Akt1/protein kinase Balpha is critical for ischemic and VEGF-mediated angiogenesis

Akt, or protein kinase B, is a multifunctional serine-threonine protein kinase implicated in a diverse range of cellular functions including cell metabolism, survival, migration, and gene expression. However, the in vivo roles and effectors of individual Akt isoforms in signaling are not explicitly clear. Here we show that the genetic loss of Akt1, but not Akt2, in mice results in defective ischemia and VEGF-induced angiogenesis as well as severe peripheral vascular disease. Akt1 knockout (Akt1-/-) mice also have reduced endothelial progenitor cell (EPC) mobilization in response to ischemia, and reintroduction of WT EPCs, but not EPCs isolated from Akt1-/- mice, into WT mice improves limb blood flow after ischemia. Mechanistically, the loss of Akt1 reduces the basal phosphorylation of several Akt substrates, the migration of fibroblasts and ECs, and NO release. Reconstitution of Akt1-/- ECs with Akt1 rescues the defects in substrate phosphorylation, cell migration, and NO release. Thus, the Akt1 isoform exerts an essential role in blood flow control, cellular migration, and NO synthesis during postnatal angiogenesis.     

Ultra-rapid detection of Chlamydia trachomatis by real-time PCR in the LightCycler using SYBR green technology or 5'-nuclease probes

Chlamydia trachomatis infections are among the most common sexually transmitted diseases and of great epidemiological importance world-wide. Identification of this pathogen can be difficult, and it is highly desirable to have a rapid and accurate nucleic acid based detection method. Several commercial PCR test systems are available (e.g. CobasAmplicor, Roche, Mannheim, Germany) but they require post-amplification detection by hybridization resulting in extended work-up time and possible cross-contamination. The objective of our study was to develop a routine diagnostic method for the sensitive, specific and rapid detection of C. trachomatis. The obvious choice is real-time PCR without any post-amplification procedures. The dye SYBR Green I (intercalating in dsDNA) provides a simple and fast real-time PCR in the LightCycler. Specific primer design combined with melting curve analysis allows a reliable and sensitive identification of C. trachomatis. In addition, a new commercial real-time PCR system (RealArt C. trachomatis LC PCR Reagents, artus, Hamburg, Germany) was evaluated, that combines sequence-specific primers and fluorescence-labelled (FRET) 5'-nuclease probes. An internal control integrated in this system detects false negative results and erroneous PCR conditions. All results were compared with the corresponding data from an analysis using the CobasAmplicor system (Roche). (Clin     

Preemptive treatment of early donor‐specific antibodies with IgA‐ and IgM‐enriched intravenous human immunoglobulins in lung transplantation

This retrospective study presents our 4‐year experience of preemptive treatment of early anti‐HLA donor specific antibodies with IgA‐ and IgM‐enriched immunoglobulins. We compared outcomes between patients with antibodies and treatment (case patients) and patients without antibodies (control patients). Records of patients transplanted at our institution between March 2013 and November 2017 were reviewed. The treatment protocol included one single 2 g/kg immunoglobulin infusion followed by successive 0.5 g/kg infusions for a maximum of 6 months, usually combined with a single dose of anti‐CD20 antibody and, in case of clinical rejection or positive crossmatch, with plasmapheresis or immunoabsorption. Among the 598 transplanted patients, 128 (21%) patients formed the case group and 452 (76%) the control group. In 116 (91%) patients who completed treatment, 106 (91%) showed no antibodies at treatment end. Fourteen (13%) patients showed antibody recurrence thereafter. In case versus control patients and at 4‐year follow‐up, respectively, graft survival (%) was 79 versus 81 (P = .59), freedom (%) from biopsy‐confirmed rejection 57 versus 53 (P = .34), and from chronic lung allograft dysfunction 82 versus 78 (P = .83). After lung transplantation, patients with early donor‐specific antibodies and treated with IgA‐ and IgM‐enriched immunoglobulins had 4‐year graft survival similar to patients without antibodies and showed high antibody clearance.     

