Hepatitis C-specific effector and regulatory CD4 T-cell responses are associated with the outcomes of primary infection.

Clearance of primary hepatitis C virus (HCV) infection has been associated with strong and broadly targeted cellular immune responses. This study aimed to characterize HCV-specific CD4+ effector and regulatory T-cell numbers and cytokine production during primary infection. Antigen-specific CD4+ T-cell responses were investigated in a longitudinal cohort of subjects from pre-infection to postoutcome, including subjects who cleared [n=12] or became chronically infected [n=17]. A cross-sectional cohort with previously cleared, or chronic infection [n=15 for each], was also studied. Peripheral blood mononuclear cells were incubated with HCV antigens and surface stained for T-effector (CD4+CD25high CD134+CD39-) and T-regulatory (CD4+CD25high CD134+CD39+) markers, and culture supernatants assayed for cytokine production. Contrary to expectations, the breadth and magnitude of the HCV-specific CD4+ T-cell responses were higher in subjects who became chronically infected. Subjects who cleared the virus had HCV-specific CD4+ T-cell responses dominated by effector T cells and produced higher levels of IFN-γ, in contrast to HCV-specific CD4+ T-cell responses dominated by regulatory T cells and more IL-10 production in those who became chronically infected. Better understanding of the role of antigen-specific CD4+ T-cell responses in primary HCV will further define pathogenesis and help guide development of a preventative vaccine.

Exploration of genetically determined resistance against hepatitis C infection in high-risk injecting drug users.

Genetic resistance to specific infections is well recognized. In hepatitis C virus (HCV) infection, genetic polymorphisms in IL-28B and the killer cell immunoglobulin-like receptors (KIR) and their HLA class I ligands have been shown to affect clearance of the virus following infection. There are limited data regarding resistance to established HCV infection. Reliable quantification of repeated exposure in high-risk populations, such as injecting drug users (IDU), is a key limitation of previous studies of resistance. Behavioural data and DNA from IDU (n = 210) in the Hepatitis C Incidence and Transmission Study in prisons (HITS-p) cohort were genotyped for polymorphisms in: IL-28B, peptidyl-prolyl isomerase A (PPIA), HLA-C and KIR2. To quantify risk, a composite risk index based on factors predictive of incident HCV infection was derived. Logistic regression analysis revealed the risk index was strongly associated with incident HCV infection (P < 0.0001). The upper tertile of the uninfected individuals had risk indices comparable to the incident cases, but remained uninfected. There were no significant differences in the frequencies of IL-28B or PPIA polymorphisms between these exposed-uninfected cases, or in the frequencies of KIR2-DL3, HLA-C1, or their combination. A framework for the investigation of genetic determinants of resistance to HCV infection has been developed. Several candidate gene associations were investigated and excluded. Further investigation of genetic determinants of resistance to HCV infection is warranted.

Rare occurrence of occult hepatitis C virus in apparently uninfected injecting drug users: a two-centre, masked, case-control study.

Occult hepatitis C virus (HCV) is a phenomenon where serum HCV RNA is not detected by sensitive commercial assays, but viral RNA is detected by ultrasensitive techniques. Occult HCV infection has not previously been studied in highly exposed, but apparently uninfected (EU) individuals. Two studies examining occult infection in EU subjects were undertaken - an initial two-centre, masked, case-control study based on cross-sectional samples (n = 35 subjects) and a single-centre confirmatory study based on longitudinal samples (n = 32 subjects). Plasma and peripheral blood mononuclear cells were tested for HCV RNA using an ultrasensitive nested polymerase chain reaction assays. Two EU subjects in the first study (10%) and one in the second study (3%) were found to have consistently detectable HCV RNA. Occult HCV infection occurs in high-risk, apparently uninfected subjects.

Continuing warfarin during cutaneous surgery.

