Immune responses against enterotoxigenic Escherichia coli (ETEC) were examined in Bangladeshi adults with naturally acquired disease and compared to responses in age-matched Bangladeshi volunteers who had been orally immunized with a vaccine consisting of inactivated ETEC bacteria expressing different colonization factor antigens (CFs) and the B subunit of cholera toxin. B-cell responses in duodenal biopsy samples, feces, intestinal washings, and blood were determined. Because most of the patients included in the study were infected with ETEC expressing CS5, immune responses to this CF were studied most extensively. Vaccinees and patients had comparable B-cell responses against this antigen in the duodenum: the median numbers of antibody-secreting cells (ASC) were 3,300 immunoglobulin A (IgA) ASC/10(7) mononuclear cells (MNC) in the patient group (n = 8) and 1,200 IgA ASC/10(7) MNC in the vaccinees (n = 13) (not a significant difference). Similarly, no statistically significant differences were seen in the levels of duodenal B cells directed against enterotoxin among vaccinees and patients. A comparison of the capacities of the various methods used to assess mucosal immune responses revealed a correlation between numbers of circulating B cells and antibody levels in saponin extracts of duodenal biopsy samples (r = 0.58; n = 13; P = 0.04) after vaccination. However, no correlation was seen between blood IgA ASC and duodenal IgA ASC after two doses of vaccine. Still, a correlation between numbers of CF-specific B cells in blood sampled from patients early during infection and numbers of duodenal B cells collected 1 week later was apparent (r = 0.70; n = 10; P = 0.03).

BACKGROUND

Pseudomonas aeruginosa is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). With chronicity of infection, the organism resides as a biofilm, shows multi-drug resistance, diversifies its colony morphology and becomes auxotrophic. The patients have been found to be colonized with multiple genotypes. The present work was carried out to characterize P. aeruginosa isolated from children with cystic fibrosis using phenotypic and genotypic methods.

RESULTS

We studied 56 patients with CF attending the Pediatric Chest clinic at All India Institute of Medical Sciences, New Delhi, India during August 1998-August 2001. These patients were regularly followed up at the clinic. Out of 56 patients, 27 were culture positive for P. aeruginosa where 8 were chronically infected (Group1) and 19 were intermittently colonized with the organism (Group2). Patients under Group1 had significantly higher rates of hospitalization, death and colonization with different colony morphotypes (p < 0.05). The isolates from Group1 patients were the positive producers of extended spectrum beta lactamase. A total of 5 auxotrophs were recovered from 2 patients where one was chronically infected with P. aeruginosa and the other was a recently enrolled patient. The auxotrophs had the specific requirement for methionine and arginine. Molecular typing revealed 33 ERIC-PCR (E1-E33) and 5 PCR-ribotyping (P1-P5) patterns. By ERIC-PCR, 4 patients were colonized with 2-4 genotypes and the remaining 23 patients were colonized with the single genotype.

CONCLUSIONS

With chronicity of infection, P. aeruginosa becomes multidrug resistant, diversifies its colony morphology, acquires mucoidity and shows auxotrophy for amino acids. The chronically infected patients can be colonized with multiple genotypes. Thus in a particular clinical set up, high index of suspicion should be there for diagnosis of CF patients so as to prevent the delay in diagnosis and management of CF patients.

BACKGROUND

Cystic fibrosis (CF) is a multi-system disease affecting multiple organs and cells besides the respiratory system. Metabolomic profiling allows simultaneous detection of biochemicals originating from cells, organs, or exogenous origin that may be valuable for monitoring of disease severity or in diagnosis.

OBJECTIVE

We hypothesized that metabolomics using serum from children would differentiate CF from non-CF lung disease subjects and would provide insight into metabolism in CF.

METHODS

Serum collected from children with CF (n = 31) and 31 age and gender matched children with other lung diseases was used for metabolomic profiling by gas- and liquid-chromatography. Relative concentration of metabolites was compared between the groups using partial least square discriminant analyses (PLS-DA) and linear modeling.

RESULTS

A clear separation of the two groups was seen in PLS-DA. Linear model found that among the 459 detected metabolites 92 differed between CF and non-CF. These included known biochemicals in lipid metabolism, oxidants, and markers consistent with abnormalities in bile acid processing. Bacterial metabolites were identified and differed between the groups indicating intestinal dysbiosis in CF. As a novel finding several pathways were markedly different in CF, which jointly point towards decreased activity in the β-oxidation of fatty acids. These pathways include low ketone bodies, low medium chain carnitines, elevated di-carboxylic acids and decreased 2-hydroxybutyrate from amino acid metabolism in CF compared to non-CF.

