BACKGROUND

Burkholderia cenocepacia is an endemic soil dweller and emerging opportunistic pathogen in patients with cystic fibrosis (CF). The identification of virulence factors and potential therapeutic targets has been hampered by the genomic diversity within the species as many factors are not shared among the pathogenic members of the species.

RESULTS

In this study, global identification of putative virulence factors was performed by analyzing the transcriptome of two related strains of B. cenocepacia (one clinical, one environmental) under conditions mimicking cystic fibrosis sputum versus soil. Soil is a natural reservoir for this species; hence, genes induced under CF conditions relative to soil may represent adaptations that have occurred in clinical strains. Under CF conditions, several genes encoding proteins thought to be involved in virulence were induced and many new ones were identified. Our analysis, in combination with previous studies, reveals 458 strain-specific genes, 126 clinical-isolate-specific, and at least four species-specific genes that are induced under CF conditions. The chromosomal distribution of the induced genes was disproportionate to the size of the chromosome as genes expressed under soil conditions by both strains were more frequent on the second chromosome and those differentially regulated between strains were more frequent on the third chromosome. Conservation of these induced genes was established using the 11 available Bcc genome sequences to indicate whether potential therapeutic targets would be species-wide.

CONCLUSIONS

Comparative transcriptomics is a useful way to identify new potential virulence factors and therapeutic targets for pathogenic bacteria. We identified eight genes induced under CF conditions that were also conserved in the Bcc and may constitute particularly attractive therapeutic targets due to their signal sequence, predicted cellular location, and homology to known therapeutic targets.

The in vitro activities of doripenem, imipenem, levofloxacin, piperacillin, ceftazidime, aztreonam, tobramycin, and cefepime were determined for 160 isolates of Pseudomonas aeruginosa (82 from cystic fibrosis [CF] patients) and 34 isolates of Burkholderia cepacia. Doripenem MIC90s were lower than those of all other comparative agents against all isolates combined and against all P. aeruginosa isolates. Doripenem was as active as levofloxacin and 2- to 32-fold more active than the other comparative agents against B. cepacia.

Neutrophil activation may play an important role in the pathogenesis of respiratory disease in Burkholderia cepacia-colonized cystic fibrosis (CF) patients. As bacterial lipopolysaccharides (LPS) are potent immunostimulatory molecules, we investigated the role of B. cepacia LPS in neutrophil activation processes. LPS extracted from a highly transmissible and virulent strain of B. cepacia (J2315) was found to increase neutrophil surface expression of the beta2 integrin, complement receptor 3, and to prime neutrophil respiratory burst responses to the neutrophil-activating agent fMet-Leu-Phe. By contrast, LPS extracted from a nonmucoid Pseudomonas aeruginosa strain isolated from a patient with CF showed little or no priming activity. As B. cepacia is currently being developed as a biocontrol agent for large-scale agricultural release, we compared LPS molecules from a range of bacterial strains for their proinflammatory ability. Priming activity was demonstrated in LPS extracts from all B. cepacia strains tested, with one environmental strain, J2552, showing the highest activity. These findings indicate (i) that B. cepacia LPS may contribute to the inflammatory nature of B. cepacia infection in CF patients, both by promoting increased neutrophil recruitment and by priming neutrophil respiratory burst responses, and (ii) that environmental strains of B. cepacia may have considerable inflammatory potential in susceptible individuals.

