A rationale for family therapy specialization in early intervention

The complete nucleotide sequence of a naturally occurring 5.36-kb streptomycin and sulphonamide resistance plasmid, designated pIG1, isolated from type D Pasteurella multocida was determined. A 1.6-kb noncoding region and a 1.4-kb region encoding three putative proteins were shown by sequence homologies and functional characterizations to be involved in the replication and mobilization of pIG1, respectively. The remaining sequence carried an unusual arrangement of streptomycin- and sulphonamide-resistant genes when compared to various other plasmids. It appears that the antibiotic resistance region of pIG1 may have evolved by recombination between three different short direct repeat DNA sequences. A 4.5-kb recombinant plasmid was constructed by replacing the antibiotic resistance genes of pIG1 with a kanamycin resistance gene and seven unique restriction sites. The resulting plasmid, designated pIG112, stably replicates in P. multocida, Pasteurella haemolytica, Actinobacillus pleuropneumoniae, and Escherichia coli and can be introduced into these organisms by either transformation or conjugation. This vector exists at approximately 70 copies per cell in P. multocida and approximately 20 copies per cell in E. coli. To demonstrate plasmid-borne gene expression in P. multocida, the P. multocida dermonecrotic toxin gene, toxA, and a genetically modified form of this gene were cloned into pIG112 and expressed in high amounts in a nontoxigenic P. multocida strain. Cell culture assays demonstrated that nontoxigenic P. multocida expressing toxA was cytopathic, whereas a strain expressing the modified toxA derivative was not.     

Bacterial flora of the respiratory tract in patients with long term tracheostomy--colonization of the lower respiratory tract by Pseudomonas aeruginosa

Throat secretions (TS) and bronchial secretions aspirated from tracheostomy sites (TSTA) from six subjects with long term tracheostomy were simultaneously collected and then cultured every two weeks from January, 1990, to December, 1992. Isolated bacteria were mainly alpha-streptococci (96.2%) and Neisseria (69.6%) in TS, and Pseudomonas aeruginosa (75.7%) in TSTA. In all cases, P. aeruginosa was isolated from and colonization of the lower respiratory tract by this organism was apparent 24.4 months, on average, after tracheostomy. There were ten episodes of respiratory infection in five cases, eight of which occurred after colonization. P. aeruginosa was the causative organism in seven of these episodes. Findings in patients with long term tracheostomy indicated separate colonization of the upper and lower respiratory tracts and that P. aeruginosa colonized the lower respiratory tract. The colonization of the lower respiratory tract by P. aeruginosa would thus appear to be an important factor inducing respiratory infection.     

Homoeopathic Belladonna

Distribution of Corynebacterium kutscheri was determined in 41 rats housed in a conventionally managed colony that were infected naturally and subclinically. At 2, 5, 10, 20 and 25 months after initial isolation of C. kutscheri, attempts were made to isolate C. kutscheri from 17 sites, with a new selective medium, FNC agar. In total, the prevalence (97.6%) of C. kutscheri isolation was significantly (P < 0.001) higher than the frequency (70.7%) of antibody detection. None of the rats manifested any distinct clinical signs of disease and macroscopic lesions caused by C. kutscheri were not detected. In 40 rats with subclinical infection, the organisms were most frequently isolated from the oral cavity, esophagus, cecal contents, and colon and rectum (> 95.0%). The isolation rate was next highest in the trachea, submaxillary lymph nodes, and nasal cavity (47.5 to 52.5%). The organisms hardly colonized the lung, liver, and kidney. Mean numbers of organisms found in the esophagus, cecal contents, and colon and rectum ranged from 10(3.9) to 10(4.2) CFU/g, and were significantly (P < 0.05, P < 0.01) high in comparison with those in the lung. These results indicated that many healthy rats in the naturally infected colony harbored C. kutscheri, and the organisms colonized the oral cavity, esophagus, cecal contents, and colon and rectum most frequently.     

