Development and characterization of three recombinant single chain antibody fragments (scFvs) directed against the CD19 antigen

Antibodies that recognize antigens restricted to leukemia, lymphoma, and normal hematopoietic cells represent a unique opportunity to develop therapeutics, because they have the potential for relatively selective treatment of these diseases. Antibodies that recognize the CD19 antigen found on normal and malignant B cells, but not on stem cells, have been used to develop immunoconjugates. However, these conjugates are large and might be suboptimal in tumor penetration when compared to molecules using smaller single chain Fv (scFv) antibody fragments. scFv has the advantage of being a molecularly engineered homogeneous molecule. In this report, we demonstrate the cloning, expression, and binding of three anti-CD19 antibodies as scFvs. All three scFvs were successfully cloned and expressed. FVS191, derived from cell line B43, and FVS192, derived from SJ25C1, were properly refolded and bound CD19 antigen in FACS competition assays. These anti-CD19 scFv should be useful in the further development of diagnostic and therapeutic molecules.     

Oxidative damage and metabolic dysfunction in Huntington's disease: selective vulnerability of the basal ganglia

The etiology of the selective neuronal death that occurs in Huntington's disease (HD) is unknown. Several lines of evidence implicate the involvement of energetic defects and oxidative damage in the disease process, including a recent study that demonstrated an interaction between huntingtin protein and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using spectrophotometric assays in postmortem brain tissue, we found evidence of impaired oxidative phosphorylation enzyme activities restricted to the basal ganglia in HD brain, while enzyme activities were unaltered in three regions relatively spared by HD pathology (frontal cortex, parietal cortex, and cerebellum). Citrate synthase-corrected complex II-III activity was markedly reduced in both HD caudate (-29%) and putamen (-67%), and complex IV activity was reduced in HD putamen (-62%). Complex I and GAPDH activities were unaltered in all regions examined. We also measured levels of the oxidative damage product 8-hydroxydeoxyguanosine (OH8dG) in nuclear DNA, and superoxide dismutase (SOD) activity. OH8dG levels were significantly increased in HD caudate. Cytosolic SOD activity was slightly reduced in HD parietal cortex and cerebellum, whereas particulate SOD activity was unaltered in these regions. These results further support a role for metabolic dysfunction and oxidative damage in the pathogenesis of HD.     

Comparison of red-green, blue-yellow and achromatic losses in glaucoma

Achromatic losses in glaucoma would be expected to be greater than, or equal to, red-green chromatic losses if the following assumptions are made: (1) the function of the remaining axons is either unchanged or non-selectively reduced; (2) red-green chromatic information is signaled by the midget ganglion cell system; and (3) the function of the magnocellular system is reduced at least as much as that of the midget ganglion cells. This prediction was tested by measuring red-green (along with blue-yellow) mixture thresholds for 1 deg, 0.2 sec test spots presented on a color monitor on a white background of 50 cd/m2. Ellipses were fitted to plots of green contrast as a function of red contrast (or yellow as a function of blue), and major and minor axes of these ellipses were taken as measures of chromatic and achromatic thresholds, respectively. The study population consisted of 29 eyes in 29 patients with early glaucoma; control data were derived from a data bank of 83 normal eyes. Red-green losses were significantly (P < 0.05) greater than achromatic losses in 6 out of the 11 eyes which showed significant losses of either chromatic or achromatic sensitivity (or both). It is concluded that, for these eyes, at least one of the above three assumptions is incorrect.     

In vitro release of acyclovir from semisolid dosage forms: effect of cyclodextrin and polyethylene glycol

Presynaptic depolarization of trigemino-thalamic (TGT) terminals may contribute to modulation of ascending oro-facial somatosensory information during active (or rapid eye movement) sleep. The relative excitability of TGT terminals was inferred from changes in the current required to maintain an antidromic firing probability of 50% (EC50) during quiet wakefulness as compared to active sleep. Depolarization or hyperpolarization of TGT terminals was defined as a decrease or increase, respectively, in the EC50. Overall, the EC50 of 8 TGT terminals was reduced by a mean 8.8+/-3.6 microA during active sleep relative to quiet wakefulness. This result suggests that depolarization of TGT terminals, which may act to suppress the transfer of sensory information from the trigeminal nucleus to the thalamus, occurs during active sleep.     

