Interleukin-3, GM-CSF, and TPA induce distinct phosphorylation events in an interleukin 3-dependent multipotential cell line

The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hematopoietic cell line, B6SUtA1. Murine granulocyte- macrophage colony-stimulating factor (mGM-CSF) was as potent as mIL-3 in stimulating these cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-O-tetradecanoyl- phorbol-13-acetate (TPA), a known activator of protein kinase C, also stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF. To assess this possibility, intact B6SUtA1 cells exposed for brief periods to mIL-3, mGM-CSF, and TPA were analyzed for changes in phosphorylation patterns using metabolic 32P-labeling and antibodies to phosphotyrosine. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a 68-Kd cytosolic protein, whereas all three agents stimulated the serine-specific phosphorylation of a 68-Kd membrane protein. Furthermore, mIL-3 stimulated tyrosine phosphorylation of the 68-Kd membrane protein, as well as of 140-, 90-, 55, and 40-Kd proteins. The 90-Kd protein was also tyrosine phosphorylated in response to mGM-CSF. These phosphotyrosine containing proteins were not detected in TPA-treated cells. These results indicate that protein phosphorylations on tyrosine and serine residues occur in B6SUtA1 cells following short-term incubation with mIL-3 or mGM-CSF and that most of these phosphorylation events are mediated by kinases other than protein kinase C (PkC).     

Procoagulant activity of reversibly acylated human factor Xa

The plasma clotting factors used to treat hemophiliacs who have developed inhibitory antibodies have a shared history of limited clinical safety and utility. To improve on existing bypass factors, we have developed a reversibly acylated form of human plasma factor Xa capable of providing a time-dependent release of procoagulant activity. Factor Xa was treated with p-amidinophenyl p'-anisate to generate anisoyl Xa. The chemical modification of the protein involves acylation of the active site serine residue of factor Xa. Anisoyl Xa deacylated in a time, pH, and temperature-dependent manner. Active factor Xa generated on deacylation of anisoyl Xa exhibited amidolytic and prothrombinase complex activities in in vitro assays, the level being comparable to those of untreated factor Xa. When Anisoyl Xa was infused into rabbits, active factor Xa was generated on deacylation of the acylated enzyme, which shortened the activated partial thromboplastin time (APTT) in a dose-dependent manner. The duration of effect on rabbit APTT could be directly correlated to the level of human plasma factor Xa. Because anisoyl Xa bypasses the "tenase" complex that is compromised in hemophilia A and B and is unaffected by inhibitory antibodies, it has the potential to be used as an effective bypass therapy.     

Duration of Lactation and Incidence of the Metabolic Syndrome in Women of Reproductive Age According to Gestational Diabetes Mellitus Status: A 20-Year Prospective Study in CARDIA (Coronary Artery Risk Development in Young Adults)

OBJECTIVE

The objective of the study was to prospectively assess the association between lactation duration and incidence of the metabolic syndrome among women of reproductive age.

RESEARCH DESIGN AND METHODS

Participants were 1,399 women (39% black, aged 18–30 years) in the Coronary Artery Risk Development in Young Adults (CARDIA) Study, an ongoing multicenter, population-based, prospective observational cohort study conducted in the U.S. Women were nulliparous and free of the metabolic syndrome at baseline (1985–1986) and before subsequent pregnancies, and reexamined 7, 10, 15, and/or 20 years after baseline. Incident metabolic syndrome case participants were identified according to National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria. Complementary log-log models estimated relative hazards of incident metabolic syndrome among time-dependent lactation duration categories by gestational diabetes mellitus (GDM) adjusted for age, race, study center, baseline covariates (BMI, metabolic syndrome components, education, smoking, physical activity), and time-dependent parity.

RESULTS

Among 704 parous women (620 non-GDM, 84 GDM), there were 120 incident metabolic syndrome case participants in 9,993 person-years (overall incidence rate 12.0 per 1,000 person-years; 10.8 for non-GDM, 22.1 for GDM). Increased lactation duration was associated with lower crude metabolic syndrome incidence rates from 0–1 month through >9 months (P < 0.001). Fully adjusted relative hazards showed that risk reductions associated with longer lactation were stronger among GDM (relative hazard range 0.14–0.56; P = 0.03) than non-GDM groups (relative hazard range 0.44–0.61; P = 0.03).

