There has been much interest recently in the antimicrobial properties of cationic peptides called beta-defensins from epithelial cells. Human beta-defensin (hBD)-1 and -2 have been particularly implicated in cystic fibrosis (CF) patients, where their inhibition by high salt concentrations may explain in part the susceptibility of the CF lung to bacterial infection. In this work, we have employed a simple co-culture system using the 16-HBE human bronchial epithelial cell line to assess growth inhibitory activity against Pseudomonas aeruginosa and Burkholderia cepacia. In medium alone, P. aeruginosa proliferated more than 100,000-fold, whereas in the presence of 16-HBE cells or 16-HBE-conditioned medium, bacterial proliferation was less than 100-fold. Raising the salt concentration of cell-free 16-HBE conditioned medium to approximately 200 mM significantly reduced this growth inhibitory activity. In contrast, there was no evidence of epithelial-derived growth inhibitory activity against two strains of B. cepacia. RT-PCR analysis indicated expression of the hBD-2 mRNA in 16-HBE cells, but not hBD-1. These data demonstrate for the first time that B. cepacia is resistant to epithelial-derived antimicrobial substances and argue against them being important in the defense against this organism in the lung.

PCR amplification of the recA gene followed by restriction fragment length polymorphism (RFLP) analysis was investigated for the rapid detection and identification of Burkholderia cepacia complex genomovars directly from sputum. Successful amplification of the B. cepacia complex recA gene from cystic fibrosis (CF) patient sputum samples containing B. cepacia genomovar I, Burkholderia multivorans, B. cepacia genomovar III, Burkholderia stabilis, and Burkholderia vietnamiensis was demonstrated. In addition, the genomovar identifications determined directly from sputum were the same as those obtained after selective culturing. Sensitivity experiments revealed that recA-based PCR could reliably detect B. cepacia complex organisms to concentrations of 10(6) CFU g of sputum(-1). To fully assess the diagnostic value of the method, sputum samples from 100 CF patients were screened for B. cepacia complex infection by selective culturing and recA-based PCR. Selective culturing identified 19 samples with presumptive B. cepacia complex infection, which was corroborated by phenotypic analyses. Of the culture-positive sputum samples, 17 were also detected directly by recA-based PCR, while 2 samples were negative. The isolates cultured from both recA-negative sputum samples were subsequently identified as Burkholderia gladioli. RFLP analysis of the recA amplicons revealed 2 patients (12%) infected with B. multivorans, 11 patients (65%) infected with B. cepacia genomovar III-A, and 4 patients (23%) infected with B. cepacia genomovar III-B. These results demonstrate the potential of recA-based PCR-RFLP analysis for the rapid detection and identification of B. cepacia complex genomovars directly from sputum. Where the sensitivity of the assay proves a limitation, sputum samples can be analyzed by selective culturing followed by recA-based analysis of the isolate.

Over a 6-year period, Burkholderia cepacia complex species were isolated from cystic fibrosis (CF) patients receiving care at The University of North Carolina Hospitals (clinic CF patients) and from those referred from other treatment centers. Fifty-six isolates collected from 30 referred patients and 26 clinic CF patients were characterized by pulsed-field gel electrophoresis (PFGE) and were assayed by PCR to detect the cable pilin gene, cblA. PFGE results indicated that six separate clusters (clusters A to F) were present among the 56 isolates and that three clusters (clusters A, B, and E) consisted only of isolates from referred patients infected with B. cepacia complex isolates prior to referral. However, one cluster (cluster C) consisted of isolates from four CF patients, and hospital records indicate that this cluster began with an isolate that came from a referred patient and that spread to three clinic CF patients. Cluster D consisted of two isolates from clinic CF patients, and hospitalization records are consistent with nosocomial, patient-to-patient spread. cblA was present in only 4 of the 56 isolates and included isolates in cluster E from the referred patients. Our results indicate a lack of spread of a previously characterized, transmissible clone from referred patients to our clinic CF population. Only two instances of nosocomial, patient-to-patient spread could be documented over the 6-year period. An additional spread of an isolate (cluster F) from a referred patient to a clinic patient could not be documented as nosocomial and may have been the result of spread in a nonhospitalized setting. The majority (36 of 56) of our B. cepacia complex-infected CF patients harbor isolates with unique genotypes, indicating that a diversity of sources account for infection. These data suggest that CF patients infected with B. cepacia complex and referred for lung transplantation evaluation were not a major source of B. cepacia complex strains that infected our resident CF clinic population.

