Synergistic Effect between Amoxicillin and TLR Ligands on Dendritic Cells from Amoxicillin-Delayed Allergic Patients.

Amoxicillin, a low-molecular-weight compound, is able to interact with dendritic cells inducing semi-maturation in vitro. Specific antigens and TLR ligands can synergistically interact with dendritic cells (DC), leading to complete maturation and more efficient T-cell stimulation. The aim of the study was to evaluate the synergistic effect of amoxicillin and the TLR2, 4 and 7/8 agonists (PAM, LPS and R848, respectively) in TLR expression, DC maturation and specific T-cell response in patients with delayed-type hypersensitivity (DTH) reactions to amoxicillin. Monocyte-derived DC from 15 patients with DTH to amoxicillin and 15 controls were cultured with amoxicillin in the presence or absence of TLR2, 4 and 7/8 agonists (PAM, LPS and R848, respectively). We studied TLR1-9 gene expression by RT-qPCR, and DC maturation, lymphocyte proliferation and cytokine production by flow cytometry. DC from both patients and controls expressed all TLRs except TLR9. The amoxicillin plus TLR2/4 or TLR7/8 ligands showed significant differences, mainly in patients: AX+PAM+LPS induced a decrease in TLR2 and AX+R848 in TLR2, 4, 7 and 8 mRNA levels. AX+PAM+LPS significantly increased the percentage of maturation in patients (75%) vs. controls (40%) (p=0.036) and T-cell proliferation (80.7% vs. 27.3% of cases; p=0.001). Moreover, the combinations AX+PAM+LPS and AX+R848 produced a significant increase in IL-12p70 during both DC maturation and T-cell proliferation. These results indicate that in amoxicillin-induced maculopapular exanthema, the presence of different TLR agonists could be critical for the induction of the innate and adaptive immune responses and this should be taken into account when evaluating allergic reactions to these drugs.

Use of fluorescent proteins to monitor the regulation of antifungal activity in root-associated Pseudomonas fluorescens CHA0

SUMMARY Roots of crop plants are the target of soil-borne pathogens, mainly fungi that cause considerable damage to plant health. By antagonizing these pathogens, some root-colonizing pseudomonads provide plants with efficient biological protection from disease. Pseudomonas fluorescens CHAO is a soil bacterium with the ability to suppress a considerable range of root diseases. A major characteristic conferring biocontrol capacity to this strain is the production of antifungal compounds, in particular 2,4-diacetyphloroglucinol (DAPG) and pyoluteorin (PLT). The regulation of the biosyntheses of these metabolites is complex and involves several regulatory systems responding to multiple environmental signals. In the present work, we have developed reporter systems based on green (GFP) and red fluorescent (DsRed) proteins to monitor regulation of antifungal gene expression in vitro and on plant roots. Stable and unstable GFP-based reporter fusions to the DAPG and PLT biosynthetic genes allowed us to demonstrate that P. fluorescens CHAO keeps the two antifungal compounds at a fine-tuned balance that can be affected by environmental signals. A GFP-based screening technique helped us to identify two novel regulators of balanced antibiotic production, i.e. MvaT and MvaV that are functionally and structurally related to the nucleoid-binding protein H-NS. They act in concert as global regulators of DAPG and PLT production and other biocontrol-related traits in P. fluorescens CHAO, and are essential for the bacterium's capacity to control a root disease caused by Pythium. The combined use of autofluorescent reporters, flow cytometry, and epifluorescence microscopy permitted us to visualize and quantify the expression of DAPG and PLT biosynthetic genes on roots. A GFP- and DsRed-based two-color approach was then developed to further improve the sensitivity of the flow cytometric quantitation method. The findings of this study shed more light on the complex regulatory mechanisms controlling antifungal activity of P. filuorescens in the rhizosphere. RESUME 4 e Les racines de plantes de culture sont la cible de divers pathogènes, principalement des champignons, qui nuisent gravement à la santé des plantes. Certains pseudomonades colonisant les racines peuvent avoir un effet antagoniste sur les pathogènes et protéger ainsi les plantes de manière efficace. Pseudomonas fluorescens CHAO est une bactérie du sol ayant la capacité de supprimer une gamme considérable de maladies racinaires. Une des caractéristiques principales conférant la capacité de biocontrôle à cette souche, est la production de composés antifongiques, en particulier le 2,4-diacétyphloroglucinol (DAPG) et la pyolutéorine (PLT). La régulation de la biosynthèse de ces métabolites est complexe et implique plusieurs systèmes régulateurs répondant à de multiples signaux environnementaux. Dans ce travail, nous avons développé des systèmes rapporteurs basés sur des protéines fluorescentes verte (GFP) et rouge (DsRed), afin d'étudier la régulation de l'expression des gènes d'antifongiques in vitro et sur les racines des plantes. Des fusions GFP stables et instables rapportrices de l'expression des gènes de biosynthèse du DAPG et de la PLT nous ont permis de démontrer que P. fluorescens CHAO gère les deux antifongiques dans une balance finement régulée pouvant être affectée par des signaux environnementaux. Une technique de criblage basée sur la GFP nous a permis d'identifier deux nouveaux régulateurs de la production d'antibiotiques, MvaT et MvaV, apparentés à la protéine H-NS liant l'ADN, Elles agissent de concert en tant que régulateurs globaux sur la production de DAPG et de PLT, ainsi que sur d'autres éléments relatifs au biocontrôle chez P. fluorescens CHAO. De plus, elles sont essentielles à la bactérie pour contrôler une maladie racinaire causée par Pythium. L'utilisation combinée de rapporteurs autofluorescents, de cytométrie de flux et de microscopie à épifluorescence nous a permis de visualiser et de quantifier l'expression des gènes de biosynthèse du DAPG et de la PLT sur les racines. Une approche utilisant simultanément la GFP et la DsRed a ensuite été développée afin d'améliorer la sensibilité de la méthode de quantification par cytométrie de flux. Les résultats de cette étude ont apporté plus de lumière sur les mécanismes régulateurs complexes contrôlant l'activité antifongique de P. fluorescens dans la rizosphère.

