Research-based flow cytometry assays for pathogenic assessment in the human B-cell biology of gene variants revealed in the diagnosis of inborn errors of immunity: a Bruton's tyrosine kinase case-study.

Introduction: Inborn errors of immunity (IEI) are an expanding group of rare diseases whose field has been boosted by next-generation sequencing (NGS), revealing several new entities, accelerating routine diagnoses, expanding the number of atypical presentations and generating uncertainties regarding the pathogenic relevance of several novel variants. Methods: Research laboratories that diagnose and provide support for IEI require accurate, reproducible and sustainable phenotypic, cellular and molecular functional assays to explore the pathogenic consequences of human leukocyte gene variants and contribute to their assessment. We have implemented a set of advanced flow cytometry-based assays to better dissect human B-cell biology in a translational research laboratory. We illustrate the utility of these techniques for the in-depth characterization of a novel (c.1685G>A, p.R562Q) de novo gene variant predicted as probably pathogenic but with no previous insights into the protein and cellular effects, located in the tyrosine kinase domain of the Bruton's tyrosine kinase (BTK) gene, in an apparently healthy 14-year-old male patient referred to our clinic for an incidental finding of low immunoglobulin (Ig) M levels with no history of recurrent infections. Results and discussion: A phenotypic analysis of bone marrow (BM) revealed a slightly high percentage of pre-B-I subset in BM, with no blockage at this stage, as typically observed in classical X-linked agammaglobulinemia (XLA) patients. The phenotypic analysis in peripheral blood also revealed reduced absolute numbers of B cells, all pre-germinal center maturation stages, together with reduced but detectable numbers of different memory and plasma cell isotypes. The R562Q variant allows Btk expression and normal activation of anti-IgM-induced phosphorylation of Y551 but diminished autophosphorylation at Y223 after anti IgM and CXCL12 stimulation. Lastly, we explored the potential impact of the variant protein for downstream Btk signaling in B cells. Within the canonical nuclear factor kappa B (NF-κB) activation pathway, normal IκBα degradation occurs after CD40L stimulation in patient and control cells. In contrast, disturbed IκBα degradation and reduced calcium ion (Ca2+) influx occurs on anti-IgM stimulation in the patient's B cells, suggesting an enzymatic impairment of the mutated tyrosine kinase domain.

Virulence properties and antimicrobial resistance of Pseudomonas aeruginosa isolated from cave waters at Roraima Tepui, Guayana Highlands.

Pseudomonas aeruginosa is a prominent member of emerging waterborne pathogens. The environmental reservoirs of multi-resistant phenotypes and other virulence factors in this bacterium are poorly understood. Our study aimed to determine the virulence properties of P. aeruginosa isolated from Roraima Sur Cave (RSC) waters at Guayana Highlands. Based on the best identification at species level by biochemical tests, 16S rRNA sequencing and phylogenetic inferences, one RSC isolate named LG11 was characterized for virulence properties in comparison with P. aeruginosa reference strains. PCR amplification of alginate, elastase, exoenzyme S, exotoxin A, neuraminidase and Quorum-Sensing genes showed a high virulence potential in LG11. This isolate demonstrated multi-resistance to ceftriaxone, tigecycline and imipenem. Pyocyanin production was greater in LG11 (0·478 µg ml-1 ) than the strain ATCC 10145 (0·316 µg ml-1 ), but the highest pigment concentration (2·140 µg ml-1 ) was displayed by the clinical strain CVCM 937 (P = 0·000175). Pronounced biomass production on granite and glass (P < 0·05) and well-developed biofilms indicated the ability of P. aeruginosa from RSC to colonize surfaces found in human and healthcare environments. These data suggest that waters from pristine ecosystems such as RSC could be reservoirs of this opportunistic bacterium carrying important virulence properties with potential epidemiological implications. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows for the first time the occurrence of virulence genes and multi-resistance to antimicrobials in Pseudomonas aeruginosa isolated from cave waters at Guayana Highlands. These findings, together with the biofilm formation on surfaces found in human and healthcare settings, suggest public health risks and the potential of these virulence properties to be transferred from or to native populations in waters. Our results provide important insights to the current knowledge of P. aeruginosa in the environment, setting the basis for future studies driven to assess reservoirs of multi-resistant bacteria and virulence features unknown in pristine ecosystems.

Species composition and abundance of Brevipalpus spp. on different citrus species in Mexican orchards.

