Genetic predisposition to leprosy: A major gene reveals novel pathways of immunity to Mycobacterium leprae

The elucidation of the genetic control of susceptibility to common infectious diseases is expected to provide new and more effective tools for prevention and control of some of the most pressings health needs on a global scale. A major advantage of whole genome based genetic approaches is that no a priori assumptions about mechanisms of pathogenesis need to be made in these studies. Hence, genetic studies can identify previously unrecognized pathways of disease susceptibility and tag critical pathogenic events for further biochemical, immunological or physiological analysis. We have applied this strategy to leprosy, a disease that still claims 400,000 new cases each year. We identified genetic variants in the shared promoter region of the PARK2 and PACRG genes as major risk factors of leprosy susceptibility. Both encoded proteins are part of the cellular ubiquitination system. Specifically, PARK2, the cause of early onset Parkinson's disease, is an E3 ligase that likely is involved in controlled proteolysis, the cellular anti-oxidants response and the regulation of innate immune responsiveness. In addition, numerous E3 ligases have recently been shown to be critical regulators of immunity. While the specific role of PARK2/PACRG in leprosy pathogenesis remains unknown, a number of experimentally testable scenarios can be developed to further explore the role of these proteins in anti-Mycobacterium leprae host responsiveness.     

Segmental heterogeneity of swelling-induced Cl transport in rat small intestine

The effect of cell swelling induced by hypotonic media was studied in segments of rat small intestine. In the Ussing chamber, exposure to a hypotonic medium caused a decrease in short-circuit current (I _sc) and potential difference (V ^ms) in the jejunum, whereas the ileum responded with an increase in I _sc and V ^ms. The transition from one pattern to the other was located about in the middle of the small intestine. Tissue conductance decreased in both segments, probably due to a reduction of paracellular shunt conductance induced by the cell swelling. Voltage scanning experiments revealed that the observed decrease in total tissue conductance in the ileum was caused solely by a decrease in local conductance in the villus region while the crypt conductance did not change, suggesting that the decrease in paracellular conductance of the crypts is compensated by an increase in cellular conductance. The response in both segments was dependent on the presence of Cl⁻ and was blocked by the Cl⁻ channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). It was not affected by the neurotoxin tetrodotoxin. In the jejunum the swelling-induced decrease in I _sc was reduced in the presence of the cyclooxygenase inhibitor, indomethacin, or the lipoxygenase inhibitor, nordihydroguaiaretic acid. In the ileum the Cl⁻ secretion induced by hypotonicity was blocked by the K⁺ channel blocker quinine and was reversed into a decrease in I _sc when serosal Ca²⁺ was zero. We conclude that the observed volume regulatory changes are initiated in the jejunum by an eicosanoid-mediated opening of basolateral Cl⁻ channels and in the ileum by a Ca²⁺-mediated opening of K⁺ channels which enhances apical Cl⁻ efflux. Received: 27 June 1995/Received after revision: 8 December 1995/Accepted: 28 December 1995     

Behavioral Characterization of Alcohol-Tolerant and Alcohol-Nontolerant Rat Lines and an F2 Generation

The Alcohol Tolerant (AT) and Alcohol Nontolerant (ANT) rats, selectively bred for ethanol-induced ataxia on the inclined plane at ALKO in Finland, were moved to the University of Colorado in 1998. The selection phenotype was tested on generation 60 animals in Colorado. In week one, ataxia was measured on the inclined plane 30 minutes after an intraperitoneal dose of 2 g/kg 15% w/v ethanol. Differences in ethanol-induced ataxia between the AT and ANT lines at the University of Colorado were similar to those in the original lines in Finland. In week two, ataxia was measured on the inclined plane at 5 and 30 minutes, and tolerance was measured as the time to regain the original angle of sliding. The AT rats rapidly developed tolerance to 2 g/kg ethanol on the inclined plane; tolerance development was significantly slower in the ANT rats. In week three, the animals were tested for the duration of loss of righting reflex (LORR) and blood ethanol concentration at regain of the righting reflex (BEC_RRR) following a dose of 3.5 g/kg. The AT rats had a significantly higher BEC_RRR than did the ANT rats, but did not differ in LORR. A separate experiment with previously untreated rats demonstrated that naïve animals of the two lines did not differ in BEC_RRR or LORR. AT and ANT rats were genotyped for the mutation that occurs in the gene for the α6 subunit of the GABA_A receptor, a natural mutation that is known to affect benzodiazepine responses. All ANT animals tested carried the mutant allele, whereas some AT families carried the mutation and others were wild type. There was no effect of the mutation in AT rats for any of the phenotypes that were tested. After several generations of brother–sister mating, the AT and ANT lines were more than 90% inbred as determined by genotyping. One AT (wild-type) line and one ANT (mutant) line were selected for breeding an F₂ intercross generation of 1200 animals. They were phenotyped for sensitivity and tolerance to ethanol on each of three consecutive weeks. Order of testing had a modest effect on some of the phenotypes: when tested during the third week as compared to weeks one or two, BEC_RRR was increased, 30-minute sensitivity was increased, and development of acute tolerance was increased. Statistically significant correlations were found between tolerance and sensitivity at both 5 and 30 minutes, and between LORR and BEC_RRR. The smaller (or absence of) significant correlations between others of the phenotypes indicate(s) that they are most likely controlled by different sets of genes.     