Increase in plasma leptin and Lep mRNA concentrations by food intake is dependent on insulin

The difference in pregnancy rates following intrauterine insemination (IUI) for 1 vs. 2 days in the periovulatory period has been reported as either inconsequential or favoring the use of two consecutive inseminations, 24 hours apart. Our study compared the monthly fecundity and cumulative probability of pregnancy in a large group of women (n = 123) undergoing controlled ovarian hyperstimulation and 1- or 2-day inseminations with donor sperm prepared from frozen-thawed samples. All patients underwent controlled ovarian hyperstimulation employing either clomiphene citrate in 217 cycles or human menopausal gonadotropin in 185 cycles. The choice of single or double insemination was decided by the day of the week each patient received human chorionic gonadotropin for ovulation induction. Approximately 80% of all the patients underwent both single and double insemination treatments during the 2.5-year study period. Ninety-three patients received single inseminations in 180 cycles, whereas 103 patients received double inseminations in 222 cycles. Nine clinical pregnancies were achieved in the 1-day group (5% per cycle, 9.7% per patient), while 39 pregnancies occurred in the 2-day group (17.9% per cycle, 37.9% per patient). Two and five spontaneous abortions occurred in the 1- and 2-day groups, yielding take-home baby rates of 3.9% per cycle (7.5% per patient) and 15.3% per cycle (33.0% per patient), respectively. The cumulative probability of conception over 15 cycles of treatment was consistently twice as high or higher for the 2-day group. The results of this study support the use of 2-day IUI treatment cycles when using frozen-thawed donor sperm.     

A proton magnetic resonance spectroscopic study in ALS: correlation with clinical findings

OBJECTIVE: To evaluate neuronal dysfunction in the motor region subcortical white matter in ALS using volumetric localized proton magnetic resonance spectroscopy (1H-MRS). METHODS: Sixteen patients with E1 Escorial definite, probable, or possible ALS and eight healthy age-matched control subjects were studied. The ALS patients were divided into those with limb onset (n = 8) and those with bulbar onset (n = 8). Measurements of the metabolic ratios N-acetylaspartate (NAA)/creatine and phosphocreatine (Cr+PCr), NAA/choline (Cho), and Cho/(Cr+PCr) were correlated with clinical assessments. RESULTS: We found no differences in the metabolic peak area ratios in the motor region when comparing the total ALS group and the control subjects. However, correlations were found between the NAA/(Cr+PCr) ratio and the E1 Escorial category (p = 0.03), the ALS severity scale (p = 0.01), and the Medical Research Council score (p = 0.06). No correlations were found between the NAA/(Cr+PCr) ratio and the Ashworth Spasticity Scale, reflex score, or disease duration (p > 0.16). Bulbar-onset patients had a lower NAA/(Cr+PCr) ratio in the motor region compared with limb-onset patients (p = 0.03). CONCLUSION: In vivo 1H-MRS of the subcortical white matter in the motor region is unlikely to be sensitive enough to detect early disease changes in ALS because there is considerable overlap between the metabolic peak area ratios from patients with ALS and normal control subjects. However, changes in the NAA/(Cr+PCr) metabolic peak area ratios correlate with clinical measures of disease severity, and this measure may be useful in monitoring disease progression.     

Osteoporosis and coronary atherosclerosis in asymptomatic postmenopausal women

The single-dose disposition kinetics of danofloxacin were determined in clinically normal lactating cows after intravenous (i.v.) and intramuscular (i.m.) administration of the drug at 1.25 mg/kg. The drug concentrations in blood serum and milk were determined by microbiological assay methods and the data were subjected to kinetic analysis. The mean i.v. and i.m. elimination half-lives (t1/2el) in serum were 54.9 and 135.7 min, respectively. The steady-state volume of distribution (Vss) was 2.04 L/kg. The drug was quickly absorbed after i.m. injection but a 'flip flop' effect was clearly evident and bioavailability was > 100%. Penetration of danofloxacin from blood into milk was rapid and extensive with drug concentrations in milk exceeding those in serum beginning 90-120 min after i.v. and i.m. administration and onwards. Milk danofloxacin concentrations equal to or higher than the minimal inhibitory concentrations (MIC) for pathogenic Gram-negative bacteria and Mycoplasma species were maintained over approximately 24 h. Concentrations greater than the MIC for Staphylococcus aureus were maintained in the milk for 12 h.     

Metabolism and metabolic actions of 6-methylpurine and 2-fluoroadenine in human cells

Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer. Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently. The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively. F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells. Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses. CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively. The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr. Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells. These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E. coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E. coli cytosine deaminase. These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment.