Efficacy and toxicity of 9-beta-D-arabinofuranosylguanine (araG) as an agent to purge malignant T cells from murine bone marrow: application to an in vivo T-leukemia model

9-beta-D-Arabinofuranosylguanine (araG), an analog of deoxyguanosine which is not degraded by purine nucleoside phosphorylase, has been previously shown in in vitro studies by our laboratory to be effective in purging malignant T cells from human bone marrow (1). We now describe studies in a murine model of T-cell acute lymphoblastic leukemia (ALL) in which we tested whether bone marrow, contaminated with malignant T cells and purged ex vivo with araG, could reconstitute both the lymphoid and myeloerythroid lineages in the absence of leukemic relapse. The model utilized 6C3HED tumor cells, derived from a Thy 1.2+ malignant murine T-cell line, which were shown to cause lethal leukemia in C3H/HeN mice. Intravenous injection of 10(6) 6C3HED cells resulted in 100% mortality within 18 days, with autopsy revealing tumor infiltration of multiple organs. 100% of non-leukemia bearing lethally irradiated C3H/HeN mice transplanted with syngeneic bone marrow, treated ex vivo with 100 microM of araG for 18 hours, survived > 365 days post-transplant with full lymphohematopoietic reconstitution. Evidence of araG's ability to purge bone marrow of malignant tumor cells without causing significant toxicity to normal marrow derived hematopoietic progenitor cells was documented in experiments in which 75% of lethally irradiated mice transplanted with syngeneic bone marrow, contaminated with 6C3HED tumor cells and treated ex vivo with 100 microM araG for 18 hours, survived for > 250 to > 400 days. Death in 25% of mice was secondary to infection which developed before marrow reconstitution occurred. Reconstitution of the lymphoid, myeloid, and erythroid lineages with donor cells in surviving mice was documented. The data presented indicate that araG may effectively purge bone marrow of malignant T cells without irreversible toxicity to hematopoietic stem cells. This purging regimen is recommended for consideration for clinical trials in patients with T-cell malignancies undergoing autologous bone marrow transplantation and may also be a viable option for T-cell depletion as a strategy to prevent graft-versus-host disease.     

The potential roles of intraovarian peptides in normal and abnormal mechanisms of reproductive physiology

Normal and abnormal follicular growth and steroidogenesis depend on gonadotropins as well as intraovarian peptide and polypeptide growth factors, which may mediate or potentiate gonadotropin action. Epidermal growth factor is mitogenic to ovarian granulosa and is a potent inhibitor of granulosa aromatase. It may be involved in the apparent arrest of follicular development commonly seen in women with polycystic ovarian syndrome as well as in the blunted response to gonadotropins seen in this syndrome. Insulin-like growth factors are also mitogenic to ovarian granulosa, but in contrast to epidermal growth factor, insulin-like growth factor-I, both alone and in synergy with gonadotropins, is a potent stimulus of aromatase and granulosa estradiol production. Insulin-like growth factor binding proteins-2 and -4, known inhibitors of insulin-like growth factor action, are higher in follicular fluid from atretic and polycystic ovarian syndrome follicles compared with estrogenic follicles and may be inhibitors of gonadotropin action in follicle selection and in polycystic ovarian syndrome. Cytokines including interleukins, tumor necrosis factor-alpha and interferon-gamma also appear to play a role in modulating ovarian steroidogenesis. Activins, inhibins, and follistatin (activin-binding protein) also affect follicular development and steroidogenesis and may play a role in dominant follicle selection and follicular atresia.     

Effect of Altitude and Management System on the Iron Content of Beef

Iron content of bovine longissimus (LD) and semimembranosus muscle (SM) from steers raised at two altitudes was compared. Total iron content of fresh muscle was not different in concentrate-fed steers reared at 2145 m (HIFD) when compared to that of steers reared at 549 m (LOFD). Iron content of muscle from steers reared at 2145 m and pasture-grazed (HIGR) was lower than that from older HIFD and LOFD steers. Steaks from the HIFD and HIGR groups contained more heme iron (P<0.05) than those from LOFD steers. The nutritional significance of the differences in heme iron must be further investigated.     

Congenital muscular dystrophy: report of one case

A case is reported of a newborn who presented with generalized hypotonia shortly after delivery. Creatine kinase (CK) was highly elevated. Muscle biopsy of the rectus femoris muscle revealed varying sized muscle fibers, displacement by fat and connective tissues, necrosis and regeneration of the muscle fibers. Magnetic resonance imaging (M.R.I.) of the brain showed normal development, compatible with the patient's age. Congenital muscular dystrophy was diagnosed from clinical manifestations, laboratory findings, and the results of muscle biopsy.     