Cells derived from the coelomic epithelium contribute to multiple gastrointestinal tissues in mouse embryos.

Gut mesodermal tissues originate from the splanchnopleural mesenchyme. However, the embryonic gastrointestinal coelomic epithelium gives rise to mesenchymal cells, whose significance and fate are little known. Our aim was to investigate the contribution of coelomic epithelium-derived cells to the intestinal development. We have used the transgenic mouse model mWt1/IRES/GFP-Cre (Wt1(cre)) crossed with the Rosa26R-EYFP reporter mouse. In the gastrointestinal duct Wt1, the Wilms' tumor suppressor gene, is specific and dynamically expressed in the coelomic epithelium. In the embryos obtained from the crossbreeding, the Wt1-expressing cell lineage produces the yellow fluorescent protein (YFP) allowing for colocalization with differentiation markers through confocal microscopy and flow cytometry. Wt1(cre-YFP) cells were very abundant throughout the intestine during midgestation, declining in neonates. Wt1(cre-YFP) cells were also transiently observed within the mucosa, being apparently released into the intestinal lumen. YFP was detected in cells contributing to intestinal vascularization (endothelium, pericytes and smooth muscle), visceral musculature (circular, longitudinal and submucosal) as well as in Cajal and Cajal-like interstitial cells. Wt1(cre-YFP) mesenchymal cells expressed FGF9, a critical growth factor for intestinal development, as well as PDGFRα, mainly within developing villi. Thus, a cell population derived from the coelomic epithelium incorporates to the gut mesenchyme and contribute to a variety of intestinal tissues, probably playing also a signaling role. Our results support the origin of interstitial cells of Cajal and visceral circular muscle from a common progenitor expressing anoctamin-1 and SMCα-actin. Coelomic-derived cells contribute to the differentiation of at least a part of the interstitial cells of Cajal.

Strain-transcending Fc-dependent killing of Plasmodium falciparum by merozoite surface protein 2 allele-specific human antibodies.