Repeat administration of gene therapy for cystic fibrosis is likely to be essential for long-term clinical efficacy. This may be minimized by the use of slow-release gene transfer preparations with more prolonged expression and longer dosing intervals for the patient. Poly(D-L-lactide-co-glycolide) (PLG) is a biodegradable and biocompatible polymer that has been used to encapsulate plasmid DNA. PLG-DNA microspheres were generated and characterized with respect to morphology, size (80% of particles <5.2 microm), and encapsulation efficiency (50.7+/-2.3%, n=6). Gel electrophoresis of DNA re-extracted from the microspheres confirmed that despite a decrease in the proportion of supercoiled conformation, it had not been degraded by the preparation process. Gene transfer efficiency was tested using microspheres encapsulating the reporter gene beta-galactosidase in vitro on Cos 7 cells and a CF airway epithelial line (CFTEo approximately ) and ex vivo in a sheep tracheal (s.t.) model. In both cases, transgene expression was significantly (P<0.01) lower at the first time point tested (24 h in vitro, 48 h ex vivo) compared to lipid-#67-mediated gene transfer. However, PLG-mediated expression in vitro was sustained at 48 h, while lipid #67-mediated expression levels had dropped significantly (P<0.05) to 50.3+/-13.7 and 38.2+/-2.7% (Cos 7 and CFTEo approximately cells, respectively) of the 24-h level. This pattern was also seen in the s.t. model where at 72 h, PLG-mediated expression was 125.4+/-7.2% of the 48-h level demonstrating significantly (P<0.05) better retention of transfection efficiency than lipid #67, where levels had fallen to approximately half the 48 h level. By 96 h, expression was still retained in the PLG-transfected group (87.3+/-12.5% of 48 h expression) but was undetectable in the lipid -#67-transfected s.t. Finally, PLG microspheres, encapsulating the reporter gene chloramphenicol transferase (CAT, 80 microg) were instilled intranasally into Balb/C mice. Compared to lipid-#67-mediated delivery, where whole lung CAT expression was highest at 48 h (13.7 x 10(3)+/-0.05 CAT U/microg protein, n=6) and then not detectable at further time points, CAT expression was not detectable in PLG-transfected mice at 48 h, but was detectable at 7, 14 and 21 days after transfection. These data demonstrate that PLG-mediated gene transfer can produce prolonged gene expression in airway epithelia. However, gene transfer efficiency still requires significant improvement.

Barriers to infection act at multiple levels to prevent viruses, bacteria, and parasites from commandeering host cells for their own purposes. An intriguing hypothesis is that if a cell experiences stress, such as that elicited by inflammation, endoplasmic reticulum (ER) expansion, or misfolded proteins, then subcellular barriers will be less effective at preventing viral infection. Here we have used models of cystic fibrosis (CF) to test whether subcellular stress increases susceptibility to adeno-associated virus (AAV) infection. In human airway epithelium cultured at an air/liquid interface, physiological conditions of subcellular stress and ER expansion were mimicked using supernatant from mucopurulent material derived from CF lungs. Using this inflammatory stimulus to recapitulate stress found in diseased airways, we demonstrated that AAV infection was significantly enhanced. Since over 90% of CF cases are associated with a misfolded variant of Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR), we then explored whether the presence of misfolded proteins could independently increase susceptibility to AAV infection. In these models, AAV was an order of magnitude more efficient at transducing cells expressing ΔF508-CFTR than in cells expressing wild-type CFTR. Rescue of misfolded ΔF508-CFTR under low temperature conditions restored viral transduction efficiency to that demonstrated in controls, suggesting effects related to protein misfolding were responsible for increasing susceptibility to infection. By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. The presence of misfolded proteins did not affect cell surface attachment of virus or influence expression levels from promoter transgene cassettes in plasmid transfection studies, indicating exploitation occurs at the level of virion trafficking or processing. Thus, we surmised that factors enlisted to process misfolded proteins such as ΔF508-CFTR in the secretory pathway also act to restrict viral infection. In line with this hypothesis, we found that AAV trafficked to the microtubule organizing center and localized near Golgi/ER transport proteins. Moreover, AAV infection efficiency could be modulated with siRNA-mediated knockdown of proteins involved in processing ΔF508-CFTR or sorting retrograde cargo from the Golgi and ER (calnexin, KDEL-R, β-COP, and PSMB3). In summary, our data support a model where AAV exploits a compromised secretory system and, importantly, underscore the gravity with which a stressed subcellular environment, under internal or external insults, can impact infection efficiency.

Transforming growth factor <em>β</em>1 (TGF-<em>β</em>1) is not only elevated in airways of cystic fibrosis (<em>CF</em>) patients, whose airways are characterized by abnormal ion transport and mucociliary clearance, but TGF-<em>β</em>1 is also associated with worse clinical outcomes. Effective mucociliary clearance depends on adequate airway hydration, governed by ion transport. Apically expressed, large-conductance, Ca(2+)- and voltage-dependent K(+) (BK) channels play an important role in this process. In this study, TGF-<em>β</em>1 decreased airway surface liquid volume, ciliary beat frequency, and BK activity in fully differentiated <em>CF</em> bronchial epithelial cells by reducing mRNA expression of the BK γ subunit leucine-rich repeat-containing protein 26 (LRRC26) and its function. Although LRRC26 knockdown itself reduced BK activity, LRRC26 overexpression partially reversed TGF-<em>β</em>1-induced BK dysfunction. TGF-<em>β</em>1-induced airway surface liquid volume hyper-absorption was reversed by the BK opener mallotoxin and the clinically useful TGF-<em>β</em> signaling inhibitor pirfenidone. The latter increased BK activity via rescue of LRRC26. Therefore, we propose that TGF-<em>β</em>1-induced mucociliary dysfunction in <em>CF</em> airways is associated with BK inactivation related to a LRRC26 decrease and is amenable to treatment with clinically useful TGF-<em>β</em>1 inhibitors.

