Intracellular survival and saprophytic growth of isolates from the Burkholderia cepacia complex in free-living amoebae

Members of the taxonomically diverse Burkholderia cepacia complex have become a major health risk for patients with cystic fibrosis (CF). Although patient-to-patient transmission of B. cepacia strains has been well-documented, very little is known about possible vehicles of transmission and reservoirs for these micro-organisms. In this work, it is shown that strains of the B. cepacia complex can survive within different isolates of the genus Acanthamoeba. Trophozoites containing bacteria developed profuse cytoplasmic vacuolization. Vacuolization was not detected in trophozoites infected with live Escherichia coli or heat-killed B. cepacia, or by incubation of trophozoites with filter-sterilized culture supernatants, indicating that metabolically active intracellular bacteria are required for the formation of vacuoles. Experiments with two different B. cepacia strains and two different Acanthamoeba isolates revealed that bacteria display a low level of intracellular replication approximately 72-96 h following infection. In contrast, extracellular bacteria multiplied efficiently on by-products released by amoebae. The findings suggest that amoebae may be a reservoir for B. cepacia and possibly a vehicle for transmission of this opportunistic pathogen among CF patients.

Steroid injection for inferior heel pain: a randomised controlled trial

BACKGROUND:

Plantar fasciitis is a common cause of heel pain. The aim of this study was twofold: to compare steroid injection with placebo injection and to compare ultrasound guided with unguided steroid injection in the management of this condition.

METHODS:

65 patients with inferior heel pain were recruited between November 2008 and June 2011. Heel pain was measured using a visual analogue scale (VAS) at baseline and follow-up 6 and 12 weeks after injection.

RESULTS:

22 patients were randomised to ultrasound guided steroid injection, 21 patients to palpation guided steroid injection and 22 to ultrasound guided placebo injection. There was a significant difference in VAS scores between the groups at 6 and 12 weeks (p=0.018 and p=0.004, respectively). There was a 19.7 (95% CI 2.5 to 37.0) difference in mean VAS scores at 6 weeks between the ultrasound guided steroid group and the placebo group and a 24.0 (95% CI 6.6 to 41.3) difference between the unguided steroid group and the placebo group at 6 weeks. At 12 weeks, the mean difference was 25.1 (95% CI 6.5 to 43.6) and 28.4 (95% CI 11.1 to 45.7) respectively between both steroid injection groups and the placebo group. There was no difference in VAS scores following steroid injection between the ultrasound guided and the unguided groups at either time point. Plantar fascia thickness was significantly reduced after injection in both active treatment groups (p=0.00).

CONCLUSIONS:

In this study, steroid injection showed a clear benefit over placebo at 6 weeks and this difference was maintained at 12 weeks.Trial Registration No ISRCTN79628180 (www.controlled-trials.com).

Preparation and Evaluation of Sustained-Release Matrix Tablets Based on Metoprolol and an Acrylic Carrier Using Injection Moulding

Sustained-release matrix tablets based on Eudragit RL and RS were manufactured by injection moulding. The influence of process temperature; matrix composition; drug load, plasticizer level; and salt form of metoprolol: tartrate (MPT), fumarate (MPF) and succinate (MPS) on ease of processing and drug release were evaluated. Formulations composed of 70/30% Eudragit RL/MPT showed the fastest drug release, substituting part of Eudragit RL by RS resulted in slower drug release, all following first-order release kinetics. Drug load only affected drug release of matrices composed of Eudragit RS: a higher MPT concentration yielded faster release rates. Adding triethyl citrate enhanced the processability, but was detrimental to long-term stability. The process temperature and plasticizer level had no effect on drug release, whereas metoprolol salt form significantly influenced release properties. The moulded tablets had a low porosity and a smooth surface morphology. A plasticizing effect of MPT, MPS and MPF on Eudragit RS and Eudragit RL was observed via DSC and DMA. Solubility parameter assessment, thermal analysis and X-ray diffraction demonstrated the formation of a solid solution immediately after production, in which H-bonds were formed between metoprolol and Eudragit as evidenced by near-infrared spectroscopy. However, high drug loadings of MPS and MPF showed a tendency to recrystallise during storage. The in vivo performance of injection-moulded tablets was strongly dependent upon drug loading. © 2012 American Association of Pharmaceutical Scientists.

