Post-hypoglycaemic hyperketonaemia does not contribute to brain metabolism during insulin-induced hypoglycaemia in humans

Summary It is controversial as to whether ketone bodies are utilized by the human brain as a fuel alternative to glucose during hypoglycaemia. To clarify the issue, we studied 10 normal volunteers during an experimental hypoglycaemia closely mimicking the clinical hypoglycaemia of patients with Type 1 (insulin-dependent) diabetes mellitus or insulinoma. Hypoglycaemia was induced by a continuous infusion of insulin (0.40 mU·kg^–1·min^–1 for 8 h, plasma insulin 180 pmol/l) which decreased the plasma glucose concentration to approximately 3.1 mmol/l during the last 3 h of the studies. Subjects were studied on two occasions, i. e. spontaneous, counterregulatory-induced post-hypoglycaemic increase in 3--hydroxybutyrate (from 0.2 to 1.1 mmol/l at 8 h), or prevention of post-hypoglycaemic hyperketonaemia (plasma -hydroxybutyrate 0.1 mmol/l throughout the study) after administration of acipimox, a potent inhibitor of lipolysis. In the latter study, glucose was infused to match the hypoglycaemia observed in the former study. The glycaemic thresholds and overall responses of counterregulatory hormones, symptoms (both autonomic and neuroglycopenic), and deterioration of cognitive function (psychomotor tests) were superimposable in the control study in which ketones increased spontaneously after onset of hypoglycaemic counterregulation, as compared to the study in which ketones were suppressed (p=NS). The fact that responses of counterregulatory hormones, symptoms and deterioration in cognitive function were not exaggerated when posthypoglycaemic hyperketonaemia was prevented, indicate that during hypoglycaemia, the counterregulatory-induced endogenous hyperketonaemia does not provide the human brain with an alternative substrate to glucose. Thus, it is concluded that during hypoglycaemia, endogenous hyperketonaemia does not contribute to brain metabolism and function.     

The effects of nitroglycerin-methoxamine combination on infarct size in conscious dogs

The effect of nitroglycerin combined with methoxamine in reducing infarct weight was studied in conscious dogs. Ten minutes after permanent left anterior descending (LAD) coronary artery occlusion, 10 dogs received nitroglycerin (450 micrograms bolus IV, then 300 micrograms/min for 4 hours) and methoxamine as needed to maintain blood pressure and heart rate. No dogs in heart failure. Ten control dogs received saline solution. Dogs were sacrificed 3 days later. The region at risk of infarction was delineated by simultaneously perfusing the aortic root with Evans blue and the distal LAD artery with saline solution under equal pressures. Slices of stained hearts were incubated with tetrazolium to identify infarct. Total weight of left ventricle (LV), risk region, and infarct was measured. Nitroglycerin-treated dogs showed no difference from control dogs in infarct weight (26.2 +/- 5.9 gm +/- SE vs 27.7 +/- 5.6 gm), percent risk region/LV (36.0 +/- 1.4% vs 37.9 +/- 3.1%), or present infarct/LV (23.5 +/- 5.2% vs 24.8 +/- 4.9%). In a subgroup with risk region/LV less than or equal to 35%, nitroglycerin reduced infarct weight by 45% (8.8 +/- 8.5% vs 15.9 +/- 7.9%), percent infarct/LV by 49% (7.1 +/- 6.8% vs 13.8 +/- 6.6%), and percent infarct/risk region by 41% (23.0 +/- 22.0% vs 38.9 +/- 15.9%). Because of the small number of dogs in the study, differences were not significant. In dogs with risk region/LV greater than 35%, nitroglycerin had no effect. Thus, in dogs without overt heart failure, nitroglycerin may salvage ischemic tissue within small areas at risk of infarction, but the results are not definitive. However, our results clearly demonstrate that in the absence of failure, nitroglycerin does not reduce the size of large infarcts.     

