Kenneth A. Schwartz

Androgenic-Anabolic Steroid Abuse and Platelet Aggregation: A Pilot Study in Weight Lifters

The abuse of anabolic-androgenic steroids by athletes has recently been associated with the development of myocardial infarction and stroke. Because platelets play a pathogenic role in these disorders, the authors hypothesized that androgenic steroid abuse among weight lifters was associated with increased platelet aggregation as measured in vitro. Twenty-eight study participants were recruited. Twelve denied current androgen use. However, 8 of these 12 tested positive for urinary androgens. Nonsignificant trends toward increased platelet counts and increased platelet aggregation to adenosine diphosphate were noted when androgen users were compared to nonusers. However, when stratified by age, older (greater than 22 years) androgen users required lower concentrations of collagen to produce 50% aggregation of test platelets than did younger (less than or equal to 22 years) androgen users (1.47 versus 3.35 micrograms/ml; p = .01). Further subgroup analysis revealed nonsignificant trends toward increased adenosine diphosphate-induced aggregability and nonsignificant trends in the platelet count in older weight lifters. Subsequent studies using collagen threshold aggregometry revealed no age-dependent effect in 17 other men (aged 18 to 46 years) not specifically selected for activity (r = .17). This study suggests an association between androgen use, age, and increased platelet sensitivity to collagen in weight lifters and may be helpful in explaining recent thrombotic disease in androgen users. It additionally calls into question the validity of subjective reporting when assessing androgen use among weight lifters.

Immune-mediated platelet destruction and thrombocytopenia in patients with solid tumours

Autoimmune mediated platelet destruction with severe thrombocytopenia was documented in eight patients with solid tumours. The patients had reduced platelet lifespan, positive platelet antibody tests, increased numbers of megakaryocytes, and a rise in platelet count following treatment with steroids, splenectomy or immunosuppressive therapy. Intravascular coagulation was excluded as the predominant cause of thrombocytopenia by near normal 125I‐fibrinogen survival; thrombocytopenia secondary to marrow suppression was ruled out by increased platelet turnover. Thus, like patients with lymphoproliferative disorders, patients with solid tumours may be thrombocytopenic because of immune mediated platelet destruction.

Aluminum-Induced Anemia

Although many questions still remain unanswered, it is clear that aluminum causes a microcytic hypoproliferative anemia and is one factor responsible for worsening anemia in patients with end-stage renal disease. Time course studies in a rat model have shown that the anemia is preceded by microcytosis; this has not yet been examined in man. The exact mechanism of aluminum-induced anemia is unknown, however it appears to involve inhibition of heme synthesis, either by inhibition of enzyme activity or interference with iron incorporation or utilization. The interrelationship between aluminum and iron, zinc, lead, or other metals in this anemia is also unknown, as are the effects of aluminum on erythroid colony forming units. The role of parathyroid hormone on aluminum-induced anemia has not been examined. Presently treatment of aluminum-induced anemia involves removal of the source of the aluminum, although recent studies with desferrioxamine show promise. It is unclear, however, exactly how desferrioxamine improves this anemia. It is clear, however, that aluminum in the dialysate can cause clinical problems including anemia, and that these problems can be substantially reduced if not eliminated by water treatment.

Non-compliance is the predominant cause of aspirin resistance in chronic coronary arterial disease patients

