Complementary and antagonistic effects of IL-3 in the early development of human megakaryocytes in culture

The effect of IL-3 on the early steps in the growth and development of megakaryocytes (MK) in culture has been studied. Although thrombopoietin (TPO) by itself could support the development of mature CD41⁺ MK from pre-MK, the number of cells produced was greatly augmented by the addition of IL-3 and SCF. IL-3 was also able to support the growth of MK colonies in semi-solid media (CFU-MK). The CD41⁺ cells that developed in suspension cultures containing IL-3 differed phenotypically from those that developed without this agent. Cells grown in the presence of IL-3 lost CD34 expression more rapidly, expressed lower levels of the platelet glycoproteins gpIIb-IIIa and Ib and achieved lower degrees of polyploidy than in the absence of IL-3. The inhibitory effects of IL-3 were not a consequence of the dilution of the mature cells by increased numbers of immature cells since it was observed under conditions in which IL-3 did not stimulate MK growth. The results obtained in these cultures suggest that IL-3 plays an important role in early MK development, but inhibits further maturation after endoreduplication begins. Thus, prolonged contact with IL-3 results in the appearance of cells that do not mature normally.     

Functional recovery after hip fracture in old-old elderly patients

Hip fracture is epidemic and prevalence increased with advanced age. Impact of comorbid and cognitive status, gender, type of fracture, operative delay and pre-fracture ambulatory levels on functional outcome was shown in previous studies. We studied functional outcome after rehabilitation for hip fracture in old-old elderly (85 years and older) and compared it to young elderly (65–74 years) community-dwelling patients. Before the fracture, old-old elderly patients were more functional dependent, has had more comorbid diseases, and more of them live alone than young elderly. The waiting time to surgery and mean length of stay in orthopedic ward were longer than in young elderly. On admission to rehabilitation treatment, old-old patients presented with more depressed mood, were more cognitive impaired, and more suffer from pain. Old-old patients presented with laboratory data of malnutrition (decreased serum levels of albumin, cholesterol, hemoglobin, hematocrit, lymphocyte count) and inflammation (increased of transferrin and C-reactive protein). Improvement in Functional Independence Measurement (FIM) scale was found in both groups but significantly better in young elderly than in old-old elderly. The change in FIM during the rehabilitation period (ΔFIM) were in FIM total and in those parts of FIM concerning locomotion. The mean duration of rehabilitation stay was significantly longer in old-old elderly patients. On discharge old-old elderly patients more suffer from pain and difference between the groups according to the laboratory and to the cognitive data increased. Age per se is indicator of frailty and determinate functional recovery after hip fracture.     

Breaking the connection: caspase 6 disconnects intermediate filament-binding domain of periplakin from its actin-binding N-terminal region

Periplakin is a member of the plakin family of cytolinkers that connect cytoskeletal networks to each other as well as to the cell junctional complexes. Here, we demonstrate a direct molecular interaction between actin and periplakin. Furthermore, the oligomerization state of periplakin was shown to determine specificity of its binding to intermediate filaments (IF) in vitro. Both the filament association and the cell membrane localization of periplakin were confirmed in the cells overexpressing human periplakin. Double labeling of the N- and C-terminally tagged periplakin revealed unexpected lack of co-localization of periplakin ends in a confluent culture, and separation of the periplakin ends was even more pronounced in apoptotic cells. Western analysis revealed that after induction of apoptosis, periplakin becomes cleaved close to its C-terminal tail. Only the distinct cleavage products, but not the full-length periplakin, were present in the cells detached from the solid support during the apoptotic process. We show that caspase 6 cleaves periplakin at an unconventional recognition site, amino acid sequence TVAD. Thus, the separation of periplakin ends disconnects the actin-binding head-rod domain from the IF-binding C-terminal domain. We show that specific cleavage products co-exist with the full-length periplakin in cells, suggesting physiological consequences due to their altered binding specificities.     

