Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V

Gln506-factor V (FV) was purified from plasma of an individual homozygous for an Arg506Gln mutation in FV that is associated with activated protein C (APC) resistance. Purified Gln506-FV, as well as Gln506-FVa generated by either thrombin or FXa, conveyed APC resistance to FV-deficient plasma in coagulation assays. Clotting assay studies also suggested that APC resistance does not involve any abnormality in FV-APC-cofactor activity. In purified reaction mixtures, Gln506-FVa in comparison to normal FVa showed reduced susceptibility to APC, because it was inactivated approximately 10-fold slower than normal Arg506-FVa. It was previously reported that inactivation of normal FVa by APC involves an initial cleavage at Arg506 followed by phospholipid- dependent cleavage at Arg306. Immunoblot and amino acid sequence analyses showed that the 102-kD heavy chain of Gln506-FVa was cleaved at Arg306 during inactivation by APC in a phospholipid-dependent reaction. This reduced but measurable susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance is a mild risk factor for thrombosis because APC can inactivate both normal FVa and variant Gln506-FVa. In summary, this study shows that purified Gln506- FV can account for APC resistance of plasma because Gln506-FVa, whether generated by thrombin or FXa, is relatively resistant to APC.     

Paroxysmal nocturnal haemoglobinuria phenotype cells and leucocyte subset telomere length in childhood acquired aplastic anaemia

The significance of paroxysmal nocturnal haemoglobinuria (PNH^pos) cells and leucocyte subset telomere lengths in paediatric aplastic anaemia (AA) is unknown. Among 22 children receiving immunosuppressive therapy (IST) for AA, 73% (16/22) were PNH^pos, of whom 94% achieved at least a partial response (PR) to IST; 11/16 (69%) achieved complete response (CR). Only 2/6 (33%) PNH^neg patients achieved PR. PNH^pos patients were less likely to fail IST compared to PNH^neg patients (odds ratio 0·033; 95% confidence interval 0·002–0·468; P = 0·012). Children with AA had short granulocyte (P = 7·8 × 10^?9), natural killer cell (P = 6·0 × 10^?4), naïve T lymphocyte (P = 0·002) and B lymphocyte (P = 0·005) telomeres compared to age‐matched normative data.     

Preserved function of regulatory T cells in chronic HIV-1 infection despite decreased numbers in blood and tissue

Regulatory T cells (Tregs) are potent immune modulators, but their role in human immunodeficiency virus type 1 (HIV-1) pathogenesis remains poorly understood. We performed a detailed analysis of the frequency and function of Tregs in a large cohort of HIV-1-infected individuals and HIV-1 negative controls. While HIV "elite controllers" and uninfected individuals had similar Treg numbers and frequencies, the absolute numbers of Tregs declined in blood and gut-associated lymphoid tissue in patients with chronic progressive HIV-1 infection. Despite quantitative changes in Tregs, HIV-1 infection was not associated with an impairment of ex vivo suppressive function of flow-sorted Tregs in both HIV controllers and untreated chronic progressors.     

Exploiting PI3K/mTOR signaling to accelerate epithelial wound healing

The molecular circuitries controlling the process of skin wound healing have gained new significant insights in recent years. This knowledge is built on landmark studies on skin embryogenesis, maturation, and differentiation. Furthermore, the identification, characterization, and elucidation of the biological roles of adult skin epithelial stem cells and their influence in tissue homeostasis have provided the foundation for the overall understanding of the process of skin wound healing and tissue repair. Among numerous signaling pathways associated with epithelial functions, the PI3K/Akt/mTOR signaling route has gained substantial attention with the generation of animal models capable of dissecting individual components of the pathway, thereby providing a novel insight into the molecular framework underlying skin homeostasis and tissue regeneration. In this review, we focus on recent findings regarding the mechanisms involved in wound healing associated with the upregulation of the activity of the PI3K/Akt/mTOR circuitry. This review highlights critical findings on the molecular mechanisms controlling the activation of mTOR, a downstream component of the PI3K–PTEN pathway, which is directly involved in epithelial migration and proliferation. We discuss how this emerging information can be exploited for the development of novel pharmacological intervention strategies to accelerate the healing of critical size wounds.     

Growth factor regulation of intracellular pH homeostasis under hypoxic conditions in isolated equine articular chondrocytes

Hypoxia and acidosis are recognized features of inflammatory arthroses. This study describes the effects of IGF‐1 and TGF‐β₁ on pH regulatory mechanisms in articular cartilage under hypoxic conditions. Acid efflux, reactive oxygen species (ROS), and mitochondrial membrane potential were measured in equine articular chondrocytes isolated in the presence of serum (10% fetal calf serum), IGF‐1 (1, 10, 50, 100 ng/ml) or TGF‐β₁ (0.1, 1, 10 ng/ml) and then exposed to a short‐term (3 h) hypoxic insult (1% O₂). Serum and 100 ng/ml IGF‐1 but not TGF‐β₁ attenuated hypoxic regulation of pH homeostasis. IGF‐1 appeared to act through mitochondrial membrane potential stabilization and maintenance of intracellular ROS levels in very low levels of oxygen. Using protein phosphorylation inhibitors PD98059 (25 µM) and wortmannin (200 nM) and Western blotting, ERK1/2 and PI‐3 kinase pathways are important for the effect of IGF‐1 downstream to ROS generation in normoxia but only PI‐3 kinase is implicated in hypoxia. These results show that oxygen and growth factors interact to regulate pH recovery in articular chondrocytes by modulating intracellular oxygen metabolites. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 197–203, 2013