In vivo effect of Pasteurella haemolytica infection on bovine neutrophil morphology

OBJECTIVE: To determine whether characteristic changes in neutrophil morphology caused in vitro by Pasteurella haemolytica leukotoxin (LKT) can be observed in vivo by electron microscopic examination of infected tissue chamber fluids and pneumonic lungs. ANIMALS: 7 mixed-breed beef calves. PROCEDURE: Tissue chambers were implanted subcutaneously in 3 calves and were inoculated with P haemolytica or phosphate-buffered saline solution. Chamber fluid samples, obtained at 8 and 32 hours after inoculation, were examined, using electron microscopy. Experimental pneumonia was induced in an additional 4 calves by transthoracic inoculation with P haemolytica. These calves were euthanatized at 6, 12, 24, and 36 hours after inoculation and lung sections were examined, using transmission electron microscopy. RESULTS: On examination, using transmission electron microscopy, neutrophils in lung sections and tissue chamber fluids had cytoplasmic and nuclear changes indicative of irreversible cell injury, including cell swelling, loss of plasma membrane ruffling, mitochondrial swelling, autolytic vacuolation, disruption of plasma membrane, nuclear pyknosis, karyolysis, and karyorrhexis. On examination, using scanning electron microscopy, leukocytes obtained from tissue chambers did not have their typical convoluted surfaces, but appeared rounded and swollen or shrunken with pitted surfaces. CONCLUSIONS: Pasteurella haemolytica-induced changes in neutrophil morphology in vivo were similar to those previously induced by in vitro exposure of neutrophils to LKT. Changes were suggestive of injury initiated by damage to the plasma membrane, which is consistent with the mechanism of action of pore-forming cytolysins. CLINICAL RELEVANCE: Pasteurella haemolytica LKT appears to be an important virulence factor in vivo; a fact that should be addressed in the development of vaccines.     

Immunohistochemical characterization of intraepithelial and subepithelial mononuclear cells of the upper airways

The upper airway is the first site of exposure to inhaled antigens and the site of initiation of mucosal immunity to certain antigens; however, the intraepithelial lymphoid populations of this region have not been well characterized. We studied 6-mu frozen tissue sections from tonsils, adenoids, and nasal mucosae using immunohistochemistry and a panel of antibodies to mononuclear antigens to determine whether nasal mucosa contained distinctive populations of mononuclear cells. Intraepithelial lymphocytes (IELs) of nasal mucosa were CD3+, CD8+, and mainly CD5+. Tonsil and adenoid both showed diffuse CD8+ IELs; clusters of CD4+ IELs were associated with B cells within the crypt epithelium. All nasal IELs were uniformly negative for Leu8 (homing receptor analog of Mel14). Scattered Leu8-positive cells were present within tonsil and adenoid crypt epithelium only. Nasal IELs rarely expressed HML1 and were often CD7-, whereas the majority of tonsillar and adenoidal IELs were HML1+ and variably CD7+. In nasal mucosa and in deep submucosa of tonsil and adenoid, 80 to 90% of T cell receptor expression was of alpha/beta type. There was a concentration of gamma/delta T cell receptor-positive cells in intraepithelial and subepithelial zones of tonsil and adenoid, with areas of up to 30% gamma/delta T cell receptor positivity. A population of intraepithelial dendritic cells was identified in all three tissues expressing mononuclear phagocyte system antigens CD14 and KiM1P, but lacking CD1a. Virtually no B cells and no organized subepithelial lymphoid tissue were identified in nasal mucosa. Nasal mucosal lymphoid tissue seems to differ from that of endodermally derived mucosae, tonsil, and adenoids to share similarities with both mucosa-associated lymphoid tissue and peripheral lymph nodes.     