Phylogenetic relationships of Irkut and West Caucasian bat viruses within the Lyssavirus genus and suggested quantitative criteria based on the N gene sequence for lyssavirus genotype definition

The nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) genes of Irkut and West Caucasian bat viruses (WCBV) were sequenced and compared with those of other lyssaviruses. N gene nucleotide identities provided unequivocal separation of all lyssavirus genotypes with an identity threshold of 82%. On this basis, Irkut virus should be considered as a new genotype with particular relatedness to genotypes 4 and 5 (78.0-78.6% identity for N gene nucleotides and 90.4-92.6% for amino acids). Furthermore, genotypes 4-6, together with Aravan, Khujand and Irkut viruses, present a solid phylogroup of Old World bat lyssaviruses. This relationship is apparent using all three viral genes, and causes overlap between intragenotype and intergenotype identities for the P gene (Aravan, Khujand viruses and genotype 6) and for the G gene (Aravan, Khujand, genotypes 5 and 6). WCBV is the most divergent of known lyssaviruses with only limited relatedness to genotypes 2 and 3.     

Esters of 6-aminohexanoic acid as skin permeation enhancers: The effect of branching in the alkanol moiety

In order to investigate the effect of branching and cyclization in the hydrophobic part of skin permeation enhancers, 17 novel branched-chain and cyclic 6-aminohexanoic acid esters were prepared. Their permeation enhancing activity was evaluated in vitro using human skin and theophylline as a model drug, and compared to that of the corresponding linear-chain analogues. The results showed that chain branching and cyclization has a negative influence on the enhancing activity of 6-aminohexanoates. For example, the enhancement ratios (ERs) of dodecan-1-yl, dodecan-2-yl, dodecan-4-yl, and cyclododecyl ester were 39.7, 29.3, 3.1, and 2.2, respectively. No significant change in the optimum length of the chain was observed. Dodecan-2-yl 6-aminohexanoate, the most active branched derivative, still maintains a remarkable enhancing activity (ER 29.3). Presumably, the relatively small degree of branching of these molecules does not prevent them from interacting with the lipid components of the stratum corneum. However, a higher degree of branching, cyclization of the chain, and presence of an aromatic ring resulted in a loss of activity.     

Direct PCR detection of Burkholderia cepacia complex and identification of its genomovars by using sputum as source of DNA

We developed a nested PCR assay that detects the recA gene of the Burkholderia cepacia complex in sputum. The product of the first PCR round is also used to identify the genomovar of the pathogen. The protocol achieves high sensitivity and specificity with simple interpretation of genomovar status.     

Clinical and laboratory prognostic factors in patients with metastatic renal cell carcinoma treated with sunitinib and sorafenib after progression on cytokines

ObjectivesThe aim of this retrospective study was to analyze prognostic factors in patients with metastatic renal cell carcinoma treated with tyrosine kinase inhibitors (TKIs) sunitinib or sorafenib after progression on cytokine therapy.Materials and methodsA national database of patients treated with targeted agents was used as the data source. A total of 319 patients treated with sunitinib (n = 181) or sorafenib (n = 138) after progression on cytokine therapy were analyzed.ResultsPrognostic factors significantly associated with poor overall survival in a multivariable Cox model included the time from diagnosis to the start of treatment with TKIs<1 year, increased neutrophil counts, increased lactate dehydrogenase, and Eastern Oncology Cooperative Group performance status 2 or higher. The parameters showing statistically significant association with progression-free survival included time from diagnosis to the beginning of treatment with TKI<1 year, increased lactate dehydrogenase, and Eastern Oncology Cooperative Group performance status 2 or higher. We have also validated the International Metastatic Renal Cell Carcinoma Database Consortium prognostic model in our cohort of patients.ConclusionWe demonstrate that the International Database Consortium prognostic model performs well for European patients treated with TKIs, including sunitinib or sorafenib, after progression on cytokines and suggest that a reduction from original 6 down to 4 parameters is possible.     

Remote ischaemic preconditioning does not protect the heart in patients undergoing coronary artery bypass grafting

Remote ischaemic preconditioning (RIPC) gained attention as a possibility to reduce myocardial injury after a subsequent sustained episode of myocardial ischaemia. This prospective randomized study was carried out to assess whether RIPC reduces myocardial injury in coronary artery bypass grafting patients. Eighty patients were assigned to remote preconditioning or control treatment. Ischaemic preconditioning was induced by three 5-min cycles of upper limb ischaemia and reperfusion after anaesthesia induction. Haemodynamic and markers of myocardial damage were analysed preoperatively and over 48 h postoperatively. The cardiac index was higher immediately after remote preconditioning in the main group. There were no differences in other haemodynamic, troponin I and creatine kinase-MB concentrations at any time point between groups. Thus, short-term remote preconditioning improves haemodynamics and does not reduce myocardial injury after coronary artery bypass surgery. Further study of high-risk patients may be needed to fully evaluate the clinical effect of RIPC.