Hematopoietic growth factor expression and ATRA sensitivity in acute promyelocytic blast cells

Acute promyelocytic leukemia (APL) is a homogeneous subgroup of acute myeloid leukemias (AMLs) characterized by the presence of the t(15,17) translocation and the resulting promyelocytic myeloid leukemia/retinoic acid receptor alpha (PML/RAR alpha) fusion proteins. To date APL is the only AML that is sufficiently sensitive to all-trans retinoic acid's (ATRA) differentiating effect. In vivo ATRA alone achieves complete remission in most APL patients. However, failure or partial responses are observed and the molecular basis of the absence of ATRA response in these patients has not been determined. To gain insights in the cell growth and differentiation of APL cells, expression of hematopoietic growth factors (HGF) shown to be produced by leukemic cells (interleukin-1 beta [IL-1 beta], IL-6, tumor necrosis factor alpha (TNF alpha), granulocyte colony-stimulating factor [G-CSF], granulocyte- macrophage colony-stimulating factor [GM-CSF], and IL-3) was studied in 16 APL samples. Twelve APL cases expressed IL-1 beta, IL-6, and TNF alpha, but not G-CSF, GM-CSF, and IL-3. These cases achieved complete remission with ATRA therapy. The four remaining patients (either TNF alpha negative or G-CSF, GM-CSF or IL-3 positive) did not achieve complete remission with ATRA. In all cases, in vivo response to ATRA therapy was correlated to the in vitro differentiation effect of all- trans retinoic acid 10(-6) mol/L. Thus, ATRA differentiation induction was strongly correlated to the HGF expression (P < .0001). These results suggest that the presence or absence of HGF's expression by APL cells may contribute to the therapeutic effect of ATRA in this disease.     

IgMs produced by two acquired immune deficiency syndrome lymphoma cell lines: Ig binding specificity and VH-gene putative somatic mutation analysis [published erratum appears in Blood 1994 Aug 1;84(3):995]

Two B-cell lines, 2F7 and 10C9, were established by single cell cloning from biopsies obtained from two acquired immune deficiency syndrome patients with Burkitt's lymphoma. Representation of the original tumors was verified by demonstration of (1) identical biallelic rearrangement of Ig genes for 2F7 and (2) shared idiotype for 10C9. Both cell lines displayed cell-surface Ig and secreted Ig (IgM lambda for 2F7, IgM kappa for 10C9). IgMs from both cell lines immunoprecipitated actin; in addition, 2F7 IgM lambda immunoprecipitated recombinant human immunodeficiency virus type 1 (HIV-1) gp 160. 2F7 IgM lambda did not react with other autoantigens (double-stranded and single-stranded DNA, actin, bovine serum albumin, IgG), whereas 10C9 IgM kappa reacted with human IgG. The 2F7 IgM heavy-chain variable region (VH) showed a 95% nucleotide homology with a previously sequenced VHIII germline gene, hv3019b9, whereas the 10C9 IgM VH showed a 95% homology with a previously sequenced VHIV germline gene, VH4.21. Use of minimally modified VH genes and demonstration of reactivity with chronically present antigens (ie, actin, HIV-1 gp 160, or human IgG) suggests that B cells in HIV-1-infected individuals proliferating in response to chronic antigenic stimulation may be at increased risk for lymphomagenesis.     

Platelets inhibit the induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells

We have investigated the role of platelets in regulating the hemostatic and vasomotor properties of vascular smooth muscle. Experiments were performed to examine the effect of the releasate from activated platelets on the production of nitric oxide from interleukin-1 beta (IL- 1 beta)-treated cultured rat aortic smooth muscle cells. Treatment of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite in the culture media and in marked elevation of intracellular cyclic guanosine monophosphate (GMP) levels. The releasate from collagen-aggregated platelets blocked the IL-1 beta- mediated production of nitrite and the accumulation of cyclic GMP in smooth muscle cells in a platelet number-dependent manner. In functional assays, the perfusates from columns containing IL-1 beta- treated smooth muscle cells relaxed detector blood vessels without endothelium and the addition of IL-1 beta-treated smooth muscle cells to suspensions of platelets inhibited their thrombin-induced aggregation. The simultaneous treatment of smooth muscle cells with IL- 1 beta and the platelet releasate abolished both the vasorelaxing activities of the perfusates and the inhibition of platelet aggregation. Platelet releasates treated with a neutralizing antibody to platelet-derived growth factor (PDGF) failed to block IL-1 beta- induced nitric oxide production by the smooth muscle cells, as measured by both biochemical and functional assays. The platelet releasate from a patient with gray platelet syndrome likewise failed to block IL-1 beta-induced nitrite release by smooth muscle cells. These results demonstrate that platelets downregulate the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells through the release of PDGF. This effect may represent a novel mechanism by which platelets regulate vasomotor tone and thrombus formation at sites of vascular injury.     