Cystic fibrosis (CF) is caused by loss-of-function mutations in the CFTR chloride channel. Wild type and mutant CFTR channels can be activated by curcumin, a well tolerated dietary compound with some appeal as a prospective CF therapeutic. However, we show here that curcumin has the unexpected effect of cross-linking CFTR polypeptides into SDS-resistant oligomers. This effect occurred for CFTR channels in microsomes as well as in intact cells and at the same concentrations that are effective for promoting CFTR channel activity (5-50 mum). Both mature CFTR polypeptides at the cell surface and immature CFTR protein in the endoplasmic reticulum were cross-linked by curcumin, although the latter pool was more susceptible to this modification. Curcumin cross-linked two CF mutant channels (Delta F508 and G551D) as well as a variety of deletion constructs that lack the major cytoplasmic domains. In vitro cross-linking could be prevented by high concentrations of oxidant scavengers (i.e. reduced glutathione and sodium azide) indicating a possible oxidation reaction with the CFTR polypeptide. Importantly, cyclic derivatives of curcumin that lack the reactive beta diketone moiety had no cross-linking activity. One of these cyclic derivatives stimulated the activities of wild type CFTR channels, Delta 1198-CFTR channels, and G551D-CFTR channels in excised membrane patches. Like the parent compound, the cyclic derivative irreversibly activated CFTR channels in excised patches during prolonged exposure (>5 min). Our results raise a note of caution about secondary biochemical effects of reactive compounds like curcumin in the treatment of CF. Cyclic curcumin derivatives may have better therapeutic potential in this regard.

The role of the Pseudomonas aeruginosa OxyR-controlled antioxidants alkyl hydroperoxide reductase CF (AhpCF) and catalase B (KatB) was evaluated in biofilm vs. planktonic culture upon exposure to hydrogen peroxide. AhpCF was found to be critical for survival of biofilm bacteria while KatB was more important for survival of planktonic free-swimming organisms.

OBJECTIVE

Radiation oncologists are often faced with patients with advanced non-small-cell lung cancer (NSCLC), who are not suitable candidates for state-of-the-art radical treatment, but who also are not judged to have a very short life expectancy. Some physicians treat these patients palliatively, whereas others advocate more intensive treatment. To find out if there is a substantial difference in outcome between these approaches, we performed a randomized prospective study.

METHODS

Between 1994 and 1998, 152 eligible patients with advanced NSCLC Stage III (n = 121) or minimal Stage IV (n = 31) were randomized to receive conventionally fractionated (cf; A: 60 Gy, 6 weeks, n = 79) or short-term treatment (PAIR; B: 32 Gy, 2 Gy b.i.d.; n = 73) of tumor and mediastinum.

RESULTS

One-year survival rate for all patients was 37% with no significant difference between the two treatment arms (A: 36%; B: 38%; p = 0.76). As far as can be judged from limited data available, palliation was adequate and similar for the two treatment arms. Apart from expected differences in the time course of esophagitis, acute side effects were moderate and equally distributed. No severe late effects were observed.

CONCLUSIONS

In the present randomized trial, survival and available data on palliation were not different after cf to 60 Gy compared to the palliative PAIR regimen. Therefore, for patients who are not suitable for radical treatment approaches, the prescription of a palliative short-term irradiation appears preferable compared to cf over several weeks.

BACKGROUND

Monitoring potential changes in the epidemiology of cystic fibrosis (CF) pathogens furthers our understanding of the potential impact of interventions.

METHODS

We performed a retrospective analysis using data reported to the Cystic Fibrosis Foundation Patient Registry (CFFPR) from 2006 to 2012 to determine the annual percent changes in the prevalence and incidence of selected CF pathogens. Pathogens included Pseudomonas aeruginosa, methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S aureus (MRSA), Haemophilus influenzae, Burkholderia cepacia complex, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans. Changes in nontuberculous mycobacteria (NTM) prevalence were assessed from 2010 to 2012, when the CFFPR collected NTM species.

RESULTS

In 2012, the pathogens of highest prevalence and incidence were MSSA and P aeruginosa, followed by MRSA. The prevalence of A xylosoxidans and B cepacia complex were relatively low. From 2006 to 2012, the annual percent change in overall (as well as in most age strata) prevalence and incidence significantly decreased for P aeruginosa and B cepacia complex, but significantly increased for MRSA. From 2010 to 2012, the annual percent change in overall prevalence of NTM and Mycobaterium avium complex increased.

