The innate immune system plays a critical role in the defense of areas exposed to microorganisms. There is an increasing body of evidence indicating that antimicrobial peptides and proteins (APs) are one of the most important weapons of this system and that they make up the protective front for the respiratory tract. On the other hand, it is known that pathogenic organisms have developed countermeasures to resist these agents such as reducing the net negative charge of the bacterial membranes. Here we report the characterization of a novel mechanism of resistance to APs that is dependent on the bacterial capsule polysaccharide (CPS). Klebsiella pneumoniae CPS mutant was more sensitive than the wild type to human neutrophil defensin 1, ␤-defensin 1, lactoferrin, protamine sulfate, and polymyxin B. K. pneumoniae lipopolysaccharide O antigen did not play an important role in AP resistance, and CPS was the only factor conferring protection against polymyxin B in strains lacking O antigen. In addition, we found a significant correlation between the amount of CPS expressed by a given strain and the resistance to polymyxin B. We also showed that K. pneumoniae CPS mutant bound more polymyxin B than the wild-type strain with a concomitant increased in the self-promoted pathway. Taken together, our results suggest that CPS protects bacteria by limiting the interaction of APs with the surface. Finally, we report that K. pneumoniae increased the amount of CPS and upregulated cps transcription when grown in the presence of polymyxin B and lactoferrin.

Early urinary continence rates were high in patients undergoing single or double layer urethrovesical anastomosis. We found no improvement in early continence rates with reconstruction of the periprostatic tissues.

Antimicrobial peptides (APs) impose a threat to the survival of pathogens, and it is reasonable to postulate that bacteria have developed strategies to counteract them. Polymyxins are becoming the last resort to treat infections caused by multidrug-resistant Gram-negative bacteria and, similar to APs, they interact with the anionic lipopolysaccharide. Given that polymyxins and APs share the initial target, it is possible that bacterial defense mechanisms against polymyxins will be also effective against host APs. We sought to determine whether exposure to polymyxin will increase Klebsiella pneumoniae resistance to host APs. Indeed, exposure of K. pneumoniae to polymyxin induces cross-resistance not only to polymyxin itself but also to APs present in the airways. Polymyxin treatment upregulates the expression of the capsule polysaccharide operon and the loci required to modify the lipid A with aminoarabinose and palmitate with a concomitant increase in capsule and lipid A species containing such modifications. Moreover, these surface changes contribute to APs resistance and also to polymyxin-induced cross-resistance to APs. Bacterial loads of lipid A mutants in trachea and lungs of intranasally infected mice were lower than those of wild-type strain. PhoPQ, PmrAB, and the Rcs system govern polymyxin-induced transcriptional changes, and there is a cross talk between PhoPQ and the Rcs system. Our findings support the notion that Klebsiella activates a defense program against APs that is controlled by three signaling systems. Therapeutic strategies directed to prevent the activation of this program could be a new approach worth exploring to facilitate the clearance of the pathogen from the airways.

The means by which airway epithelial cells sense a bacterial infection and which intracellular signalling pathways are activated upon infection are poorly understood. A549 cells and human primary airway cells (NHBE) were used to investigate the response to infection with Klebsiella pneumoniae. Infection of A549 and NHBE with K. pneumoniae 52K10, a capsule polysaccharide (CPS) mutant, increased the surface levels of ICAM-1 and caused the release of IL-8. By contrast, the wild-type strain did not elicit these responses. Consistent with a functional role for these responses, there was a correlation between ICAM-1 levels and the number of adherent leukocytes on the epithelial cell surface. In addition, treatment of neutrophils with IL-8 enhanced their ability to kill K. pneumoniae. Strain 52K10 was internalized by A549 cells more efficiently than the wild-type, and when infections with 52K10 were performed in the presence of cytochalasin D the inflammatory response was abrogated. These findings suggest that cellular activation is mediated by bacterial internalization and that CPS prevents the activation through the blockage of bacterial adhesion and uptake. Collectively, the results indicate that bacterial internalization by airway epithelial cells could be the triggering signal for the activation of the innate immune system of the airway. Infection of A549 cells by 52K10 was shown to trigger the nuclear translocation of NF-kB. Evidence is presented showing that 52K10 activated IL-8 production through Toll-like receptor (TLR) 2 and TLR4 pathways and that A549 cells could use soluble CD14 as TLR co-receptor. INTRODUCTIONOne of the largest epithelial surfaces of humans is located in the respiratory tract, which needs to be protected without compromising its functionality. In addition to serving as a physical barrier, airway epithelial cells react against insults such as cigarette smoke or air pollutants, mounting an inflammatory response. This response includes the production of pro-inflammatory and chemoattractant cytokines and the cell-surface expression of the adhesion molecule ICAM-1 (CD54) (Bayram et al., 1998;Hellermann et al., 2002;Hoshino et al., 2001;Mio et al., 1997;Pathmanathan et al., 2003;. Airway epithelial cells also interact with microbial pathogens, which should be rapidly killed or otherwise they may easily reach the bloodstream. There are reports showing that infection of airway epithelial cells by pathogens such as Streptococcus pneumoniae, Mycoplasma pneumoniae and group B streptococci triggers the secretion of IL-8 and increases the surface levels of ICAM-1 (Doran et al., 2002;Mikamo et al., 2004;Murdoch et al., 2002;Yang et al., 2002). These responses have been related to the recruitment of neutrophils into airways (Look et al., 1992;Tosi et al., 1992Tosi et al., , 1992, which is a common histological finding in patients with pneumonia independently of the infecting micro-organism. The current evidence shows that IL-8 targets neutrophils to sites of attack through its chemoattractant and activat...

