Influence of nanofibers on growth and gene expression of human tendon derived fibroblast

Background  

Rotator cuff tears are a common and frequent lesion especially in older patients. The mechanisms of tendon repair are not fully understood. Common therapy options for tendon repair include mini-open or arthroscopic surgery. The use of growth factors in experimental studies is mentioned in the literature. Nanofiber scaffolds, which provide several criteria for the healing process, might be a suitable therapy option for operative treatment. The aim of this study was to explore the effects of nanofiber scaffolds on human tendon derived fibroblasts (TDF's), as well as the gene expression and matrix deposition of these fibroblasts.     

BPIFB1 (LPLUNC1) is upregulated in cystic fibrosis lung disease

Although the biology the PLUNC (recently renamed BPI fold, BPIF) family of secreted proteins is poorly understood, multiple array based studies have suggested that some are differentially expressed in lung diseases. We have examined the expression of BPIFB1 (LPLUNC1), the prototypic two-domain containing family member, in lungs from CF patients and in mouse models of CF lung disease. BPIFB1 was localized in CF lung samples along with BPIFA1, MUC5AC, CD68 and NE and directly compared to histologically normal lung tissues and that of bacterial pneumonia. We generated novel antibodies to mouse BPIF proteins to conduct similar studies on ENaC transgenic (ENaC-Tg) mice, a model for CF-like lung disease. Small airways in CF demonstrated marked epithelial staining of BPIFB1 in goblet cells but staining was absent from alveolar regions. BPIFA1 and BPIFB1 were not co-localised in the diseased lungs. In ENaC-Tg mice there was strong staining of both proteins in the airways and luminal contents. This was most marked for BPIFB1 and was noted within 2?weeks of birth. The two proteins were present in distinct cells within epithelium. BPIFB1 was readily detected in BAL from ENaC-Tg mice but was absent from wild-type mice. Alterations in the expression of BPIF proteins is associated with CF lung disease in humans and mice. It is unclear if this elevation of protein production, which results from phenotypic alteration of the cells within the diseased epithelium, plays a role in the pathogenesis of the disease.     

A THEMIS:SHP1 complex promotes T‐cell survival

THEMIS is critical for conventional T‐cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr‐phosphorylation‐independent fashion. Rather, SHP1 and THEMIS engage with the N‐SH3 and C‐SH3 domains of GRB2, respectively, a configuration that allows GRB2‐SH2 to recruit the complex onto LAT. Consistent with THEMIS‐mediated recruitment of SHP to the TCR signalosome, THEMIS knock‐down increased TCR‐induced CD3‐ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock‐down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK‐mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T‐cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T‐cell development and differentiation.     

Tyrosine phosphorylation of myosin heavy chain during skeletal muscle differentiation: an integrated bioinformatics approach

Background  

Previously it has been shown that insulin-mediated tyrosine phosphorylation of myosin heavy chain is concomitant with enhanced association of C-terminal SRC kinase during skeletal muscle differentiation. We sought to identify putative site(s) for this phosphorylation event.     

Periodontal therapy increases neutrophil extracellular trap degradation

In periodontitis, polymorphonuclear leucocytes (PMNs) are activated. They entrap and eliminate pathogens by releasing neutrophil extracellular traps (NETs). Abnormal NET degradation is part of a pro-inflammatory status, affecting co-morbidities such as cardiovascular disease. We aimed to investigate the ex vivo NET degradation capacity of plasma from periodontitis patients compared to controls (part 1) and to quantify NET degradation before and after periodontal therapy (part 2). Fresh NETs were obtained by stimulating blood-derived PMNs with phorbol 12-myristate 13-acetate. Plasma samples from untreated periodontitis patients and controls were incubated for 3 h onto freshly generated NETs (part 1). Similarly, for part 2, NET degradation was studied for 91 patients before and 3, 6 and 12 mo after non-surgical periodontal therapy with and without adjunctive systemic antibiotics. Finally, NET degradation was fluorospectrometrically quantified. NET degradation levels did not differ between periodontitis patients and controls, irrespective of subject-related background characteristics. NET degradation significantly increased from 65.6 ± 1.7% before periodontal treatment to 75.7 ± 1.2% at 3 mo post periodontal therapy, and this improvement was maintained at 6 and 12 mo, irrespective of systemic usage of antibiotics. Improved NET degradation after periodontitis treatment is another systemic biomarker reflecting a decreased pro-inflammatory status, which also contributes to an improved cardiovascular condition.     