The variable severity of Burkholderia cepacia complex infections in cystic fibrosis (CF) has recently been ascribed to differences in the virulence between genomovars. Specifically, genomovar III isolates have been associated with higher transmission rates and adverse outcomes compared to other B cepacia genomovars, and consequently further segregation between genomovar III and non-genomovar III B cepacia infected patients is advocated in some centres. The important role of non-genomovar III isolates is presented in the context of a clinical case whereby a patient with long-standing pulmonary infection with B multiovorans developed bacteremic infection reminiscent of the fatal 'cepacia syndrome'.

The cyanobacterial genus Lyngbya includes free-living, benthic, filamentous cyanobacteria that form periodic nuisance blooms in lagoons, reefs, and estuaries. Lyngbya spp. are prolific producers of biologically active compounds that deter grazers and help blooms persist in the marine environment. Here, our investigations reveal the presence of three distinct Lyngbya species on nearshore reefs in Broward County, FL, sampled in 2006 and 2007. With a combination of morphological measurements, molecular biology techniques, and natural products chemistry, we associated these three Lyngbya species with three distinct Lyngbya chemotypes. One species, identified as Lyngbya cf. confervoides via morphological measurements and 16S rRNA gene sequencing, produces a diverse array of bioactive peptides and depsipeptides. Our results indicate that the other two Lyngbya species produce either microcolins A and B or curacin D and dragonamides C and D. Results from screening for the biosynthetic capacity for curacin production among the three Lyngbya chemotypes in this study correlated that capacity with the presence of curacin D. Our work on these bloom-forming Lyngbya species emphasizes the significant phylogenetic and chemical diversity of the marine cyanobacteria on southern Florida reefs and identifies some of the genetic components of those differences.

The time taken to cross the rabbit corneal epithelium and stroma was estimated for fluorescein (F), carboxyfluorescein (CF), rhodamine B (RB), and sulforhodamine B (SRB). Paired corneas were mounted in vitro; one was intact and the dye solution was kept in continuous contact with its epithelial surface; the epithelium was scraped from the other and the dye was applied as a pulse to the bare stroma. The time course of the dye appearing in a solution rapidly passing over the endothelial surface was determined by fluorometry. This rate of appearance was compared in the two cases and used to estimate the diffusional lag time introduced by the epithelium. For the very hydrophilic CF and SRB, the delay was too short to measure; this is compatible with the passage of these dyes taking place through the paracellular spaces. For the very lipophilic RB, the delay was about 2 min; this was rather too slow for it to be explained as being controlled entirely by diffusion in the cytosol. For the intermediate F, the delay was 5 min; it is suggested that this is a result of it partitioning between the spaces and the cytosol during its passage. The experiments also led to determinations of the permeability of the epithelial and endothelial layers to the dyes. In both cases lipophilicity was a strong determinant of penetration, but not the only one. The permeability of the endothelium to F was unchanged from its in vivo value in these experiments, but that of the epithelium was increased four-fold. The diffusion rate of the dyes across the stroma could also be determined. There was no clear relationship with molecular size or partition coefficient. The rate of diffusion of F across the tissue was about half that in its plane, as determined in previous experiments. This is possibly a result of the anisotropic structure of the tissue.

To better understand peptide-induced membrane fusion at a molecular level, we set out to determine the structure of the fusogenic peptide FP23 from the HIV-1 protein gp41 when bound to a lipid bilayer. An established solid-state (19)F nuclear magnetic resonance (NMR) approach was used to collect local orientational constraints from a series of CF(3)-phenylglycine-labeled peptide analogues in macroscopically aligned membranes. Fusion assays showed that these (19)F-labels did not significantly affect peptide function. The NMR spectra were characteristic of well-behaved samples, without any signs of heterogeneity or peptide aggregation at 1:300 in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC). We can conclude from these NMR data that FP23 has a well-defined (time-averaged) conformation and undergoes lateral diffusion in the bilayer plane, presumably as a monomer or small oligomer. Attempts to evaluate its conformation in terms of various secondary structures, however, showed that FP23 does not form any type of regular helix or β-strand. Therefore, all-atom molecular dynamics (MD) simulations were carried out using the orientational NMR constraints as pseudo-forces to drive the peptide into a stable alignment and structure. The resulting picture suggests that FP23 can adopt multiple β-turns and insert obliquely into the membrane. Such irregular conformation explains why the structure of the fusion peptide could not be reliably determined by any biophysical method so far.