We studied the abundance of Brevipalpus spp. in citrus orchards in the Mexican states of Yucatan, Quintana Roo and Campeche. Mites were collected from 100 trees containing a mixture of citrus species where sweet orange was always the main species. Eight collections were made at each location from February 2010 to February 2011. Mites from the genus Brevipalpus were separated from other mites surveyed and their abundance and relationships with the different citrus species were quantified throughout the collection period. A subsample of 25% of the total Brevipalpus mites collected were identified to species level and the interaction of mite species and citrus species were described. Brevipalpus spp. were present on all collection dates and their relative abundance was similar on all citrus species studies. The smallest number of mites collected was during the rainy season. Brevipalpus phoenicis (Geijskes) and Brevipalpus californicus (Banks) were the only two species present and they were found in all locations except Campeche, where only B. phoenicis was present. Yucatan and Campeche are at greater risk of leprosis virus transmission than Quintana Roo because the main vector, B. phoenicis, was more abundant than B. californicus. The implications of our results for the design of more accurate sampling and control methods for Brevipalpus spp. are discussed.

Phytophthora Crown and Root Rot of Zucchini Squash in Almería, Spain.

Zucchini (Cucurbita pepo) is intensively cropped in approximately 4,500 ha of plastic houses in southern Spain. In 2008 to 2009, Consul, Cronos, and Tosca zucchini plants showed symptoms of leaf wilting, basal stem necrosis, and plant death. Incidences of dead plants were 20 to 30% and these plants were distributed in clusters. Phytophthora capsici Leonian was isolated from the basal stems of symptomatic plants, using PDA and cornmeal agar amended with a pimaricin, ampicillin, and rifampicin. Five resultant isolates (PCl-211, PCl-221, PCl-611, PCl-612, and PCl-811) on lima beans agar (LBA) produced white mycelia with lemon-shaped and papillate sporangia borne on long pedicels, but no oospores or chlamydospores. These isolates had an identical ribosomal DNA ITS sequence, matching with that of P. capsici in GenBank. The sequences of two representative isolates, PCl-211 and PCl-811, were deposited in GenBank with accession nos. KC662328 and KC688317, respectively. The pathogenicity of these five isolates was tested on zucchini cv. Consul in 1-liter containers filled with vermiculite in May and September of 2009. Plants were inoculated at the 2 to 3 true-leaf stage. Plates with LBA fully covered with colony of each isolate were separately blended and homogenized with 300 ml of sterile distilled water. Inocula were poured around stem at 50 ml per plant. Each experiment had three replicates and four plants per replicate. Treatments with different isolates were arranged in a randomized complete block design. In both experiments, 12 uninoculated plants served as controls. Test plants were maintained for a month following inoculation in a greenhouse with mean temperatures ranging from 21.9 to 27.9°C and from 20.7 to 24.6°C for the May and September experiments, respectively. The first wilting occurred 5 days after inoculation. At the end of the May experiment, all control plants and those inoculated with PCl-221 remained asymptomatic while 83.3% of those inoculated with PCl-211 and 100% of those with the other isolates were dead. Inoculated plants exhibited crown and root rots, excluding the secondary roots. In the September experiment, 83.3% and 33.3% of plants inoculated with PCl-211 and PCl-221, respectively, were symptomatic, while all plants inoculated with the other isolates were dead. The control plants remained healthy. The pathogen was consistently recovered from symptomatic plants in both experiments. Although P. capsici was reported in peppers (Capsicum annuum) in several provinces of Spain (1), to our knowledge, this is the first report of P. capsici as the causal agent of crown rot in zucchini plants in plastic houses in the Almería Province of Spain, one of the world's largest concentrations of greenhouses. Reference: (1) J. L. Andrés et al. Span J Agric Res 3:326, 2005.

Online monitoring of aluminium in drinking water with fluorimetric detection.

We report a procedure for the online monitoring of aluminium in drinking water by flow injection analysis. The reaction used is the formation of a complex with morin. Under the working conditions, this can be accomplished in an ethanol-rich hydroalcoholic medium, which modifies the fluorescent characteristics of the complex, allowing the determination of aluminium concentrations higher than 3.1 microgl(-1), with a linear application range between 2 and 250 microgl(-1), an R.S.D. of 2.3% (n=10, 120 microgl(-1)) and a sampling frequency of 90 h(-1). The method can thus be considered one of the most sensitive and fastest for the continuous determination of aluminium. In the presence of anionic surfactants, the sensitivity of the determination is increased. In this form, aluminium is detected at concentrations higher than 2.8 microgl(-1), with a linear application range of 2-50 microgl(-1). The procedure was applied to the analysis of aluminium in drinking, river, and underground water. Under the proposed working conditions, only Fe(III), fluoride and phosphates interfere. The interference of Fe(III) can be avoided with hydroxylamine and that of phosphates and polyphosphates by acid digestion of the samples.

