Roles of protein kinase C, Ca2+, Pyk2, and c-Src in agonist activation of rat lacrimal gland p42/p44 MAPK

PURPOSE: Although p42/p44 mitogen-activated protein kinase (MAPK) negatively modulates protein secretion stimulated by cholinergic and alpha(1D)-adrenergic agonists, it does not play a role in epidermal growth factor (EGF)-stimulated protein secretion. Therefore, this study was conducted to determine the roles that protein kinase C (PKC), intracellular Ca(2+) ([Ca(2+)](i)), and nonreceptor tyrosine kinases Pyk2 and Src play in the activation of agonist- and EGF-stimulated MAPK activation. METHODS: Lacrimal gland acini were isolated by collagenase digestion and incubated with phorbol 12-myristate 13-acetate (PMA) to activate PKC or ionomycin, a Ca(2+) ionophore. Acini were preincubated with the PKC inhibitors calphostin C or Ro-31-8220, EGTA to chelate Ca(2+), or the c-Src inhibitor PP1 before stimulation with the cholinergic agonist carbachol, the alpha(1D)-adrenergic agonist phenylephrine, or EGF. Activated MAPK, Pyk2, and c-Src amounts were measured by Western blot analysis. RESULTS: PMA and ionomycin significantly increased the activation of MAPK in a time- and concentration-dependent manner. Inhibition of PKC partially inhibited carbachol-stimulated MAPK activation while completely inhibiting phenylephrine- and EGF-stimulated MAPK activation. Chelation of Ca(2+) also partially inhibited carbachol-stimulated MAPK with no effect on phenylephrine- and EGF-stimulated MAPK activation. Carbachol increased the phosphorylation of Pyk2 on tyrosine 402 and c-src on tyrosine 416 in a time-dependent manner. The c-src inhibitor PP1 inhibited carbachol-stimulated phosphorylation of Pyk2. CONCLUSIONS: It was concluded that cholinergic agonists use Ca(2+) and PKC to phosphorylate Pyk2 and c-Src, which subsequently stimulate MAPK activity. In contrast, alpha(1D)-adrenergic agonists and EGF do not use Pyk2 and Src but do use PKC to activate MAPK.     

Recovery of sickle cell disease ischemic maculopathy after erythropheresis: a clinical case study

The authors report a case of a young patient with a recent decrease in unilateral vision. He had homozygote sickle cell disease with multiple general complications. Fundus examination was normal apart from a mild alteration of the macular reflect in the left eye, but fluorescein angiography showed multiple arteriolar macular occlusions, explaining the decrease in vision in the left eye. After erythropheresis, vision acuity improved and fluorescein angiography showed reperfusion. This case suggests that transfusional exchange may improve acute macular ischemia secondary to sickle cell disease.     

Evolution of the postoperative pericardial effusion after day 15: the problem of the late tamponade

STUDY OBJECTIVES: To evaluate, through clinical and transthoracic echocardiography (TTE) follow-up, the natural history of persistent pericardial effusion (PE) after postoperative day 15 in patients who were given and were not given anticoagulant therapy. DESIGN AND PATIENTS: We retrospectively studied a cohort of 1,277 patients who were hospitalized between May 1997 and May 1999 in our center a mean (+/- SD) time period of 15 +/- 3 days after undergoing coronary artery bypass graft (CABG) surgery (856 patients) or valve replacement (VR) surgery (421 patients). MEASUREMENTS: TTE was performed on mean (+/- SD) postoperative day 20 +/- 1 (TTE(1)) and postoperative day 30 +/- 2 (TTE(2)). PE severity was classified on a scale from grade 1 to grade 4. RESULTS: On postoperative day 20 +/- 1, PE was present in 22% of the 1,277 patients and was more frequent after patients underwent CABG surgery than after undergoing VR surgery (25% vs 17%, respectively; p < 0.01). On postoperative day 30 +/- 2, the overall incidence of late tamponade in patients with PE was 4%. The incidence increased with the severity grade of PE at TTE(1) (p < 0.001). The negative predictive value of a severity grade < 2 at TTE(1) for late tamponade was 100%. Late tamponade incidence was higher after VR surgery than after CABG surgery (11% vs 2%, respectively; p < 0.01), and was higher in patients who had received anticoagulation therapy than in those who had not (8% vs 2%, respectively; p < 0.05). CONCLUSION: Persisting PE is common after postoperative day 15 and is more frequent after undergoing CABG surgery than after undergoing VR surgery. The incidence of late tamponade is usually underestimated, and it increases with the presence of VR, anticoagulation therapy, and/or higher postoperative TTE severity grade. Our data suggest that only patients with a PE severity grade of >/= 2 (< 10% of patients) require TTE follow-up after postoperative day 20.     

Heart failure and cachexia

Cachexia is related to a malnutrition state related to hypercatabolism. Initially described in cancer, it is also related to several chronic diseases including heart failure. Defined by an unintentional weight loss exceeding 7.5% of body mass during more than 6 months, it is presented by the association of nutritional deficiencies, digestive and/or urinary losses as well as metabolic abnormalities causing fat and lean mass loss and is associated to a poor prognosis. The pathophysiology of cachexia and heart failure presented some similarities associating especially neuro-hormonal activation, a cortisol/DHEA ratio imbalance, as well as pro-inflammatory cytokines activation. Currently the treatment of cachexia is mainly preventive, based on ACE-inhibitors and beta-blockers therapy and physical reconditioning. The benefits of hormonal and nutritional substitutes remains to be evidenced.     