It is widely accepted that antibody responses against the human parasitic pathogen Plasmodium falciparum protect the host from the rigors of severe malaria and death. However, there is a continuing need for the development of in vitro correlate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the gene PFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines.

Efecto de los contenidos de C, Mn y Si en el comportamiento a fluencia en caliente de aceros de construcción al carbono y microaleados: aplicación a la obtención de grano ultrafino en productos largos laminados

Després d’aplicar alguns tractaments d’elaboració i conservació als aliments, queden bacteris lesionats. Aquests bacteris perden la capacitat de créixer en els medis de cultiu selectiu convencionals, de manera que se’n subestima el recompte. Malgrat això, poden recuperar-se als aliments i suposar un risc per la salut, ja que alguns encara poden mantenir activitat metabòlica i integritat estructural. En aquest projecte, es van optimitzar protocols de preparació de mostres per citometria de flux (CF) per avaluar l’estat fisiològic de patògens alimentaris (Escherichia coli O157:H7, Salmonella Enteritidis i Listeria monocytogenes) sotmesos a estrès. Es van estudiar principalment dos paràmetres fisiològics: la integritat de membrana, mitjançant iodur de propidi i fluorocroms de la família SYTO; i l’activitat respiratòria, per la reducció intracel•lular d’una sal de tetrazole, el CTC. En primer lloc, es van avaluar variables de protocol, com la concentració de colorant, la ràtio entre colorants, la solució de tinció i el temps d’incubació, en mostres control (cèl•lules sanes i mortes). A continuació, els protocols optimitzats es van aplicar a suspensions bacterianes en medi de cultiu que prèviament havien estat sotmeses a estressos físics i fisicoquímics. Durant l’etapa final del projecte, els coneixements adquirits sobre la preparació de mostres per CF es van aplicar a l’anàlisi de mostres de matriu complexa: amanides comercials inoculades amb E. coli O157:H7. Als assajos amb indicadors d’integritat de membrana en suspensions bacterianes sotmeses a estrès, es van poder quantificar cèl•lules amb la membrana parcialment danyada (presumptes cèl•lules lesionades). El recompte de cèl•lules que mantingueren l’activitat respiratòria després de ser sotmeses a estrès va ser superior al que es va obtenir mitjançant recompte en placa convencional, cosa que va evidenciar la presència de cèl•lules actives però no cultivables. La introducció d’estratègies per reduir les interferències provocades per les partícules alimentàries i l’ús d’un anticòs amb marcatge fluorescent va permetre detectar selectivament les cèl•lules d’E. coli O157:H7 i avaluar-ne la integritat de membrana simultàniament. L’anàlisi de cèl•lules bacterianes per CF requereix de la exhaustiva optimització dels protocols, que són específics per cada soca i matriu. Malgrat això, i a diferència del mètode convencional per recompte en placa, ofereix la possibilitat d’obtenir una gran quantitat d’informació sobre el sovint complex estat fisiològic d’una mostra.

Productive HIV-1 infection of primary CD4+ T cells induces mitochondrial membrane permeabilization leading to a caspase-independent cell death.

We have explored in vitro the mechanism by which human immunodeficiency virus, type 1 (HIV-1) induces cell death of primary CD4+ T cells in conditions of productive infection. Although HIV-1 infection primed phytohemagglutinin-activated CD4+ T cells for death induced by anti-CD95 antibody, T cell death was not prevented by a CD95-Fc decoy receptor, nor by decoy receptors of other members of the TNFR family (TNFR1/R2, TRAILR1/R2/OPG, TRAMP) or by various blocking antibodies, suggesting that triggering of death receptors by their cognate ligands is not involved in HIV-induced CD4 T cell death. HIV-1 induced CD4 T cell shrinkage, cell surface exposure of phosphatidylserine, loss of mitochondrial membrane potential (Deltapsim), and mitochondrial release of cytochrome c and apoptosis-inducing factor. A typical apoptotic phenotype (nuclear chromatin condensation and fragmentation) only occurred in around half of the dying cells. Treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a broad spectrum caspase inhibitor, prevented nuclear chromatin condensation and fragmentation in HIV-infected CD4+ T cells and in a cell-free system (in which nuclei were incubated with cytoplasmic extracts from the HIV-infected CD4+ T cells). Nevertheless, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not prevent mitochondrial membrane potential loss and cell death, suggesting that caspases are dispensable for HIV-mediated cell death. Our findings suggest a major role of the mitochondria in the process of CD4 T cell death induced by HIV, in which targeting of Bax to the mitochondria may be involved.