The incidence and role of actinomyces in recurrent acute tonsillitis

Although actinomyces has been identified in between 1.77% and 37% of resected tonsils its possible role in recurrent acute tonsillitis has received little attention. A histological and bacteriological study of 129 pairs of tonsils from patients with recurrent acute tonsillitis showed actinomyces to be present in 29.5%. The organism, however, was also present in 40% of tonsils from 10 patients with no history of tonsillar disease. In neither of these groups was there any specific evidence of tissue reaction to actinomyces nor was there a male preponderance as in clinical actinomycosis. The presence of actinomyces in the tonsil was not favoured by the concurrence of beta-lactamase producing bacteria. These data indicate that actinomyces does not have a causal role in recurrent acute tonsillitis.     

Domestic violence. A clinician''s strategy

Pasteurella haemolytica was isolated from three of 18 grass samples and four of 18 water samples collected from two grazing fields occupied by sheep. This microorganism was also isolated from three of nine straw bedding samples collected from a pen housing ewes affected by mastitis caused by P. haemolytica. The same ewes developed scabbed papilloma-like lesions on the teat and udder skin. These lesions were colonized by P. haemolytica of various serotypes. Colder, wetter weather seems to prolong the survival of P. haemolytica in the environment of sheep. Survival of virulent strains of P. haemolytica in the environment could accumulatively increase the bacterial count, contributing to their transmission from animal to animal. The preference of P. haemolytica for colder, wetter conditions was confirmed in the laboratory where this microorganism survived longer in distilled water, phosphate-buffered saline, Todd-Hewitt broth, and ewe's milk kept at 4 degrees C.     

Expression of recombinant DNA introduced into Chlamydia trachomatis by electroporation

Electroporation was used to introduce DNA into the elementary bodies of the obligate parasitic bacterium Chlamydia trachomatis. The source of DNA for these experiments was the chimeric plasmid pPBW100, which was constructed from the well-characterized 7.5-kb plasmid of C. trachomatis and the Escherichia coli plasmid pBGS9. To select directly for C. trachomatis carrying pPBW100, an in-frame gene fusion between the chlamydial promoter P7248 and a promoterless chloramphenicol acetyltransferase (cat) cassette was incorporated into the plasmid. After infection of McCoy cells with electroporated elementary bodies containing pPBW100, the following were observed: (i) the plasmid DNA was detected inside the chloramphenicol-resistant chlamydial inclusions by in situ and Southern hybridization analyses; (ii) both physical and biochemical evidence showed that chloramphenicol acetyltransferase was synthesized by the electroporated C. trachomatis; (iii) expression of P7248::cat was developmentally regulated and occurred during the early stages of chlamydial reticulate body development; and (iv) although the expression from P7248::cat was mainly transient, there were rare instances where chloramphenicol-resistant C. trachomatis were observed after four passages.     

Homology throughout the multiple 32-kilobase circular plasmids present in Lyme disease spirochetes

We have characterized seven different 32-kb circular plasmids carried by Borrelia burgdorferi isolate B31. Restriction endonuclease recognition site mapping and partial sequencing of these plasmids indicated that all seven are probably closely related to each other throughout their lengths and have substantial relationships to cp8.3, an 8.3-kb circular plasmid of B. burgdorferi sensu lato isolate Ip21. With the addition of the seven 32-kb plasmids, this bacterial strain is known to carry at least 10 linear and 9 circular plasmids. Variant cultures of B. burgdorferi B31 lacking one or more of the 32-kb circular plasmids are viable and, at least in some cases, infectious. We have examined a number of different natural isolates of Lyme disease borreliae and found that all of the B. burgdorferi sensu stricto isolates and most of the B. burgdorferi sensu lato isolates tested appear to carry multiple 32-kb circular plasmids related to those of B. burgdorferi B31. The ubiquity of these plasmids suggests that they may be important in the natural life cycle of these organisms. They may be highly conjugative plasmids or prophage genomes, which could prove to be useful in genetically manipulating B. burgdorferi.     