CONCLUSIONS

Serum metabolomics discriminated CF from non-CF and show altered cellular energy metabolism in CF potentially reflecting mitochondrial dysfunction. Future studies are indicated to examine their relation to the underlying CF defect and their use as biomarkers for disease severity or for cystic fibrosis transmembrane regulator (CFTR) function in an era of CFTR modifying drugs.

The study was designed to explore the role and possible mechanisms of hydrogen sulfide (H2S) in the regulation of myocardial collagen remodeling in spontaneously hypertensive rats (SHRs). We treated nine-week-old male SHRs and age- and sex-matched Wistar-Kyoto rats (WKYs) with NaHS (90 μmol/kg(-1)·day(-1)) for 9 wks. At 18 wks, plasma H2S, tail arterial pressure, morphology of the heart, myocardial ultrastructure and collagen volume fraction (CVF), myocardial expressions of collagen I and III protein and procollagen I and III mRNA, transforming growth factor-<em>β</em>1 (TGF-<em>β</em>1), TGF-<em>β</em> type I receptor (T<em>β</em>R-I), type II receptor (T<em>β</em>R-II), p-Smad2 and 3, matrix metalloproteinase (MMP)-13 and tissue inhibitors of MMP (TIMP)-1 proteins were determined. TGF-<em>β</em>1-stimulated cultured cardiac fibroblasts (<em>CFs</em>) were used to further study the mechanisms. The results showed that compared with WKYs, SHRs showed a reduced plasma H2S, elevated tail artery pressure and increased myocardial collagen, TGF-<em>β</em>1, T<em>β</em>R-II, p-Smad2 and p-Smad3 expressions. However, NaHS markedly decreased tail artery pressure and inhibited myocardial collagen, TGF-<em>β</em>1, T<em>β</em>R-II, p-Smad2 and p-Smad3 protein expressions, but H2S had no effect on the expressions of MMP-13 and TIMP-1. Hydralazine reduced blood pressure but had no effect on myocardial collagen, MMP-13 and TIMP-1 expressions and TGF-<em>β</em>1/Smad signaling pathway. H2S prevented activation of the TGF-<em>β</em>1/Smad signaling pathway and abnormal collagen synthesis in <em>CFs</em>. In conclusion, the results suggested that H2S could prevent myocardial collagen remodeling in SHR. The mechanism might be associated with inhibition of collagen synthesis via TGF-<em>β</em>1/Smad signaling pathway.

A comparative study of the phase-locked response of auditory nerve fibers was performed in two frog species, Eleutherodactylus coqui and Bombina orientalis. From the tuning characteristics and phase response of single auditory nerve fibers to low frequency tones (0.08-1.0 kHz) we attempt to deduce the mechanics of the auditory organ responsible for low-frequency hearing in the frog, the amphibian papilla (a.p.). The phase-locked responses of auditory nerve fibers in B. orientalis were essentially identical to those from cells with similar CFs in E. coqui, despite the presence of a conspicuous caudal extension of the a.p. in E. coqui (an apparently derived morphology), a feature completely absent in B. orientalis. The fine structure of the frequency-dependent phase behavior was examined in both species with a residual phase analysis. The most significant non-linear phase behavior was confined to neurons with CFs less than 0.3 kHz. The intensity dependence of the phase response in E. coqui revealed that the preferred firing phase of an auditory nerve fiber depends upon the relation of test frequency (TF) and CF of the neuron examined. For TFs greater than CF there was a progressive phase lag as stimulus level was increased; the inverse was true for TFs less than CF. Click latencies measured in E. coqui were inversely related to CF and were similar though systematically shorter than the response latencies estimated from the phase-frequency functions. The click response was similar to that documented in other species, showing a significant level dependence and the presence of multiple peaks, with the time between peaks related to the period of the neuron's CF. A 'neurogram' was compiled for a.p. fiber responses in both species in response to several pure tones. Based on the known tonotopy of the a.p. this measure reflects the phase response of the a.p. over the extent of its length. The population phase response in anurans is quite similar to that obtained from mammalian auditory nerve fibers for the same range of test frequencies (0.08-1.0 kHz). The similarity between the responses of auditory fibers in these two anuran species suggests the micromechanics of the a.p. rostral to the tectorial curtain is similar in both species and that it is the likely site for the origin of the CF-dependent time delays.

