Altered glycosaminoglycan production by HL-60 cells treated with 4- methylumbelliferyl-beta-D-xyloside

Glycosaminoglycans, mainly chondroitin 4-sulfate, are located in the primary granules of human myeloid cells. These polyanionic carbohydrates are believed to play an important role in leukocyte maturation and function. To study the effect of altered chondroitin sulfate metabolism on human promyelocytic leukemia cells, we have treated HL-60 cells with 4-methylumbelliferyl-beta-D-xyloside. beta-D- Xylosides initiate the synthesis of free chondroitin sulfate chains. Cytochemical studies of treated cells demonstrated a marked increase in cytoplasmic granules stained with cationic dyes. This was confirmed by radiolabeled precursor incorporation studies that demonstrated a 344% increase in 35S-sulfate uptake into glycosaminoglycans associated with the cells and a 39% increase in incorporation into glycosaminoglycans released into the media. Chromatographic analyses of these glycosaminoglycans from treated cells demonstrated that the newly formed chondroitin sulfate chains were not attached to protein core and were of shorter length, but of greater charge density than chondroitin sulfate produced by control cells. Thus, beta-D-xyloside appears to alter the protein linkage, chain length, and sulfation of chondroitin sulfate produced by HL-60 cells, and these changes are morphologically evident. These biochemically altered cells may provide important information concerning the role of these macromolecules in myeloid development.     

Enteropathogenic Escherichia coli-induced myosin light chain phosphorylation alters intestinal epithelial permeability

BACKGROUND & AIMS: Infection of epithelial cells with enteropathogenic Escherichia coli (EPEC) induces phosphorylation of the 20-kilodalton myosin light chain (MLC20). The physiological consequence of this biochemical observation, however, has not been discerned. The aim of this study was to determine if EPEC-induced phosphorylation of MLC20 was involved in the associated perturbation of intestinal epithelial barrier function. METHODS: Cultured intestinal epithelial cells, T84, were infected with EPEC. The effects of protein kinase inhibitors on EPEC-induced perturbation of barrier function were assessed using electrophysiological techniques. Alterations in MLC20 phosphorylation were correlated with functional responses. RESULTS: Inhibition of myosin light chain kinase, but not protein kinase C or tyrosine kinase, prevented the decrease in resistance caused by EPEC infection and significantly diminished EPEC-induced MLC20 phosphorylation. Epithelial cell monolayers genetically manipulated to constitutively increase MLC20 phosphorylation were relatively resistant to the effects of EPEC on barrier function. CONCLUSIONS: For the first time, these data show that a physiological consequence of the long-recognized increase in MLC20 phosphorylation by EPEC is perturbation of intestinal epithelial barrier function, which probably contributes to the diarrhea associated with this infection. (Gastroenterology 1997 Dec;113(6):1873-82)     

Pseudo-von Willebrand disease: a mutation in the platelet glycoprotein Ib alpha gene associated with a hyperactive surface receptor

Pseudo (platelet-type)-von Willebrand disease is an autosomal dominant bleeding disorder caused by the hyperfunction of a receptor on the platelet surface. The abnormal receptor, glycoprotein Ib, displays increased affinity for its ligand, von Willebrand factor. Four members (normal mother/affected father/two affected daughters) of a family with pseudo-von Willebrand disease were studied to determine the molecular genetic basis for their congenital platelet defect. Segments of the platelet glycoprotein Ib alpha gene were amplified by means of the polymerase chain reaction, cloned, and sequenced. A point mutation (A to G, codon 239) was found in segments from the affected individuals but not from the normal. The mutation results in a single amino acid substitution (valine-mutant for methionine-normal) at residue 239 within the Ib alpha binding site for von Willebrand factor. Both the mutant and the normal sequence were found in affected individuals, suggesting a heterozygous state. Amplified DNA from family members and from 58 normal individuals was analyzed by allele-specific oligonucleotide hybridization. Only the normal sequence was found in the mother and the normal individuals, whereas both the normal and the mutant alleles were found in the affected family members. The described mutation is associated with the pseudo-von Willebrand disease phenotype seen in this kindred. The resultant single amino acid substitution in glycoprotein Ib alpha relates to increased receptor function and to excessive binding of von Willebrand factor to the platelet surface.     

Physical activity and metabolic health in chronic kidney disease: a cross-sectional study

Background

Patients with chronic kidney disease (CKD) are at high risk of progression to end stage renal disease and cardiovascular events. Physical activity may reduce these risks by improving metabolic health. We tested associations of physical activity with central components of metabolic health among people with moderate-severe non-diabetic CKD.