The risk of haemorrhage from minor cutaneous surgical procedures has long been a concern in the treatment of patients receiving warfarin as anti-coagulation therapy. Interruption, alteration, hospital admission and monitoring have resource implications as well as the potential for complications. Therefore, we wanted to determine whether it was feasible to undertake typical minor plastic surgery procedures without altering patients' warfarin dosage regimens. We undertook a prospective study of 51 patients (age range 36 to 86), with 78 wounds, undergoing a range of minor cutaneous surgical procedures including excision biopsies, local flaps and skin grafts. The patients continued their normal warfarin regimen and the INR was checked on the day of surgery, ranging from 1.1 to 4.0. There were no problems encountered during surgery, but two patients presented with bleeding from a wound a few days post-operatively. We feel that it is unnecessary to modify warfarin regimens for minor cutaneous surgery. However, a well-briefed patient and experienced surgical management with good support facilities are a prerequisite for this.

Glycogen synthase kinase 3 (GSK3) in the heart: a point of integration in hypertrophic signalling and a therapeutic target? A critical analysis.

Glycogen synthase kinase 3 (GSK3, of which there are two isoforms, GSK3alpha and GSK3beta) was originally characterized in the context of regulation of glycogen metabolism, though it is now known to regulate many other cellular processes. Phosphorylation of GSK3alpha(Ser21) and GSK3beta(Ser9) inhibits their activity. In the heart, emphasis has been placed particularly on GSK3beta, rather than GSK3alpha. Importantly, catalytically-active GSK3 generally restrains gene expression and, in the heart, catalytically-active GSK3 has been implicated in anti-hypertrophic signalling. Inhibition of GSK3 results in changes in the activities of transcription and translation factors in the heart and promotes hypertrophic responses, and it is generally assumed that signal transduction from hypertrophic stimuli to GSK3 passes primarily through protein kinase B/Akt (PKB/Akt). However, recent data suggest that the situation is far more complex. We review evidence pertaining to the role of GSK3 in the myocardium and discuss effects of genetic manipulation of GSK3 activity in vivo. We also discuss the signalling pathways potentially regulating GSK3 activity and propose that, depending on the stimulus, phosphorylation of GSK3 is independent of PKB/Akt. Potential GSK3 substrates studied in relation to myocardial hypertrophy include nuclear factors of activated T cells, beta-catenin, GATA4, myocardin, CREB, and eukaryotic initiation factor 2Bvarepsilon. These and other transcription factor substrates putatively important in the heart are considered. We discuss whether cardiac pathologies could be treated by therapeutic intervention at the GSK3 level but conclude that any intervention would be premature without greater understanding of the precise role of GSK3 in cardiac processes.

Technical considerations of fibular osteocutaneous flap dissection.

SUMMARY: Fibula flap dissection is a reliable, often first-choice, means of osteocutaneous free tissue transfer. Experience from many centres has shown this flap to be ideally suited to the reconstruction of mandibular defects, which is its main indication in our unit. Despite its popularity, harvesting the fibula osteocutaneous flap remains technically challenging. We describe modifications of the standard flap dissection technique which we employ in our unit. In our hands, these help to simplify the dissection of the flap pedicle and maximise the pedicle length available for microvascular anastomoses. This has helped to ensure that the flap can be raised safely and expediently.

Onychocryptosis-phenol burn fiasco.

Ingrowing toenails, onychocryptosis, are a common condition that can be treated conservatively or surgically. Conservative treatment often fails necessitating operative intervention. Surgical treatments include wedge resection, Zadik's procedure and nail removal with ablation of the germinal matrix by phenol. The following report describes a 15-year-old male footballer who sustained burns following phenol treatment, resulting in amputation of his left great toe, as well as suggestions to avoid any similar complications.

Argyria caused by an earring.

The staining of skin by silver is termed argyria and is grey-blue in colour. This may be caused by a number of mechanisms such as ingestion and direct implantation. We report an unusual case, caused by an impacted earring, where the skin discoloration was not entirely typical of argyria. This may have been due to copper impurities present in the earring. The literature on the subject is also reviewed.