CONCLUSIONS

Longer duration of lactation was associated with lower incidence of the metabolic syndrome years after weaning among women with a history of GDM and without GDM, controlling for preconception measurements, BMI, and sociodemographic and lifestyle traits. Lactation may have persistent favorable effects on women''s cardiometabolic health.Lactation has favorable effects on cardiometabolic risk factors in women with and without a history of gestational diabetes mellitus (GDM), a strong predictor of type 2 diabetes (1,2) and the metabolic syndrome after pregnancy (3). In the general population, lactating compared with nonlactating women exhibit a less atherogenic lipid profile (4) and lower blood glucose and insulin concentrations (5). Consistent with these findings, lactating women with recent GDM experience lower fasting plasma glucose and insulin levels, higher plasma HDL cholesterol levels, and 50% lower prevalence of type 2 diabetes at 12–16 weeks postpartum (6,7).Yet, few studies have investigated whether lactation''s favorable effects on cardiometabolic risk factors persist after weaning to protect women against future disease. The only study, to our knowledge, to measure changes from preconception to after weaning reported 6-mg/dl higher average HDL cholesterol levels among women who lactated for ≥3 months versus <3 months independent of preconception plasma HDL cholesterol levels and weight gain (8). Epidemiologic studies have reported weak to modest protective associations between lactation and disease risk in midlife to late life, including lower prevalence of the metabolic syndrome (9,10) or cardiovascular risk factors (11) and lower incidence of myocardial infarction (12) and type 2 diabetes (13). Yet, evidence is lacking that directly links risk factor changes that persist after weaning to subsequent disease onset, because disease status and lactation history were ascertained decades after pregnancy, and preconception and/or postweaning risk factor measurements were not available (9–13). Other limitations include classification of outcomes via self-report only (11–13), and failure to account for mediating or confounding effects of lifestyle habits during the reproductive years. Lastly, lactation duration in relation to disease risk has not been examined separately among women with a history of GDM, with the exception of one study reporting a null association with incident diabetes (13).To our knowledge, studies have never examined lactation and incidence of the metabolic syndrome, or variation in disease risk by GDM status. To address these gaps, we prospectively examined whether increasing duration of lactation was associated with lower incidence of the metabolic syndrome during a 20-year period among women of childbearing age. We examined incidence rates for GDM and non-GDM pregnancies and controlled for preconception risk factor levels, sociodemographics, and follow-up behavioral attributes.     

HLA class I antibodies in patients awaiting kidney transplantation and the association with renal graft survival

The presence of alloantibodies against human leukocyte antigens (HLA) in the circulation of a transplant recipient shows a significant negative impact on the outcome of solid-organ transplantations. The aim of this study was to examine the impact on renal graft survival of various patterns of alloantibodies detected among patients awaiting kidney transplantation. Among more than 2000 patients awaiting kidney transplantations between July 1992 and March 2006, were 683 patients who displayed anti-HLA alloantibodies, 318 of whom were enrolled in this study. Each patient was followed for at least 9 months; the presence of HLA alloantibodies was checked every 3 months by an enzyme-linked immunosorbent assay. Among these 318 patients, 55 patients underwent kidney transplantations. Their median follow-up time was 69 (range, 9-129) months, including 267 (84%) who displayed persistent class I HLA alloantibodies. The intermittent presence of class I HLA alloantibodies was seen in 20 (6.3%) patients. Serum class I HLA antibodies which was positive at first then became undetectable in 4 (1.3%) patients. Three (0.9%) patients were unsensitized at first and then developed class I HLA alloantibodies later; & 24 (7.5%) patients had class I HLA alloantibodies only once during the follow-up period. Among these patients, 55 patients received renal transplantations. The median survival time was shortest in the patients with persistent class I HLA alloantibodies (59.9 months) and longest among patients who were positive at first and then became negative thereafter or in whom class I HLA alloantibodies was detected only once (132 months). There was a significant difference in graft survival times between patients who had persistent HLA alloantibodies and those in whom to have class I HLA alloantibodies were detected only once (P < .05). In this study, the persistent presence of class I HLA alloantibodies among pretransplantation patients was associated with poorer renal graft outcomes. Surveys of various patterns of sensitization to class I HLA antigen may help us to perform risk stratification. High-risk patients may need more aggressive approaches to deplete antibody or complement levels.     

Sirolimus conversion experience in a single center

OBJECTIVE: One major cause of graft loss is chronic allograft nephropathy (CAN), which may relate to calcineurin inhibitors (CNIs). We converted CAN cases from CNIs to sirolimus and observed the outcomes. METHOD: From January 2004 to August 2007, there were 28 kidney recipients in our center with creeping creatinine levels compatible with CAN. We started sirolimus at 2 mg/d and reduced the CNIs gradually. Sirolimus trough levels were kept between 5 and 8 ng/mL. Mycophenolic acid was cut in half; there was no adjustment on prednisolone dose. RESULTS: The mean switch time was 47.3 months after transplantation. One case discontinued sirolimus due to severe drug-induced pneumonitis. Twelve of the 27 (45%) patients showed improvements in graft function. The most frequent complications were anemia (13/28), hyperlipidemia (13/28), and pneumonitis (4/28). A baseline serum creatinine level less than 2.2 mg/dL seemed to forecast a response to sirolimus conversion. Most of the graft functional improvement occurred within 6 months after the switch. No graft or patient loss was encountered. CONCLUSION: Our experience suggested that 45% of patients with sirolimus conversion showed improved graft function. Among patients within 1 year after transplantation, those with a creatinine level less than 2.2 mg/dL, no proteinuria, and no hyperlipidemia seemed to be better candidates for Sirolimus conversion.     