Serologic responses of young colostrum fed dairy calves to antigens of Pasteurella haemolytica A1

The potential and limitations of early calfhood vaccination to induce active immunity to Pasteurella haemolytica A1 in conventional colostrum fed calves were investigated. Holstein dairy calves (n = 29) were vaccinated at 2 and 4 weeks of age, or at 6 and 8 weeks of age with a commercial culture supernatant vaccine (Presponse, Langford Inc., Guelph, Ont., Canada), or remained unvaccinated as controls. Serum antibody titres were measured using an indirect bacterial agglutination assay, a leukotoxin neutralization assay, and enzyme immunoassays for antibodies of the IgM, IgG1, and IgG2 isotypes binding purified capsular polysaccharide of P. haemolytica A1. Seroconversion (fourfold or greater increase in serum antibody titre) rates were compared using Fisher's exact test. The effects of passive antibody titres and age on response to vaccination were assessed by linear modelling. Vaccination at 2 and 4 weeks of age was associated with 40%, and 0% of calves seroconverting on the basis of agglutinating antibody titres, and leukotoxin neutralizing titres respectively, and 50%, 0%, and 0% seroconverting on the basis of IgM, IgG1 and IgG2 antibodies to capsular polysaccharide, respectively. Agglutinating antibody responses were not related to prevaccination antibody titres, or to age at vaccination. Higher responses (p = 0.08) to leukotoxin were observed in older calves (after taking differences in prevaccination titres into account). Statistical analyses of responses to capsular polysaccharide among calves with comparable prevaccination IgG1 antibody titres revealed significantly higher IgM, IgG1 and IgG2 responses in older calves. Rising titres of IgM antibodies in nonvaccinated calves after 5 weeks of age suggest natural exposure to P. haemolytica A1 or antigens which result in serologic cross-reactions as a means of priming immune responses.     

Antimicrobial susceptibilities and molecular epidemiology of Salmonella enterica serotype enteritidis strains isolated in Hong Kong from 1986 to 1996

The incidence of salmonellosis has been increasing in Hong Kong since 1989. The most common Salmonella enterica serotype isolated in 1994 was S. enteritidis. The antimicrobial susceptibilities and molecular epidemiology of 275 S. enteritidis strains isolated in this locality between 1986 and 1996 were studied. Over 99% of the isolates were susceptible to 17 of the 19 antimicrobial agents tested. One isolate harbored an autotransferring plasmid that confers resistance to tetracycline and trimethoprim-sulfamethoxazole. Another isolate harbored a mobilizable plasmid that confers resistance to ampicillin and cephalothin. This isolate was found to produce a beta-lactamase with a pI of 5.2. A total of 264 isolates (96%) were found to harbor one to five plasmids, and the majority (254) harbored a 60-kb plasmid. Of these isolates, 94% contained identical 60-kb plasmids. Based on plasmid profiles, plasmid and chromosomal fingerprints, ribotypes, and randomly amplified polymorphic DNA (RAPD) patterns, 170 (62%) isolates were allocated to group 1b. About 90% of isolates had identical or similar DNA fingerprints, ribotypes, and RAPD patterns, suggesting that a predominant clone of S. enteritidis was circulating in Hong Kong during the period being studied.     

Nickel content of human palatine tonsils: analysis of small tissue samples by flameless atomic absorption spectrophotometry

We describe a method for determining the nickel content of small tissue samples by flameless atomic absorption spectrophotometry in this case biopsy specimens from human palatine tonsils. Contact between tissue samples and metallic objects was avoided, except for the use of biopsy forceps (Type No. 8150.00 Wolf, stainless steel) for collecting samples, to imitate the actual procedure when small biopsy specimens are removed from the nasal mucosal membranes in nickel workers for histopathological and chemical investigations. Nickel contamination from this instrument was insignificant at the precision of the present procedures. The mean concentration of nickel in 15 tonsils was 13.5 +/- 7.0 (SD) microng/100g (wet wt). The mean nickel concentration in eight different samples of the same tonsil was 5.6 +/- 2.7 (SD microng/100 g.     