1. Single cochlear nerve fiber recordings from unexposed chinchillas show spatial distributions of amplitude and phase of the distortion products f2 - f1 and 2f1 - f2 similar to those previously reported for the cat (35, 37, 42). 2. Damaging the organ of Corti in the region corresponding to the frequencies of a two-tone stimulus substantially reduces the amplitude of these distortion products at their characteristic places. 3. The distortion products 2f - f1 and 2f1 - f2 thus appear to be generated in the organ of Corti in the region of the primary-frequency places. 4. The neural responses suggest that the distortion products are propagated in the motion of the cochlear partition like externally applied stimulus tones at the distortion frequencies wih a similar spatial distribution of distortion product amplitude and phase. Models of the cochlea that assume nonlinear cochlear-partition dynamics can account for the similarity by demonstrating that distortion products generated by cochlear-partition nonlinearity can propagate apicalward in the motion of the cochlear partition. 5. Models of the cochlea using a linear-system model for cochlear partition motion, in cascade with a nonlinear transduction stage and a subsequent sharp filter, are inadequate to account for present observations, unless two currently implausible assumptions are made: a) stimulus tones near 4 kHz must propagate in normal cochleas at least as far apically as the 300-Hz place with sufficient amplitude to generate f2 - f1 there, and b) damage to the organ of Corti must interfere with this propagation of 4-kHz stimulus tones to the 300-Hz place. 6. Distortion generation in the cochlea is sensitive to delicate alterations of the organ of Corti. Short moderate-intensity exposures to sound can reversibly reduce the amplitudes of the distortion products f2 - f1 and 2f1 - f2 seen in responses from cochlear nerve fibers with characteristic frequencies (CF) near the distortion frequencies. Since such exposures do ot produce permanent structural changes visible under light microscopy, it seems most reasonable to believe that subtle changes in the organ of Corti (most likely in the hair cells themselves) in the region most responsive to f1 and f2 reduce the generation of mechanically present distortion products.

Desensitization of P2Y2 receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2 receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current (Isc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the Isc with a potency profile of ATP = UTP>> 2-methylthioATP>>) alpha,beta-methylene-ATP. RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2 receptor agonist UTP and increased in a concentration-dependent manner (IC50 approximately 10(-6) M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (<10 min) at preincubation concentrations less than the EC50 (1.9 x 10(-6) M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2 receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10(-5) M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism.

G-protein coupled receptors (GPCRs) constitute the largest family of intercellular signaling molecules and are estimated to be the target of more than 50% of all modern drugs. As with most integral membrane proteins (IMPs), a major bottleneck in the structural and biochemical analysis of GPCRs is their expression by conventional expression systems. Cell-free (CF) expression provides a relatively new and powerful tool for obtaining preparative amounts of IMPs. However, in the case of GPCRs, insufficient homogeneity of the targeted protein is a problem as the in vitro expression is mainly done with detergents, in which aggregation and solubilization difficulties, as well as problems with proper folding of hydrophilic domains, are common. Here, we report that using CF expression with the help of a fructose-based polymer, NV10 polymer (NVoy), we obtained preparative amounts of homogeneous GPCRs from the three GPCR families. We demonstrate that two GPCR B family members, corticotrophin-releasing factor receptors 1 and 2β are not only solubilized in NVoy but also have functional ligand-binding characteristics with different agonists and antagonists in a detergent-free environment as well. Our findings open new possibilities for functional and structural studies of GPCRs and IMPs in general.

BACKGROUND

In cancer, an extracellular and membrane bound localization of cathepsins contribute to the invasion of tumor cells at the basement membrane.

METHODS

This is the first study that explored levels of cathepsins B (CatB), L (CatL) and their inhibitor cystatin C (CysC) in the cystic fluid (CF) of ovarian tumors (n = 110).

RESULTS

CF contained considerable amounts of CatB, CatL and CysC. Remarkable differences in CatB and CatL and CysC CF levels were found between different histopathological tumor subtypes. Levels of CatB and CysC were significantly higher in CF of malignant serous tumors compared to those found in benign serous tumors (p = 0.010 and p = 0.001 respectively), whereas levels of CatL were significantly higher in CF of malignant mucinous tumors compared to those found in benign mucinous tumors (p = 0.035). CatB and CysC showed a strong correlation in the group of patients with malignant serous tumors (p < 0.001; R = 0.921) suggesting that the increase in CatB might be balanced by a corresponding increase in CysC.