Failure of recombinant human interleukin-3 therapy to induce erythropoiesis in patients with refractory Diamond-Blackfan anemia [see comments]

In two previous studies, we observed that recombinant human interleukin- 3 (IL-3) induced an increase in marrow burst-forming unit-erythroid- derived colonies in vitro in some patients with Diamond-Blackfan anemia (DBA). To determine whether a similar erythropoietic response could be induced in vivo, we treated 13 patients with DBA (aged 4 to 19 years) with two preparations of IL-3. All patients had absent absolute reticulocyte counts and markedly reduced to absent recognizable bone marrow erythroid elements; patients with circulating reticulocytes in the previous 12 months were excluded from study. All patients except 1 had failed steroid therapy and had been transfusion-dependent since infancy; 1 patient was maintained on high-dose prednisone at the time of enrollment. On the first arm of the study, IL-3 (Immunex Corp, Seattle, WA) was administered subcutaneously using a dose escalation regimen of 125 to 500 micrograms/m2/day in divided dosage at 12-hour intervals, coadministered with 1.5 mg/kg/d of oral ferrous sulphate. Of the 13 patients that entered the trial, 4 stopped prematurely because of adverse side effects. In the other 9 evaluable cases, reticulocytes increased transiently in 1 patient from 0 to 65 x 10(9)/L after 35 days of IL-3 therapy at 250 micrograms/m2, but transfusion dependency persisted. One transient peak in absolute reticulocyte count was noted in 6 other patients, but no erythroid response was observed after completion of a full course of IL-3. Oral prednisone at 0.5 mg/kg/d was then coadministered with IL-3 at 500 micrograms/m2 to 5 of the patients without effect, and treatment was stopped. In 2 patients, a second preparation of IL-3 (Sandoz Canada Inc, Dorval, Quebec, Canada) was initiated in a dose escalation regimen of 2.5 to 10 micrograms/kg and was coadministered with ferrous sulphate. No erythroid response was observed in either patient, and in one of the two, alternate-day subcutaneous recombinant erythropoietin at 300 U/kg was administered for 3 weeks in combination with daily IL-3 at 10 micrograms/kg, but no increased erythropoiesis was seen. Significant increases in white blood cell and eosinophil counts during administration of both preparations of IL-3 were observed in all patients. These data show that the response of DBA patients to IL-3 in vivo is heterogeneous and cannot be predicted from in vitro studies. The absence of a corrective effect of IL-3 in these patients with DBA indicates that a deficiency of the cytokine is not central in the pathogenesis of the disorder.     

Diamond-Blackfan anemia: promotion of marrow erythropoiesis in vitro by recombinant interleukin-3

To clarify the defective erythropoiesis in eight patients with Diamond- Blackfan anemia, we studied their bone marrow response in vitro to recombinant human interleukin-3 (IL-3) and recombinant granulocyte- macrophage colony-stimulating factor (GM-CSF). In an erythropoietin- containing assay system, specimens from six of the eight patients yielded low numbers of erythroid colonies compared to control values, and in five of these no erythropoietin dose-response could be elicited. Addition of IL-3, GM-CSF or both to cultures from the six patients had no effect on CFU-E-derived colonies. In contrast, IL-3 but not GM-CSF induced a marked increase in the number (183%) and size of the BFU-E- derived colonies in five of the six cases and partially corrected the impaired dose-response to erythropoietin in four. Bone marrow from the other two patients yielded numbers of CFU-E and BFU-E colonies comparable to controls and manifested similar increments in colonies with increasing concentrations of erythropoietin. When IL-3 was added to these cultures, further increments were observed in the number and size of BFU-E colonies. We conclude that IL-3 enhanced the marrow erythropoiesis in most of the patients and exerted a corrective effect on the aberrant colony formation in the presence of erythropoietin. The data raise the possibility of IL-3 as a therapeutic agent in Diamond- Blackfan anemia.