Dysregulated intracellular signaling impairs CTGF-stimulated responses in human mesangial cells exposed to high extracellular glucose

High ambient glucose activates intracellular signaling pathways to induce the expression of extracellular matrix and cytokines such as connective tissue growth factor (CTGF). Cell responses to CTGF in already glucose-stressed cells may act to transform the mesangial cell phenotype leading to the development of glomerulosclerosis. We analyzed cell signaling downstream of CTGF in high glucose-stressed mesangial cells to model signaling in the diabetic milieu. The addition of CTGF to primary human mesangial cells activates cell migration which is associated with a PKC-zeta-GSK3beta signaling axis. In high ambient glucose basal PKC-zeta and GSK3beta phosphorylation levels are selectively increased and CTGF-stimulated PKC-zeta and GSK3beta phosphorylation was impaired. These effects were not induced by osmotic changes. CTGF-driven profibrotic cell signaling as determined by p42/44 MAPK and Akt phosphorylation was unaffected by high glucose. Nonresponsiveness of the PKC-zeta-GSK3beta signaling axis suppressed effective remodeling of the microtubule network necessary to support cell migration. However, interestingly the cells remain plastic: modulation of glucose-induced PKC-beta activity in human mesangial cells reversed some of the pathological effects of glucose damage in these cells. We show that inhibition of PKC-beta with LY379196 and PKC-beta siRNA reduced basal PKC-zeta and GSK3beta phosphorylation in human mesangial cells exposed to high glucose. CTGF stimulation under these conditions again resulted in PKC-zeta phosphorylation and human mesangial cell migration. Regulation of PKC-zeta by PKC-beta in this instance may establish PKC-zeta as a target for constraining the progression of mesangial cell dysfunction in the pathogenesis of diabetic nephropathy.

Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1ß, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1ß processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1ß release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1ß release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1ß response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1ß processing and release.

Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis

The excretory-secretory (ES) proteins of nematode parasites are of major interest as they function at the host-parasite interface and are likely to have roles crucial for successful parasitism. Furthermore, the ES proteins of intracellular nematodes such as Trichinella spiralis may also function to regulate gene expression in the host cell. In a recent proteomic analysis we identified a novel secreted cystatin-like protein from T. spiralis L1 muscle larva. Here we show that the protein, MCD-1 (multi-cystatin-like domain protein 1), contains three repeating cystatin-like domains and analysis of the mcd-1 gene structure suggests that the repeated domains arose from duplication of an ancestral cystatin gene. Cystatins are a diverse group of cysteine protease inhibitors and those secreted by parasitic nematodes are important immuno-modulatory factors. The cystatin superfamily also includes cystatin-like proteins that have no cysteine protease inhibitory activity. A recombinant MCD-1 protein expressed as a GST-fusion protein in Escherichia coli failed to inhibit papain in vitro suggesting that the T. spiralis protein is a new member of the non-inhibitory cystatin-related proteins. MCD-1 secreted from T. spiralis exists as high- and low-molecular weight isoforms and we show that a recombinant MCD-1 protein secreted by HeLa cells undergoes pH-dependent processing that may result in the release of individual cystatin-like domains. Furthermore, we found that mcd-1 gene expression is largely restricted to intracellular stages with the highest levels of expression in the adult worms. It is likely that the major role of the protein is during the intestinal stage of T. spiralis infections.

Evidence for a non-replicative intracellular stage of nontypable Haemophilus influenzae in epithelial cells

Nontypable Haemophilus influenzae (NTHi) is a Gram-negative, non-capsulated human bacterial pathogen, a major cause of a repertoire of respiratory infections, and intimately associated with persistent lung bacterial colonization in patients suffering from chronic obstructive pulmonary disease (COPD). Despite its medical relevance, relatively little is known about its mechanisms of pathogenicity. In this study, we found that NTHi invades the airway epithelium by a distinct mechanism, requiring microtubule assembly, lipid rafts integrity, and activation of phosphatidylinositol 3-kinase (PI3K) signalling. We found that the majority of intracellular bacteria are located inside an acidic subcellular compartment, in a metabolically active and non-proliferative state. This NTHi-containing vacuole (NTHi-CV) is endowed with late endosome features, co-localizing with LysoTracker, lamp-1, lamp-2, CD63 and Rab7. The NTHi-CV does not acquire Golgi- or autophagy-related markers. These observations were extended to immortalized and primary human airway epithelial cells. By using NTHi clinical isolates expressing different amounts of phosphocholine (PCho), a major modification of NTHi lipooligosaccharide, on their surfaces, and an isogenic lic1BC mutant strain lacking PCho, we showed that PCho is not responsible for NTHi intracellular location. In sum, this study indicates that NTHi can survive inside airway epithelial cells.

Severe visual loss after autologous blood injection for mitomycin-C- associated hypotonous maculopathy

It has been shown that mitomycin-C increases the success rate of trabeculectomy; however, a rise in the incidence of postoperative complications has also been reported. Consequently, the use of antimetabolite is usually reserved for patients who are at high risk of surgical failure or for patients with advanced glaucoma in whom low intraocular pressure is desired. This report describes a patient who suffered severe visual loss which was a direct result of hypotonous maculopathy after trabeculectomy with mitomycin-C and various other complications from the subsequent interventions.