Fluoride and hard cheese exposure on etched enamel in neck-irradiated patients in situ

Oral anaerobic treponemes are assoicated with active periodontal disease and may comprise up to 57% of the microbiota in periodontal pockets. Four treponeme strains (designated U2a, U2b, U9b, and U9c) isolated from clincial cases were found to harbor a new 4.2-kb plasmid when plasmid DNA was extracted and purified employing the Qiagen Plasmid Kit. This plasmid differs from the smaller plasmids (2.0-, 2.6-, and 2.7-Kb) reported previously by others in Treponema denticola. The newly discovered 4.2-kb plasmid was found to be the same in all four treponeme strains by restriction endonuclease analysis. It is a circular plasmid since restriction with PstI, Pvu II, Sma I, Xma I, Ava 1 or Bam HI produced a single band of the same size. Bacterial strain U2b was shown to be Treponema socranskii and U9c to be T. denticola. The plasmid is designated "pTS1". The presence of the same plasmid in different species of the treponemes isolated from the same patient suggests the possibility of a naturally occurring genetic transfer system within the oral spirochetes or their ability to take up and maintain mobilizable plasmids.     

Cytokine and antibody responses in women infected with Neisseria gonorrhoeae: effects of concomitant infections

The levels of interleukin (IL)-1, IL-6, IL-8, IL-10, and transforming growth factor-beta in sera and genital tract secretions from women with gonococcal cervicitis and other genital infections were examined. Cytokines were not elevated in genital secretions from gonococcus-infected compared with uninfected patients. The level of serum IL-6 was higher in gonococcus-infected than in uninfected patients at recruitment. Serum, but not local, IL-1 and IL-6 levels were elevated in patients concomitantly infected with Trichomonas vaginalis or Chlamydia trachomatis in addition to Neisseria gonorrhoeae compared with levels in patients infected with any single organism. Concomitant infection altered neither the total immunoglobulin concentrations nor the levels of antigonococcal antibodies in serum or local secretions. The results suggest that N. gonorrhoeae induces only a limited cytokine and antibody response during uncomplicated cervical infections; however, the presence of other sexually transmitted disease-causing organisms can alter the systemic cytokine but not the antigonococcal antibody levels.     

Differential kinetics and distribution of antibodies in serum and nasal and vaginal secretions after nasal and oral vaccination of humans

Although nasal vaccination has emerged as an interesting alternative to systemic or oral vaccination, knowledge is scarce about the immune responses after such immunization in humans. In the present study, we have compared the kinetics and organ distribution of the antibody responses after nasal and oral vaccination. We immunized female volunteers nasally or orally with cholera toxin B subunit (CTB) and determined the specific antibody levels in serum and nasal and vaginal secretions, as well as the number of circulating antibody-secreting cells, before immunization and 1, 2, 3, 6, and 26 weeks thereafter. Nasal vaccination induced 9-fold CTB-specific immunoglobulin A (IgA) and 56-fold specific IgG antibody increases in nasal secretions, whereas no significant IgA increase was seen after oral vaccination. Both oral and nasal vaccination resulted in 5- to 6-fold CTB-specific IgA and 20- to 30-fold specific IgG increases in vaginal secretions. Strong serum responses to CTB were also induced by both routes of vaccination. A notable difference between nasal and oral vaccination was that the nasal route elicited a specific antibody response with a later onset but of much longer duration than did the oral route. We conclude from this study that the nasal route is superior to the oral route for administering at least nonliving vaccines against infections in the upper respiratory tract, whereas either oral or nasal vaccination might be used for eliciting antibody responses in the female genital tract.     

Experimental contact of bighorn sheep (Ovis canadensis) with horses and cattle, and comparison of neutrophil sensitivity to Pasteurella haemolytica cytotoxins

