Innate-like γδ T cell responses to mycobacterium Bacille Calmette-Guerin using the public Vγ2 repertoire in Macaca fascicularis 
The Vγ2Vδ2 T cell subset responds to Bacille Calmette-Guerin (BCG) immunization in macaques and may be a component of protective immunity against tuberculosis. We characterized the effects of BCG on the Vγ2Vδ2 T cell receptor repertoire by comparing the starting population of Vγ2 chains in cynomolgus macaques with the repertoire found after priming or booster immunization with BCG. The starting repertoire was dominated by public Vγ2 chain sequences that were found repeatedly among unrelated animals. Primary exposure to BCG triggered expansion of cells expressing public Vγ2 chains and booster immunization was often associated with contraction of these same subsets. Thus, BCG-reactive Vγ2 chains were present at high frequency in the repertoire of mycobacteria-naïve macaques and they comprised the major response to primary or booster immunization. Normal selection processes that created the naïve Vγ2 repertoire in macaques, also encoded the capacity for rapid responses to mycobacteria. The unusual composition of a normal Vγ2 repertoire helps to explain the powerful γδ T cell responses to BCG immunization.
PMCID: PMC2958528  PMID: 17292671
Vγ2Vδ2; repertoire; BCG; M. fascicularis
17.  Altered cord blood γδ T cell repertoire in Nigeria: possible impacts of environmental factors on neonatal immunity 
Molecular immunology  2008;45(11):3190-3197.
Infectious diseases during pregnancy can impact the development of fetal immunity, leading to reduced neonatal resistance to infection and decreased responses to pediatric vaccines. P. falciparum causes placental infection in low parity pregnant women and is among the pathogens that affect fetal immunity. Recognizing the relationship between malaria and γδ T lymphocytes in adults, we asked whether neonatal γδ T cells would be altered in malaria-endemic regions as a marker for changes in fetal immunity. Our initial studies compared cord blood γδ T cells from deliveries to HIV- mothers in Jos (Nigeria) where malaria is endemic, or in Rome (Italy). We noted substantial differences in the Vγ2 repertoire for cord blood collected in Jos or Rome; differences were consistent with a negative selection mechanism operating on the fetal Vγ2 chain repertoire in neonates from Jos. A specific disruption affected the fraction of γδ T cells that we expect will respond to Bacille Calmette-Guerin (BCG). Fetal γδ T cell depletion might be a mechanism for impaired neonatal immunity and lowered responses to pediatric vaccines.
PMCID: PMC2601555  PMID: 18440637
cord blood; gammadelta T lymphocytes; Vγ2 repertoire; Jγ1.2; Nigeria
19.  Analysis of the Shotgun Expression Library of the Mycobacterium tuberculosis Genome for Immunodominant Polypeptides: Potential Use in Serodiagnosis 
A recombinant DNA strategy was applied to analyze and screen the shotgun expression library from a clinically confirmed local virulent isolate of Mycobacterium tuberculosis with sera from tuberculosis patients, which led to expression and purification of highly immunoreactive and specific mycobacterial antigens expressed during the course of active disease which could be of diagnostic significance. An enzyme-linked immunoassay for diagnosis of tuberculosis was devised by using a shotgun immunoexpression library in the λgt11 vector. DNA from a virulent M. tuberculosis patient isolate (TBW-33) confirmed with the BACTEC 460 system was sheared and expressed to generate shotgun polypeptides. β-Galactosidase fusion proteins capable of demarcating active tuberculosis infections from Mycobacterium bovis BCG-vaccinated healthy subjects or people harboring environmental mycobacteria were selected by comparative immunoreactivity studies. Promising mycobacterial DNA cassettes were subcloned and expressed into the glutathione S-transferase (GST) fusion vector pGEX-5X-1 with a strong tac promoter and were expressed in Escherichia coli BL21. These fusion proteins were severed at a built-in factor Xa recognition site to separate the GST tags and were utilized in an indirect enzyme-linked immunoassay for serodiagnosis of patients with active tuberculosis. The system offered a clear demarcation between BCG-vaccinated healthy subjects and patients with active tuberculosis and proved to be effective in detecting pulmonary as well as extrapulmonary tuberculosis, with an overall sensitivity of 84.33% and an overall specificity of 93.62%.