CONCLUSIONS

Further studies are warranted to investigate cathepsins as possible prognostic biomarkers for the aggressiveness of ovarian cancer.

The role of growth temperature and growth irradiance on the regulation of the stoichiometry and function of the photosynthetic apparatus was examined in the cyanobacterium Plectonema boryanum UTEX 485 by comparing mid-log phase cultures grown at either 29 degrees C/150 micromol m(-2) s(-1), 29 degrees C/750 micromol m(-2) s(-1), 15 degrees C/150 micromol m(-2) s(-1), or 15 degrees C/10 micromol m(-2) s(-1). Cultures grown at 29 degrees C/750 micromol m(-2) s(-1) were structurally and functionally similar to those grown at 15 degrees C/150 micromol m(-2) s(-1), whereas cultures grown at 29 degrees C/150 micromol m(-2) s(-1) were structurally and functionally similar to those grown at 15 degrees C/10 micromol m(-2) s(-1). The stoichiometry of specific components of the photosynthetic apparatus, such as the ratio of photosystem (PS) I to PSII, phycobilisome size and the relative abundance of the cytochrome b(6)/f complex, the plastoquinone pool size, and the NAD(P)H dehydrogenase complex were regulated by both growth temperature and growth irradiance in a similar manner. This indicates that temperature and irradiance may share a common sensing/signaling pathway to regulate the stoichiometry and function of the photosynthetic apparatus in P. boryanum. In contrast, the accumulation of neither the D1 polypeptide of PSII, the large subunit of Rubisco, nor the CF(1) alpha-subunit appeared to be regulated by the same mechanism. Measurements of P700 photooxidation in vivo in the presence and absence of inhibitors of photosynthetic electron transport coupled with immunoblots of the NAD(P)H dehydrogenase complex in cells grown at either 29 degrees C/750 micromol m(-2) s(-1) or 15 degrees C/150 micromol m(-2) s(-1) are consistent with an increased flow of respiratory electrons into the photosynthetic intersystem electron transport chain maintaining P700 in a reduced state relative to cells grown at either 29 degrees C/150 micromol m(-2) s(-1) or 15 degrees C/10 micromol m(-2) s(-1). These results are discussed in terms of acclimation to excitation pressure imposed by either low growth temperature or high growth irradiance.

The membrane structure of the naturally occurring gramicidins A, B, and C was investigated using circular dichroism (CD) spectroscopy and single-channel recording techniques. All three gramicidins form channels with fairly similar properties (Bamberg, E., K. Noda, E. Gross, and P. Läuger. 1976. Biochim. Biophys. Acta. 419:223-228.). When incorporated into lysophosphatidylcholine micelles, however, the CD spectrum of gramicidin B is different from that of gramicidin A or C (cf. Prasad, K. U., T. L. Trapane, D. Busath, G. Szabo, and D. W. Urry. 1983. Int. J. Pept. Protein Res. 22:341-347.). The structural identity of the channels formed by gramicidin B has, therefore, been uncertain. We find that when gramicidins A and B are incorporated into dipalmitoylphosphatidylcholine vesicles, their CD spectra are fairly similar, suggesting that the two channel structures could be similar. In planar bilayers, gramicidins A, B, and C all form hybrid channels with each other. The properties of the hybrid channels are intermediate to those of the symmetric channels, and the appearance rates of the hybrid channels (relative to the symmetric channels) corresponds to what would be predicted if all three gramicidin molecules were to form structurally equivalent channels. These results allow us to interpret the different behavior of channels formed by the three gramicidins solely on the basis of the amino acid substitution at position 11.

We used a whole-gut perfusion technique to study subclinical gut inflammation in children with cystic fibrosis (18 elective tests, three lavages to treat distal intestinal obstruction syndrome); and in 12 control children with constipation or pre-colonoscopy. We assayed for haemoglobin, IgG, albumin, alpha-1-antitrypsin, granulocyte elastase, interleukin-1 beta (IL-1 beta) and IL-8 concentrations in whole-gut lavage fluid. Results for two children with distal intestinal obstruction syndrome, the only children in the series taking Nutrizym 22, were strikingly abnormal. This new test has revealed subclinical gut mucosal inflammation in a minority of CF children, for which distal intestinal obstruction syndrome, Nutrizym 22 treatment, or both, may be risk factors.