We have investigated the frequency of the delta F508 mutation on cystic fibrosis (CF) chromosomes in Denmark. Of 304 chromosome tested, 86.8% have the delta F508 mutation. The majority of the chromosomes with this mutation are found on chromosomes with the XV2c/KM19 haplotype B (97.3%), whereas 15/16 chromosomes with haplotype C have another mutation, confirming that only very few mutations will account for the majority of CF genes in the Danish population.

OBJECTIVE

In cystic fibrosis (CF), pancreatic disease begins in utero and progresses over time to complete destruction of the organ. Although inflammatory cells have been detected in the pancreas of humans and pigs with CF, their subtypes have not been characterized.

METHODS

Using four-color flow cytometry, we analyzed the surface antigens of leukocytes in pancreas, blood, and mesenteric lymph nodes (MLN) of newborn pigs with CF (CFTR(-/-) and CFTR(Δ)(F508/)(Δ)(F508)) and in those without CF (CFTR(+/-), CFTR(+/)(Δ)(F508), CFTR(+/+)). Pancreatic histopathology was examined with HE stain.

RESULTS

CF pig pancreas had patchy distribution of inflammatory cells with neutrophils/macrophages in dilated acini, and lymphocytes in the interstitium compared to non-CF. B cells, effector (MHC-II(+)) and cytotoxic (CD2(+)CD8(+)) γδ T cells, activated (MHC-II(+) and/or CD25(+)) and effector (CD4(+)CD8(+)) αβ T helper cells, effector natural killer cells (MHC-II(+)CD3(-)CD8(+)), and monocytes/macrophages and neutrophils were increased in the CF pig pancreas compared to pigs without CF. Blood and MLN leukocyte populations were not different between CF and non-CF pigs.

CONCLUSIONS

We discovered an activated immune response that was specific to the pancreas of newborn CF pigs; inflammation was not systemic. The presence of both innate and adaptive immune cells suggests that the disease process is complex and extensive.

OBJECTIVE

Cystic fibrosis (CF) is an autosomal recessive hereditary disease and is the frequently common lethal genetic pathology. The purposes of this study were to: (1) determine the presence of 3 different types of enamel defects: (a) demarcated opacities; (b) diffuse opacities; and (c) hypoplasia in the deciduous and permanent dentition of CF patients; and (2) compare with a control group.

METHODS

The case group was defined as 13 patients who were diagnosed with CF and enrolled in a multiprofessional project of the Catholic University of Brasília (CUB), Brasilia, Brazil. All CF subjects were compared with control subjects selected from patients at the CUB. Each CF subject was individually paired with a control subject of similar sex and age. A full-mouth examination was carried out for the developmental defects of enamel (DDE) index.

RESULTS

The most prevalent enamel defect in deciduous teeth was demarcated opacities present in 16% of the case group and in 7% of the control group. Although the defects were more prevalent in the case group, the difference was not statistically significant (P=0.57). The frequency of demarcated opacities was more prevalent in permanent teeth of the case group: 39% compared to 11% in the control group. For the control group, diffuse opacities were the more prevalent defects: 17% compared to 15% in the case group. The case group had more enamel defects in permanent teeth compared to the control (P=0.0003).

CONCLUSIONS

In this study, enamel defects were frequently found in the permanent teeth of CF patients. Therefore, professionals who treat children should be alerted to promoting oral health among these patients.

