Respiratory Profile in Patients After Liver Transplantation.

BACKGROUND: Patients in the immediate post-operative liver transplantation (LxT) period can develop respiratory and functional complications. In the postoperative months, these functions tend to improve. Nevertheless, there are few studies that evaluate precisely and specifically respiratory function in post-LxT long-term after surgery. The objective of the study was to describe the respiratory profile of patients 1 to 6 months after LxT, accompanied by LxT outpatients. METHODS: We included patients between 25 and 60 years old. We excluded patients with chronic renal or cerebrovascular impairment, severe heart disease, and history of lung surgery or liver re-transplantation. Evaluations were carried out on 3 occasions: 1 month, 3 months, and 6 months after LxT. The following evaluations were submitted: respiratory muscle strength (manuvacuometer), value flows and lung volumes (spirometer), and surface electromyography analyzing root mean square in the right (RMS-R) and left (RMS-L) diaphragm. We analyzed MELD (Model for End-Stage Liver Disease). After normality tests, we used the Friedman test (non-parametric values) and ANOVA (parametric values), P ≥ .5 with the use of SPSS 21.0. RESULTS: Patients (n = 15) had a mean age of 53.0 ± 7.5 years and 25.9 ± 4.6 MELD score. The statistically significant value obtained at the 3 occasions of evaluation was RMS-R, with a decline during periods of evaluation. This can be caused by removal of the liver, resulting in a denervation and reduction in compliance of this portion of the muscle. CONCLUSIONS: Patients between 1 and 6 months after transplantation have a specific respiratory profile, close to normal values. However, there are few studies on this subject, and we suggest that more research be done.

Regulation of fibrinolysis by C-terminal lysines operates through plasminogen and plasmin but not tissue-type plasminogen activator.

BACKGROUND: Binding of tissue-type plasminogen (Pgn) activator (t-PA) and Pgn to fibrin regulates plasmin generation, but there is no consistent, quantitative understanding of the individual contribution of t-PA finger and kringle 2 domains to the regulation of fibrinolysis. Kringle domains bind to lysines in fibrin, and this interaction can be studied by competition with lysine analogs and removal of C-terminal lysines by carboxypeptidase B (CPB). METHODS: High-throughput, precise clot lysis assays incorporating the lysine analog tranexamic acid (TA) or CPB and genetically engineered variants of t-PA were performed. In particular, wild-type (WT) t-PA (F-G-K1-K2-P) and a domain-switched variant K1K1t-PA (F-G-K1-K1-P) that lacks kringle 2 but retains normal t-PA structure were compared to probe the importance of fibrin lysine binding by t-PA kringle 2. RESULTS: WT t-PA showed higher rates of fibrinolysis than K1K1t-PA, but the inhibitory effects of TA or CPB were very similar for WT t-PA and the variant t-PA (< 10% difference). Urokinase plasminogen activator (u-PA)-catalyzed fibrinolysis was also inhibited by TA, even though Pgn activation could be stimulated. Fibrin treated with factor XIIIa (FXIIIa) generates crosslinked degradation products, but these did not affect the results obtained with WT t-PA and K1K1t-PA. CONCLUSIONS: t-PA kringle 2 has a minor role in the initial interaction of t-PA and fibrin, but stimulation of fibrinolysis by C-terminal lysines (or inhibition by carboxypeptidases or TA) operates through Pgn and plasmin binding, not through t-PA. This is also true when fibrin is crosslinked by treatment with FXIIIa.

Hindered dissolution of fibrin formed under mechanical stress.

BACKGROUND: Recent data indicate that stretching forces cause a dramatic decrease in clot volume accompanied by gross conformational changes of fibrin structure. OBJECTIVE: The present study attempts to characterize the lytic susceptibility of fibrin exposed to mechanical stress as a model for fibrin structures observed in vivo. METHODS AND RESULTS: The relevance of stretched fibrin models was substantiated by scanning electron microscopic (SEM) evaluation of human thrombi removed during surgery, where surface fibrin fibers were observed to be oriented in the direction of shear forces, whereas interior fibers formed a random spatial meshwork. These structural variations were modeled in vitro with fibrin exposed to adjustable mechanical stress. After two- and three-fold longitudinal stretching (2 × S, 3 × S) the median fiber diameter and pore area in SEM images of fibrin decreased two- to three-fold. Application of tissue plasminogen activator (tPA) to the surface of model clots, which contained plasminogen, resulted in plasmin generation which was measured in the fluid phase. After 30-min activation 12.6 ± 0.46 pmol mm(-2) plasmin was released from the non-stretched clot (NS), 5.5 ± 1.11 pmol mm(-2) from 2 × S and 2.3 ± 0.36 pmol mm(-2) from 3 × S clot and this hampered plasmin generation was accompanied by decreased release of fibrin degradation products from stretched fibrins. Confocal microscopic images showed that a green fluorescent protein-fusion variant of tPA accumulated in the superficial layer of NS, but not in stretched fibrin. CONCLUSION: Mechanical stress confers proteolytic resistance to fibrin, which is a result of impaired plasminogen activation coupled to lower plasmin sensitivity of the denser fibrin network.