Decreased memory T-cell response and function in human immunodeficiency virus-infected patients with tegumentary leishmaniasis

The effects of human immunodeficiency virus (HIV) on the immune response in patients with cutaneous leishmaniasis have not yet been fully delineated. This study quantified and evaluated the function of memory T-cell subsets in response to soluble Leishmania antigens (SLA) from patients coinfected with HIV and Leishmania with tegumentary leishmaniasis (TL). Eight TL/HIV coinfected subjects and 10 HIV seronegative subjects with TL were evaluated. The proliferative response of CD4+and CD8+T-cells and naïve, central memory (CM) and effector memory (EM) CD4+T-cells in response to SLA were quantified using flow cytometry. The median cell division indices for CD4+and CD8+T-cells of coinfected patients in response to SLA were significantly lower than those in patients with Leishmania monoinfection (p < 0.05). The proportions of CM and EM CD4+T-cells in response to SLA were similar between the coinfected patients and patients with Leishmania monoinfection. However, the median CM and EM CD4+T-cell counts from coinfected patients were significantly lower (p < 0.05). The reduction in the lymphoproliferative response to Leishmaniaantigens coincides with the decrease in the absolute numbers of both EM and CM CD4+T-cells in response to Leishmania antigens in patients coinfected with HIV/Leishmania.

Stringent V beta requirement for the development of NK1.1+ T cell receptor-alpha/beta+ cells in mouse liver.

The liver of C57BL/6 mice contains a major subset of CD4+8- and CD4-8- T cell receptor (TCR)-alpha/beta+ cells expressing the polymorphic natural killer NK1.1 surface marker. Liver NK1.1+TCR-alpha/beta+ (NK1+ T) cells require interaction with beta2-microglobulin-associated, major histocompatibility complex I-like molecules on hematopoietic cells for their development and have a TCR repertoire that is highly skewed to Vbeta8.2, Vbeta7, and Vbeta2. We show here that congenic C57BL/6.Vbeta(a) mice, which lack Vbeta8- expressing T cells owing to a genomic deletion at the Vbeta locus, maintain normal levels of liver NK1+ T cells owing to a dramatic increase in the proportion of cells expressing Vbeta7 and Vbeta2 (but not other Vbetas). Moreover, in C57BL/6 congenic TCR-V Vbeta3 and -Vbeta8.1 transgenic mice (which in theory should not express other Vbeta, owing to allelic exclusion at the TCR-beta locus), endogenous TCR-Vbeta8.2, Vbeta7, and Vbeta2 (but not other Vbetas) are frequently expressed on liver NK1+T cells but absent on lymph node T cells. Finally, when endogenous V beta expression is prevented in TCR-Vbeta3 and Vbeta8.1 transgenic mice (by introduction of a null allele at the C beta locus), the development of liver NK1+T cells is totally abrogated. Collectively, our data indicate that liver NK1+T cells have a stringent requirement for expression of TCR-Vbeta8.2, Vbeta7, or Vbeta2 for their development.

Early and Prolonged Antiretroviral Therapy Is Associated with an HIV-1-Specific T-Cell Profile Comparable to That of Long-Term Non-Progressors.