Animal danders

Animals release proteins into their surroundings through secretions, as excretions, or as dander. The quantity of dander that is dispersed by cats, dogs, or humans is sufficient to supply food for dust mites and to supply easily measurable quantities of proteins in dust. Fel d 1, Can f 1, and human IgA or IgG can be found in microgram quantities in dust samples. Allergens also can accumulate from the urine of wild or pet rodents. For cats and dogs, the accumulation of dander particles is not related to the cleanliness of the animals. All animals, including humans, provide a fully adequate supply of organic material for bacterial growth in a carpet, provided conditions are sufficiently humid. The authors' preliminary results in Virginia do not find a significant difference in endotoxin between homes with or without animals. The likely explanation for the nonallergic IgG and IgG4 response to cat, dog, or rat allergens is high exposure to proteins from these animals. If the highest levels of cat allergen in a home can result in immunologic tolerance, it is unlikely that primary avoidance would be successful at reducing exposure. The data showing that 80% of Swedish children with cat allergies never had lived with a cat imply that the concentrations of cat allergen in schools or in houses without a cat are sufficient to cause sensitization. Primary prevention would be possible only on a community basis, which is unlikely to occur. Sensitization to cat, rat, dog, or mouse allergens consistently is associated with asthma. In symptomatic children with positive skin test results, there is a strong case for allergen avoidance and a clear need for controlled trials. Controlled trials of avoidance should include houses without cats and schools. Controlling exposure to cat allergens with the cat in situ requires aggressive measures, such as removing reservoirs, washing the cat, and air cleaning. Many allergic or symptomatic children who live with a cat do not have positive skin test results or positive IgE antibodies to cats. Avoidance measures related to animals should be recommended only for individuals with positive skin test results. Increasing evidence shows that exposure to cats, dogs, rats, and other animals can induce a form of immunologic tolerance without causing allergic disease, and it is important to understand why this change occurs with dander allergens rather than with all allergens. The most probable explanations are related to the form and quantity of airborne allergens.     

Inhibition of the development of immediate hypersensitivity by staphylococcal enterotoxin B

We investigated the ability of staphylococcal enterotoxin B (SEB) to modify the immediate hypersensitivity response induced in BALB/c mice following sensitization to ovalbumin (OVA), a response mediated by OVA-reactive Vβ8 T cells. Mice were sensitized by skin painting with OVA every second day over a period of 2 weeks. SEB, a potent activator of Vβ8⁺ T cells, was administered at the same site where OVA was applied (skin of the lower abdomen) following two different protocols. In protocol (A) SEB was injected intradermally 1 day before painting with OVA and on day 7; in protocol B, SEB was injected each time OVA was applied to the skin (eight times). SEB (but not SEA) altered the development of immediate hypersensitivity to OVA, as demonstrated by the reduction in allergen-specific IgE, decreased OVA-specific immediate skin test responsiveness, and prevented the development of increased airways responsiveness after bronchial challenge with OVA. Injections of SEB did not alter the proliferative responses of local draining lymph node cells or spleen mononuclear cells to OVA, indicating that administration of SEB did not inhibit the sensitization to OVA, but shifted the immune response away from an immediate type response (IgE/IgG₁) to IgG_2a, IgG_2b and IgG₃. Although both protocols of SEB treatment did not lead to a major deletion of the Vβ8 T cell population, they did reduce the proliferative response of Vβ8⁺ T cells to OVA. These data indicate that the bacterial toxin SEB is capable of modifying the immediate hypersensitivity response induced by OVA by altering the functional capacity of antigen-reactive Vβ8 T cells.     

Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes)

Novel fluorescent oligonucleotides that contain a 3' minor groove binding group (MGB) hybridize to single-stranded targets with increased sequence-specificity compared to ordinary DNA probes. This reduces non-specific probe hybridization and results in low background fluorescence during the 5' nuclease PCR assay (TaqMan, Applied Biosystems, Foster City, CA). We developed a method for closed-tube genotyping using two allele-specific MGB probes labeled with different fluorophores in one reaction. After PCR, tubes were transported to a fluorescence plate-reader for analysis of fluorescence. Common spreadsheet software was used for automated genotype assignment. As an example, DNA samples from 172 hemochromatosis patients were selected and tested for molecular defects in the HFE gene, i.e., mutations in codon 63 and 282. Tight genotype clusters were observed for both codons and results with MGB probes were identical to conventional genotyping (PCR + restriction-fragment-length-polymorphism). We show that this fast and easy method can be used for large-scale (high-throughput) genetic studies but also for routine molecular diagnostics without post-PCR manipulation of amplicons or the need for real-time quantitative PCR machines. Hum Mutat 19:554-559, 2002.     

In vitro activity of E-4868, a new trifluoroquinolone,compared to six similar compounds

The compound E-4868 (Laboratorios Dr. Esteve) is a trifluoro, 7-azetidinyl quinolone with properties resembling those of other fluoroquinolones. Its activity in vitro was compared to that of six other similar drugs against more than 700 nosocomial isolates using standard methods. The MIC50s of E-4868 for enteric bacilli ranged from 0.015 to 0.25 µg/ml, being highest forProvidencia spp.Pseudomonas aeruginosa strains were two-fold more susceptible to E-4868 than to ofloxacin. MICs of E-4868 forHaemophilus influenzae, Moraxella catarrhalis and pathogenicNeisseria spp. were all 0.12 µg/ml. E-4868 was equal in activity to or eight-fold more active than ciprofloxacin against gram-positive cocci. The MICs of E-4868 for pneumococci were all 0.5 µg/ml but anaerobes such asBacteroides fragilis were generally less susceptible (MIC90, 4 µg/ml). There was almost complete cross-resistance to several other fluoroquinolones. Resistant mutants were selected by a multiple passage technique but the rate of mutation to resistance was very low (< 10^–8) at an 8 x MIC.     

Studies on the mammary tumor-inhibiting effects of diethylstilbestrol and its mono- and diphosphate

Summary Diethylstilbestrol (DES), diethylstilbestrol monophosphate (DES-MP) and diethylstilbestrol diphosphate (DES-DP) were tested for their estrogen receptor affinity, estrogenic potency and mammary tumor-inhibiting activity in vitro and in vivo. DES had a much higher receptor binding affinity than its mono-or diphosphate. All three compounds inhibited the growth of the hormone-dependent MCF-7 and hormone-independent MDA-MB 231 breast cancer line only at relatively high concentrations. The estrogenic potency in the immature mouse uterine weight test decreased in the order DES>DES-MPDES-DP. The hormone-dependent MXT mammary tumor of the mouse was inhibited by all three compounds at a dosage of 1.0 mg/kg per week. At a dose of 0.01 mg/kg, DES, DES-MP, and DES-DP stimulated the tumor growth. Thus, for the first time, a biphasic effect on tumor growth was demonstrated in intact mature animals. As the effects of all three compounds were similar in this assay, a cleavage of the phosphate groups is likely. A decrease in estrogenic potency concomitant with a retained antitumor effect of DES-MP and DES-DP compared to DES was not demonstrable in the mature mouse using the MXT assay, only in the uterotrophic test in the immature mouse.Dedicated to Professor Dietrich Schmähl on occasion of his 60th birthdaySupported by the Deutsche Forschungsgemeinschaft and by the Verband der Chemischen Industrie, Fonds der Chemischen Industrie. The authors thank Dr. Weigert, Asta-Werke AG, Degussa Pharma Gruppe, Bielefeld, FRG, for the analysis of DES-MP and DP     

Practice patterns of Doctor of Pharmacy graduates: educational implications

The purpose of this study was to evaluate the appropriateness of the predominantly clinical PharmD curriculum in light of actual employment patterns of PharmD graduates. Data were gathered via a survey of PharmD graduates from the seven post-baccalaureate programs active since 1972. Respondents were asked about their employment patterns and characteristics; about their formal education in the areas of management, statistics, and research methods; and to indicate whether they had completed residencies or fellowships. The results indicate that many PharmD graduates are employed in positions requiring considerable nonclinical skills, especially management skills. The results further indicate that few respondents have completed formal education, residencies, or fellowships which would prepare them for management or other nonclinical positions. Consequently, it appears that PharmD programs should require considerably more management training and education in order to adequately prepare their graduates for the positions open to them.