Methods

We performed a cross-sectional study of 47 people with CKD (estimated GFR <60 ml/min/1.73 m²) and 29 healthy control subjects. Accelerometry was used to measured physical activity over 7 days, the hyperinsulinemic-euglycemic clamp was used to measure insulin sensitivity, and DXA was used to measured fat mass. We tested associations of physical activity with insulin sensitivity, fat mass, blood pressure, serum lipid concentrations, and serum high sensitivity C-reactive protein concentration using multivariable linear regression, adjusting for possible confounding factors.

Results

Participants with CKD were less active than participants without CKD (mean (SD) 468.1 (233.1) versus 662.3 (292.5) counts per minute) and had lower insulin sensitivity (4.1 (2.1) versus 5.2 (2.0 (mg/min)/(μU/mL)), higher fat mass (32.0 (11.4) versus 29.4 (14.8) kg), and higher triglyceride concentrations (153.2 (91.6) versus 99.6 (66.8) mg/dL). With adjustment for demographics, comorbidity, medications, and estimated GFR, each two-fold higher level of physical activity was associated with a 0.9 (mg/min)/(μU/mL) higher insulin sensitivity (95% CI 0.2, 1.5, p?=?0.006), an 8.0 kg lower fat mass (?12.9, ?3.1, p?=?0.001), and a 37.9 mg/dL lower triglyceride concentration (?71.9, ?3.9, p?=?0.03). Associations of physical activity with insulin sensitivity and triglycerides did not differ significantly by CKD status (p-values for interaction >0.3).

Conclusions

Greater physical activity is associated with multiple manifestations of metabolic health among people with moderate-severe CKD.

     

Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma

Although molecular biologic techniques can now detect minimal numbers of residual cancer cells in patients in complete clinical remission, the clinical significance of minimal residual disease has never been conclusively established. If the detection of minimal residual disease predicts which patients will relapse, then therapy could be altered based upon the detection of these cells. The t(14;18) can be detected by polymerase chain reaction (PCR) amplification in 50% of patients with B-cell non-Hodgkin's lymphoma and allows detection of one lymphoma cell in up to 1 million normal cells. To determine the clinical significance of the detection of minimal residual lymphoma cells in the bone marrow (BM) PCR amplification was used to detect the presence of residual lymphoma cells after autologous BM transplantation (ABMT) in serial BM samples from 134 patients with B-cell lymphoma in whom a bcl- 2 translocation could be detected. PCR analysis was performed on a total of 542 BM samples obtained while these patients were in complete remission. Disease-free survival was markedly increased in patients with no PCR-detectable lymphoma cells in the marrow compared with those in whom residual lymphoma cells were detected (P < .00001), and the presence of detectable lymphoma cells was associated with a 48-fold increase in the risk of relapse. Of the 77 patients (57%) with no PCR- detectable lymphoma cells in their most recent BM sample, none have relapsed. In contrast, all 33 patients (25%) who have relapsed had PCR- detectable lymphoma cells detected in their BM before clinical relapse occurred. In 19 patients (14%), residual lymphoma cells in the BM were detected early following transplantation and subsequently were no longer detectable, although these patients received no further therapy. In these patients, residual lymphoma cells may already have been irreversibly damaged by the high-dose therapy or an endogenous immune mechanism may be capable of eliminating residual lymphoma cells in some patients. Therefore, although the detection of minimal residual disease by PCR following ABMT in patients with lymphoma identifies those patients at high risk of relapse, the presence of residual minimal disease early after transplantation may not be associated with poor prognosis in a small subset of patients. Confirmatory studies will be required to determine more definitively the role of minimal disease detection to identify which patients require additional therapy.     

CFU-GM content of bone marrow graft correlates with time to hematologic reconstitution following autologous bone marrow transplantation with 4- hydroperoxycyclophosphamide-purged bone marrow