Peripheral blood neutrophils from horses, cattle, and Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were evaluated for susceptibility to cytotoxin-dependent lysis of different biotypes and serotypes of Pasteurella haemolytica of domestic sheep, cattle, bighorn sheep, or mountain goat (Oreamnos americana) origin utilizing a cytotoxicity assay which measures the degree of bacteria cytotoxin-killing of neutrophils. All isolates of P. haemolytica (biotypes A and T) were noncytotoxic to horse neutrophils. Thirteen of 18 R haemolytica biotype A isolates were cytotoxic (> 50% neutrophil death in vitro) to bighorn sheep neutrophils, and four of 10 P. haemolytica biotype A isolates were cytotoxic to neutrophils of cattle; P. haemolytica biotype T (= Pasteurella trehelosi) isolates were noncytotoxic to neutrophils of bighorn sheep and cattle. When six bighorn sheep were pastured with three horses, only P. haemolytica biotype T isolates were recovered from the bighorn sheep throughout the study; Pasteurella spp. organisms were not isolated from the three horses. At initiation of a study where five bighorn sheep were pastured with three cattle, P. haemolytica biotype A, serotype 1, 2 was isolated from all three cattle, and only P. haemolytica biotype T isolates were recovered from the bighorn sheep. One bighorn sheep died in each of the horse and cattle copasturing experiments. Pasteurella haemolytica was not isolated from the bighorn sheep which died in the horse copasturing experiment. A noncytotoxic P. haemolytica biotype A, serotype 2 was isolated at necropsy from the bighorn which died in the cattle contact experiment. Based on these experiments, we believe bighorn sheep and horse association would not be detrimental to bighorns due to P. haemolytica induced pneumonia.     

Observations on health care issues in the former Soviet Union

Bacteriologic samples from 31 young men were cultured quantitatively for aerobes and anaerobes; these samples included 31 specimens of tonsils (16 infected and 15 healthy), 16 specimens from pericoronal pockets of lower third molars (11 infected and 5 symptom-free), and 6 postoperative specimens from lower-third-molar extraction sockets. Anaerobes were isolated more often from infected third molars than from infected tonsils (14.5 isolates vs. 8.4 isolates, respectively; P < .001). Infected tonsil samples contained significantly more anaerobic species if an adjacent partly erupted lower third molar was present rather than absent (10.3 isolates vs. 6.9 isolates, respectively; P < .05). Eubacterium aerofaciens, Clostridium species, Peptostreptococcus micros, and Prevotella oris were frequently isolated. Streptococcus salivarius was found more frequently in tonsillar specimens, whereas Corynebacterium species, Prevotella denticola, Capnocytophaga species, Peptostreptococcus anaerobius, and Lactobacillus species were more common in pericoronal pocket samples. Thus, partial eruption of lower third molars increases the number of anaerobic bacterial species on tonsils and many species can be isolated simultaneously from both tonsils and lower third molars.     

Susceptibility of Dall sheep (Ovis dalli dalli) to pneumonia caused by Pasteurella haemolytica

We evaluated susceptibility of Dall sheep (Ovis dalli dalli) to bacterial pneumonia induced by two strains of Pasteurella haemolytica of domestic sheep origin by evaluating the sensitivity of blood neutrophils of eight Dall sheep to lysis by cytotoxins of P. haemolytica, and by intratracheal inoculation of three Dall sheep, two bighorn sheep (Ovis canadensis), and two domestic sheep with 3.7 x 10(6) or 2.5 x 10(7) colony forming units of P. haemolytica. Neutrophils from the Dall sheep were more sensitive to lysis by cytotoxins from supernatants of a P. haemolytica, biotype A, serotype 2 (A2), of domestic sheep origin, than were neutrophils from six bighorn sheep. This cytotoxic bacterium was the same isolate that was used for intratracheal inoculation of two Dall sheep and two domestic sheep. Inoculation of this cytotoxic P. haemolytica A2 resulted in fatal fibrinopurulent pleuropneumonia in the first Dall sheep within 24 hr of inoculation, and pneumonic lesions in the second Dall sheep before it was euthanized 52 hr after inoculation. This strain of P. haemolytica A2 did not cause respiratory disease when inoculated into two domestic sheep. A noncytotoxic strain of P. haemolytica; biotype T, serotype 3,4,10 of domestic sheep origin did not result in pneumonia in the third Dall sheep or two bighorn sheep. Prior to inoculation, P. haemolytica, biotype T isolates were obtained from all three Dall sheep, but none of these isolates was cytotoxic. At necropsy, cytotoxic P. haemolytica A2 was isolated from lungs and other tissues of the two pneumonic Dall sheep. Based on these results, we conclude that Dall sheep appear to be at least as sensitive as bighorn sheep to pneumonia caused by P. haemolytica A2 of domestic sheep origin. Because in vitro and in vivo results appear closely correlated in this and other studies, we believe with additional evaluation and standardization, neutrophil cytotoxicity tests may serve as a substitute for live animal challenges in future studies of pathogenic P. haemolytica in wild sheep.     