The antiepileptic effect of progesterone, 5-alpha-pregnane-3,20-dione, 3-alpha-hydroxy-5-alpha-pregnane-20-one, and 3-alpha-hydroxy-5-beta-pregnane-20-one were tested in an experimental animal model, and compared with the effect of clonazepam. The steroids were dissolved in serum from ovariectomized cats. Ovariectomized adult cats were used and spontaneous epileptic discharges were generated by placing small pieces of penicillin-soaked filter papers on the ipsi and contralateral cerebral cortex. The frequency and amplitude of the interictal epileptiform spikes were recorded, and analysed in a computer. The changes in frequency and amplitudes were calculated. The drugs were infused during 20-s periods into one cerebral hemisphere via the ipsilateral lingual artery with speeds of 1.1, 3.4 and 6.3 ml min-1. A penicillin focus on the contralateral hemisphere served as a simultaneous control. Progesterone and clonazepam showed similar inhibitory effects on epileptiform interictal spiking (median reduction of spike frequency 21%, cf. Table I). The 5-alpha-pregnane-3, 20-dione was generally less potent than progesterone (median reduction 9%) and the 5-alpha- and 5-beta-pregnanolones were two to three times more potent than progesterone (54-66% reduction). The latency of the inhibitory effect was 4-10 s measured from the entrance of the infusion into the lingual artery. The depression lasted 10-20 min. It is concluded that the pregnanolones have strong antiepileptic properties. The rapid onset of effect indicates that the steroids may interact with the neuronal function at the membrane or synaptic levels.

Tentoxin, a natural cyclic tetrapeptide produced by phytopathogenic fungi from the Alternaria species affects the catalytic function of the chloroplast F(1)-ATPase in certain sensitive species of plants. In this study, we show that the uncompetitive inhibitor tentoxin binds to the alphabeta-interface of the chloroplast F(1)-ATPase in a cleft localized at betaAsp-83. Most of the binding site is located on the noncatalytic alpha-subunit. The crystal structure of the tentoxin-inhibited CF(1)-complex suggests that the inhibitor is hydrogen bonded to Asp-83 in the catalytic beta-subunit but forms hydrophobic contacts with residues Ile-63, Leu-65, Val-75, Tyr-237, Leu-238, and Met-274 in the adjacent alpha-subunit. Except for minor changes around the tentoxin-binding site, the structure of the chloroplast alpha(3)beta(3)-core complex is the same as that determined with the native chloroplast ATPase. Tentoxin seems to act by inhibiting inter-subunit contacts at the alphabeta-interface and by blocking the interconversion of binding sites in the catalytic mechanism.

Cystic fibrosis (CF) is caused by defects in the CF transmembrane conductance regulator (CFTR) that functions as a chloride channel in epithelial cells. The most common cause of CF is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the plasma membrane (PM) is important. The coat protein complex I (COPI) has been implicated in the anterograde and retrograde transport of proteins and lipids between the endoplasmic reticulum and the Golgi. Here, we investigated the role of COPI in CFTR trafficking. Blocking COPI recruitment to membranes by expressing an inactive form of the GBF1 guanine nucleotide exchange factor for ADP-ribosylation factor inhibits CFTR trafficking to the PM. Similarly, inhibiting COPI dissociation from membranes by expressing a constitutively active ADP-ribosylation factor 1 mutant arrests CFTR within disrupted Golgi elements. To definitively explore the relationship between COPI and CFTR in epithelial cells, we depleted beta-COP from the human colonic epithelial cell HT-29Cl.19A using small interfering RNA. Beta-COP depletion did not affect CFTR synthesis but impaired its trafficking to the PM. The arrest occurred pre-Golgi as shown by reduced level of glycosylation. Importantly, decreased trafficking of CFTR had a functional consequence as cells depleted of beta-COP showed decreased cAMP-activated chloride currents. To explore the mechanism of COPI action in CFTR traffic we tested whether CFTR was COPI cargo. We discovered that the alpha-, beta-, and gamma-subunits of COPI co-immunoprecipitated with CFTR. Our results indicate that the COPI complex plays a critical role in CFTR trafficking to the PM.