Clinical experience of mycophenolate mofetil in the treatment of chronic allograft nephropathy in kidney transplantation: three-year follow-up

BACKGROUND: Mycophenolate mofetil (MMF) in conjunction with calcineura antagonists has been shown to prevent acute rejection in renal allograft recipients. Its role in treatment of chronic rejection or allograft nephropathy is still controversial. We initiated the study to investigate the effect of adding MMF to a cyclosporine plus prednisolone regimen in renal recipients with chronic allograft nephropathy. MATERIALS AND METHODS: We retrospectively studied 36 patients with chronic allograft nephropathy, defined clinically as increased of serum creatinine, proteinuria, and hypertension. Renal function, cyclosporine level, renal biopsy, and renal scan were regularly done as indicated. MMF was added to 20 recipients after initial treatment with cyclosporine and prednisolone. The other 16 recipients were managed without adding MMF. Serum creatinine was monitored for 3 years. RESULTS: The demographic characteristics of the patients in the two groups were comparable. The average dose of prednisolone was unchanged throughout the study and the trough level of cyclosporine was maintained in the range of 100 to 150 ng/mL. The serum creatinine decreased initially in the group on MMF, but renal function deteriorated progressively after 6 months. There was a difference in serum creatinine between the two groups but this did not reach statistical significance. CONCLUSION: MMF therapy tender to improve renal function initially but did not attenuate significantly the impairment in chronic allograft nephropathy.     

Early postoperative echocardiographic studies of atrial septal defects

Fifteen consecutive adult patients with uncomplicated atrial septal defects (ASD) underwent echocardiographic examinations both before and 3-8 days (mean 7 days) after surgery to study the early postoperative changes in cardiac dimensions and interventricular septal motion. Echocardiographic analyses included patterns of interventricular septal motion, right and left ventricular dimensions at end-diastole (RVDd & LVDd), aortic root dimension at end-diastole (ARDd) and left atrial dimension at end-systole (LADs). The results showed that the septal motion was abnormal in 87% (13/15) before and 40% (6/15) after operation. RVDd decreased from 36 +/- 7 mm to 27 +/- 7 mm (p less than 0.01) while LVDd increased from 33 +/- 6 mm to 39 +/- 4 mm (p less than 0.01). There were no significant changes in LADs and ARDd after surgery. These observations suggest that in patients with ASD the ventricular dimensions and patterns of interventricular septal motion are changed significantly shortly after surgical repair.     

N-acetylcysteine attenuates ventilator-induced lung injury in an isolated and perfused rat lung model

N-acetylcysteine (NAC) suppresses the generation of reactive oxygen species (ROS) that are implicated in ventilator-induced lung injury (VILI). We thus hypothesised that NAC attenuates VILI. VILI was induced by mechanical ventilation with a tidal volume (Vt) of 15mlkg(-1) in isolated and perfused rat lung. NAC was administered in the perfusate prior to the onset of mechanical ventilation. A group ventilated with low Vt of 5mlkg(-1) served as control. Haemodynamics, lung injury indices, inflammatory responses and activation of apoptotic pathways were determined upon completion of the mechanical ventilation. There was an increase in lung permeability and lung weight gain after mechanical ventilation with high Vt, compared to low Vt. The levels of inflammatory cytokines including interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and macrophage inflammatory protein-2 (MIP-2) increased in lung lavage fluids; the concentrations of H(2)O(2) were higher in lung lavage fluids, and the expression of myeloperoxidase (MPO), JNK, P38, pAKT and caspase-3 in lung tissue was greater in the high Vt than in the low Vt group. The concentrations of glutathione (GSH) in lung tissue were higher in low Vt than those in high Vt. The administration of NAC increased GSH, attenuated ROS, cytokines, MPO, JNK, pAKT and caspase-3 and lung permeability associated with decreased activation of nuclear factor-κB. VILI is associated with inflammatory responses including the generation of ROS, cytokines and the activation of mitogen-activated protein kinase cascade. The administration of NAC attenuates the inflammatory responses, apoptosis and VILI in the isolated, perfused rat lung model.