Crown Rot of Zucchini Squash Caused by Fusarium solani f. sp. cucurbitae in Almería Province, Spain.

Cucumber, melon, watermelon, and zucchini are intensively cropped in the southern part of Spain where approximately 20,000 ha of the crops are grown in greenhouses. In the spring of 2007, zucchini plants (Cucurbita pepo) at the fruit-bearing stage in three commercial plastichouses in Almería exhibited necrosis on the basal stem, wilt, and death. The incidence of dead plants was 20 to 30%. Fusarium solani was consistently isolated from the basal stems of symptomatic plants on potato dextrose agar (PDA). Cultures of six single-hyphal transfers were identified on the basis of molecular sequences and morphological characteristics (2). Sequences of ribosomal DNA from ITS1 region, 5.8S rDNA, and ITS2 were identical for all six isolates of F. solani. The rDNA sequence of isolate Fscl-3 of F. solani was deposited as GenBank Accession No. AM940070. The pathogenicity of these six isolates of F. solani was tested in two experiments conducted in one plastichouse in Almería. Pregerminated seeds of zucchini cv. Consul were sown in 1-liter containers filled with vermiculite on 21 May and 22 June, 2007 (experiments 1 and 2, respectively). Plants at the one- to two-true-leaf stage or younger were inoculated with a soil drench of 2.0 to 8.4 × 105 propagules per ml). One colonized PDA petri plate of each isolate was blended and homogenized in 500 ml of distilled water. Inoculum (50 ml per plant) was poured around the stem of zucchini plants growing in vermiculite. The experimental design was a randomized complete block with three replicates with each plot comprising four plants (one plant per container). In both experiments, 12 uninoculated plants of the same cultivar served as controls. Plants were maintained for 1 month following inoculation in a greenhouse with mean temperatures ranging between 20.7 and 24.6°C and 23.3 to 29.8°C for experiments 1 and 2, respectively. Wilting first occurred 9 days after inoculation, and 14 days later, all plants inoculated with the F. solani isolates died. Inoculated plants exhibited lesions on the stem base without rot of secondary roots. At the end of the experiment, the uninoculated plants remained asymptomatic. Results of experiment 2, with higher temperatures, were similar. The pathogen was consistently recovered from symptomatic plants in both experiments, fulfilling Koch's postulates. Although F. solani f. sp. cucurbitae race 1 was reported in field squash (C. maxima) in the province of Valencia of east-central Spain (1), to our knowledge, this is the first report of F. solani as the causal agent of crown rot of zucchini plants in plastichouses in the Almería Province of Spain, one of the world's largest concentrations of greenhouses. References: (1) J. García-Jiménez et al. Plant Dis. 81:1216, 1997. (2) C. M. Messiaen and R. Cassini. Taxonomy of Fusarium. Page 427 in: Fusarium: Diseases, Biology, and Taxonomy. P. E. Nelson et al., eds. Pennsylvania State University, University Park, 1981.

First Report of Root and Crown Necrosis of Bean Caused by Pythium aphanidermatum in Spain.

Adult bean plants (Phaseolus vulgaris cv. Festival) growing in a commercial greenhouse in southeastern Spain developed symptoms of root necrosis, necrotic streaks on the basal stems, and plant wilt. A Pythium sp. was isolated consistently from roots and basal stems on selective agar (P5ARP). Single-hyphal transfers produced intercalary antheridia, oogonia (23 to 26 µm in diameter), oospores (18 to 20 µm in diameter), and zoospores in toruloid sporangia. Cardinal temperatures were a minimum of 10°C, an optimum of 28 to 34°C, and a maximum of 40°C. Daily growth rate on corn meal agar at 25°C was 15 mm. The internal transcribed spacer 1 (ITS 1) rDNA sequence of the isolate matched the sequences of Pythium aphanidermatum in GenBank. The sequence of isolate Py-294 was deposited in GenBank, Accession No. AM396563. This isolate was identified as P. aphanidermatum on the basis of morphological and cultural characteristics (1) and the ITS rDNA sequence. To fulfill Koch's postulates, 50 ml of inoculum of isolate Py-294 was used to inoculate bean plants (cvs. Donna and Emerite) at the five-leaf stage. The inoculum was prepared by homogenizing 2-week-old potato dextrose agar-petri plate cultures in 300 ml of distilled water. The plants were maintained in a greenhouse at temperatures of 18.8 to 30.3°C. Irrigation water had an electric conductivity of 0.5 to 0.6 dS·m-1 while the nutrient solution had 1.9 to 2.1 dS·m-1. Two months after sowing, 35.4 and 100% of cvs. Donna and Emerite, respectively, developed root necrosis, while control plants grown in bags containing noninoculated perlite remained healthy. The pathogen was reisolated from roots and basal stems of symptomatic plants. The test was repeated with similar results. To our knowledge, this is the first report of P. aphanidermatum as the causal agent of root and crown necrosis of adult bean plants in Spain. Reference: (1) A. J. Van der Plaats-Niterink. Stud. Mycol. 21:242, 1981.