BackgroundOur previous publication showed that 9% of patients with a history of myocardial infarction MI. could be labeled as aspirin resistant; all of these patients were aspirin resistant because of non-compliance. This report compares the relative frequency of aspirin resistance between known compliant and non-compliance subjects to demonstrate that non-compliance is the predominant cause of aspirin resistance.MethodsThe difference in the slopes of the platelet prostaglandin agonist (PPA) light aggregation curves off aspirin and 2 hours after observed aspirin ingestion was defined as net aspirin inhibition.ResultsAfter supposedly refraining from aspirin for 7 days, 46 subjects were judged non-compliant with the protocol. Of the remaining 184 compliant subjects 39 were normals and 145 had a past history of MI. In known compliant subjects there was no difference in net aspirin inhibition between normal and MI subjects. Net aspirin inhibition in known compliant patients was statistically normally distributed. Only 3% of compliant subjects (2 normals and 5 MI) had a net aspirin inhibitory response of less than one standard deviation which could qualify as a conservative designation of aspirin resistance. A maximum of 35% of the 191 post MI subjects could be classified as aspirin resistant and/or non-compliant: 9% aspirin resistant because of non-compliance, 23% non-compliant with the protocol and possibly 3% because of a decreased net aspirin inhibitory response in known compliant patients.ConclusionOur data supports the thesis that the predominant cause of aspirin resistance is noncompliance.

Treatment of glioma patients with ketogenic diets: report of two cases treated with an IRB-approved energy-restricted ketogenic diet protocol and review of the literature.

BackgroundBased on the hypothesis that cancer cells may not be able to metabolize ketones as efficiently as normal brain cells, the ketogenic diet (KD) has been proposed as a complementary or alternative therapy for treatment of malignant gliomas.Case presentationWe report here our experience in treating two glioma patients with an IRB-approved energy-restricted ketogenic diet (ERKD) protocol as monotherapy and review the literature on KD therapy for human glioma patients. An ERKD protocol was used in this pilot clinical study. In addition to the two patients who enrolled in this study, we also reviewed findings from 30 other patients, including 5 patients from case reports, 19 patients from a clinical trial reported by Rieger and 6 patients described by Champ. A total of 32 glioma patients have been treated using several different KD protocols as adjunctive/complementary therapy. The two patients who enrolled in our ERKD pilot study were monitored with twice daily measurements of blood glucose and ketones and daily weights. However, both patients showed tumor progression while on the ERKD therapy. Immunohistochemistry reactions showed that their tumors had tissue expression of at least one of the two critical mitochondrial ketolytic enzymes (succinyl CoA: 3-oxoacid CoA transferase, beta-3-hydroxybutyrate dehydrogenase 1). The other 30 glioma patients in the literature were treated with several different KD protocols with varying responses. Prolonged remissions ranging from more than 5 years to 4 months were reported in the case reports. Only one of these patients was treated using KD as monotherapy. The best responses reported in the more recent patient series were stable disease for approximately 6 weeks. No major side effects due to KD have been reported in any of these patients.ConclusionsWe conclude that 1. KD is safe and without major side effects; 2. ketosis can be induced using customary foods; 3. treatment with KD may be effective in controlling the progression of some gliomas; and 4. further studies are needed to determine factors that influence the effectiveness of KD, whether as a monotherapy, or as adjunctive or supplemental therapy in treating glioma patients.Trial registrationClinicalTrials.gov# NCT01535911

Glucocorticoid-induced apoptosis in early B cells from human bone marrow.

The sensitivity of normal human lymphoid precursor cells to glucocorticoid-induced apoptosis is a subject of controversy. The in vitro response of cells of the B lineage (CD19+) from the marrow of 22 adult subjects to glucocorticoids was evaluated herein using both natural steroids and dexamethasone (Dex). When exposed to 1 μM Dex, 32% of the subjects exhibited high losses of CD19+ B cells in the range of 45%. The remaining subjects exhibited more modest losses in CD19+ cells of 26%–40%. Surprisingly, cortisol, a naturally produced glucocorticoid, produced B lineage losses nearly equivalent to Dex, which reached maximum by 12 hr. It was subsequently noted that the variances in losses of CD19+ cells among the subjects correlated closely with the proportion of early CD10+ CD19+ B cells present in the initial population. The latter cells exhibited a high degree of sensitivity to glucocorticoids, with losses of 60%–80% noted. Mature B cells bearing IgD, on the other hand, were fairly resistant to glucocorticoids. Merocyanine 540, a membrane dye that fluoresces in the disordered membrane of apoptotic cells, confirmed that early or progenitor B cells in human bone marrow were indeed undergoing glucocorticoid-induced apoptosis, which could be blocked by the glucocorticoid antagonist RU38486. These data provide evidence that human marrow B cells, especially early B-cell progenitors, are quite sensitive to glucocorticoids and readily undergo apoptosis within a few hours of exposure to the steroids.