OBJECTIVE

Increasing evidence indicates that Pseudomonas aeruginosa grows as a biofilm in the lungs of cystic fibrosis (CF) patients. In contrast, the bacterial inoculum used in conventional susceptibility testing is composed of planktonic cells. As a prelude to a clinical trial of biofilm susceptibility testing in CF, simulated antibiotic regimens based on either biofilm or conventional susceptibility testing of CF patient isolates were compared.

METHODS

Biofilm and conventional susceptibilities were determined for P. aeruginosa isolate sets from 40 CF patients. An algorithm was used to assign simulated regimens of two anti-pseudomonal antibiotics for each patient/susceptibility method dataset. For agents with equivalent activity, the algorithm included a drug selection hierarchy, the rationale for which was suppression of chronic infection. Substitution of an alternative hierarchy, based on treatment of acute exacerbation, was used to evaluate the robustness of the regimen assignments.

RESULTS

For both drug-ranking schemes, all 40 simulated regimens based on conventional susceptibilities included a beta-lactam antibiotic. In contrast, based on biofilm testing, only 43% of chronic regimens and 65% of acute regimens included a beta-lactam. Moreover, the conventional and biofilm regimens assigned to individual patients were discordant, with only 20% and 40% of chronic and acute regimens, respectively, consisting of drugs in the same two mechanistic classes by both methods.

CONCLUSIONS

Biofilm susceptibility testing of CF P. aeruginosa isolate sets leads to different antibiotic assignments than conventional testing, with no single two-drug regimen predicted to provide optimal anti-biofilm activity against the majority of isolate sets.

Green mutant cells of sycamore (Acer pseudoplatanus L.), which had been selected by mutagenic treatment of the white wild type, grow photoheterotrophically in auxin-depleted culture medium. In contrast to the wild-type cells, mutant cells exhibit photosynthetic O(2)-evolution activity during their growth coincident with increases of (a) chlorophyll, (b) protein, and (c) ribulose-1,5-bisphosphate (RuBP) carboxylase activity. Functionally competent chloroplasts were isolated from the green cells. Mechanism(s) governing gene expression of amyloplast DNA in the heterotrophically grown white cells were compared with those of the chloroplast DNA isolated from the mutant cells. We have demonstrated in both amyloplast and chloroplast DNAs the presence of sequences homologous to the maize chloroplast genes for photosynthesis, including the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)(rbcL), the 32 kDa Q(B) protein (PG32) (psbA), the apoprotein of P700 (psaA) and subunits of CF(1) (atpA, atpB, and atpE). However, employing either enzyme assays or immunological techniques, RuBisCO and CF(1) cannot be detected in the white wild type cells. Northern blot hybridization of the RNA from the white cells showed high levels of transcripts for the 16S rRNA gene and low level of transcripts for psbA; based on comparison with results obtained using the green mutant cells, we propose that the amyloplast genome is mostly inactive except for the 16S rRNA gene and psbA which is presumably regulated at the transcriptional level.

We have studied the relationship between the cytokine production induced in vivo by prolonged isometric exercise and the symptom complex marked by fatigue in patients with chronic fatigue syndrome (CFS). Twelve male patients and 13 matched male control subjects undertook an isometric hand-grip exercise protocol utilizing dynamometers. Subjects undertook 30 minutes of exercise, for which the target force was set at 40% of the maximal voluntary contraction and the duty cycle was 50%. Prior to, during, and for 24 hours following the exercise, blood samples were collected and assayed for the presence of cytokines, including interferon-gamma and interferon-alpha, interleukin-1 beta, and tumor necrosis factor-alpha. At those times subjects also completed the Profile of Mood States (POMS) questionnaire, which served as a measure of changes in subjective fatigue. No significant alteration in the level of any of the cytokines in the plasma of patients or control subjects was detected before, during, or after exercise. Surprisingly, the patients' levels of fatigue, depression, and confusion, as measured by the POMS, decreased in response to the exercise. These data do not confirm the presence of an immunologic process correlating with the exacerbation of fatigue after exercise experienced by patients with CFS. Limitations in the study design and in the sensitivity of the cytokine assays may have affected our results.

OBJECTIVE

Vascular endothelial growth factor (VEGF) is the prime regulator of angiogenesis and vascular permeability and its serum levels increase in cystic fibrosis (CF). The mechanisms of VEGF overproduction and its impact on CF lung pathology and pulmonary vascular permeability during lung transplantation are not fully understood.