Regulation of mitogen-activated protein kinases in cardiac myocytes through the small G protein Rac1.

Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.

Signalling pathways in cardiac myocyte hypertrophy.

In response to a requirement for increased contractile power in vivo, mammalian cardiac myocytes adapt through a hypertrophic response (cell enlargement in the absence of cell division). This response can be simulated by exposing isolated myocytes in primary culture to alpha-adrenergic agonists or the vasoactive peptide, endothelin-1. The signalling pathways responsible for hypertrophic growth have been actively studied, and it is likely that reversible protein phosphorylation and dephosphorylation are involved. Three signalling pathways show particular potential as regulators of the response, ie protein kinase C (PKC), mitogen-activated protein kinase (MAPK) cascades, and calcineurin. These species are thought to regulate the rate and specificity of gene transcription ultimately through modifying the transactivating activity of nuclear transcription factors. There are three pertinent MAPK cascades, the extracellular signal-regulated kinase (ERK) cascade, the c-Jun N-terminal kinase (JNK or SAPK1) cascade, and the p38-MAPK (SAPK2-5) cascade. PKC participates in the activation of the ERK cascade but does not contribute significantly to the activation of the two remaining cascades. Calcineurin (or protein phosphatase 2B) is activated by increases in [Ca2+i] through the [Ca2+]-sensing protein, calmodulin. In this review, I discuss the evidence for and against the involvement of these signalling proteins in the induction of myocyte hypertrophy and emphasize that the ERK cascade should perhaps feature more widely in the collective consciousness.

Regulation of protein kinase B and 4E-BP1 by oxidative stress in cardiac myocytes.

Stimulation of phosphatidylinositol 3'-kinase (PI3K) and protein kinase B (PKB) is implicated in the regulation of protein synthesis in various cells. One mechanism involves PI3K/PKB-dependent phosphorylation of 4E-BP1, which dissociates from eIF4E, allowing initiation of translation from the 7-methylGTP cap of mRNAs. We examined the effects of insulin and H(2)O(2) on this pathway in neonatal cardiac myocytes. Cardiac myocyte protein synthesis was increased by insulin, but was inhibited by H(2)O(2). PI3K inhibitors attenuated basal levels of protein synthesis and inhibited the insulin-induced increase in protein synthesis. Insulin or H(2)O(2) increased the phosphorylation (activation) of PKB through PI3K, but, whereas insulin induced a sustained response, the response to H(2)O(2) was transient. 4E-BP1 was phosphorylated in unstimulated cells, and 4E-BP1 phosphorylation was increased by insulin. H(2)O(2) stimulated dephosphorylation of 4E-BP1 by increasing protein phosphatase (PP1/PP2A) activity. This increased the association of 4E-BP1 with eIF4E, consistent with H(2)O(2) inhibition of protein synthesis. The effects of H(2)O(2) were sufficient to override the stimulation of protein synthesis and 4E-BP1 phosphorylation induced by insulin. These results indicate that PI3K and PKB are important regulators of protein synthesis in cardiac myocytes, but other factors, including phosphatase activity, modulate the overall response.

Extracellular regulated kinase, but not protein kinase C, is an antiapoptotic signal of insulin-like growth factor-1 on cultured cardiac myocytes.

This study aims to elucidate the signaling pathway for insulin-like growth factor-1 (IGF-1) in cultured neonatal rat cardiomyocytes and particularly the role of IGF-1 in cardiac apoptosis. IGF-1 stimulated polyphosphoinositide turnover, translocation of protein kinase C (PKC) isoforms (alpha, epsilon, and delta) from the soluble to the particulate fraction, activation of phospholipid-dependent and Ca(2+)-, phospholipid-dependent PKC, and activation of the extracellular-regulated kinase (ERK). IGF-1 attenuated sorbitol-induced cardiomyocyte viability and nuclear DNA fragmentation. These antiapoptotic effects of IGF-1 were blocked by PD-098059 (an MEK inhibitor) but not by bisindolylmaleimide I (BIM, a specific PKC inhibitor). The ERK pathway may therefore be an important component in the mechanism whereby IGF-1 exerts its antiapoptotic effect on the cardiomyocyte.