CONCLUSIONS

The epidemiology of CF pathogens continues to change. The causes of these observations are most likely multifactorial and include improvements in clinical care and infection prevention and control. Data from this study will be useful to evaluate the impact of new therapies on CF microbiology.

BACKGROUND

Human herpesvirus 6 (HHV-6) and 7 (HHV-7) have been suggested as possible triggering agents for chronic fatigue syndrome (CFS).

OBJECTIVE

To determine the possible association of HHV-6 and HHV-7 infections with CFS.

METHODS

The prevalence of latent/persistent and active viral infections by nPCR, characteristic of HHV-6 variants using restriction endonuclease analysis and changes of lymphocyte subsets in peripheral blood by laser flow-cytometry in 17 CFS patients was examined. In addition, 12 patients with unexplained chronic fatigue and 20 blood donors (BD) were studied.

RESULTS

No difference in prevalence of latent/persistent single viral infections between the patients and BD was found but dual infection rate was significantly higher in CFS patients. Active HHV-6 and dual (HHV-6 + HHV-7) infections were detected in CFS patients only and frequency of HHV-7 reactivation was also significantly higher in these patients. HHV-6 variant B was predominant in CFS patients (12/13). The changes of immunological parameters in CFS patients with active dual infection were characterized by significant decrease of CD3+ and CD4+ T cells, significant increase of CD95+ cells and decrease of CD4+/CD8+ ratio.

CONCLUSIONS

HHV-6 and HHV-7 may be involved in the pathogenesis of CFS and reactivation of both viruses may provoke changes in the phenotype of circulating lymphocytes.

BACKGROUND

While cystic fibrosis (CF) is the most common cause of bronchiectasis in childhood, non-CF bronchiectasis is associated with a wide variety of disorders. The objective of this study was to determine the relative prevalence and specific etiologies on non-CF bronchiectasis in childhood.

METHODS

EMBASE, Medline, OVID Cochrane Reviews, Directory of Open Access Journals, Open Science Directory, EPSCO information services, and OAlster were searched electronically and the bibliographies of selected studies were searched manually. The search was conducted independently by 2 authors.

METHODS

(1) any clinical trial, observational study or cross-sectional case series of 10 or more patients with a description of the conditions associated with bronchiectasis; (2) subjects aged 21 years or younger; (3) cystic fibrosis was excluded and; (4) the diagnosis was confirmed by computed tomography of the chest.

METHODS

Patient number, age range, inclusion criteria, diagnostic criteria, patient source, and categorical and specific etiology.

RESULTS

From 491 studies identified, 12 studies encompassing 989 children with non-CF bronchiectasis were selected. Sixty-three percent of the subjects had an underlying disorder. Infectious (17%), primary immunodeficiency (16%), aspiration (10%), ciliary dyskinesia (9%), congenital malformation (3%), and secondary immunodeficiency (3%) were the most common disease categories; 999 etiologies were identified. Severe pneumonia of bacterial or viral etiology and B cell defects were the most common disorders identified.

CONCLUSIONS

The majority of children with non-CF bronchiectasis have an underlying disorder. A focused history and laboratory investigated is recommended.

Poorly sporulating Aspergillus isolates from patients with cystic fibrosis (CF) are generally identified in routine procedures as Aspergillus spp. In this study, we identified and characterized 11 isolates belonging to two unusual Aspergillus species of the section Fumigati (A. lentulus and Neosartorya pseudofischeri) recovered from four different patients. Aspergillus lentulus was found occasionally during a 10-year follow-up study of one CF patient colonized by A. fumigatus. Neosartorya pseudofischeri was isolated from three patients followed in different European hospitals. This species was recovered from two sputum samples of one patient, and from four successive samples of the two other patients, suggesting that it may be responsible for chronic colonization. Both species were isolated together with A. fumigatus. Isolates from both species did not grow at 50°C, and DNA sequence analysis, together with further morphological observations permitted identification at the species level. Growth at different temperatures and antifungal susceptibility were also investigated. All the isolates of N. pseudofischeri exhibited a very low susceptibility to voriconazole (VRZ) whereas a very low susceptibility to VRZ and amphotericin B was seen with the A. lentulus isolates.

A copper-catalyzed decarboxylative trifluoromethylation of various α,β-unsaturated carboxylic acids by using a stable and inexpensive solid, sodium trifluoromethanesulfinate (CF(3)SO(2)Na, Langlois reagent), was developed. In addition, an iron-catalyzed difluoromethylation of aryl-substituted acrylic acids by using zinc difluoromethanesulfinate (DFMS, (CF(2)HSO(2))(2)Zn, Baran reagent) via a similar radical process was also achieved.