Studies on the resting behaviour and host choice of Anopheles gambiae and An. arabiensis from Muleba,Tanzania

The relative efficacy of a mechanical (Prokopack) collection method vs. manual aspiration in the collection of resting mosquitoes was evaluated in northern Tanzania before and after an intervention using indoor residual spraying and longlasting insecticide‐treated nets. In smoke‐free houses mosquitoes were collected from the roof and walls, but in smoky houses mosquitoes were found predominantly on the walls. Anopheles gambiae (Diptera: Culicidae) constituted 97.7% of the 312 An. gambiae complex specimens identified before but only 19.3% of the 183 identified after the intervention. A single sampling with the Prokopack collected a third of the available insects. Anopheles gambiae completed its gonotrophic development indoors, whereas Anopheles arabiensis did so outdoors. In both species gonotrophic development took 2 days. Most unfed resting An. arabiensis collected outdoors were virgins, whereas the majority of engorged insects were parous (with well‐contracted sacs). Daily survival was estimated to be 80.0%. Only 9.4% of the engorged An. arabiensis collected outdoors and 47.1% of those collected indoors had fed on humans. Using the Prokopack sampler is more efficient than manual methods for the collection of resting mosquitoes. Malaria transmission may have been affected by a change in vector composition resulting from a change in feeding, rather than reduced survival. Monitoring the proportions of members of the An. gambiae complex may provide signals of an impending breakdown in control.     

Characterization of Pdgfrb-Cre transgenic mice reveals reduction of ROSA26 reporter activity in remodeling arteries

With the intention to modulate gene expression in vascular mural cells of remodeling vessels, we generated and characterized transgenic mouse lines with Cre recombinase under the control of the platelet-derived growth factor receptor-β promoter, referred to as Tg(Pdgfrb-Cre)(35Vli) . Transgenic mice were crossed with the Gt(ROSA)26Sor(tm1Sor) strain and examined for Cre activation by β-galactosidase activity, which was compared with endogenous Pdgfrb expression. In addition, Pdgfrb-Cre mice were used to drive expression of a conditional myc-tagged Cthrc1 transgene. There was good overlap of β-galactosidase activity with endogenous Pdgfrb immunoreactivity. However, dedifferentiation of vascular mural cells induced by carotid artery ligation revealed a dramatic discrepancy between ROSA26 reporter activity and Pdgfrb promoter driven Cre dependent myc-tagged Cthrc1 transgene expression. Our studies demonstrate the capability of the Pdgfrb-Cre mouse to drive conditional transgene expression as a result of prior Cre-mediated recombination in tissues known to express endogenous Pdgfrb. In addition, the study shows that ROSA26 promoter driven reporter mice are not suitable for lineage marking of smooth muscle in remodeling blood vessels.     

Multidimensional prognostic indices for use in COPD patient care. A systematic review

Background

A growing number of prognostic indices for chronic obstructive pulmonary disease (COPD) is developed for clinical use. Our aim is to identify, summarize and compare all published prognostic COPD indices, and to discuss their performance, usefulness and implementation in daily practice.

Methods

We performed a systematic literature search in both Pubmed and Embase up to September 2010. Selection criteria included primary publications of indices developed for stable COPD patients, that predict future outcome by a multidimensional scoring system, developed for and validated with COPD patients only. Two reviewers independently assessed the index quality using a structured screening form for systematically scoring prognostic studies.

Results

Of 7,028 articles screened, 13 studies comprising 15 indices were included. Only 1 index had been explored for its application in daily practice. We observed 21 different predictors and 7 prognostic outcomes, the latter reflecting mortality, hospitalization and exacerbation. Consistent strong predictors were FEV₁ percentage predicted, age and dyspnoea. The quality of the studies underlying the indices varied between fairly poor and good. Statistical methods to assess the predictive abilities of the indices were heterogenic. They generally revealed moderate to good discrimination, when measured. Limitations: We focused on prognostic indices for stable disease only and, inevitably, quality judgment was prone to subjectivity.

Conclusions

We identified 15 prognostic COPD indices. Although the prognostic performance of some of the indices has been validated, they all lack sufficient evidence for implementation. Whether or not the use of prognostic indices improves COPD disease management or patients'' health is currently unknown; impact studies are required to establish this.     

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency

Background

Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.

Methods

Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.

Results

All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.

Conclusion

First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.