Colonization with Aspergillus fumigatus (Af) constitutes a common finding in children with cystic fibrosis (CF). The relationship between sensitization to Af and lung functon (LF) was studied in 118 patients with CF (61 girls and 57 boys; mean age: 14.3 yr; SD: 7 yr). Mean follow up was 2.2 yr. On average, 8.1 (SD: 4.8) LF tests were performed per patient. Measurement of total IgE and specific IgE antibodies to Af, and a skin prick test (SPT) for Af, were done once a year. Thirty-one children (26%) were sensitized to Af. On average, LF parameters were not significantly different in Af-sensitized children than in nonsensitized children. Linear regression analyses were performed, using the repeated measures design. With adjustment for gender, age, height, and weight, sensitization to Af was associated with lower values of FEV1 (beta = -0.209; p < 0.05) and FEF(25-75) (beta = -0.356; p < 0.01). Analysis of different subgroups of sensitization demonstrated the effect on LF only in Af-sensitized patients with elevated total IgE levels, and not in Af-sensitized patients with normal IgE levels. Furthermore, there was evidence for a more rapid decline in LF for Af-sensitized patients with elevated total IgE levels than in those with normal IgE levels. We conclude that sensitization to Af in the presence of increased IgE values is associated with lower LF values in children with CF.

OBJECTIVE

Present investigation deals with an extensive approach incorporating in vitro and in vivo experimentation to treat chronic osteomyelitis, using hydroxyapatite porous scaffolds.

METHODS

Hydroxyapatite was synthesized in the laboratory by wet chemical method, different porous scaffolds have been fabricated. In vitro studies include variation of porosity with interconnectivity, pore-drug interfacial studies by SEM-EDAX and drug elution studies (by HPLC) both in contact with PBS and SBF at approximately 37 degrees C. In vivo trials were based on experimental osteomyelitis in rabbit model induced in tibia by Staphylococcus aureus. Characterizations included observation of histopathology, radiology and estimation of drug in both bone and serum for 42 days by HPLC method and subsequent bone-biomaterial interface by SEM.

RESULTS

It was established that lower pore percentage with a distribution of mainly micro-pores were found to be superior over the higher pore percentage both in vitro and in vivo. The criteria was matched with the 50N50H samples which had 50-55% porosity with an average pore size approximately 110 microm, having higher interconnectivity (10-100 microm), moderately high adsorption efficiency (approximately 50%) when loaded with CFS (drug combinations consisting of irreversible b-lactamase inhibitor and b-lactam antibiotic). CFS release from HAp implants were faster in PBS than SBF. Further, both the results of in vitro and in vivo drug elution after 42 days showed release higher than minimum inhibitory concentration of CFS against Staphylococcus aureus. In vivo studies also proved the superiority of CFS loaded HAp implants than parenteral group based on eradication of infection and new bone formation.

CONCLUSIONS

HAp based porous scaffold loaded with CFS and designed porosity (in terms of micro- and macro-porosity, interconnectivity) was found to be an ideal delivery system which could locally, sustainably release the composite antibiotic in reliable manner both in terms of in vitro drug elution behaviour in contact with SBF and in vivo animal trial.

Screening for colorectal carcinoma (CRC) was organized for 236 asymptomatic family members in 22 Finnish cancer family syndrome (CFS) kindreds, and 58% (137) of the subjects accepted the invitation. Double-contrast colonography and sigmoidoscopy or colonoscopy were used. In 137 subjects aged 20-65 years, with 3 or more first-degree relatives with CRC, one screening visit showed a colonic neoplasm in 12 (9%) subjects. Two had carcinoma (Dukes A and B), and 10 subjects one or more adenomas. Two of the subjects not attending screening (2%) developed Dukes C colon carcinoma during the study period, and one of them died of cancer. Continued screening of 34 patients with a previously identified CFS showed metachronous colorectal tumours in 12 (35%) cases: 9 operable carcinomas and 9 adenomas within 3 years. The preliminary result of screening on the basis of CFS was encouraging. Effective and continuous screening, however, requires centralized organization. The continued follow-up of identified CFS cases effectively revealed metachronous colorectal tumours at a curable stage, but its benefit was burdened by a high rate of advanced malignancies other than CRC.