METHODS

The expression of VEGF, its receptors, hypoxia inducible factor (HIF)-1alpha, beta, angiopoietins, and endothelial cell marker CD31 were studied in lung biopsies of CF and COPD patients and controls, using real time reverse transcription (RT)-PCR and Western blotting. DNA binding activity of HIF-1 to VEGF-A promoter was assessed by electrophoretic mobility shift assay (EMSA) and wet-to-dry lung weight ratios as well as microvascular density (MVD) were determined. Serum VEGF-A concentrations in enzyme-linked immunosorbent assay (ELISA) and wet-to-dry weight ratios of donor lungs were monitored during transplantation in CF and COPD patients. Primary graft dysfunction (PGD) was diagnosed and graded according to the guidelines of the International Society for Heart and Lung Transplantation.

RESULTS

VEGF-A165 and Flt-1 mRNA expression (P<0.05), VEGF-A (P<0.05), and HIF-1alpha (P<0.05) protein levels, DNA binding activity of HIF-1 to VEGF promoter (P<0.001) and extravascular lung water content (P<0.05) were increased in CF lungs versus controls, whereas MVD was unchanged. Before and during lung transplantation, VEGF-A serum concentrations were higher in CF versus COPD patients (P<0.05) and 60 min following reperfusion donor lungs transplanted to CF patients had higher tissue water contents than in COPD patients (P<0.05). PGD grade 3 occurred more frequently in CF (22.7%) versus COPD patients (4%). PGD grade 3 patients had significantly higher VEGF serum concentrations versus PGD grade 0-2 patients (P<0.001).

CONCLUSIONS

These data indicate that upregulated VEGF-A levels are most likely induced by enhanced HIF-1 binding to VEGF-A promoter, possibly contributing to elevated serum VEGF-A levels in CF. Furthermore, CF patients undergoing lung transplantation are possibly more susceptible to PGD because of increased VEGF-A expression that mediates increased lung graft vascular permeability.

Administration of recombinant adenoviral (AdV) vectors to animals can lead to inflammatory and immune responses. For therapeutic indications in which repeated treatment is necessary, such as cystic fibrosis (CF), these responses can limit the therapeutic usefulness of the vector. In principle, the utility of the vector can be improved by increasing its therapeutic index, that is, by either increasing its efficacy or decreasing its toxicity. A strategy that would enhance the efficacy of an adenoviral approach would allow the use of fewer virus particles to achieve a given level of transgene expression, and thereby also reduce unwanted effects such as immune responses. Following up on our observation that treating polarized normal human bronchial epithelial cells with calcium (Ca(2+))-free medium or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) significantly enhanced the subsequent transfection of these cells with cationic lipid:pDNA complexes, we have now asked whether such a treatment protocol might also improve the ability of AdV to infect these cells. Treating polarized airway epithelial cells with EGTA led to a dramatic increase in AdV-mediated transduction, as demonstrated by an approximately 50-fold increase in transgene expression. This strategy was also tested in vivo and resulted in substantial increases (up to 50-fold) in the ability of AdV vectors to infect mouse tracheal epithelium. Transfection of mouse trachea with an AdV aerosol was also significantly increased by pretreatment with EGTA. The enhancing effects of EGTA could not be duplicated with hypo- or hyperosmotic treatments. Light microscopy of mouse trachea that had been EGTA treated and then infected with AdV demonstrated an EGTA-mediated AdV infection of airway epithelial cells. The apparent enhanced potency of AdV for airway cells resulting from this strategy provides a significant increase in the therapeutic index of this gene delivery vector, and may increase the likelihood that it can be used for clinical indications requiring chronic administration of the vector.

BACKGROUND

High-dose ibuprofen (HDI) is a clinically beneficial anti-inflammatory regimen that may be a useful reagent to study induced sputum inflammatory marker changes over short study periods appropriate for early-phase CF clinical trials.

METHODS

We conducted a 28-day, open-label, randomized, controlled trial among 72 clinically stable CF subjects (FEV1≥40% predicted) randomized to HDI or routine care that assessed IL-6, IL-8, TNF-α, IL-1-β, free neutrophil elastase, and white cell counts with differentials change from baseline in induced sputum.

RESULTS

IL-6 was the only biomarker with significant within-group change: 0.13 log10 pg/mL mean reduction among ibuprofen-treated subjects (p=0.04); and no change in the control group. IL-6 change between groups was statistically significant (p=0.024). No other inflammatory biomarker differences were observed between groups after 28 days.

