Longitudinal changes in liver fibrosis in children with sickle cell disease undergoing chronic transfusion therapy.

BACKGROUND AND STUDY AIMS: The progression of liver injury from transfusional iron overload in sickle cell disease (SCD) is poorly understood. We sought to identify predictors liver fibrosis development over time. PATIENTS AND METHODS: We performed a retrospective cohort study of chronically transfused SCD patients who had > or = 2 serial liver biopsies. Core biopsies were scored for fibrosis in a blinded fashion. Primary analyses evaluated longitudinal changes in liver fibrosis and changes in surrogate markers. Secondary analyses determined the relationship between liver iron concentration (LIC) and serum biomarkers. RESULTS: 26 people had > or = 2 serial biopsies for evaluation (n = 70 biopsies total). Fibrosis was Ishak grade 0 or 1 in all biopsies. Evaluation of the first 2 biopsies showed fibrosis regression (n = 6), development (n = 2), persistence (n = 1), and absence (n = 17). There was no consistent association of fibrosis with LIC over time, or between changes in fibrosis status and surrogate markers. For predicting fibrosis on a cross-sectional basis, ALT and ferritin performed moderately (AUCs 0.80 and 0.63, respectively) but LIC performed poorly (AUC 0.30). The highest positive likelihood ratios for fibrosis were for ferritin cutoff of 5000 ng/mL (LR + 5.7) and ALT cutoff of 65 U/L (LR + 5.2). CONCLUSIONS: Liver fibrosis progression is minimal in chronically transfused SCD. LIC does not correlate well with fibrosis development. We propose routine liver biopsies are not necessary components in the standard monitoring of chronically transfused SCD patients.

Autologous stem cell transplantation for acute myeloid leukemia in first remission.

We studied the feasibility, toxicity, and efficacy of a 2-step approach to autologous stem cell transplantation for patients with acute myeloid leukemia in first remission. Step 1 consisted of consolidation chemotherapy including cytarabine 2000 mg/m2 twice daily for 4 days concurrent with etoposide 40 mg/kg by continuous infusion over 4 days. During the recovery from this chemotherapy, peripheral blood stem cells were collected under granulocyte colony-stimulating factor stimulation. Step 2, autologous stem cell transplantation, involved the preparative regimen of busulfan 16 mg/kg followed by etoposide 60 mg/kg and reinfusion of unpurged peripheral blood stem cells. A total of 128 patients were treated. During step 1, there was 1 treatment-related death. A median CD34+ cell dose of 14 (x10(6)/kg) was collected in 3 aphereses. Ten patients suffered relapse before transplantation, and 117 patients (91%) proceeded to transplantation. During step 2, there were 2 treatment-related deaths, and 35 patients subsequently suffered relapse. With median follow-up of 30 months, 5-year disease-free survival for all patients entered in the study is projected to be 55%. By cytogenetic risk group, 5-year disease-free survival is 73% for favorable-risk patients, 51% for intermediate-risk patients, and 0% for poor-risk patients. We conclude that this 2-step approach to autologous transplantation produces excellent stem cell yields and allows a high percentage of patients to receive the intended therapy. Preliminary efficacy analysis is very encouraging, with outcomes that appear superior to those of conventional chemotherapy.

Improved folding yields of a model protein using protein disulfide isomerase.

PURPOSE: To study the effects of recombinant human protein disulfide isomerase (rhPDI) concentration, reduced glutathione:oxidized glutathione ratio (GSH:GSSG) and temperature on the efficiency of oxidative folding of a model protein, recombinant human interleukin 2 (C125A mutation) (C125A rhIL-2). METHODS: C 125A rhIL-2 inclusion bodies were reduced and denatured by guanidium hydrochloride (Gdm.Cl) and 100 mM GSH. The solution was diluted 10 times into folding buffer, allowing C125A rhIL-2 to fold either in the absence or presence of rhPDI. The renatured and unfolded C125A rhIL-2 species were quantitated by reversed phase-HPLC. RESULTS: The initial folding rate of C125A rhIL-2 linearly increased with rhPDI:C125A rhIL-2 molar ratio in the first 2.5 minutes, and reached the highest rate when the rhPDI:C125A rhIL-2 ratio was 1:1. The oxidative folding of C125A rhIL-2 linearly increased as the GSH:GSSG molar ratio decreased from 10:0 to 10:3. The folding of C125A rhIL-2 was also dependent on temperature, and optimum folding was realized at 23 degrees C. CONCLUSIONS: These results demonstrate that under optimal redox potential and temperature, rhPDI enhances the oxidative folding of C125A rhIL-2. In the oxidative folding of C125A rhIL-2, rhPDI exerts its effect on folding by the acceleration of thiol/disulfide interchange.