Seven toluene-oxidizing bacterial strains (Pseudomonas mendocina KR1, Burkholderia cepacia G4, Pseudomonas putida F1, Pseudomonas pickettii PKO1, and Pseudomonas sp. strains ENVPC5, ENVBF1, and ENV113) were tested for their ability to degrade chloroform (CF). The greatest rate of CF oxidation was achieved with strain ENVBF1 (1.9 nmol/min/mg of cell protein). CF also was oxidized by P. mendocina KR1 (0.48 nmol/min/mg of cell protein), strain ENVPC5 (0.49 nmol/min/mg of cell protein), and Escherichia coli DH510B(pRS202), which contained cloned toluene 4-monooxygenase genes from P. mendocina KR1 (0.16 nmol/min/mg of cell protein). Degradation of [14C]CF and ion analysis of culture extracts revealed that CF was mineralized to CO2 (approximately 30 to 57% of the total products), soluble metabolites (approximately 15%), a total carbon fraction irreversibly bound to particulate cellular constituents (approximately 30%), and chloride ions (approximately 75% of the expected yield). CF oxidation by each strain was inhibited in the presence of trichloroethylene, and acetylene significantly inhibited trichloroethylene oxidation by P. mendocina KR1. Differences in the abilities of the CF-oxidizing strains to degrade other halogenated compounds were also identified. CF was not degraded by B. cepacia G4, P. putida F1, P. pickettii PKO1, Pseudomonas sp. strain ENV113, or P. mendocina KRMT, which contains a tmo mutation.

OBJECTIVE

Over 80% of patients with cystic fibrosis (CF) have bronchodilator therapy prescribed, yet bronchodilator use in CF remains controversial. The development of long-acting beta-agonist drugs for clinical use has provided additional rationale for considering bronchodilator therapy in CF. This paper will review recent developments in bronchodilator use in CF patients, with emphasis on the long-acting beta agonists.

RESULTS

It is reported that 50 to 60% of CF patients demonstrate significant intermittent airway hyperreactivity in response to bronchodilators or challenges. The beta-agonist drugs are the most commonly prescribed bronchodilators. Several mechanisms may be implicated in therapeutic response of CF patients to bronchodilators including direct smooth muscle relaxation, increased mucociliary clearance, direct effects on inflammatory cells and bacterial adherence, and possible direct effects on CF transmembrane conductance regulator (CFTR) function. Several recent studies have shown improved outcomes with long-acting bronchodilators.

CONCLUSIONS

In spite of the widespread use of bronchodilators, there are very few long-term studies of their effects in CF patients. However, there are clearly clinical benefits in certain situations. Further research into the most appropriate utilization of these medications to improve outcomes in patients with CF would be helpful.

The etiology of Crohn's disease (CD) is unresolved, but it is likely that an interplay of host genetic factors and environmental triggers is relevant. Mycobacterium paratuberculosis (MAP) has been focused upon as one of these triggers because it causes a similar chronic inflammatory bowel disease in animals. However, the differences among MAP antigens isolated from humans (H-MAP) and cattle (B-MAP) have not been well characterized. In this study, culture filtrate (CF) proteins from MAP isolates were tested with sera from CD patients and healthy controls in enzyme-linked immunosorbent assay (ELISA). Antibody produced by seven CD patients reacted differently according to the antigen source: strong reactivity was seen to H-MAP CF, but not to B-MAP CF. Six proteins, ModD, PepA, transaldolase, EchA9, MAP2120c, and MAP2950c, in H-MAP CF reacting specifically with CD patient sera were identified by liquid chromatography-electrospray ionization-MS. Bioinformatic analysis revealed that ModD and PepA were the same proteins reacting with sera from cattle infected with MAP. The elevated antibody responses of CD patients to rModD and rPepA were confirmed by ELISA (P<0.001). These results support previous studies showing ModD and PepA as key antigens for the diagnosis of MAP infections. The study also identified additional proteins potentially useful in the design of assays for human MAP infections.