The efficacy of cefmetazole and flomoxef (CF) for the treatment of patients with extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) bacteremia (ESBL-CF group) was compared with that of carbapenem treatment for ESBL-EC patients (ESBL-carbapenem group) and with that of CF treatment in patients with non-ESBL-EC bacteremia (non-ESBL-CF group). Adult patients treated for E. coli bacteremia in four hospitals were retrospectively evaluated. The 30-day mortality rates in patients belonging to the ESBL-CF, ESBL-carbapenem, and non-ESBL-CF groups were compared as 2 (empirical and definitive therapy) cohorts. The adjusted hazard ratios (aHRs) for mortality were calculated using Cox regression models with weighting according to the inverse probability of propensity scores for receiving CF or carbapenem treatment. The empirical-therapy cohort included 104 patients (ESBL-CF, 26; ESBL-carbapenem, 45; non-ESBL-CF, 33), and the definitive-therapy cohort included 133 patients (ESBL-CF, 59; ESBL-carbapenem, 54; non-ESBL-CF, 20). The crude 30-day mortality rates for patients in the ESBL-CF, ESBL-carbapenem, and non-ESBL-CF groups were, respectively, 7.7%, 8.9%, and 3.0% in the empirical-therapy cohort and 5.1%, 9.3%, and 5.0% in the definitve-therapy cohort. In patients without hematological malignancy and neutropenia, CF treatment for ESBL-EC patients was not associated with mortality compared with carbapenem treatment (empirical-therapy cohort: aHR, 0.87; 95% confidence interval [CI], 0.11 to 6.52; definitive therapy cohort: aHR, 1.04; CI, 0.24 to 4.49). CF therapy may represent an effective alternative to carbapenem treatment for patients with ESBL-EC bacteremia for empirical and definitive therapy in adult patients who do not have hematological malignancy and neutropenia.

Animal models used to study the pathogenesis of non-alcoholic fatty liver disease (NAFLD) are, in general, either genetically altered, or fed with a diet that is extremely high in fat or carbohydrates. Recent findings support the role of oxidative stress, lipid peroxidation and inflammation as probable causative factors. We hypothesize that not only the amount of dietary fat, but the quality of fat is also important in inducing NAFLD. Based on previous observations that female rats fed a diet comprising unsaturated fatty acids are susceptible to liver injury, we proposed that female rats fed with a diet containing fish oil and dextrose would develop pathological and biochemical features of NAFLD. We fed a highly unsaturated fat diet (30% fish oil) to female Sprague-Dawley rats (180-200g), consumed ad libitum for 8 weeks (NAFLD; n=6-8 ). Control animals (CF; n=6-8) were fed with an isocaloric regular rat chow. At killing, blood and liver samples were collected for serum alanine aminotransferase (ALT), histology and molecular analysis. Each histological sample was evaluated for fatty liver (graded from 0 to 4+ according to the amount of fatty change), necrosis (number of necrotic foci (no./mm2) and inflammation (cells per mm2). The amount of collagen formation was estimated based on the amount of Sirius Red staining. Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out for tumor necrosis factor alpha (TNF-alpha), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), adiponectin, glutathione peroxidase (GPx), superoxide dismutase (Cu/Zn SOD) and catalase (CAT). Western Blot analysis was done for cyclooxygenases-2 (COX-2), inducible nitric oxide synthase (iNOS) and nitrotyrosine. Electrophoretic mobility shift assay was performed for nuclear factor-kappa B (NF-kB) activity. NAFLD rats had a significantly higher serum ALT level, amount of collagen formation, fatty liver, necrosis and inflammation when compared with the chow-fed control rats. mRNA and protein levels of NF-kB regulated genes, which included TNF-alpha, COX-2 and iNOS were also significantly (p<0.01; p<0.01; p<0.05 respectively) upregulated in the NAFLD group when compared with the chow-fed control rats. mRNA levels of antioxidants CAT and GPX were reduced by 35% and 50% respectively in the NAFLD group. However, Cu/Zn SOD mRNA was similar in both groups. The mRNA level of adiponectin was also reduced in NAFLD group. NF-kB activity was markedly increased in the NAFLD rats (p<0.01). The level of oxidative stress, represented by the formation of nitrotyrosine, was significantly elevated in the NAFLD rats (p<0.01). We conclude that NAFLD rats demonstrated several features of NAFLD, which included fatty liver, inflammation, necrosis, increased oxidative stress, an imbalance between pro and antioxidant enzymes mRNAs, reduced adiponectin levels and upregulation of pro-inflammatory mediators. We propose that female rats fed with a diet containing highly unsaturated fatty acids are an extremely useful model for the study of NAFLD.