Immunosuppressive effects of streptozotocin-induced diabetes result in absolute lymphopenia and a relative increase of T regulatory cells

OBJECTIVE

Streptozotocin (STZ) is the most widely used diabetogenic agent in animal models of islet transplantation. However, the immunomodifying effects of STZ and the ensuing hyperglycemia on lymphocyte subsets, particularly on T regulatory cells (Tregs), remain poorly understood.

RESEARCH DESIGN AND METHODS

This study evaluated how STZ-induced diabetes affects adaptive immunity and the consequences thereof on allograft rejection in murine models of islet and skin transplantation. The respective toxicity of STZ and hyperglycemia on lymphocyte subsets was tested in vitro. The effect of hyperglycemia was assessed independently of STZ in vivo by the removal of transplanted syngeneic islets, using an insulin pump, and with rat insulin promoter diphtheria toxin receptor transgenic mice.

RESULTS

Early lymphopenia in both blood and spleen was demonstrated after STZ administration. Direct toxicity of STZ on lymphocytes, particularly on CD8⁺ cells and B cells, was shown in vitro. Hyperglycemia also correlated with blood and spleen lymphopenia in vivo but was not lymphotoxic in vitro. Independently of hyperglycemia, STZ led to a relative increase of Tregs in vivo, with the latter retaining their suppressive capacity in vitro. The higher frequency of Tregs was associated with Treg proliferation in the blood, but not in the spleen, and higher blood levels of transforming growth factor-β. Finally, STZ administration delayed islet and skin allograft rejection compared with naive mice.

CONCLUSIONS

These data highlight the direct and indirect immunosuppressive effects of STZ and acute hyperglycemia, respectively. Thus, these results have important implications for the future development of tolerance-based protocols and their translation from the laboratory to the clinic.Many animal models of diabetes depend on the administration of diabetogenic drugs such as streptozotocin (STZ) or alloxan. These are toxic glucose analogs that target pancreatic β-cells via GLUT2 transporter uptake. Because of the simplicity and reproducibility of diabetes induction with STZ, a highly stable glucosamine-nitrosourea compound, it represents by far the most widely used model (1). Indeed, among 131 analyzed articles published in 2010 on murine islet transplantation, 100 (76.3%) used STZ to induce diabetes, whereas 21 (16%) were performed in models of spontaneous diabetes (mostly NOD mice), 3 (2.3%) involved alloxan, 5 (3.8%) relied on transplanted islets in nondiabetic mice, and 2 (1.5%) used alternative ways for diabetes induction. Whereas STZ is known to target β-cells via the transporter GLUT2, it is not specific, since the kidney and liver are also susceptible to STZ toxicity (1). Moreover, several groups have described immunomodifying effects of STZ both in vitro and in vivo, although the confounding impact of the induced hyperglycemia was not clearly distinguished (2–6). Luo et al. (7) reported that hyperglycemia, potentially via an increased level of corticosteroids, causes a rapid depletion in thymocytes and splenic T cells followed by homeostatic T-cell proliferation. However, the exact mechanisms leading to the observed immunosuppression after STZ administration, especially the effect on different lymphocyte subpopulations, including T regulatory cells (Tregs), remain elusive.One of the major goals in islet transplantation (Tx) research is to find strategies to obviate the need for lifelong immunosuppression that is toxic to the β-cells and detrimental to the host (8). Indeed, using STZ-induced diabetes models, novel immunosuppressive and tolerance induction protocols for allogeneic as well as xenogeneic islet Tx have demonstrated long-term graft survival (9,10). However, many costimulation blockade–based Tx tolerance protocols that are successful in chemically induced diabetic mice failed to produce long-term graft tolerance in NOD mice or in other Tx models such as skin Tx, with the latter being considered as one of the most stringent models. The underlying autoimmunity directed against β-cells, the mode of Tx, and the skin-specific antigen–presenting cells all have been put forward to explain the observed resistance to Tx tolerance (10,11). Another possible explanation for these apparent differences is that immunosuppression related to STZ-induced diabetes could result in an overestimation of the efficacy of tolerance induction. Consistent with this notion, a protocol targeting costimulation that induced tolerance in STZ-induced diabetic recipients was unable to induce tolerance in nondiabetic recipients (7). Thus, STZ and/or the ensuing hyperglycemia seem to downregulate the adaptive immune response against islet grafts.In the past few years, Tregs were reported to play a key role in long-term islet graft tolerance (12–15). However, none of these studies addressed the possible effect of STZ administration and the ensuing hyperglycemia on Tregs. Using a rat-to-mouse islet Tx model, we recently reported an increase of Tregs in lymphoid organs and within grafts of tolerant mice treated with rapamycin and anti-CD154 monoclonal antibody (mAb), suggesting a critical role for Tregs in the induction phase of tolerance (9,16). The aim of the current study was to analyze whether STZ-induced diabetes leads to changes in Treg numbers and function and whether this affects subsequent immune responses using models of islet and skin Tx.