We used differential-display PCR of peripheral blood mononuclear cells (PBMCs) to search for candidate biomarkers for chronic fatigue syndrome (CFS). PBMCs were collected from a subject with CFS and an age- and sex-matched control before and 24 h after exercise. RNA expression profiles were generated using 46 primer combinations, and the similarity between the individuals was striking. Differentially expressed bands were excised, reamplified, and sequenced, yielding 95 nonredundant sequences, of which 50 matched to known gene transcripts, 38 matched to genes with unknown functions, and 7 had no similarity to any database entry. Most (86%) of the differences between the two subjects were present at baseline. Differential expression of ten genes was verified by real-time reverse-transcription PCR: five (cystatin F, MHC class II, platelet factor 4, fetal brain expressed sequence tag, and perforin) were downregulated, and the remaining five genes (cathepsin B, DNA polymerase epsilon4, novel EST PBMC191MSt, heparanase precursor, and ORF2/L1 element) were upregulated in the subject with CFS. Many of these genes have known functions in defense and immunity, thus supporting prior suggestions of immune dysregulation in the pathogenesis of CFS. Differential-display PCR is a powerful tool for identification of candidate biomarkers. Investigation of these markers in samples from well-designed epidemiological studies of CFS will be required to determine the validity of these candidate biomarkers. The real-time reverse-transcription PCR assays that we developed for assay of these biomarkers will facilitate high-throughput testing of these additional samples.

A graft-vs.-host reaction (GVHR) was induced in young male CAF(I) and CBBALB/cJ spleen cells. A proportion of such mice subsequently developed lymphoreticular rumors. Cell-free extracts (CFEs) prepared from the reticular tissues of CAF(1) mice killed at intervals after the induction of the GVHR were tested for their capacity to produce the same tumors in a litter of syngeneic mice inoculated at birth. 12 of 29 (41.4%) such extracts were positive, causing lymphoreticular tumors in one or more littermate recipients. The positive CFEs came from donors killed at all stages of the GVHR, from tumor-bearing mice as well as from non-tumor-bearing mice. However, whereas less than 30% of CFEs from mice killed within 12 mo of GVHR induction were oncogenic, the incidence of oncogenic extracts from mice killed 12-15 mo after GVHR induction rose to 75%. None of the CFEs prepared from nine normal uninjected male CAF(1) mice killed between the ages of 8 and 18 mo transmitted tumors to recipients. CFEs prepared from CAF(1) mice with the GVHR were tested for infectious murine leukemia virus (MuLV) using the XC assay and also for complement-fixing (CF) group-specific MuLV antigen. Substantial titers of B-tropic MuLV and CF antigen were detected in at least half the extracts from mice killed 11-14 mo after GVHR induction. During the first few months of GVHR induction, MuLV titers were low and CF antigen was absent. Neither infectious MuLV nor CF antigen were detected in CFEs prepared from normal control mice. Serially passed CFEs originating from a CBCF MuLV antigen, and C-type particles. These data together provide evidence that MuLV is activated during the GVHR and that it is responsible for the eventual development of lymphoreticular tumors.

Cell walls of Histoplasma capsulatum yeast-form chemotypes 1 (chem 1) and 2 (chem 2) treated sequentially with several polysaccharolytic enzymes and Pronase yielded soluble, nondialyzable polysaccharides at each step, which were analyzed for monosaccharides, protein composition, and serological activity. Polysaccharide recovered after digestion of chem 1 walls with beta(1-->3)-glucanase contained glucose>> mannose>> glucosamine>> galactose. This fraction (chem 1 betaG(1)) was analyzed by polyacrylamide gel electrophoresis and contained a component having an apparent molecular weight of 120,000. The chem 1 betaG(1) fraction was reactive in immunodiffusion (ID), producing an immune precipitate not identical to the H and M factors of histoplasmin. In a side-by-side ID comparison with extracts of chem 2, the chem 1 betaG(1) antigen contained an additional determinant not found in chem 2 extracts when tested with goat antiserum to H. capsulatum. Therefore, the chem 1 antigen gave preliminary ID evidence of antigenic group specificity. A chemical difference observed was the absence of glucosamine from chem 2 polysaccharide. In complement fixation (CF) tests, 9 of 17 sera from human histoplasmosis patients reacted with chem 1 betaG(1), but some cross-reactivity with sera of patients with other systemic mycoses occurred. The immunoelectrophoretic patterns of chem 1 wall-derived polysaccharides showed a marked shift in mobility after Pronase digestion, implying the presence of covalent peptides. The ultrastructural appearance and serological activity of intact walls and enzyme-resistant mural cores were also studied. The surface of the mural cores of both chemotypes was perforated and frayed. In shadow-cast preparations both fibrillar and globular areas persisted in the mural cores. The CF end point serum dilutions showed an increase after alpha- and beta-glucanase extractions of chem 2 walls and fourfold reduction after Pronase digestion. The mural cores of both chemotypes were still reactive in CF tests and retained some ability to bind fluorescent antibody. The chem 1 mural core reacted with specific fluorescein-labeled H. capsulatum antiglobulins produced by adsorption with Blastomyces dermatitidis, thus indicating at least partial retention of H. capsulatum-specific factors. The presence of galactose, mannose, and glucose was detected in the mural cores as well as enriched levels of amino sugar, despite exposure to chitinase.