Blockade of GRP receptors inhibits gastric emptying and gallbladder contraction but accelerates small intestinal transit.

BACKGROUND & AIMS: This study was designed to characterize [D-F(5)Phe(6)D-Ala(11)]Bn(6-13)OMe (BIM26226) as a gastrin-releasing peptide (GRP)-preferring bombesin receptor antagonist and to determine whether GRP physiologically regulates gastrointestinal motility. Intravenous BIM26226 (5-500 microg. kg(-1). h(-1)) inhibits GRP-induced gallbladder contraction and plasma cholecystokinin (CCK) release in a dose-dependent fashion. METHODS: Gastric emptying and small bowel transit of a solid meal were quantified using scintigraphy. Meal-stimulated gallbladder contraction was measured by sonography in a 2-period crossover design. RESULTS: Intravenous BIM26226 potently inhibited gastric lag time (114 +/- 7 vs. 41 +/- 6 minutes [control]) and gastric emptying rate (0.11 +/- 0.02%/min vs. 0.26 +/- 0.04%/min [control]), whereas concomitant infusion of BIM26226 accelerated small bowel transit time (153 +/- 41 vs. 262 +/- 20 minutes [control]). A continuous liquid meal perfusion into the duodenum induced complete gallbladder contraction (t(50%), 35 +/- 4 minutes), which BIM26226 inhibited significantly (t(50%), 64 +/- 8 minutes). BIM26226 did not alter plasma CCK response, indicating that circulating CCK did not mediate these effects. CONCLUSIONS: These data show that BIM26226 is a potent antagonist of exogenous and endogenous GRP and suggest that GRP is a major physiologic regulator of gastric emptying, small bowel transit, and gallbladder contraction.

Sex trading and psychological distress among women recruited from the streets of Harlem.

OBJECTIVES: This study examines the relationship between sex trading and psychological distress and the implications of that relationship for prevention of human immunodeficiency virus among a sample of young women recruited from the streets of Harlem. METHODS: Interviews were conducted with 346 predominantly drug-using women, aged 18 to 29 years, of whom 176 had exchanged sex for money or drugs in the previous 30 days and were categorized as "sex traders." Psychological distress was measured by using the Brief Symptom Inventory. RESULTS: Sex traders scored significantly higher than non-sex traders on the General Severity Index and on eight of the nine subscales of the Brief Symptom Inventory. Multivariate analysis indicated that after adjustments were made for age; ethnicity; pregnancy; recent rape; perceived risk for acquired immunodeficiency syndrome; current, regular crack use; and current, regular alcohol use, sex traders scored 0.240 units higher on the General Severity Index than non-sex traders. CONCLUSIONS: Poor mental health and drug dependence may under-mine the motivation and ability of these sex traders to adopt safer sex behavior. Therefore, interventions need to be integrated with mental health services and drug treatment to reduce risk behavior in this population.

Crack cocaine smokers who turn to drug injection: characteristics, factors associated with injection, and implications for HIV transmission. The Multicenter Crack Cocaine and HIV Infection Study Team.

A survey of 1220 street-recruited crack cocaine smokers revealed that crack smokers may turn to drug injection to ease crack withdrawal. Crack smokers who later injected tended to smoke crack more heavily and for longer periods than those who did not inject. The initiation of injection was significantly associated with ever snorting heroin (prevalence ratio [PR] = 3.4, 95% confidence interval [CI] = 2.0-5.9) or snorting heroin specifically while smoking crack (PR = 2.3, 95% CI = 1.3-4.0), suggesting that snorted heroin use may mediate the transition to injection among crack smokers. Programs to prevent and treat crack dependence may prevent later injection and injection-related infections including HIV.

Crack cocaine smoking and oral sores in three inner-city neighborhoods.