Background: Intervention with antiretroviral treatment (ART) and control of viral replication at the time of HIV-1 seroconversion may curtail cumulative immunological damage. We have therefore hypothesized that ART maintenance over a very prolonged period in HIV-1 seroconverters could induce an immuno-virological status similar to that of HIV-1 long-term non-progressors (LTNPs).Methodology/Principal Findings: We have investigated a cohort of 20 HIV-1 seroconverters on long-term ART (LTTS) and compared it to one of 15 LTNPs. Residual viral replication and reservoirs in peripheral blood, as measured by cell-associated HIV-1 RNA and DNA, respectively, were demonstrated to be similarly low in both cohorts. These two virologically matched cohorts were then comprehensively analysed by polychromatic flow cytometry for HIV-1-specific CD4(+) and CD8(+) T-cell functional profile in terms of cytokine production and cytotoxic capacity using IFN-gamma, IL-2, TNF-alpha production and perforin expression, respectively. Comparable levels of highly polyfunctional HIV-1-specific CD4(+) and CD8(+) T-cells were found in LTTS and LTNPs, with low perforin expression on HIV-1-specific CD8+ T-cells, consistent with a polyfunctional/non-cytotoxic profile in a context of low viral burden.Conclusions: Our results indicate that prolonged ART initiated at the time of HIV-1 seroconversion is associated with immuno-virological features which resemble those of LTNPs, strengthening the recent emphasis on the positive impact of early treatment initiation and paving the way for further interventions to promote virological control after treatment interruption.

Tuberculin skin test and interferon-gamma release assay values are associated with antimicrobial peptides expression in polymorphonuclear cells during latent tuberculous infection

It has been reported that patients with progressive tuberculosis (TB) express abundant amounts of the antimicrobial peptides (AMPs) cathelicidin (LL-37) and human neutrophil peptide-1 (HNP-1) in circulating cells, whereas latent TB infected donors showed no differences when compared with purified protein derivative (PPD) and QuantiFERON®-TB Gold (QFT)-healthy individuals. The aim of this study was to determine whether LL-37 and HNP-1 production correlates with higher tuberculin skin test (TST) and QFT values in TB household contacts. Twenty-six TB household contact individuals between 26-58 years old TST and QFT positive with at last two years of latent TB infection were recruited. AMPs production by polymorphonuclear cells was determined by flow cytometry and correlation between TST and QFT values was analysed. Our results showed that there is a positive correlation between levels of HNP-1 and LL-37 production with reactivity to TST and/or QFT levels. This preliminary study suggests the potential use of the expression levels of these peptides as biomarkers for progression in latent infected individuals.

Assessment of immunological changes in Epstein-Barr virus co-infection in Egyptian chronic HCV patients

Epstein-Barr virus (EBV) plays a major role in liver pathology. Similar to other members of the herpesvirus family, EBV establishes a persistent infection in more than 90% of adults. The aim of this study was to evaluate the impact of EBV and chronic hepatitis C co-infection (HCV) on biochemical and immunological responses in patients. The study was conducted in 62 patients and 33 apparently healthy controls. Patients were divided into three groups: group I, consisting of 31 patients with chronic hepatitis C infection (CHC), group II, consisting of eight patients with EBV infection and without HCV infection and group III, consisting of 23 patients with EBV and chronic HCV. The percentage of CD3+ cells, helper CD4+ cells and CD19+ B-cells was measured by flow cytometry. Human interferon-γ (IFN-γ) and interleukin (IL)-15 levels were measured by an ELISA. The levels of liver alanine aminotransferase and aspartate aminotransferase enzymes were higher in EBV/HCV patients compared to that in EBV and HCV mono-infected patients. EBV/HCV patients had significantly reduced percentages of CD3+ and CD4+ cells compared to EBV patients. Serum IFN-γ levels were significantly reduced in EBV/HCV patients (3.86 pg/mL) compared to CHC patients (6.76 pg/mL) and normal controls (4.69 pg/mL). A significant increase in serum IL-15 levels was observed in EBV/HCV patients (67.7 pg/mL) compared to EBV patients (29.3 pg/mL). Taken together, these observations suggest that HCV and EBV co-infection can potentiate immune response dampening in patients.