Rare homologous gene targeting in Histoplasma capsulatum: disruption of the URA5Hc gene by allelic replacement

URA5 genes encode orotidine-5'-monophosphate pyrophosphorylase (OMPpase), an enzyme involved in pyrimidine biosynthesis. We cloned the Histoplasma capsulatum URA5 gene (URA5Hc) by using a probe generated by PCR with inosine-rich primers based on relatively conserved sequences in OMPpases from other organisms. Transformation with this gene restored uracil prototrophy and OMPpase activity to UV-mutagenized ura5 strains of H. capsulatum. We attempted to target the genomic URA5 locus in this haploid organism to demonstrate homologous allelic replacement with transforming DNA, which has not been previously done in H. capsulatum and has been challenging in some other pathogenic fungi. Several strategies commonly used in Saccharomyces cerevisiae and other eukaryotes were unsuccessful, due to the frequent occurrence of ectopic integration, linear plasmid formation, and spontaneous resistance to 5-fluoroorotic acid, which is a selective agent for URA5 gene inactivation. Recent development of an efficient electrotransformation system and of a second selectable marker (hph, conferring hygromycin B resistance) for this fungus enabled us to achieve allelic replacement by using transformation with an insertionally inactivated Deltaura5Hc::hph plasmid, followed by dual selection with hygromycin B and 5-fluoroorotic acid, or by screening hygromycin B-resistant transformants for uracil auxotrophy. The relative frequency of homologous gene targeting was approximately one allelic replacement event per thousand transformants. This work demonstrates the feasibility but also the potential challenge of gene disruption in this organism. To our knowledge, it represents the first example of experimentally directed allelic replacement in H. capsulatum, or in any dimorphic systemic fungal pathogen of humans.     

Prostaglandin D2 measurement in nasal secretions is not a reliable marker for mast cell activation in atopic patients

BACKGROUND: Prostaglandin D2 (PGD2) is a very important mast cell product during the early-phase nasal allergic reaction. However, the quantification of PGD2 in nasal secretions has not yet been well established. OBJECTIVE: Quantitative determination of PGD2 in nasal secretions of atopic patients (n = 17) after nasal allergen challenge (NAC) and in non-allergic healthy volunteers (n = 10). METHODS: The nasal microsuction sampling technique was used to obtain the nasal secretions with an exactly known and minimally diluted volume. A sensitive and specific enzyme immunoassay was chosen to measure the more stable 11-methoxime derivative of PGD2, which was obtained after extraction in acetone/ethanol and conversion using methoxamine-HCl. The concentrations of PGD2 in nasal secretions obtained from 10 non-allergic healthy volunteers were used as reference values. RESULTS: There was no significant difference in the concentrations of PGD2 between men (median: 569 pg/mL) and women (median: 407 pg/mL), nor between the baseline concentrations from atopic patients (median: 410 pg/mL) and non-allergic controls (median: 477 pg/mL). In the atopic patient group, PGD2 did not significantly increase during the entire sampling period after NAC. The absence of PGD2 response contrasted with the nasal symptoms manifested by sneezing, increased nasal airway resistance, and the significant increases of the concentrations of histamine, tryptase, and leukotriene C4 (LTC4) 5 min after NAC. CONCLUSION: This observation suggests that the measurement of PGD2 alone in the nasal secretions does not give reliable information on mast cell activation during a nasal allergic reaction.