The oral microbiota associated with the initiation and progression of dental caries has yet to be fully characterized. The Human Oral Microbe Identification Using Next-Generation Sequencing (HOMINGS) approach was used to analyze the microbiomes of site-specific supragingival dental plaques from children with different caries status. Fifty-five children (2 to 7 years of age) were assessed at baseline and at 12 months and grouped as caries free (CF), caries active with enamel lesions (CAE), and caries active with dentin carious lesions (CA). Plaque samples from caries-free tooth surfaces (PF) and from enamel carious lesions (PE) and dentin carious lesions (PD) were collected. 16S community profiles were obtained by HOMINGS, and 408 bacterial species and 84 genus probes were assigned. Plaque bacterial communities showed temporal stability, as there was no significant difference in beta diversity values between the baseline and 12-month samples. Irrespective of collection time points, the microbiomes of healthy tooth surfaces differed substantially from those found during caries activity. All pairwise comparisons of beta diversity values between groups were significantly different (P < 0.05), except for comparisons between the CA-PF, CAE-PE, and CA-PE groups. Streptococcus genus probe 4 and Neisseria genus probe 2 were the most frequently detected taxa across the plaque groups, followed by Streptococcus sanguinis, which was highly abundant in CF-PF. Well-known acidogenic/aciduric species such as Streptococcus mutans, Scardovia wiggsiae, Parascardovia denticolens, and Lactobacillus salivarius were found almost exclusively in CA-PD. The microbiomes of supragingival dental plaque differ substantially among tooth surfaces and children of different caries activities. In support of the ecological nature of caries etiology, a steady transition in community species composition was observed with disease progression.

A spectroscopic characterization is presented of the minor photosystem II chlorophyll a/b-binding protein CP29 (or the Lhcbby a modified form of a published protocol [Henrysson, T., Schroder, W. P., Spangfort, M. & Akerlund, H.-E. (1989) Biochim. Biophys. Acta 977, 301-308]. The isolation procedure represents a quicker, cheaper means of isolating this minor antenna protein to an equally high level of purity to that published previously. The pigment-binding protein shows similarities to other related light-harvesting complexes (LHCs), including the bulk complex LHCIIb but more particularly another minor antenna protein CP26 (Lhcbbinds about six chlorophyll a and two chlorophyll b molecules. Two chlorophyll b absorption bands are present at 638 and 650 nm and they are somewhat more pronounced than in a recent report [Giuffra, E., Zucchelli, G., Sandonà, D., Croce, R., Cugini, D., Garlaschi, F.M., Bassi, R. & Jennings, R.C. (1997) Biochem. 36, 12984-12993]. The bands give rise to positive and negative linear dichroism, respectively; both show negative CD bands (cf. bands with similar properties at 637 and 650 nm in CP26). Chlorophyll a absorption is dominated by a large contribution at 674 nm which also shows similarities to the major band in LHCIIb and CP26, while (as for CP26) a reduction in absorption around 670 nm is observed relative to the bulk complex. Principal differences from LHCIIb and CP26, and from other CP29 preparations, occur in the carotenoid region.

Enzyme immunoassay (EIA), immunodiffusion (ID), and complement fixation (CF) tests for antibody to the A antigen of Blastomyces dermatitidis were assessed in 47 patients in an epidemic of blastomycosis and in 89 control subjects with lower respiratory tract illness. Antibody was detected by EIA, ID, and CF in 77%, 28%, and 9% of the patients, respectively. EIA titers ranged from 1:8-1:512 (median titer, 1:128). Antibody detected by ID or CF was always detectable by EIA. Antibody was detected by EIA 13 days after illness onset, and the peak seroprevalence rate and geometric mean titer occurred 50-70 days after onset. Antifungal therapy produced a significant decline in antibody titer by approximately six months after onset. Seven (8%) control subjects had detectable antibody, six had EIA titers of 1:8, and one had a titer of 1:16. The specificities for EIA, ID, and CF were 92%, 100%, and 100%, respectively. The EIA provides a significant advance in serodiagnostic testing for blastomycosis and can be used in an outbreak setting as an epidemiological tool to identify acute B. dermatitidis infection; titers greater than or equal to 1:32 strongly support the diagnosis, whereas titers of 1:8 or 1:16 are suggestive.