Lymphocyte subsets in human immunodeficiency virus-unexposed Brazilian individuals from birth to adulthood

Ethnic origin, genetics, gender and environmental factors have been shown to influence some immunologic indices, so that development of reference values for populations of different backgrounds may be necessary. We have determined the distribution of lymphocyte subsets in healthy Brazilian individuals from birth to adulthood. Lymphocyte subsets were determined using four-colour cytometry in a cross-sectional study of 463 human immunodeficiency virus-unexposed children and adults from birth through 49 years of age. Lymphocyte subsets varied according to age, as previously observed in other studies. However, total CD4+ T cell numbers were lower than what was described in the Pediatric AIDS Clinical Trials Group P1009 (PACTG P1009), which assessed an American population of predominantly African and Hispanic backgrounds until the 12-18 year age range, when values were comparable. Naïve percentages and absolute values of CD8+ T cells, as assessed by CD45RA expression, were also lower than the PACTG P1009 data for all analysed age ranges. CD38 expression on both CD4+ and CD8+ T cells was lower than the PACTG P1009 values, with a widening gap between the two studies at older age ranges. Different patterns of cell differentiation seem to occur in different settings and may have characteristic expression within each population.

Monitoring of CD4(+)CD25(high)IL-7R alpha(high) activated T Cells in Kidney Transplant Recipients

Background and objectives In humans, circulating CD4(+)CD25(high) T cells contain mainly regulatory T cells (Treg; FoxP3(+)IL-7R alpha(low)), but a small subset is represented by activated effector T cells (Tact; FoxP3(-)IL-7R alpha(high)). The balance between Tact and Treg may be important after transplantation. The aim of this study was first to analyze and correlate CD4(+)CD25(high) Tact and Treg with the clinical status of kidney transplant recipients and second to study prospectively the effect of two immunosuppressive regimens on Tact/Treg during the first year after transplantation.Design, setting, participants, & measurements CD4(+)CD25(high) Tact and Treg were analyzed by flow cytometry, either retrospectively in 90 patients greater than 1 year after kidney transplantation (cross-sectional analysis) or prospectively in 35 patients receiving two immunosuppressive regimens after kidney transplantation (prospective analysis).Results A higher proportion of Tact and a lower proportion of Treg were found in the majority of kidney recipients. In chronic Immoral rejection, a strikingly higher proportion of Tact was present. A subgroup of stable recipients receiving calcineurin inhibitor-free immunosuppression (mycophenolate mofetil, azathioprine, or sirolimus) had Tact values that were similar to healthy individuals. In the prospective analysis, the proportion of Tact significantly increased in both immunosuppression groups during the first year after transplantation.Conclusions These data highlight distinct patterns in the proportion of circulating Tact depending on the clinical status of kidney recipients. Moreover, the prospective analysis demonstrated an increase in the proportion of Tact, regardless of the immunosuppressive regimen. The measurement of Tact, in addition to Treg, may become a useful immune monitoring tool after kidney transplantation. Clin J Am Soc Nephrol 6: 2025-2033, 2011. doi: 10.2215/CJN.09611010

Gain-of-function mutations in PDR1, a regulator of antifungal drug resistance in Candida glabrata, control adherence to host cells.

Candida glabrata is an emerging opportunistic pathogen that is known to develop resistance to azole drugs due to increased drug efflux. The mechanism consists of CgPDR1-mediated upregulation of ATP-binding cassette transporters. A range of gain-of-function (GOF) mutations in CgPDR1 have been found to lead not only to azole resistance but also to enhanced virulence. This implicates CgPDR1 in the regulation of the interaction of C. glabrata with the host. To identify specific CgPDR1-regulated steps of the host-pathogen interaction, we investigated in this work the interaction of selected CgPDR1 GOF mutants with murine bone marrow-derived macrophages and human acute monocytic leukemia cell line (THP-1)-derived macrophages, as well as different epithelial cell lines. GOF mutations in CgPDR1 did not influence survival and replication within macrophages following phagocytosis but led to decreased adherence to and uptake by macrophages. This may allow evasion from the host's innate cellular immune response. The interaction with epithelial cells revealed an opposite trend, suggesting that GOF mutations in CgPDR1 may favor epithelial colonization of the host by C. glabrata through increased adherence to epithelial cell layers. These data reveal that GOF mutations in CgPDR1 modulate the interaction with host cells in ways that may contribute to increased virulence.