Characterization of the molecular chaperone calnexin in the channel catfish, Ictalurus punctatus, and its association with MHC class II molecules

Folding and assembly of MHC molecules in mammals occurs in the endoplasmic reticulum (ER), but has not been studied in teleosts. Calnexin (CNX) is an ER chaperone that associates with glycoproteins bearing a monoglucosylated N-linked oligosaccharide side chain. Here we report the first identification and characterization of a full-length CNX cDNA clone in a teleost, and the association of the CNX chaperone with MHC class II in a channel catfish T cell line. The 1.8 kb CNX clone encodes a protein of 607 amino acids that is 72% identical to the consensus sequence of mammalian CNXs. The association of CNX with class II is of particular interest because the native MHC class II alpha chain of Ictalurus punctatus does not bear any N-linked oligosaccharide consensus glycosylation sequences. Thus the assembly of class II molecules in the catfish probably proceeds via different steps than occurs in mammals.     

β-Adrenergic stimulation does not regulate Na pump function in voltage-clamped ventricular myocytes of the rat heart

Summary Na pump current was measured in rat ventricular myocytes to determine if -adrenergic stimulation can directly modulate Na,K-ATPase activity. Enzymatically-isolated heart cells were voltage-clamped with a single patch electrode and Na pump current was briefly activated by rapidly increasing extracellular [K⁺] from 0 to 15 mM for 3–5 s after other ionic currents were blocked or inactivated. The salt solution in the voltage-clamping electrode included (in mM): (1) 100 Na⁺, 10 EGTA, (2) 5 Na⁺, 10 EGTA, or (3) 100 Na⁺, 7.5 Ca²⁺, 10 EGTA (free [Ca²⁺]=480 nM). With all three electrode solutions, Na pump current was not significantly changed after 2–4 min in the presence of 10 M isoprenaline. -adrenergic pathways were still intact as evidenced by the two-fold increase in Cd²⁺-sensitive Ba²⁺ current through calcium channels that was observed in the presence of isoprenaline. Thus, -adrenergic stimulation does not appear to directly regulate Na,K-ATPase activity in rat ventricular myocytes.     

The Immune Reactivity Role of HCV-Induced Liver Infiltrating Lymphocytes in Hepatocellular Damage

Liver infiltrating lymphocytes (LIL) were isolated from HCV-positive (+) and HCV-negative (–) end-stage livers. Phenotypic analysis and functional studies using proliferative and lymphocytotoxic assays were performed with the isolated LIL. Two CD3+ lymphocyte populations were found in LIL using FITC anti-CD3 monoclonal antibodies (mAb). One was a bright fluorescence intensity population (as in PBL), and the other dim. We calculated the number of FITC-anti-CD3 mAbs bound per lymphocyte on PBL and LIL and found 80,040 ± 4628 and 39,615 ± 3932, respectively. Therefore, HCV+ and HCV– patient PBL contained approximately twice the number of CD3 molecules per cell than patient CD3+ LIL. LIL also contained approximately a threefold higher concentration of TCR+, CD4–CD8–, and CD56,16 (NK) cells than the patient PBL. Thus, a major subset of LIL is phenotypically similar to mouse NK1.1+ intermediate T cells. LIL freshly isolated from HCV+ livers exhibited weak CTL activity against EBV- or Con A-transformed lymphoblast targets infected with vaccinia–HCV recombinant virus (rHCV) or primary hepatocyte cultured cells. However, after in vitro coculture of LIL with rHCV, these cells developed a strong cytotoxicity for the above targets. In contrast, LIL from HCV– livers were not cytotoxic against the same targets. Histochemical studies (in situ) demonstrated that these hepatocytes express CD95, and stains demonstrated apoptosis. The HCV+ hepatocytes also express class I MHC molecules and ICAM-1. The addition of mAb specific for these adhesion molecules inhibited CML activity. Short-term cultured hepatocytes (targets) from HCV+ and HCV– patients produced low levels of cytokines IL-1, IL-2, IL-6, TNF, and IFN- but a high level of IL-8. It is speculated that LIL expressing reduced numbers of CD3 molecules may even function as immune regulators as proposed for intermediate T cells in mice.     

Effect of the late complement components C5b-9 on human monocytes: release of prostanoids, oxygen radicals and of a factor inducing cell proliferation

Recently, we reported stimulation of rat macrophages and human platelets by isolated C5b-9 to synthesize prostaglandin E (PGE) or thromboxane B2 (TXB2). In the present study, we tested whether besides prostanoids, C5b-9 also would induce the production of other mediators. We found that C5b-9 in sublytic concentrations stimulated human granulocytes (polymorphonuclear leukocytes) or monocytes to release oxygen radicals. Furthermore, monocytes release interleukin-1 in response to C5b-9. Thus, besides having a lytic capacity, C5b-9 also functions as a stimulator of various cells.     