Ketolytic and glycolytic enzymatic expression profiles in malignant gliomas: implication for ketogenic diet therapy

BackgroundRecent studies in animal models, based on the hypothesis that malignant glioma cells are more dependent on glycolysis for energy generation, have shown promising results using ketogenic diet (KD) therapy as an alternative treatment strategy for malignant glioma, effectively starving glioma cells while providing ketone bodies as an energy source for normal neurons and glial cells. In order to test this treatment strategy in humans, we investigated the relative expression of several key enzymes involved in ketolytic and glycolytic metabolism in human anaplastic glioma (WHO grade III) and glioblastoma (GBM, WHO grade IV).MethodsImmunohistochemistry was performed on formalin fixed paraffin embedded sections from 22 brain biopsies (17 GBM, 3 anaplastic astrocytoma and 2 anaplastic oligoastrocytoma) using antibodies raised against glycolytic and ketolytic enzymes. The glycolytic enzymes included hexokinase-II (HK2) and pyruvate kinase M2 isoform (PKM2). The ketone body metabolic enzymes included: succinyl CoA: 3-oxoacid CoA transferase (OXCT1), 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and BDH2), and acetyl-CoA acetyltransferase 1 (ACAT1). The immunoreactivities were graded using a semi-quantitative scale based on the percentage of positive cells: POS (>20%), LOW (5-20%), and very low (VLOW) (<5%). Focal non-neoplastic “normal” brain tissue within the biopsy specimens served as internal controls.ResultsThe rate limiting mitochondrial ketolytic enzymes (OXCT1 and BDH1) were either LOW or VLOW, concordantly in 14 of the 17 GBMs and in 1 of 5 anaplastic gliomas, whereas at least one of the glycolytic enzymes was POS in 13 of these 17 GBMs and all 5 anaplastic gliomas. Cytosolic BDH2 and mitochondrial ACTAT1 were, surprisingly, POS in most of these tumors.ConclusionOur results showing that malignant gliomas have differential expression of ketolytic and glycolytic enzymes are consistent with previous studies that have shown that these are genetically heterogeneous tumors. It seems reasonable to hypothesize that patients with low or very low expression of key ketolytic enzymes in their malignant gliomas may respond better to the KD therapy than those patients with positive expression of these enzymes. Further studies in animal models and/or a large-scale clinical trial would be needed to test this hypothesis.

Type I Glanzmann's thrombasthenia in a Great Pyrenees dog.

An 8-month-old female Great Pyrenees dog with chronic epistaxis and a history of gingival bleeding during shedding of deciduous teeth was evaluated for platelet function. Platelet morphology was normal at both the light and electron microscopic level. Platelet number and mean platelet volume were also normal. Platelet aggregation responses to adenosine diphosphate, collagen, platelet activating factor, and thrombin were markedly reduced, although shape change responses were normal. Clot retraction was markedly impaired. Monoclonal antibody (MoAb) Y2/51, a murine anti-human platelet β3 antibody that cross-reacts with canine platelet β3 , and MoAb 5G11, a murine anti-dog platelet αIIbβ3 antibody, bound minimally to affected dog platelets, as demonstrated by flow cytometry. Binding of MoAb Y2/51 was not detectable by immunoblot. MoAb CAP1, a murine anti-dog fibrinogen receptor-induced binding site antibody, failed to bind to affected dog platelets, as demonstrated by flow cytometry. A reduction in glycoproteins αIIb and β3 was demonstrated by two-dimensional protein electrophoresis. This is the first reported case of type I Glanzmanns thrombasthenia in the dog that closely resembles the clinical syndrome and the platelet morphology described in type I Glanzmanns thrombasthenia of human beings.