Degradation of antiflammin 2 under acidic conditions.

Antiflammin 2 (HDMNKVLDL, AF2) is a synthetic peptide derived from the region of highest sequence similarity of lipocortin I and uteroglobin, and is a potent antiinflammatory agent without any known side effects of corticosteroids. The antiinflammatory activity of AF2 has been demonstrated, but is not reproducible between laboratories. It has been suggested that the chemical instability of this peptide is responsible for the loss of activity. The degradation of AF2 in aqueous solutions at a pH range of 3 to 10 has been reported. In this study, the degradation of AF2 at acidic pHs was monitored by reversed-phase HPLC. The reactions were studied as functions of buffer concentration and temperature. The rates of loss of AF2 followed apparent pseudo-first-order kinetics. Several products were isolated and identified by fast atom bombardment mass spectroscopy and tandem mass spectroscopy, and were the result of C- and N-terminus hydrolyses of aspartyl peptide bonds in AF2. The peptide bonds at C-termini of the aspartyl residues were most susceptible to hydrolysis, resulting in the formation of major degradation products, HDMNKVLD, MNKVLDL, and MNKVLD. The minor products from the N-terminus hydrolysis were HDMNKVL and MNKVL and formed at much slower rates.

Covalent association of protein disulfide isomerase with recombinant human interleukin 2 in vitro.

Protein disulfide isomerase has broad specificity in the catalysis of the formation and rearrangement of native disulfide bonds in proteins. This enzyme has two independent thioredoxin-like active sites (-CGHC-) and a peptide binding site. However, the mechanisms involving the catalytic processes are not clearly understood. It was reported that the enzyme associates with scrambled pancreatic ribonuclease A in vitro, and with misfolded human lysozyme in vivo. In the present study, recombinant human interleukin 2 has been chosen to probe the reaction intermediate in the reaction with the enzyme. We have identified and characterized a covalent associate formed in vitro by SDS-PAGE and Western blot analysis. This associate has a molecular weight of 71-72 kDa, the approximate sum of the molecular weights of the enzyme and the substrate. Western blot analysis confirmed that it formed via an intermolecular disulfide bond. Upon treatment with 2-mercaptoethanol, this bond was cleaved.

Oxidative degradation of antiflammin 2.

PURPOSE: To study the oxidation of the methionine residue of antiflammin 2 (HDMNKVLDL, AF2) as a function of pH, buffer concentration, ionic strength, and temperature using different concentrations of hydrogen peroxide and to determine the accessibility of methionine residue to oxidation. METHODS: Reversed-phase high-performance liquid chromatography (RPHPLC) was used as the main analytical method in determining the oxidation rates of AF2. Calibration curves for AF2 and the oxidation product, methionine sulfoxide of AF2 (Met(O)-3-AF2), were constructed for each measurement using standard materials. Fast Atom Bombardment Mass Spectroscopy (FABMS) was used to characterize the product. RESULTS: Met(O)-3-AF2 was the only oxidation product detected at pH 3.0 to 8.0. The oxidation rates were independent of buffer concentrations, ionic strength, and pH from 3.0 to 7.0. However, there was an acceleration of the rates at basic pHs, and small amounts of degradation products other than Met(O)-3-AF2 were observed in this alkaline region. CONCLUSIONS: Oxidation of methionine in AF2 does not cause the biological inactivation reported by other laboratories since this drug is relatively stable under neutral conditions in the absence of oxiding agent.

The behavior of chickens following embryonic treatment with testosterone propionate.

This study was designed to investigate an initial response and the flocking behavior of chicks treated embryonically with testosterone propionate (TP). The high (1.28 gm. %) and low (0.32 gm. %) levels of TP interfered with the response of these chicks to a specific stimulus. However, only the high level TP depressed the flocking response. TP administered prior to day 13 of embryonic development will depress sexual behavior in the chicken. These data suggest that TP influences a variety of behavioral patterns in the chicken.

Agnonistic behavior in organized and disorganized cotton rat populations.

Agonistic interaction rate is significantly lower in groups of caged cotton rats (Sigmodon hispidus) from naturally occurring organized populations than in groups composed of strangers. Some type of social structure is apparently present between animals sharing a common area under natural conditions. After a 24-hour period, there is no significant difference in the behavior of the two groups, an indication that a social structure is rapidly formed in the disorganized groups.