Solubilized nonstructural antigen III from Saint Louis encephalitis (SLE)-Japanese B encephalitis (JBE)-West Nile (WN) and dengue-2 arbovirus-infected pig kidney cells was purified by employing Brij-58 solubilization, organic solvent extraction, and column chromatography. Diethylaminoethyl-cellulose column peak C eluate contained only intracellular viral envelope protein designated antigen I. Antigen I of SLE virus absorbed homologous neutralizing antibody; however, the intracellular nonstructural protein designated antigen III did not. The antigenic relationships of solubilized antigens I and III prepared from SLE, JBE, and WN virus-infected cells were determined by complement-fixation (CF) and immunodiffusion analyses. Solubilized antigen I of each virus cross-reacted broadly with both homologous and heterologous antibody at high antigen concentrations in the CF test. Antigen III of each virus reacted with only homologous antibody by both CF and immunoprecipitation. This study demonstrates that during SLE, JBE, WN, and dengue-2 infections, virus-specific proteins containing both type-specific and group-reactive determinants are synthesized. Antigen III, a nonstructural protein, is serologically virus type specific and may be useful as a type-specific diagnostic reagent.

Airway surface hydration depends on the balance between transepithelial Na(+) absorption and Cl(-) secretion. In adult mice, absence of functional cystic fibrosis transmembrane conductance regulator (Cftr) fails to recapitulate human cystic fibrosis (CF) lung disease. In contrast, overexpression of the epithelial Na(+) channel β subunit in transgenic mice (βENaC-Tg) produces unregulated Na(+) hyperabsorption and results in CF-like airway surface dehydration, mucus obstruction, inflammation, and increased neonatal mortality. To investigate whether the combination of airway Na(+) hyperabsorption and absent Cftr-mediated Cl(-) secretion resulted in more severe lung pathology, we generated double-mutant ΔF508 CF/βENaC-Tg mice. Survival of ΔF508 CF/βENaC-Tg mice was reduced compared with βENaC-Tg or ΔF508 CF mice. Absence of functional Cftr did not affect endogenous or transgenic ENaC currents but produced reduced basal components of Cl(-) secretion and tracheal cartilaginous defects in both ΔF508 CF and ΔF508 CF/βENaC-Tg mice. Neonatal ΔF508 CF/βENaC-Tg mice exhibited higher neutrophilic pulmonary inflammation and club cell (Clara cell) necrosis compared with βENaC-Tg littermates. Neonatal ΔF508 CF/βENaC-Tg mice also exhibited spontaneous bacterial infections, but the bacterial burden was similar to that of βENaC-Tg littermates. Adult ΔF508 CF/βENaC-Tg mice exhibited pathological changes associated with eosinophilic crystalline pneumonia, a phenotype not observed in age-matched βENaC-Tg mice. Collectively, these data suggest that the combined abnormalities in Na(+) absorption and Cl(-) secretion produce more severe lung disease than either defect alone. Airway cartilage abnormalities, airway cell necrosis, and exaggerated neutrophil infiltration likely interact with defective mucus clearance caused by βENaC overexpression and absent CFTR-mediated Cl(-) secretion to produce the increased neonatal mortality observed in ΔF508 CF/βENaC-Tg mice.

This paper investigates the usefulness of two mitochondrial genes (16S rRNA and cytochrome b) to solve taxonomical difficulties within the genus Hylomyscus and to infer its evolutionary history. Both genes proved to be suitable molecular markers for diagnosis of Hylomyscus species. Nevertheless the resolving powers of these two genes differ, and with both markers (either analyzed singly or in combination), some nodes remain unresolved. This is probably related to the fact that the species emerged during a rapid diversification event that occurred 2-6 Myr ago (4-5 Myr ago for most divergence events). Our molecular data support the recognition of an "aeta" group, while the "alleni" and "parvus" groups are not fully supported. Based on tree topology and genetic divergence, two taxa generally recognized as subspecies should be elevated at the species level (H. simus and H. cf kaimosae). H. stella populations exhibit ancient haplotype segregation that may represent currently unrecognized allopatric species. The existence of cryptic species within H. parvus is questioned. Finally, three potentially new species may occur in West Central Africa. The Congo and Oubangui Rivers, as well as the Volta and Niger Rivers and/or the Dahomey gap could have formed effective barriers to Hylomyscus species dispersal, favoring their speciation in allopatry. The pronounced shifts in African climate during the late Pliocene and Miocene, which resulted in major changes in the distribution and composition of the vegetation, could have promoted speciation within the genus (refuge theory). Future reports should focus on the geographic distribution of Hylomyscus species in order to get a better understanding of the evolutionary history of the genus.

We have combined kinematic and electromyogram (EMG) analysis of running Blaberus discoidalis to examine how middle and hind leg kinematics vary with running speed and how the fast depressor coxa (Df) and fast extensor tibia (FETi) motor neurons affect kinematic parameters. In the range 2.5-10 Hz, B. discoidalis increases step frequency by altering the joint velocity and by reducing the time required for the transition from flexion to extension. For both Df and FETi the timing of recruitment coincides with the maximal frequency seen for the respective slow motor neurons. Df is first recruited at the beginning of coxa-femur (CF) extension. FETi is recruited in the latter half of femur-tibia (FT) extension during stance. Single muscle potentials produced by these fast motor neurons do not have pronounced effects on joint angular velocity during running. The transition from CF flexion to extension was abbreviated in those cycles with a Df potential occurring during the transition. One effect of Df activity during running may be to phase shift the beginning of joint extension so that the transition is sharpened. FETi is associated with greater FT extension at higher running speeds and may be necessary to overcome high joint torques at extended FT joint angles.

Serological diagnosis and follow-up of paracoccidioidomycosis (PCM) patients have relied mainly on the detection of antibody responses by using techniques such as complement fixation (<em>CF</em>) and immunodiffusion. We recently described a novel inhibition enzyme-linked immunosorbent assay (inh-ELISA) which proved to be useful in the diagnosis of PCM via the detection of an 87-kDa determinant in patient sera (<em>B</em>. L. Gomez, J. I. Figueroa, A. J. Hamilton, <em>B</em>. Ortiz, M. A. Robledo, R. J. Hay, and A. Restrepo, J. Clin. Microbiol. 35:3278-3283, 1997). This test has now been assessed as a means of following up PCM patients. A total of 24 PCM patients, classified according to their clinical presentation (6 with the acute form of the disease, of whom two had AIDS, 12 with the multifocal form of the disease, and 6 with the unifocal form of the disease), were studied. The four human immunodeficiency virus-negative patients with acute PCM showed a statistically significant decrease in circulating antigen levels after the start of antifungal therapy. Antigen levels in this group became negative by our criteria (</=2.3 microgram/ml) before week 20 and remained so in three of four of these patients. In contrast, the two AIDS patients who also presented with the acute form of PCM showed no statistically significant decrease in circulating antigen levels even after 68 weeks of therapy. Taken together as a group, the patients with the multifocal form showed a statistically significant decrease in antigenemia after 28 weeks of therapy. In addition, five of six patients with the unifocal form became antigen negative by week 40. Antigen level decrease mirrored clinical cure in the majority of patients in all clinical groups; in contrast, measurement of anti-PCM antibodies via the <em>CF</em> test showed wide fluctuations in titers during the follow-up period. The inh-ELISA for the detection of the 87-kDa Paracoccidioides brasiliensis determinant would appear to be a valuable additional tool in the follow-up of PCM patients.