Methaemoglobin formation in the koala and the possum

Measurements were made of glutathione (GSH) levels, catalase activity and the oxidant sensitivity of the erythrocytes from the koala (Phascolarctos cinereus) and the common brushtail possum (Trichosurus vulpecula). The oxidant sensitivity was tested by treating the haemolysates with either 0.55 him H₂O₂ or 1.4mm NaNO₂. The erythrocytes of the koala had greater levels of GSH and catalase and yet were found to be more susceptible to oxidation induced by both these oxidants.     

Osteoclastogenesis inhibitory factor exhibits hypocalcemic effects in normal mice and in hypercalcemic nude mice carrying tumors associated with humoral hypercalcemia of malignancy

Osteoclastogenesis inhibitory factor (OCIF) is a novel secreted protein that inhibits osteoclastogenesis both in vitro and in vivo. In this study, we examined the effects of OCIF on serum calcium (Ca) concentrations in normal mice and in hypercalcemic nude mice carrying tumors associated with humoral hypercalcemia of malignancy. In normal mice, a single intraperitoneal injection of OCIF reduced serum Ca levels in a dose-dependent manner. Significant decrease in serum Ca (by 1.6 ± 0.3 mg/dL, n = 5) was observed 2 h after the injection of OCIF at 20 mg/kg and the hypocalcemic effect continued for up to 12 h. Serum phosphate (Pi) concentrations also decreased in response to OCIF. Urinary excretion of Ca, Pi, and creatinine did not change significantly after injection of OCIF or vehicle. In hypercalcemic, tumor-bearing nude mice, a single intraperitoneal injection of OCIF at 20 mg/kg resulted in a dramatic decrease in serum Ca (maximal decrease 2.8 ± 0.37 mg/dL, n = 11), which continued for up to 24 h. The results suggest that OCIF decreased serum Ca through its inhibitory effect on bone resorption. Furthermore, it is suggested that OCIF has therapeutic potential for the treatment of hypercalcemic conditions such as malignancy-associated hypercalcemia.     

Ultrastructural detection of neuronally transported choleragenoid by postembedding immunocytochemistry in freeze-substituted Lowicryl HM20™ embedded tissue

Choleragenoid (cholera toxin B-fragment; CTB) is an anterograde, retrograde and transganglionic neuronal tracer. We describe a method for detecting CTB-labeled neuronal cell bodies, neurites and boutons at the ultrastructural level, using postembedding immunogold techniques on freeze-substituted Lowicryl HM20™ embedded nervous tissue. Primary afferents and motoneurons were labeled by injection of CTB in the dorsal ramus of the C2 spinal nerve of the rat. Following fixation with paraformaldehyde (4%) and glutaraldehyde (0.25%), tissue sections from the spinal cord C2 segment were freeze-substituted and embedded in Lowicryl HM20™ and subsequently processed with postembedding immunocytochemistry for CTB and glutamate. Immunogold particles indicating CTB immunoreactivity were found over primary afferents and motoneurons. In primary afferents in the central cervical nucleus (CCN) and motor nuclei, immunogold labeling was seen in boutons over vesicle-containing axoplasm and to a lesser extent over axoplasm devoid of vesicles, but not over mitochondria or axolemma. In motoneurons, immunogold particles were seen over the Golgi apparatus in the soma and over lysosomes in both soma and dendrites. Quantification of glutamate-like immunoreactivity in 20 CTB-labeled and 20 CTB-negative boutons in the neuropil was found similar, indicating that CTB does not interfere with the immunocytochemical detection of neuronal epitopes such as the transmitter substance glutamate.     

Serum p55 and p75 tumour necrosis factor receptors as markers of disease activity in juvenile chronic arthritis.