Objectives To evaluate the efficacy of an individualised acupuncture protocol for patients with fibromyalgia. Methods Randomised controlled multicentre trial, blinded to participants and to data analysts. Conducted in three primary care centres in southern Spain. A total of 164 participants aged over 17 years and diagnosed with fibromyalgia were enrolled in this trial; 153 participants completed the study. Participants were randomly assigned to either the real intervention (individualised acupuncture, IA) or the sham intervention (sham acupuncture, SA). In both the IA and SA groups, one session per week (lasting 20 min) was provided, in addition to usual pharmacological treatment. The primary outcome was change in pain intensity at 10 weeks. Results Intention-to-treat analysis revealed that the decrease in pain intensity at 10 weeks was greater (p=0.001) in the IA group (−41.0%, 95% CI −47.2% to −34.8%) than in the SA group (−27.1%, 95% CI −33.2% to −20.9%). During the follow-up period, significant differences (p<0.01) in favour of the IA group persisted at 12 months (IA: −19.9%, 95% CI −24.6% to −15.1%; vs SA: −6.2%, 95% CI −11.2% to −1.2%). Conclusions Individualised acupuncture treatment in primary care in patients with fibromyalgia proved efficacious in terms of pain relief, compared with placebo treatment. The effect persisted at 1 year, and its side effects were mild and infrequent. Therefore, the use of individualised acupuncture in patients with fibromyalgia is recommended. Trial Registration Number ISRCTN60217348.

Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whether Klebsiella pneumoniae upregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection with K. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively. Klebsiella-dependent upregulation of TLR expression occurred via a positive IB␣-dependent NF-〉 pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed that Klebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation.

Objective To determine the effect of bovine lactoferrin on prevention of diarrhea in children. Study design We conducted a community-based randomized double-blind placebo controlled trial comparing supplementation with bovine lactoferrin versus placebo. Previously weaned children were enrolled at 12–18 months and followed for 6 months with daily home visits for data collection and supplement administration. Anthropometric measures were done monthly. Results 555 children were randomized: 277 to lactoferrin and 278 to placebo; 65 dropped out; 147,894 doses were administered (92% compliance). Overall there were 91,446 child-days of observation and 1,235 diarrhea episodes lasting 6,219 days. The main pathogens isolated during diarrheal episodes were norovirus (35.0%), enteropathogenic E. coli (11.4%), Campylobacter (10.6%), enteroaggregative E. coli (8.4%), enterotoxigenic E. coli (6.9%) and Shigella (6.6%). The diarrhea incidence was not different between groups: 5.4 vs. 5.2 episodes/child/year for lactoferrin and placebo, respectively (p=0.375). However, the diarrhea longitudinal prevalence was lower in the lactoferrin group (6.6% vs. 7.0%, p=0.017) as well as the median duration of episodes (4.8 vs. 5.3 days, p=0.046), proportion of episodes with moderate or severe dehydration (1.0% vs. 2.6%, p=0.045) and liquid stools load (95.0 vs. 98.6) liquid stools/child/year, p<0.001). There were no adverse events related to the intervention. Conclusions Although there was no decrease in diarrhea incidence, longitudinal prevalence and severity were decreased with lactoferrin.

This paper presents the first published report of a national-level effort to implement the Integrated Management of Childhood Illness (IMCI) strategy at scale. IMCI was introduced in Peru in late 1996, the early implementation phase started in 1997, with the expansion phase starting in 1998. Here we report on a retrospective evaluation designed to describe and analyze the process of taking IMCI to scale in Peru, conducted as one of five studies within the Multi-Country Evaluation of IMCI Effectiveness, Cost and Impact (MCE) coordinated by the World Health Organization. Trained surveyors visited each of Peru's 34 districts, interviewed district health staff and reviewed district records. Findings show that IMCI was not institutionalized in Peru: it was implemented parallel to existing programmes to address acute respiratory infections and diarrhoea, sharing budget lines and management staff. The number of health workers trained in IMCI case management increased until 1999 and then decreased in 2000 and 2001, with overall coverage levels among doctors and nurses calculated to be 10.3%. Efforts to implement the community component of IMCI began with the training of community health workers in 2000, but expected synergies between health facility and community interventions were not realized because districts where clinical training was most intense were not those where community IMCI training was strongest. We summarize the constraints to scaling up IMCI, and examine both the methodological and policy implications of the findings. Few monitoring data were available to document IMCI implementation in Peru, limiting the potential of retrospective evaluations to contribute to programme improvement. Even basic indicators recommended for national monitoring could not be calculated at either district or national levels. The findings document weaknesses in the policy and programme supports for IMCI that would cripple any intervention delivered through the health service delivery system. The Ministry of Health in Peru is now working to address these weaknesses; other countries working to achieve high and equitable coverage with essential child survival interventions can learn from their experience.