Autologous bone marrow transplants (BMTs) can repopulate the hematologic system of patients treated with marrow-ablative chemotherapy and/or radiotherapy. However, treatment of the bone marrow graft to eliminate residual tumor cells prior to reinfusion can delay the return of peripheral blood elements, presumably from damage to or loss of hematopoietic stem cells responsible for hematologic recovery. To develop a model predictive of hematologic recovery, we studied the progenitor cell contents of 4-hydroperoxycyclophosphamide (100 micrograms/mL)-purged bone marrow grafts of 40 consecutive patients undergoing autologous BMT at this center. Granulocyte-macrophage colonies (CFU-GM) were grown from all grafts after treatment with this chemotherapeutic agent, but erythroid (BFU-E) and mixed (CFU-GEMM) colonies were grown from only 44% and 33% of the grafts respectively. The recovery of CFU-GM after purging ranged from 0.07% to 23%. The logarithm of CFU-GM content of the treated grafts was linearly correlated with the time to recovery of peripheral blood leukocytes (r = -0.80), neutrophils (r = -0.79), reticulocytes (r = -0.60), and platelets (r = -0.66). The CFU-GM content of purged autologous bone marrow grafts may reflect the hematopoietic stem cell content of the grafts and thus predict the rate of hematologic recovery in patients undergoing autologous BMT.     

A phase I trial of recombinant human interleukin-6 in patients with myelodysplastic syndromes and thrombocytopenia

To evaluate the hematologic effects of recombinant human interleukin-6 (rhIL-6, Escherichia coli, SDZ ILS 969, IL-6), and determine its toxicity profile, we performed a phase I trial of IL-6 in 22 patients with various myelodysplastic syndromes (MDS), platelet counts < 100,000/microL, and < 5% bone marrow (BM) blasts. Patients received one of four doses of IL-6 (1.0, 2.5, 3.75, and 5.0 micrograms/kg/d) as a subcutaneous injection on day 1, followed by a 7-day wash-out period, and then 28 days of IL-6 therapy. Dose-limiting toxicities of fatigue, fever, and elevated alkaline phosphatase were seen at 5.0 micrograms/kg/d; the maximum tolerated dose was 3.75 micrograms/kg/d. All patients experienced at least grade II fever and all had an increase in acute phase proteins. Eight patients (36%) experienced at least a transient improvement in platelet counts; three fulfilled the criteria for response, whereas five others had clinically significant increases that failed to meet response criteria. Various IL-6-related toxicities prevented more than three patients from receiving maintenance therapy. Two of the three patients who received maintenance IL-6 therapy had a persistent increase in platelet counts, during 3 and 12 months of IL-6 therapy, respectively. Laboratory studies indicated that IL-6 increased the frequency of higher ploidy megakaryocytes but did not significantly increase the number of assayable megakaryocytic progenitor cells, suggesting that IL-6 acts as a maturational agent rather than a megakaryocyte colony-stimulating factor. Although IL-6 therapy can promote thrombopoiesis in some MDS patients, its limited activity and significant therapy-related toxicity preclude its use as a single agent in this patient population. Further studies, combining low doses of IL-6 with other hematopoietic growth factors, are underway.     

Recombinant human factor IX: replacement therapy, prophylaxis, and pharmacokinetics in canine hemophilia B

Recombinant human factor IX (rFIX) has been expressed in transduced cultured cell systems since 1985. Because there has been limited in vivo testing of rFIX in hemophilia B subjects, this study was undertaken using the severe hemophilia B canines of the Chapel Hill strain. Three groups of hemophilic dogs received either 50, 100, or 200 IU/kg of rFIX. As a control, a fourth group of hemophilic dogs received 50 IU/kg of a high purity, plasma-derived human FIX (pdFIX). The coagulant and hemostatic effects of rFIX and pdFIX were similar with all comparative dosing regimens. Based on activity data, the elimination half-life of rFIX was 18.9 +/- 2.3 hours and pdFIX was 17.9 +/- 2.1 hours. A prophylactic regimen administering rFIX daily resulted in a continuous therapeutic level of plasma FIX and was accompanied by a two-fold increase in recovery levels by day 5, compared to that observed with administration of a single bolus. The mechanisms of the high to complete recovery of FIX with the prophylactic regimen could depend not only on the degree of saturation of the vascular endothelial binding sites but also on the altered dynamics of the balance of FIX distribution between the intravascular and extravascular compartments. The pharmacokinetic (PK) parameters for rFIX and pdFIX were similar. However, the relative PK values for V1 and V5s of both products on day 5 differed greatly from day 1 and may reflect the changing equilibrium of FIX between compartments with elevated levels of plasma FIX. Neutralizing antihuman FIX antibodies resulting from human FIX antigen being administered to FIX deficient dogs were observed beginning at 14 days. The antigenicity of rFIX and pdFIX appeared to be comparable. Despite the very different procedures used for production of rFIX and pdFIX products, in vivo testing in hemophilia B dogs showed the functional behavior of these products is similar; they are highly effective for replacement therapy and for prophylaxis.