Small guanine nucleotide-binding proteins and myocardial hypertrophy.

The small (21 kDa) guanine nucleotide-binding protein (small G protein) superfamily comprises 5 subfamilies (Ras, Rho, ADP ribosylation factors [ARFs], Rab, and Ran) that act as molecular switches to regulate numerous cellular responses. Cardiac myocyte hypertrophy is associated with cell growth and changes in the cytoskeleton and myofibrillar apparatus. In other cells, the Ras subfamily regulates cell growth whereas the Rho subfamily (RhoA, Rac1, and Cdc42) regulates cell morphology. Thus, the involvement of small G proteins in hypertrophy has become an area of significant interest. Hearts from transgenic mice expressing activated Ras develop features consistent with hypertrophy, whereas mice overexpressing RhoA develop lethal heart failure. In isolated neonatal rat cardiac myocytes, transfection or infection with activated Ras, RhoA, or Rac1 induces many of the features of hypertrophy. We discuss the mechanisms of activation of the small G proteins and the downstream signaling pathways involved. The latter may include protein kinases, particularly the mitogen-activated or Rho-activated protein kinases. We conclude that although there is significant evidence implicating Ras, RhoA, and Rac1 in hypertrophy, the mechanisms are not fully understood.

Activation of the small GTP-binding protein Ras in the heart by hypertrophic agonists.

The small (21-kDa) guanine nucleotide-binding protein Ras plays a central role in the regulation of cell growth and division. In the cardiac myocyte, it has been implicated in the hypertrophic adaptation. We have recently examined the ability of hypertrophic agonists such as endothelin-1, phenylephrine and phorbol esters to increase the "activity" (GTP loading) of Ras. We have also studied the signaling events that lead to activation of Ras and the processes that respond to Ras activation. In this brief review, we describe these studies and set them within the context of the hypertrophic response.

Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential.

Cardiac myocyte apoptosis is potentially important in many cardiac disorders. In other cells, Bcl-2 family proteins and mitochondrial dysfunction are probably key regulators of the apoptotic response. In the present study, we characterized the regulation of antiapoptotic (Bcl-2, Bcl-xL) and proapoptotic (Bad, Bax) Bcl-2 family proteins in the rat heart during development and in oxidative stress-induced apoptosis. Bcl-2 and Bcl-xL were expressed at high levels in the neonate, and their expression was sustained during development. In contrast, although Bad and Bax were present at high levels in neonatal hearts, they were barely detectable in adult hearts. We confirmed that H(2)O(2) induced cardiac myocyte cell death, stimulating poly(ADP-ribose) polymerase proteolysis (from 2 hours), caspase-3 proteolysis (from 2 hours), and DNA fragmentation (from 8 hours). In unstimulated neonatal cardiac myocytes, Bcl-2 and Bcl-xL were associated with the mitochondria, but Bad and Bax were predominantly present in a crude cytosolic fraction. Exposure of myocytes to H(2)O(2) stimulated rapid translocation of Bad (<5 minutes) to the mitochondria. This was followed by the subsequent degradation of Bad and Bcl-2 (from approximately 30 minutes). The levels of the mitochondrial membrane marker cytochrome oxidase remained unchanged. H(2)O(2) also induced translocation of cytochrome c from the mitochondria to the cytosol within 15 to 30 minutes, which was indicative of mitochondrial dysfunction. Myocytes exposed to H(2)O(2) showed an early loss of mitochondrial membrane potential (assessed by fluorescence-activated cell sorter analysis) from 15 to 30 minutes, which was partially restored by approximately 1 hour. However, a subsequent irreversible loss of mitochondrial membrane potential occurred that correlated with cell death. These data suggest that the regulation of Bcl-2 and mitochondrial function are important factors in oxidative stress-induced cardiac myocyte apoptosis.