Synthesis and evaluation of 5-aminosalicyl-glycine as a potential colon-specific prodrug of 5-aminosalicylic acid

OBJECTIVE: To estimate more precisely the risk of fetal loss and congenital abnormalities after maternal parvovirus B19 infection, and to assess the long term outcome for surviving infants. DESIGN: Prospective cohort study of pregnant women with confirmed B19 infection with follow up of the surviving infants. The rate of fetal loss in the study cohort was compared with that in pregnant women with varicella. SETTING: Cases reported by laboratories in England and Wales between 1985-1988 and 1992-1995. SAMPLE: Four hundred and twenty-seven pregnant women with B19 infection and 367 surviving infants of whom 129 were followed up at 7-10 years of age. METHODS: Questionnaires to obstetricians and general practitioners on outcome of pregnancy and health of surviving infants. Maternal infection confirmed by B19-specific IgM assay and/or IgG seroconversion. RESULTS: The excess rate of fetal loss in women with B19 infection was confined to the first 20 weeks of gestation and averaged 9%. Seven cases of fetal hydrops followed maternal infections between 9 and 20 weeks of gestation (observed risk 2.9%, 95% CI 1.2-5.9). No abnormalities attributable to B19 infection were found at birth in surviving infants (observed risk 0%, upper 95% CI 0.86%). No late effects were found at 7-10 years. CONCLUSIONS: Around 1 in 10 women infected before 20 weeks of gestation will suffer a fetal loss due to B19. The risk of an adverse outcome of pregnancy after this stage is remote. Infected women can be reassured that the maximum possible risk of a congenital abnormality due to B19 is under 1% and that long term development will be normal.     

Increased MPTP neurotoxicity in vesicular monoamine transporter 2 heterozygote knockout mice

The neurotoxic action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been proposed to be attenuated by sequestration into intracellular vesicles by the vesicular monoamine transporter (VMAT2). The purpose of this study was to determine if mice with genetically reduced levels of VMAT2 (heterozygote knockout; VMAT2 +/-) were more vulnerable to MPTP. Striatal dopamine (DA) content, the levels of DA transporter (DAT) protein, and the expression of glial fibrillary acidic protein (GFAP) mRNA, a marker of gliosis, were assessed as markers of MPTP neurotoxicity. In all parameters measured VMAT2 +/- mice were more sensitive than their wild-type littermates (VMAT2 +/+). Administration of MPTP (7.5, 15, or 30 mg/kg, b.i.d.) resulted in dose-dependent reductions in striatal DA levels in both VMAT2 +/- and VMAT2 +/+ animals, but the neurotoxic potency of MPTP was approximately doubled in the VMAT2 +/- mice: 59 versus 23% DA loss 7 days after 7.5 mg/kg dose for VMAT2 +/- and VMAT2 +/+ mice, respectively. Dopaminergic nerve terminal integrity, as assessed by DAT protein expression, also revealed more drastic reductions in the VMAT2 +/- mice: 59 versus 35% loss at 7.5 mg/kg and 95 versus 58% loss at 15 mg/kg for VMAT2 +/- and VMAT2 +/+ mice, respectively. Expression of GFAP mRNA 2 days after MPTP was higher in the VMAT2 +/- mice than in the wild-type: 15.8- versus 7.8-fold increase at 7.5 mg/kg and 20.1- versus 9.6-fold at 15 mg/kg for VMAT2 +/- and VMAT2 +/+ mice, respectively. These observations clearly demonstrate that VMAT2 +/- mice are more susceptible to the neurotoxic effects of MPTP, suggesting that VMAT2-mediated sequestration of the neurotoxin into vesicles may play an important role in attenuating MPTP toxicity in vivo.     