A single amino acid substitution in the capsid protein VP1 of coxsackievirus B3 (CVB3) alters plaque phenotype in Vero cells but not cardiovirulence in a mouse model

We previously described a large plaque attenuant (p14V-1) derived from a cardiovirulent Coxsackievirus B3 (CVB3) and showed that there were no major determinants of either attenuation or plaque phenotype in the 5' nontranslated region (5'NTR). Part of the region encoding the last 124 amino acids of VP3 and the first 106 amino acids of VP1 of the attenuant was then sequenced and compared to the wild-type. Three nucleotide changes were found in the VP1 coding region: a silent single base change at nucleotide position 2467 (C to U) and a double-base change at position 2690-1 (AA to GT), which leads to a change from lysine to serine at amino acid position 80. This mutation maps to the begining of B-C loop of the three-dimensional structure of VP1 of CVB3, where a distinct surface projection is formed. Two infectious chimeric cDNA clones were constructed, based on a cardiovirulent cDNA construct. In one construct, the 5'NTR and the VP3-VP1 region were from p14V-1 and in the other, only the VP3-VP1 region was from this attenuant. Both chimeric viruses produced large plaques on Vero cell monolayers, similar to p14V-1 but larger than the prototypic cardiovirulent virus. In vivo experiments showed that both chimeric viruses induced myocarditis in a murine model, similar to wild-type virus. We conclude that mutation serine-80 in capsid protein VP1 of p14V-1 is a determinant of the large plaque phenotype but is not responsible for attenuation.     

Effects of amino acids and gastric inhibitory polypeptide on insulin release in dogs

Insulin release following intravenous administration of an amino acid solution with and without a simultaneous infusion of varying amounts of porcine gastric inhibitory polypeptide (GIP) was studied in dogs. Group I received a 10-amino acid mixture (300 mosmol/kg iv) at 16.6 ml/min for 1 h; group II, amino acid mixture plus 0.5 micrograms.kg-1.h-1 porcine GIP; group III, amino acid mixture plus 1.0 micrograms.kg-1.h-1 of GIP; group IV (a and b) received either 0.5 or 1.0 micrograms.kg-1.h-1 of GIP alone. Compared to group I, groups II and III had a greater insulin response during the first 30 min of the infusion. Group] IV (a and b) showed no insulin release. Glucose concentrations showed no significant change in all groups. From these results, it is concluded that insulin release after intravenous infusion of an amino acid mixture plus GIP is greater than after amino acids or GIP alone. It appears that this effect is more pronounced in the early phase of insulin release.     

Relative value of three laboratory methods in the diagnosis of multiple sclerosis

We compared three methods of analysis for IgG in cerebrospinal fluid, using samples from 158 patients with clinically suspected multiple sclerosis and from 200 neurological controls. The tests were: search for oligoclonal bands, calculation of rate of synthesis of IgG in the cerebrospinal fluid, and determination of the IgG/albumin ratio. Paired cerebrospinal fluid and serum samples were collected and their IgG and albumin concentrations measured. Oligoclonal bands were detected by electrophoresis on agarose. Positive results were obtained in 94, 75, and 67% of patients with probable or definite multiple sclerosis by the three respective methods. In contrast, for patients for whom the clinical diagnosis of multiple sclerosis was considered possible, positive results were obtained in 10, 43, and 13%, respectively. Evidently, detection of oligoclonal bands remains the best single test for the presence of abnormal IgG in suspected multiple sclerosis patients. A combination of the first two tests is most sensitive for both probable and definite multiple sclerosis (97%) and possible multiple sclerosis (50%). Some infectious or immunologic disorders can also produce these IgG abnormalities, but they can usually be distinguished from multiple sclerosis by other clinical and laboratory data.     

Glutamate receptor antagonists inhibit calpain-mediated cytoskeletal proteolysis in focal cerebral ischemia

Excitatory amino acids may promote microtubular proteolysis observed in ischemic neuronal degeneration by calcium-mediated activation of calpain, a neutral protease. We tested this hypothesis in an animal model of focal cerebral ischemia without reperfusion. Spontaneously hypertensive rats were treated with 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)quinoxaline (NBQX), a competitive antagonist of the neuronal receptor for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or cis-4-[phosphono-methyl]-2-piperidine carboxylic acid (CGS 19755), a competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor. After treatment, all animals were subjected to permanent occlusion of the middle cerebral artery for 6 or 24 h. Infarct volumes measured in animals pretreated with CGS 19755 after 24 h of ischemia were significantly smaller than those quantified in ischemic controls. Rats pretreated with NBQX showed partial amelioration of cytoskeletal injury with preserved immunolabeling of microtubule-associated protein 2 (MAP 2) at 6 and 24 h and reduced accumulation of calpain-cleaved spectrin byproducts only at 6 h. Prevention of cytoskeletal damage was more effective after pretreatment with CGS 19755, as shown by retention of MAP 2 immunolabeling and significant restriction of calpain activity at both 6 and 24 h. Preserved immunolabeling of tau protein was observed at 6 and 24 h only in animals pretreated with CGS 19755. Western analysis performed on ischemic cortex taken from controls or rats pretreated with either NBQX or CGS 19755 suggested that loss of tau protein immunoreactivity was caused by dephosphorylation, rather than proteolysis. These results demonstrate a crucial link between excitotoxic neurotransmission, microtubular proteolysis, and neuronal degeneration in focal cerebral ischemia.