OBJECTIVE

To determine whether patients with "pure" chronic fatigue syndrome (neurasthenia) have sleep abnormalities which may contribute to subjective measures of daytime fatigue.

METHODS

Sleep characteristics of 20 patients meeting research criteria for chronic fatigue syndrome (CFS) but not depression, anxiety, or sleep disorder were compared with sleep characteristics of 20 healthy subjects matched for age and sex. Measures of sleep included a) subjective interview reports and sleep diaries and b) home-based polysomnography.

RESULTS

Patients with CFS complained of poor quality unrefreshing sleep. They also napped during the day. Polysomnograph data showed no difference in actual nocturnal sleep time between the two groups although patients with CFS spent significantly longer in bed (p < .01), slept less efficiently (p < .03), and spent longer awake after sleep onset (p < .05). The polysomnographs of seven patients with CFS and one healthy subject were regarded as significantly abnormal. Five patients and one healthy subject had difficulty maintaining sleep. One patient had a disorder of both initiating and maintaining sleep and one patient woke early.

CONCLUSIONS

Patients with "pure" CFS complain of unrefreshing sleep but only a minority have a clearly abnormal polysomnograph. The most common abnormality is of long periods spent awake after initial sleep onset. Although sleep abnormalities may play a role in the etiology of CFS, they seem to be unlikely to be an important cause of daytime fatigue in the majority of patients. However, pharmacological and behavioral methods that improve sleep quality may be an important component of a pragmatically based treatment package for patients who do have abnormal sleep.

The contractile response to beta-adrenoceptor stimulation has been found to be reduced in single myocytes from failing human ventricle. A portion of that reduction was statistically related to the age of the patient. We have developed a method for obtaining viable contracting myocytes from small biopsies of human ventricle, enabling experiments to be performed on non-failing left ventricle from patients undergoing coronary artery surgery. These subjects are generally in an older age range than those we have previously obtained from donor hearts. The present study compares responses to isoproterenol and high Ca2+ between myocytes from older >> 50 years, n = 8) and younger (< 40 years, n = 5) subjects. Myocytes from older patients did not differ in their contraction amplitude (10.7 +/- 1.0 cf. 10.8 +/- 1.1% cell shortening), or contraction and relaxation velocities in high Ca2+ at a driving frequency of 0.2 Hz (32 degrees C). The sensitivity to isoproterenol, as determined from the EC50 value (concentration for half-maximal effect), was also similar between age groups (1.16 +/- 0.64 nM young, cf. 2.76 +/- 2.0 nM old), although higher than in myocytes from failing hearts (22.3 +/- 7.5 nM, n = 11). However, for the older group there was a significant depression in maximum contraction amplitude with isoproterenol (8.5 +/- 0.6 cf. 11.5 +/- 1.0% cell shortening, P < 0.05) and in the ratio between this and the maximum Ca2+ response (isoproterenol/Ca2+ ratio, 0.79 +/- 0.05 cf. 1.16 +/- 0.12, P < 0.05). Concomitantly, the maximum contraction and relaxation velocities achieved in the presence of isoproterenol were also depressed in older subjects (P < 0.02 for both). We conclude that age and/or coronary disease with unimpaired left ventricular function selectively reduces the maximum effect of isoproterenol but not the concentration at which this occurs.

Chronic fatigue syndrome is a condition that affects women in disproportionate numbers, and that is often exacerbated in the premenstrual period and following physical exertion. The signs and symptoms, which include fatigue, myalgia, and low-grade fever, are similar to those experienced by patients infused with cytokines such as interleukin-1. The present study was carried out to test the hypotheses that (1) cellular secretion of interleukin-1 beta (IL-1 beta), interleukin-1 receptor antagonist (IL-1Ra), and soluble interleukin-1 receptor type II (IL-1sRII) is abnormal in female CFS patients compared to age- and activity-matched controls; (2) that these abnormalities may be evident only at certain times in the menstrual cycle; and (3) that physical exertion (stepping up and down on a platform for 15 min) may accentuate differences between these groups. Isolated peripheral blood mononuclear cells from healthy women, but not CFS patients, exhibited significant menstrual cycle-related differences in IL-1 beta secretion that were related to estradiol and progesterone levels (R2 = 0.65, P < 0.01). IL-1Ra secretion for CFS patients was twofold higher than controls during the follicular phase (P = 0.023), but luteal-phase levels were similar between groups. In both phases of the menstrual cycle, IL-1sRII release was significantly higher for CFS patients compared to controls (P = 0.002). The only changes that might be attributable to exertion occurred in the control subjects during the follicular phase, who exhibited an increase in IL-1 beta secretion 48 hr after the stress (P = 0.020). These results suggest that an abnormality exists in IL-1 beta secretion in CFS patients that may be related to altered sensitivity to estradiol and progesterone. Furthermore, the increased release of IL-1Ra and sIL-1RII by cells from CFS patients is consistent with the hypothesis that CFS is associated with chronic, low-level activation of the immune system.