PMCID: PMC262431  PMID: 14607866
20.  Vγ9Vδ2 T-Cell Anergy and Complementarity-Determining Region 3-Specific Depletion during Paroxysm of Nonendemic Malaria Infection  
Infection and Immunity  2003;71(5):2945-2949.
Vγ9Vδ2 T lymphocytes strongly respond to phosphoantigens from Plasmodium parasites. Thus, we analyzed the changes in Vγ9Vδ2 T-cell function and repertoire during the paroxysm phase of nonendemic malaria infection. During malaria paroxysm, Vγ9Vδ2 T cells were early activated but rapidly became anergic and finally loose Jγ1.2 Vγ9 complementarity-determining region 3 transcripts.
PMCID: PMC153242  PMID: 12704176
21.  Computer-based design of an HLA-haplotype and HIV-clade independent cytotoxic T-lymphocyte assay for monitoring HIV-specific immunity. 
Molecular Medicine  2002;8(12):798-807.
BACKGROUND: Human immunodeficiency virus (HIV)- specific CD8-positive cytotoxic T-lymphocytes (CTL) play a key role in controlling HIV infection. Monitoring CTL response could be clinically relevant during structured therapy interruption (STI), HIV exposure, and vaccine trials. However, HLA patients' restriction and HIV variability limited the development of a CTL assay with broad specificity. MATERIALS AND METHODS: We designed an HLA-class I/HIV-1 clade independent assay for assessing HIV- specific CTL by using a computer-assisted selection ofthe CTL epitopes. Twenty-eight 15-mers were selected by peptide-binding motifs analysis using different databases (HIV-Immunology Database, SYFPEITHI, BIMAS). Altogether they putatively bind to more than 90% of HLA haplotypes in different populations, with an overall HIV-1 variability below 9%. The peptide pool was used as an antigen in an intracellular cytokine staining (ICS) assay for quantifying HIV-specific CTL response. RESULTS: The test can be performed using both fresh and cryopreserved peripheral blood mononuclear cells (PBMC), whereas GAG protein as antigen works only on fresh PBMC. A significantly higher CTL response with respect to HIV-negative controls was detected in all HIV-1 infected subjects of two groups of patients with different ethnicities (Caucasians and Africans) and coming from areas with different HIV-1 clade prevalences (clade B and A/G, respectively). In Caucasian patients, after month of STI, the number of HIV-1 specific CTL (2,896 +/- 2,780 IFN-gamma specific CD8 cells/ml) was significantly higher than that found at enrolment (2,125 +/- 4,426 IFN-gamma specific CD8 cells/ml, p< 0.05). CONCLUSIONS: These data indicate that this CTL assay is broadly specific and could represent a useful clinical tool for HIV immunodiagnostic independent of HLA-haplotype and HIV-clade variabilities.
PMCID: PMC2039964  PMID: 12606814
22.  Human Macrophage Gamma Interferon Decreases Gene Expression but Not Replication of Mycobacterium tuberculosis: Analysis of the Host-Pathogen Reciprocal Influence on Transcription in a Comparison of Strains H37Rv and CMT97 
Infection and Immunity  2001;69(12):7262-7270.
Mycobacterium tuberculosis is an intracellular pathogen that readily survives and replicates in human macrophages (MΦ). Host cells have developed different mycobactericidal mechanisms, including the production of inflammatory cytokines. The aim of this study was to compare the MΦ response, in terms of cytokine gene expression, to infection with the M. tuberculosis laboratory strain H37Rv and the clinical M. tuberculosis isolate CMT97. Both strains induce the production of interleukin-12 (IL-12) and IL-16 at comparable levels. However, the clinical isolate induces a significantly higher and more prolonged MΦ activation, as shown by reverse transcription-PCR analysis of IL-1β, IL-6, IL-10, transforming growth factor beta, tumor necrosis factor alpha, and gamma interferon (IFN-γ) transcripts. Interestingly, when IFN-γ transcription is high, the number of M. tuberculosis genes expressed decreases and vice versa, whereas no mycobactericidal effect was observed in terms of bacterial growth. Expression of 11 genes was also studied in the two M. tuberculosis strains by infecting resting or activated MΦ and compared to bacterial intracellular survival. In both cases, a peculiar inverse correlation between expression of these genes and multiplication was observed. The number and type of genes expressed by the two strains differed significantly.
PMCID: PMC98810  PMID: 11705896

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