An immune-related gene expression atlas of the shrimp digestive system in response to two major pathogens brings insights into the involvement of hemocytes in gut immunity.

Much of our current knowledge on shrimp immune system is restricted to the defense reactions mediated by the hemocytes and little is known about gut immunity. Here, we have investigated the transcriptional profile of immune-related genes in different organs of the digestive system of the shrimp Litopenaeus vannamei. First, the tissue distribution of 52 well-known immune-related genes has been assessed by semiquantitative analysis in the gastrointestinal tract (foregut, midgut and hindgut) and in the hepatopancreas and circulating hemocytes of shrimp stimulated or not with heat-killed bacteria. Then, the expression levels of 18 genes from key immune functional categories were quantified by fluorescence-based quantitative PCR in the midgut of animals experimentally infected with the Gram-negative Vibrio harveyi or the White spot syndrome virus (WSSV). Whereas the expression of some genes was induced at 48 h after the bacterial infection, any of the analyzed genes showed to be modulated in response to the virus. Whole-mount immunofluorescence assays confirmed the presence of infiltrating hemocytes in the intestines, indicating that the expression of some immune-related genes in gut is probably due to the migratory behavior of these circulating cells. This evidence suggests the participation of hemocytes in the delivery of antimicrobial molecules into different portions of the digestive system. Taken all together, our results revealed that gut is an important immune organ in L. vannamei with intimate association with hemocytes.

ABCB1 polymorphisms and the concentrations of lopinavir and ritonavir in blood, semen and saliva of HIV-infected men under antiretroviral therapy.

INTRODUCTION: Lopinavir and ritonavir are frequently included in highly active antiretroviral therapy (HAART) regimens for HIV infection. These drugs are substrates, and may also inhibit and/or induce the P-glycoprotein (ABCB1) transporter, encoded by the polymorphic ABCB1 gene. We investigated the impact of three common exonic ABCB1 polymorphisms on the concentrations of lopinavir and ritonavir in blood, semen and saliva of HIV-infected men under stable HAART containing ritonavir-boosted lopinavir. MATERIALS & METHODS: Blood, semen and saliva samples were collected from 113 subjects, 30-35 minutes before the scheduled morning dose of lopinavir/ritonavir, and trough drug concentrations were measured using LC/MS/MS. The 1236C>T, 2677G>T/A and 3435C>T polymorphisms were genotyped using the single base extension-termination method and ABCB1 haplotypes were statistically inferred. RESULTS: Median (25th-75th percentile) trough concentrations (ng/ml) of lopinavir in plasma, semen and saliva were 6326 (4070-8617), 286.0 (128.4-475.5) and 72.7 (38.0-119.6), respectively. The corresponding concentrations (ng/ml) for ritonavir were 261.8 (172.2-398.6), 17.7 (9.2-27.6) and 5.3 (3.2-9.0), respectively. Univariate and multivariate regression analysis revealed no influence of ABCB1 genotypes or haplotypes on the concentrations of lopinavir and ritonavir in plasma, semen and saliva of HIV-infected men under stable HAART treatment. CONCLUSION: The ABCB1 1236C>T, 2667G>T/A and 3435C>T genotypes and haplotypes are not predictors of lopinavir and ritonavir concentrations in blood plasma, semen or saliva of HIV-infected men under stable HAART treatment. The concentrations of lopinavir and ritonavir in saliva are not reliable predictors of the concentration of these drugs in semen.

Determination of lopinavir and ritonavir in blood plasma, seminal plasma, saliva and plasma ultra-filtrate by liquid chromatography/tandem mass spectrometry detection.

A method based on liquid-liquid extraction followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the simultaneous determination of lopinavir (LPV) and ritonavir (RTV) in human blood, semen and saliva samples. The acquisition was performed in multiple reaction monitoring (MRM) mode, monitoring the transitions: m/z 629 > 447.1 for LPV, 721.18 > 268.02 for RTV and m/z 747.22 > 322.03 for the internal standard (IS). The limit of quantification was 1 ng/mL for both analytes in all matrices. The method was linear in the studied range (1-2000 ng/mL for LPV and 1-200 ng/mL for RTV), with r2 > 0.99 for each drug, and the run time was 4.5 min. The intra-assay precisions (%) were in the ranges of 0.1-14.2 (LPV) and 0.4-12.7 (RTV), the inter-assay precisions were in the ranges of 2.8-15.3 (LPV) and 1.1-12.8 (RTV) and the intra-and inter-assay recoveries were >85% for both drugs. The extraction efficiencies were 73.5-118.4% for LPV and 74.4-126.2% for RTV. The analytical method was applied to measure LPV and RTV concentrations in blood plasma (total and unbound fraction), saliva and semen of six HIV+ individuals under stable treatment with Kaletra soft gel capsules. The results were consistent with previously published data.

Distribution of ABCB1 polymorphisms among Brazilians: impact of population admixture.

INTRODUCTION: Interethnic admixture is a source of cryptic population structure that may lead to spurious genotype-phenotype associations in pharmacogenomic studies. We studied the impact of population stratification on the distribution of ABCB1 polymorphisms (1236C>T, 2677G>T/A and 3435C>T) among Brazilians, a highly admixed population with Amerindian, European and African ancestral roots. METHODS: Individual DNA from 320 healthy adults was genotyped with a panel of ancestry informative markers, and the proportions of African component of ancestry (ACA) were estimated. ABCB1 genotypes were determined by the single base extension/termination method. We describe the association between ABCB1 polymorphisms and ACA by fitting a linear proportional odds logistic regression model to the data. RESULTS: The distribution of the ABCB1 2677G>T/A and 3435C>T, but not the 1236C>T, SNPs displayed a significant trend for decreasing frequency of the T alleles and TT genotypes from White to Intermediate to Black individuals. The same trend was observed in the frequency of the T/nonG/T haplotype at the 1236, 2677 and 3435 loci. When the population sample was proportioned in quartiles, according to the individual ACA estimates, the frequency of the T allele and TT genotype at each locus declined progressively from the lowest (< 0.25 ACA) to the highest (> 0.75 ACA) quartile. Linear proportional odds logistic regression analysis confirmed that the odds of having the T allele at each locus decreases in a continuous manner with the increase of the ACA, throughout the ACA range (0.13-0.94) observed in the overall population sample. A significant association was also detected between the individual ACA estimates and the presence of the T/nonG/T haplotype in the overall population. CONCLUSION: Self-identification according to the racial/color categories proposed by the Brazilian Census is insufficient to properly control for population stratification in pharmacogenomic studies of ABCB1.