Background: Social and health inequities predispose vulnerable populations to adverse morbidity and mortality outcomes of epidemics and pandemics. While racial disparities in cumulative incidence (CmI) and mortality from the influenza pandemics of 1918 and 2009 implicated Blacks with survival disadvantage relative to Whites in the United States, COVID-19 currently indicates comparable disparities. We aimed to: (a) assess COVID-19 CmI by race, (b) determine the Black-White case fatality (CF) and risk differentials, and (c) apply explanatory model for mortality risk differentials.

Methods: COVID-19 data on confirmed cases and deaths by selective states health departments were assessed using a cross-sectional ecologic design. Chi-square was used for CF independence, while binomial regression model for the Black-White risk differentials.

Results: The COVID-19 mortality CmI indicated Blacks/AA with 34% of the total mortality in the United States, albeit their 13% population size. The COVID-19 CF was higher among Blacks/AA relative to Whites; Maryland, (2.7% vs. 2.5%), Wisconsin (7.4% vs. 4.8%), Illinois (4.8% vs. 4.2%), Chicago (5.9% vs. 3.2%), Detroit (Michigan), 7.2% and St. John the Baptist Parish (Louisiana), 7.9%. Blacks/AA compared to Whites in Michigan were 15% more likely to die, CmI risk ratio (CmIRR) = 1.15, 95% CI, 1.01-1.32. Blacks/AA relative to Whites in Illinois were 13% more likely to die, CmIRR = 1.13, 95% CI, 0.93-1.39, while Blacks/AA compared to Whites in Wisconsin were 51% more likely to die, CmIRR = 1.51, 95% CI, 1.10-2.10. In Chicago, Blacks/AA were more than twice as likely to die, CmIRR = 2.24, 95% CI, 1.36-3.88.

Conclusion: Substantial racial/ethnic disparities are observed in COVID-19 CF and mortality with Blacks/AA disproportionately affected across the United States.

Keywords: COVID-19 (SARS-COV2); United States; case fatality; health disparities; mortality; race/ethnicity.

BACKGROUND

Chronic lung inflammation with increased susceptibility to bacterial infections cause much of the morbidity and mortality in patients with cystic fibrosis (CF), the most common severe, autosomal recessively inherited disease in the Caucasian population. Exogenous inhaled hyaluronan (HA) can exert a protective effect against injury and beneficial effects of HA have been shown in experimental models of chronic respiratory diseases. Our objective was to examine whether exogenous administration of nebulized HA might interfere with lung inflammation in CF.

METHODS

F508del homozygous mice (Cftr(F508del) ) and transgenic mice overexpressing the ENaC channel β-subunit (Scnn1b-Tg) were treated with nebulized HA (0.5 mg/mouse/day for 7 days). Tumor necrosis factor-alpha (TNFα), macrophage inflammatory protein-2 (MIP-2), myeloperoxidase (MPO) levels, and macrophage infiltration were assessed on lung tissues. IB3-1 and CFBE41o-epithelial cell lines were cultured with HA (24 hr, 100 µg/ml) and Reactive Oxygen Species (ROS), Tissue Transglutaminase (TG2) SUMOylation and Peroxisome Proliferator Activated Receptor gamma (PPARγ) and phospho-p42/p44 levels were measured by dichlorodihydrofluorescein assay, or fluorescence resonance energy transfer (FRET) microscopy or immunoblots.

RESULTS

Nebulized HA reduced TNFα expression (P < 0.005); TNFα, MIP-2, and MPO protein levels (P < 0.05); MPO activity (P < 0.05); and CD68+ cells counts (P < 0.005) in lung tissues of Cftr(F508del) and Scnn1b-Tg mice, compared with saline-treated mice. HA reduced ROS, TG2 SUMOylation, TG2 activity, phospho-p42-44, and increased PPARγ protein in both IB3-1 and CFBE41o cells (P < 0.05).

CONCLUSIONS

Nebulized HA is effective in controlling inflammation in vivo in mice CF airways and in vitro in human airway epithelial cells. We provide the proof of concept for the use of inhaled HA as a potential anti-inflammatory drug in CF therapy.