The influence of the causes and contexts of medical errors on emergency medicine residents' responses to their errors: an exploration.

PURPOSE: To determine emergency medicine residents' emotional and behavioral responses to their medical errors and examine associations between residents' responses to medical error and perceptions of their training. METHOD: In 2003, 55 residents at two U.S. residency programs were asked to complete questionnaires about their errors and responses to their errors in three domains: emotional response, learning behavior, and disclosure. The questions were a mixture of free text, yes/no responses, and some were rated using a five-point Likert scale. Based on a conceptual framework, the authors constructed scales to describe the various domains and associations between the residents' responses to medical error and perceptions of their training were examined using Somers' D. RESULTS: A total of 43 residents returned questionnaires (80%); 40 of these residents described errors. Thirty-three (83%) residents discussed the error with someone; 27 (71%) with the attending and 10 (28%) with the patient/family. Negative emotions were common: 27 (68%) felt remorse, 21 (53%) guilt, 23 (58%) inadequacy, and 22 (55%) frustration. Residents' negative emotional responses were associated with their personal characteristics [26% (95% CI, 5-47%) association with lack of experience] and residents' perceptions of their training environment: 15% association with job overload (95% CI, -8-38%) and 23% association with lack of institutional support (95% CI, 5-41%). While 32 (81%) residents increased attention to detail, only 2 (5%) increased their use of evidence-based medicine. CONCLUSIONS: Errors committed by emergency medicine residents often resulted in negative emotions, limited constructive system-based improvements, and inadequate disclosure. Negative perceptions of the training environment are associated with negative emotional responses.     

Induction of heme oxygenase-1 expression inhibits platelet-dependent thrombosis

Heme oxygenase-1 (HO-1) plays a key role in protecting tissue from oxidative stress. Although some studies implicate HO-1 in modulating thrombosis after vascular injury, the impact of HO-1 on the rate of clot formation in vivo is poorly defined. This study examined the potential function of HO-1 in regulating platelet-dependent arterial thrombosis. Platelet-rich thrombi were induced in C57BL/6J mice by applying 10% ferric chloride to the exposed carotid artery. Mean occlusion time of wild-type mice (n = 10) was 14.6 +/- 1.0 min versus 12.9 +/- 0.6 min for HO-1-/- mice (n = 11, p = 0.17). However, after challenge with hemin, mean occlusion time was significantly longer in wild-type mice (16.3 +/- 1.2 min, n = 15) than HO-1-/- mice (12.0 +/- 1.0 min, n = 9; p = 0.021). Hemin administration induced an approximately twofold increase in oxidative stress, measured as plasma thiobarbituric acid reactive substances. Immunohistochemical analysis revealed that hemin induced a robust increase in HO-1 expression within the carotid arterial wall. Ex vivo blood clotting within a collagen-coated perfusion chamber was studied to determine whether the accelerated thrombosis observed in HO-1-/- mice was contributed to by effects on the blood itself. Under basal conditions, mean clot formation during perfusion of blood over collagen did not differ between wild-type mice and HO-1-/- mice. However, after hemin challenge, mean clot formation was significantly increased in HO-1-/- mice compared with wild-type controls. These results suggest that, under basal conditions, HO-1 does not exert a significant effect on platelet-dependent clot formation in vivo. However, under conditions that stimulate HO-1 production, platelet-dependent thrombus formation is inhibited by HO-1. Enhanced HO-1 expression in response to oxidative stress may represent an adaptive response mechanism to down-regulate platelet activation under prothrombotic conditions.     

Tacrolimus and Mycophenolate Mofetil Regimens in Transplantation

Immunosuppression, although necessary to enable the graft to escape the consequences of immune surveillance, carries some risks for the patient. There is an associated increase in neoplasms, opportunistic infections and end-organ toxicity. In addition, even with excellent patient compliance, rejection (acute and chronic) remains a major limitation that contributes to the loss or decrease in the function of the allograft. New drugs have been added to the armamentarium of immunosuppressive agents to suppress allograft rejection and to rescue grafts from cyclosporin-resistant rejection. With the availability of these immunosuppressive agents, it has become increasingly difficult to choose the appropriate combination of immunosuppressants with a beneficial effect for the patient and for the allograft. We describe 2 new immunosuppressive agents and some of their different uses in solid organ transplantation.