Anti-integrins--new platelet function inhibitors for therapy and prevention of acute coronary syndrome

Acute occlusion of a large coronary artery by a platelet thrombus is a life-threatening event. Intravasal thrombus generation in most cases is caused by a disturbed interaction between platelets and the vessel wall, with accompanying platelet hyperreactivity, local adhesion to the vessel wall, activation and aggregate formation after binding of soluble fibrinogen to the activated GP IIb/IIIa-receptor. Conventional antiplatelet agents, such as aspirin or ticlopidine/clopidogrel, inhibit uncontrolled local agonist-induced signal transduction within the platelet by interfering with the thromboxane A2 and ADP pathway, respectively. This results in an activation of the platelet GP IIb/IIIa-receptor and, finally, in a reduced capacity of fibrinogen binding. Antiintegrins inhibit cell-cell and cell-matrix interactions. Antagonists of the platelet integrin alpha IIB/beta 3 (GP IIb/IIIa) (eg. Abciximab, Eptifibatide, Tirofiban) inhibit platelet adhesion and aggregation via their RGD (KGD) binding sequence, resulting in reduced fibrinogen binding. The significance of inhibition of other RGD-containing adhesion molecules (von Willebrand Factor, Vitronectin) with respect to the clinical efficacy of these compounds is stil under debate. GP IIb/IIIa-antagonists are the most effective inhibitors of platelet function and in high doses, may cause complete inhibition of platelet aggregation and maximum prolongation of bleeding time. The clinical efficacy of GP IIb/IIIa-antagonists for acute percutaneous coronary interventions and in the management of the acute coronary syndrome is established. Whether Abciximab and low-molecular weight intravenous compunds (Eptifibatide, Tirofiban, Lamifiban) are equipotent, remains to be demonstrated by controlled comparative studies. Orally active low-molecular-weight compounds (Sibrafiban, Xemilofiban and others) are currently undergoing clinical trials. Whether these substances are superior to oral aspirin and/or clopidogrel in long-term prevention of acute arterial vessel occlusions remains to be determined.     

Mechanical characteristics of articular cartilage bonds

BACKGROUND: Sutures for adaptation of articular cartilage are used in arthritis therapy techniques. However, little is known about the mechanical functionality of these sutures. The objective of the present work was to compare the mechanical properties of articular cartilage bonds either generated by suture, or, alternatively, by chemical cross-linking of the opposing surfaces or in vitro integrative repair of cartilage blocks. METHODS: Bonding was achieved by suture in varying numbers, positions and orientations, by surface cross-linking using carbodiimide in combination with pepsin or guanidine (immediate bonding), or by cultivation for 14 days, either with or without testosterone. The mechanical properties of the cartilage bonds were measured under tensile loading. FINDINGS: Suture led to the highest maximal load at failure and by far to the highest strain and lowest stiffness of the bonded samples. Immediate bonding by chemical cross-linking in combination with pepsin led to a low force at failure, but the highest stiffness, as compared to all other groups. Cultivation in the presence of testosterone led to a higher force at failure and a higher strain than chemical cross-linking. INTERPRETATION: Suture technique for bonding of cartilage surfaces leads to a very elastic adaptation which allows synovial fluid flow in between the interface of cartilage wounds. Long-term bonding of cartilage wounds would be counteracted by a fluid flow through the interface during motion of the joint. Immediate bonding of cartilage wounds by chemical cross-linking reagents might be a useful alternative tool. Even more promising, with regard to the mechanical properties, appears to be integrative repair of cartilage blocks stimulated by testosterone.     

Relationship between detection limit and bias of accuracy of quantification of RNA by RT-PCR

Evidence is presented demonstrating that the distribution of data obtained applying a given RT-PCR method deviates from a normal distribution depending on the limit of detection. The effect of this is a bias towards higher values and concomitantly a systematic error in respect to the accuracy of the evaluation due to this deviation from normality. In addition, evidence is presented that an evaluation assuming a log-normal distribution is more appropriate.     