Morphologic Investigations of the Guinea Pig Model of Iron Overload

We have developed a guinea pig model of iron overload toxicity. Animals were administered intraperitoneal iron dextran 3 times a week to achieve total body iron load of 0.25,0.5 1.0,1.5, and 2.0 g Fe/kg body weight in less than 30 days. Quantitation of tissue iron levels with atomic absorption indicated increased iron deposition in liver and heart of the iron-loaded guinea pigs (p < 0.001). Additionally, the iron-loaded pigs demonstrated decreased nuclear magnetic resonance spectropscopy T1 relaxation times in both liver and heart (p < 0.001). Serum iron, total body iron capacity, and transferrin saturation values were also determined in guinea pigs treated with 0.25,0.5, and 1.0 g Fe/kg body weight. Serum iron and total iron-binding capacity were significantly increased at 0.5 and 1.0 g Fe/kg; transferrin saturation was elevated at 0.25 and 1.0 g Fe/kg. Histologic examination of liver, heart, and bone marrow as well as ultrastructural studies on liver and heart confirmed increased iron deposition in treated animals. At the low iron dose level of 0.5 g Fe/kg, liver iron particles were primarily confined to Kupffer cells with minimal hepatocellular localization. Increased hepatocellular iron deposition was observed with larger doses of loaded iron. Myocardial iron was most prominent in interstitial cells of the epicardium, endocardium, myocardium, and coronary adipose tissue. Ultrastructurally, the presence of iron particles in perinuclear, membrane-bound structures (consistent with lysosomes) was confirmed using x-ray microanalysis. These morphological studies suggest that in this animal model siderosis of hepatic mononuclear phagocyte and myocardial interstitial cells may be the initial lesions leading to further biochemical and functional abnormalities. Correlation between tissue iron measurements and both light and electron microscopic changes, presented in this report, serve to introduce the iron-loaded guinea pig as a model for the study of iron-induced tissue damage.

Development of a sensitive immunoradiometric assay for detection of platelet surface-associated immunoglobulins in thrombocytopenic dogs

OBJECTIVE To develop a direct assay to measure platelet surface-associated immunoglobulins (PSAIg) in dogs and to determine whether the assay is useful in the diagnosis of immune-mediated thrombocytopenia (IMT). ANIMALS 20 healthy dogs were used to develop reference intervals, and 23 dogs with IMT and 17 with non-IMT were used to evaluate the clinical use of this assay. PROCEDURE After optimization of platelet collection and assay conditions, concentrations of PSAIg were measured, using radiolabeled staphylococcal protein A (SpA) and polyclonal antibodies against canine IgG (anti-gamma) and IgM (anti-micro). Concentrations of PSAIg were expressed as the percentage of radiolabeled immunoglobulin detector bound. RESULTS Cut-off values (mean + 3 SD) were as follows: SpA, 1.1%; anti-gamma, 1.3%; and anti-micro, 3.5%. Values greater than these cut-off values were considered positive. Values determined by use of radiolabeled SpA for all dogs with IMT were greater than the cut-off value; values were considered high positives (> 5 times cut-off value) for 22 of these 23 dogs. Although 9 of 17 dogs with non-IMT also had PSAIg concentrations greater than the cut-off value, values were considered high positives for only 3 of these 9 dogs. CONCLUSIONS AND CLINICAL RELEVANCE The immunoradiometric assay developed is a reliable and sensitive method to detect PSAIg in dogs. However, to obtain accurate results, optimum temperature, time, and storage conditions must be used. Detection of increased concentrations of PSAIg in dogs presumed to have non-IMT should alert clinicians to reconsider an immune-mediated basis for the thrombocytopenia.