Crack cocaine causes blisters, sores, and cuts on the lips and in the mouths of persons who smoke it, and such sores may facilitate the oral transmission of HIV. We recruited young adults aged 18-29 years, who either were current regular crack smokers, or who had never smoked crack, from inner city neighborhoods in New York, Miami, and San Francisco. Participants were interviewed for HIV risk behaviors and history of recent oral sores and were tested for HIV, syphilis, and herpes simplex virus (HSV) antibodies. Among the 2,323 participants recruited, 1,404 (60%) were crack smokers. Crack smokers (10.0%) were more likely than nonsmokers (4.5%) to report having had oral sores in the past 30 days [prevalence odds ratio (POR) 2.4, 95% confidence interval (CI) 1.7-3.4]. Sores were also more prevalent among those who had ever injected drugs (14.3%) than among those who had not (6.7%; POR 2.3, 95% CI 1.7-3.4), and among those with HIV infection (14.3%) than among those without it (8.0%; POR 1.9, 95% CI 1.3-2.8). Among the 429 participants who reported receptive oral sex, those who reported oral sores were more likely than those who did not to have HIV infection, after other HIV risk factors were controlled for (adjusted POR 1.9, 95% CI 1.0-3.6). Our results confirm that crack smokers have a high prevalence of oral sores and provides evidence that these sores, although infrequently, may facilitate oral transmission of HIV.

Intersecting epidemics--crack cocaine use and HIV infection among inner-city young adults. Multicenter Crack Cocaine and HIV Infection Study Team.

BACKGROUND AND METHODS: The smoking of "crack" cocaine is thought to be associated with high-risk sexual practices that accelerate the spread of infection with the human immunodeficiency virus (HIV). We studied 2323 young adults, 18 to 29 years of age, who smoked crack regularly or who had never smoked crack. The study participants, recruited from the streets of inner-city neighborhoods in New York, Miami, and San Francisco, were interviewed and tested for HIV. This report presents the findings for the 1967 participants (85 percent) who had never injected drugs. RESULTS: Of the 1137 crack smokers, 15.7 percent were positive for HIV antibody, as compared with 5.2 percent of the 830 nonsmokers (prevalence ratio adjusted for the city, 2.4; 99 percent confidence interval, 1.7 to 3.6). The prevalence of HIV was highest among the crack-smoking women in New York (29.6 percent) and Miami (23.0 percent). Of the 283 women who had sex in exchange for money or drugs, 30.4 percent were infected with HIV as compared with 9.1 percent of the 286 other women (prevalence ratio, 3.1; 99 percent confidence interval, 1.9 to 5.1); of the 91 men who had anal sex with other men, 42.9 percent were infected with HIV as compared with 9.3 percent of the 582 men who did not have anal sex with other men (prevalence ratio, 4.7; 99 percent confidence interval, 3.0 to 7.4). In multivariable analyses, these high-risk sexual practices accounted for the higher prevalence of HIV infection among the crack smokers, as compared with those who did not smoke crack. Women who had recently had unprotected sex in exchange for money or drugs were as likely to be infected as men who had had sex with men (40.9 percent vs. 42.9 percent). CONCLUSIONS: In poor, inner-city communities young smokers of crack cocaine, particularly women who have sex in exchange for money or drugs, are at high risk for HIV infection. Crack use promotes the heterosexual transmission of HIV.

High-risk sex behavior among young street-recruited crack cocaine smokers in three American cities: an interim report. The Multicenter Crack Cocaine and HIV Infection Study Team.

Since crack cocaine appeared in urban areas in the United States in the mid-1980s, reports have suggested that crack smokers may be at increased risk of sexually transmitted diseases (STDs), including infection with HIV, because they have multiple sex partners, trade sex for money or drugs, and rarely use condoms. A cross-sectional survey is being conducted in urban neighborhoods in Miami, New York and San Francisco--where crack use is common--to explore these issues. Indigenous street outreach workers are recruiting men and women who are either current regular crack smokers or who have never smoked crack; each group is further stratified according to whether participants had ever injected drugs. Participants were interviewed about their sexual and drug-use practices. Overall, crack smokers, whether injectors or not, engaged in higher-risk sexual behaviors than nonsmokers, reported greater numbers of sex partners than nonsmokers, and were more likely than nonsmokers to have exchanged sex for money or drugs or to have had an STD. Differences between crack smokers and nonsmokers were generally greater among non-injectors than among injectors, and generally greater among women than among men. Condom use, although somewhat more common with paying than nonpaying partners, was infrequent overall. Most of the subjects had not been in substance abuse treatment in the preceding 12 months, and a majority had never been in substance abuse treatment. Education and prevention programs specifically targeted at crack smokers not currently in substance abuse treatment are needed to reach these high-risk persons.