OBJECTIVE: To determine the expression of tumour necrosis factor alpha (TNF alpha) and its soluble receptors (p55 and p75) in the sera and synovial fluid of patients with juvenile chronic arthritis (JCA), and their correlation with disease activity parameters. METHODS: Ninety eight sera from 45 patients with JCA (14 systemic, 12 polyarticular, 19 pauciarticular), 20 sera from age matched healthy controls, and five synovial fluids from five antinuclear antibody (ANA) positive pauciarticular JCA patients were tested for the presence of TNF alpha, soluble TNF receptors p55 and p75 (sTNFRp55, sTNFRp75), and interleukin-6 (IL-6) by an enzyme amplified sensitivity immunoassay. Physician global estimate of disease activity, weekly fever score and joint score, C reactive protein (CRP), erythrocyte sedimentation rate (ESR), and haemoglobin concentration were evaluated as parameters of disease activity. The expression of p55 and p75 on peripheral mononuclear cells (MNCs) from five patients with systemic JCA and synovial MNCs from five ANA positive patients with pauciarticular JCA was evaluated by flow cytometry. RESULTS: TNF alpha serum concentrations did not differ significantly between the patients with active JCA and the control group. No correlation was found between TNF alpha and parameters of disease activity, but both p55 and p75 showed a significant positive correlation with the physician global estimate of disease activity (p < 0.001), ESR (p < 0.001), CRP (p < 0.001), and serum concentrations of IL-6 (p < 0.001). Serum concentrations of haemoglobin correlated inversely with the concentrations of p55 and p75 (p < 0.001). Synovial lymphocytes selectively expressed the p75 surface receptor. CONCLUSIONS: sTNFRp55 and sTNFRp75 each represent a sensitive marker of disease activity in JCA. Their increased expression in biological fluids may support the hypothesis that TNF alpha has a role in the pathogenesis of JCA.     

The Presence of Insulin-degrading Enzyme in Human Ileal and Colonic Mucosal Cells

The aim of this research is to characterize the presence of insulin-degrading enzyme in human colon and ileal mucosal cells. Biochemical studies, including the activity-pH profiles, the effects of enzyme inhibitors, immunoprecipitation and western blots, were conducted. The majority of insulin-degrading activity in colon mucosal cells was localized in the cytosol. In both colon and ileum, cytosolic insulin-degrading activities had a pH optimum at pH 7.5, and were extensively inhibited by each of N-ethylmaleimide, p-chloromercuribenzoate, and 1,10-phenanthroline, but were very weakly affected by each of leupeptin, chymostatin, diisopropyl phosphofluoridate and soybean trypsin inhibitor. In the colon and ileum, more than 93% and 96%, respectively, of cytosolic insulin-degrading activities were removed by the mouse monoclonal antibody to human RBC insulin-degrading enzyme, as compared with less than 20% by the normal mouse IgG for both tissues. Further, a western blot analysis revealed that a cytosolic protein of 110 kD, in both human colon and ileum, reacted with the monoclonal antibody to insulin-degrading enzyme. It is concluded that insulin-degrading enzyme is present in the cytosol of human colon and ileal mucosal cells.     

Detection of undiagnosed coagulopathies using routine rapid heparin neutralization.

OBJECTIVE: To determine the most frequent clinical causes of a prolonged activated partial thromboplastin time (APTT) result, and to determine whether a new heparin-removal device (the Hepchek, Pall Biomedical, Glen Cove, NY 11542) is capable of efficiently detecting the causes of these values. DESIGN: A combination of chart review and laboratory testing comparing the criterion standard--the heparin chromogenic substrate assay--with the Hepchek. Laboratory investigations were blinded and controlled. SETTING: Inpatient, acute-care hospital. PATIENTS: A total of 1,000 hospital patients with a variety of hemostatic disorders. MAIN OUTCOME MEASURE: The extent to which the Hepchek accurately identified the etiology of a prolonged APTT result. RESULTS: The APTT was prolonged in 25.2% of samples. The presence of heparin in the sample was confirmed by chromogenic assay or by using the Hepchek heparin-removal filter. The presence of heparin was confirmed in 12.8% of all samples and in more than 50% of all abnormal samples. The cause of the abnormal APTT was often unappreciated by the clinician. Bayesian analysis of the Hepchek's ability to diagnose heparin correctly as the cause of the abnormal APTT showed a sensitivity of 100% and specificity of 99.9%. CONCLUSION: Use of the Hepchek in the routine clinical laboratory is an efficient and rapid method of detecting heparin as a cause of isolated prolonged APTT results, and should reduce demands for unwarranted coagulation analyses and inappropriate treatment with blood products.