Efeito dos genótipos para alphaS1-caseína sobre as frações proteicas e lipídicas do leite de cabra / Effect of genotypes for alphaS1-casein on proteic and lipidic fractions in goat milk

O alto polimorfismo encontrado no lócus do gene da αS1-caseína em caprinos, classificado em quatro níveis de expressão - alto, médio, baixo e nulo -, está associado à produção de 3,6; 1,6; 0,6 e 0g/L/alelo, respectivamente. O estudo foi realizado para investigar possíveis variações na produção de leite e seus constituintes, no perfil de caseínas e na lipólise da gordura. Quarenta e quatro cabras foram distribuídas em cinco genótipos: dois homozigotos, um para alta (AA) e outro para produção intermediária (EE), e três heterozigotos chamados AE, AF e EF, para αs1-caseína. Para a lipólise, o leite foi subamostrado em quatro alíquotas que sofreram tratamento térmico no momento da ordenha e após 24h de resfriamento. Diferenças entre genótipos foram observadas para a produção de caseína e de suas frações. As demais variáveis não diferiram entre genótipos. O genótipo AA apresentou os maiores conteúdos de caseína (28,6g/L) e de αS1-cn (22,3 por cento). Os demais genótipos apresentaram média de 20,4g/L. Os grupos AE e AF apresentaram média de 12,1, EE-10,1 e EF-9,1 por cento de αS1-cn. O resfriamento do leite por 24 horas aumentou a taxa de lipólise no leite. A genotipagem das cabras para αS1-cn pode ser usada como ferramenta de seleção com objetivo de obter produtos lácteos com distintos perfis de proteínas.

A high polymorphism is found in the locus of goat αS1-casein gene and it is classified in four levels of expression, named high, medium, and low, associated with production of 3.6, 1.6, 0.6, and 0 g/L/allele, respectively. The study was conducted to investigate possible variations on milk yield and components, profile of casein, and lipolysis of fat. Forty-four goats were assigned to five distinct genotypes as two homozygous, one for high (AA) and the other for intermediate yield (EE); and three heterozygous named AE, AF, and EF for the αs1-casein. For lipolysis, milk was sampled in four aliquots which were treated soon after milking and 24 hours after cooling. Differences were observed for both casein yield and its fractions. No difference was found for other variables. The AA genotype presented the higher content of both casein (28.6g/L) and αS1-cn (22.3 percent). Other genotypes averaged 20.4g/L for casein content. Values of αS1-cn were 12.1 percent for heterozygous and 10.1 and 9.1 percent for both EE and EF genotype respectively. Cooling the milk for 24 hours increased the rate of lipolysis. Genotyping goats for the αS1-cn can be used as a tool for selecting animal targeting milk products with distinct profiles of proteins.

Biological activity of EDQM CRS for Interferon alfa-2a and Interferon alfa-2b - assessment in two in vitro bioassays.

The European Directorate for the Quality of Medicines (EDQM) supplies Chemical Reference Substances (CRS) for Interferon (IFN) alfa-2a (CRS I0320300) and for IFN alfa-2b (CRS I0320301) for specified physicochemical tests. However, no information is provided as to their biological activity. In contrast, the World Health Organization (WHO) provides the 2nd International Standards (IS) for IFN alfa-2a (code 95/650) and for IFN alfa-2b (code 95/566), with activity defined in International Units (IU) for calibration of biological activity of preparations of IFN. We have compared the EDQM CRSs with the WHO ISs in two bioassay systems, one measuring the anti-proliferative activity in the Daudi cell line and the other measuring a reporter gene activation in an A549 cell line. In each of these assay systems, the CRSs gave dose - response relations, which were similar to those for the WHO ISs. Estimates of relative activity for each CRS, in terms of the respective IS, showed specific biological activity for the CRSs of the same order as the nominal specific activity for the ISs. However, the estimates of relative activity were not consistent between the two assays systems, emphasizing the need for calibration within each system, if the CRS were to be used as a working standard for bioassays. For structure-activity studies, both physicochemical and biological activity characterisation are required for the same biopharmaceutical preparation. CRS I0320300 and CRS I0320301 may prove useful as working standards for some bioassay systems.

The effect of anisotropic architecture on cell and tissue infiltration into tissue engineering scaffolds.

A common phenomenon in tissue engineering is rapid tissue formation on the outer edge of the scaffold which restricts cell penetration and nutrient exchange to the scaffold centre, resulting in a necrotic core. To address this problem, we generated scaffolds with both random and anisotropic open porous architectures to enhance cell and subsequent tissue infiltration throughout the scaffold for applications in bone and cartilage engineering. Hydroxyapatite (HA) and poly(D,L-lactic acid) (P(DL)LA) scaffolds with random open porosity were manufactured, using modified slip-casting and by supercritical fluid processing respectively, and subsequently characterised. An array of porous aligned channels (400 microm) was incorporated into both scaffold types and cell (human osteoblast sarcoma, for HA scaffolds; ovine meniscal fibrochondrocytes, for P(DL)LA scaffolds) and tissue infiltration into these modified scaffolds was assessed in vitro (cell penetration) and in vivo (tissue infiltration; HA scaffolds only). Scaffolds were shown to have an extensive random, open porous structure with an average porosity of 85%. Enhanced cell and tissue penetration was observed both in vitro and in vivo demonstrating that scaffold design alone can influence cell and tissue infiltration into the centre of tissue engineering scaffolds.