Medium and large injured dorsal root ganglion cells increase TRPV-1, accompanied by increased alpha2C-adrenoceptor co-expression and functional inhibition by clonidine

Some electrophysiologic studies demonstrate new, excitatory alpha2-adrenoceptors on peripheral nociceptors and their dorsal root ganglion (DRG) cell bodies after nerve injury, yet administration of alpha2-adrenoceptor agonists at these sites reduces hypersensitivity rather than worsens it. Since TRPV-1 expressing nociceptor afferents are important in many pain states, we examined the expression of this channel and its co-expression with alpha2C-adrenoceptors in injured DRG cell bodies and the ability of alpha2-adrenoceptors to inhibit responses to stimulation. Rats underwent tight ligation of the left L5 and L6 spinal nerves, followed by behavioral testing, removal of L5 and L6 DRGs, and either immunostaining for TRPV-1 channels and alpha2C-adrenoceptors or intracellular calcium videomicroscopy in response to electrical field stimulation before and after perfusion with clonidine and capsaicin. Spinal nerve ligation produced tactile allodynia. In normal and sham controls, about one-third of DRG neurons were TRPV-1-immunoreactive (IR), one half were alpha2C-adrenoceptor-IR and one-fourth co-expressed both. After nerve ligation there was a reduction in the number of small, strongly TRPV-1-IR or alpha2C-adrenoceptor-IR neurons, but an increase in medium and large, lightly stained cells and in their co-expression. The proportion of clonidine inhibited cells which responded to capsaicin increased 5 fold after injury. We conclude that TRPV-1 and alpha2C-adrenoceptors are up-regulated in some injured medium and large size neurons after nerve ligation. Increased co-expression by immunocytochemistry, and increased proportion of cells inhibited by clonidine and expressing functional TRPV-1 channels suggest that these cells may play an important role in the analgesic effects of alpha2-adrenoceptor agonists in neuropathic pain.     

Combining the benefits of tele-rehabilitation and virtual reality-based balance training: a systematic review on feasibility and effectiveness

Purpose: A motivational surrounding is desirable in stroke rehabilitation considering the need to train repetitively to improve balance, even after discharge from rehabilitation facilities. This review aims to investigate whether it is feasible to combine virtual reality (VR) which allows exercising in game-like environments with tele-rehabilitation in a community-dwelling stroke population.

Methods: Literature searches were conducted in five databases, for example, PubMed and the Cochrane Library. Randomized controlled trial (RCT) and non-RCT investigating feasibility and effectiveness of VR-based tele-rehabilitation were included. Based on the risk of bias and study design, methodological quality is ranked according to the GRADE guidelines.

Results: Seven studies (n?=?120) were included, of which four are RCTs. Evidence regarding therapy adherence and perceived enjoyment of VR, as well as a cost–benefit of tele-rehabilitation emphasizes feasibility. Equal effects are reported comparing this approach to a therapist-supervised intervention in the clinical setting on balance and functional mobility.

Conclusions: Tele-rehabilitation could be a promising tool to overcome burdens that restrict accessibility to rehabilitation in the future. VR can increase motivation allowing longer and more training sessions in community-dwelling stroke survivors. Therefore, combining the benefits of both approaches seems convenient. Although evidence is still sparse, functional improvements seem to be equal compared to a similar intervention with therapist-supervision in the clinic, suggesting that for cost-efficient rehabilitation parts of therapy can be transferred to the homes.

• Implications for rehabilitation

• The use of tele-rehabilitation could be a promising tool to overcome burdens that restrict the access of stroke survivors to long-term rehabilitative care.

• VR-based interventions are game-like and therefore seem to provide a motivational environment which allows longer exercise sessions and greater adherence to therapy.