CONCLUSIONS

Although we studied only one agent, HDI, these results suggest that one month may be inadequate to assess anti-inflammatory candidates using markers from induced sputum.

We have used monoclonal antibodies to study the expression of calgranulins by keratinocytes in inflammatory dermatoses. Calgranulins are intracellular calcium binding proteins which have inflammatory cytokine activity and are composed of at least two different chains, calgranulin A and B. Antibody CF 145 and CF 557 identify calgranulin A and B, respectively. MAC 387 recognizes a molecule probably containing both calgranulins. Keratinocytes in normal skin did not contain these molecules. The keratinocytes in 52 cases of different inflammatory dermatoses showed expression of both calgranulin chains in lesional but not in non-lesional skin. Keratinocytes in inflammatory dermatoses therefore express an intracellular calcium binding protein which has cytokine activity.

Phylogenetic inference regarding the biogeography and evolution of the family Cobitidae depends in large part on the correct interpretation of transitions between the morphological states of secondary sexual characters (e.g., the scale of Canestrini or lamina circularis). Here, we use the complete mitochondrial ATP synthase 8 and 6 and cytochrome b genes to provide an independent assessment of systematics and biogeographic relationships of species in the genus Cobitis, including geographic and subgeneric sampling of species with Canestrini's scale present, duplicated, or absent. The mtDNA-based phylogeny for the genus Cobitis provides the first formal hypothesis for the group and permits a phylogenetic-based assessment of the morphological transitions demonstrated by Canestrini's scale. Our data confirm the monophyly of the genus Cobitis and indicate that European Cobitis comprise six evolutionarily independent lineages. These lineages were defined by nucleotide synapomorphies permitting bootstrapped confidence estimates of 95% or greater and mtDNA genetic distances greater than 4.5% and correspond with moderate fidelity to the Cobitis groups defined by Bacescu (1962, Rev. Roum. Biol. 4, 435-448). The Caucasian lineage, C. cf. sibirica, represents the basal sister species of the genus Cobitis, supporting an eastern Asiatic origin of the European Cobitis: Cobitis sensu stricto, Acanestrinia, Bicanestrinia, Iberocobitis, and Cobitis calderoni. Phylogenetic relationships among Cobitis subgenera and species indicate that the ancestral condition of one scale of Canestrini was duplicated once at the origin of the Bicanestrinia lineage and has been independently lost by C. calderoni and C. elongata. The absence of the scale of Canestrini is the synapomorphy defining the subgenus Acanestrinia, but the mtDNA phylogeny indicates that Acanestrinia is not a natural group and places C. calderoni as the sister lineage to the subgenus Iberocobitis, a finding that is also geographically parsimonius.

Forty Klebsormidium strains isolated from soil crusts of mountain regions (Alps, 600–3,000 m elevation) were analyzed. The molecular phylogeny (internal transcribed spacer rDNA sequences) showed that these strains belong to clades B/C, D, E, and F. Seven main (K. flaccidum, K. elegans, K. crenulatum, K. dissectum, K. nitens, K. subtile, and K. fluitans) and four transitional morphotypes (K. cf. flaccidum, K. cf. nitens, K. cf. subtile, and K. cf. fluitans) were identified. Most strains belong to clade E, which includes isolates that prefer humid conditions. One representative of the xerophytic lineage (clade F) as well as few isolates characteristic of temperate conditions (clades B/C, D) were found. Most strains of clade E were isolated from low/middle elevations (<1,800 m above sea level; a.s.l.) in the pine-forest zone. Strains of clades B/C, D, and F occurred sporadically at higher elevations (1,548–2,843 m a.s.l.), mostly under xerophytic conditions of alpine meadows. Comparison of the alpine Klebsormidium assemblage with data from other biogeographic regions indicated similarity with soil crusts/biofilms from terrestrial habitats in mixed forest in Western Europe, North America, and Asia, as well as walls of buildings in Western European cities. The alpine assemblage differed substantially from crusts from granite outcrops and sand dunes in Eastern Europe (Ukraine), and fundamentally from soil crusts in South African drylands. Epitypification of the known species K. flaccidum, K. crenulatum, K. subtile, K. nitens, K. dissectum, K. fluitans, K. mucosum, and K. elegans is proposed to establish taxonomic names and type material as an aid for practical studies on these algae, as well as for unambiguous identification of alpine strains. New combination Klebsormidium subtile (Kützing) Mikhailyuk, Glaser, Holzinger et Karsten comb. nov. is made.