NMDA receptors mediate peripheral chemoreceptor afferent input in the conscious rat

N-methyl-D-aspartate (NMDA) glutamate receptors mediate critical components of cardiorespiratory control in anesthetized animals. The role of NMDA receptors in the ventilatory responses to peripheral and central chemoreceptor stimulation was investigated in conscious, freely behaving rats. Minute ventilation (VE) responses to 10% O2, 5% CO2, and increasing intravenous doses of sodium cyanide were measured in intact rats before and after intravenous administration of the NMDA receptor antagonist MK-801 (3 mg/kg). After MK-801, eupcapnic tidal volume (VT) decreased while frequency increased, resulting in a modest reduction in VE. Inspiratory time (TI) decreased, whereas expiratory time remained unchanged. The VE responses to hypercapnia were qualitatively similar in control and MK-801 conditions, with slight reductions in respiratory drive (VT/TI) after MK-801. In contrast, responses to hypoxia were markedly attenuated after MK-801 and were primarily due to reduced frequency changes, whereas VT was unaffected. Sodium cyanide doses associated with significant VE increases were 5 and 50 microg/kg before and after MK-801, respectively. Thus 1-log shift to the right of individual dose-response curves occurred with MK-801. Selective carotid body denervation reduced VE during hypoxia by 70%, and residual hypoxic ventilatory responses were abolished after MK-801. These findings suggest that, in conscious rats, carotid and other peripheral chemoreceptor-mediated hypoxic ventilatory responses are critically dependent on NMDA receptor activation and that NMDA receptor mechanisms are only modestly involved during hypercapnia.     

A humanized form of a CD4-specific monoclonal antibody exhibits decreased antigenicity and prolonged plasma half-life in rhesus monkeys while retaining its unique biological and antiviral properties

Certain monoclonal antibodies (MAbs) directed against CD4 can efficiently block HIV-1 replication in vitro. To explore CD4-directed passive immunotherapy for prevention or treatment of AIDS virus infection, we previously examined the biological activity of a nondepleting CD4-specific murine MAb, mu5A8. This MAb, specific for domain 2 of CD4, blocks HIV-1 replication at a post-gp120-CD4 binding step. When administered to normal rhesus monkeys, all CD4+ target cells were coated with antibody, yet no cell clearance or measurable immunosuppression occurred. However, strong anti-mouse Ig responses rapidly developed in all monkeys. In the present study, we report a successfully humanized form of mu5A8 (hu5A8) that retains binding to both human and monkey CD4 and anti-AIDS virus activity. When administered intravenously to normal rhesus monkeys, hu5A8 bound to all target CD4+ cells without depletion and showed a significantly longer plasma half-life than mu5A8. Nevertheless, an anti-hu5A8 response directed predominantly against V region determinants did eventually appear within 2 to 4 weeks in most animals. However, when hu5A8 was administered to rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques, anti-hu5A8 antibodies were not detected. Repeated administration of hu5A8 in these animals resulted in sustained plasma levels and CD4+ cell coating with humanized antibody for 6 weeks. These studies demonstrate the feasibility of chronic administration of CD4-specific MAb as a potential means of treating or preventing HIV-1 infection.     

Tyrosine-dependent and -independent mechanisms of STAT3 activation by the human granulocyte colony-stimulating factor (G-CSF) receptor are differentially utilized depending on G-CSF concentration

The granulocyte colony-stimulating factor receptor (G-CSF-R) activates multiple STAT proteins. Although the membrane-proximal cytoplasmic region of the G-CSF-R is necessary and sufficient for activation of STAT1 and STAT5, activation of STAT3 requires the membrane distal region that contains four tyrosines. Although one of these (Y704) has previously been shown to be involved in STAT3 activation from a truncated G-CSF-R derived from a patient with severe chronic neutropenia (SCN), this tyrosine is not required for STAT3 activation by the full-length G-CSF-R. To investigate possible alternative mechanisms of STAT3 activation, we generated a series of Ba/F3 cell transfectants expressing the wild-type G-CSF-R or mutant receptors that either completely lack tyrosines or retain just one of the four cytoplasmic tyrosines of the G-CSF-R. We show that, at saturating G-CSF concentrations, STAT3 activation from the full-length G-CSF-R is efficiently mediated by the C-terminal domain in a manner independent of receptor tyrosines. In contrast, at low G-CSF concentrations, Y704 and Y744 of the G-CSF-R play a major role in STAT3 activation. Both tyrosine-dependent and -independent mechanisms of STAT3 activation are sensitive to the Jak2 inhibitor AG-490, follow similar kinetics, and lead to transactivation of a STAT3 reporter construct, indicating functional equivalence. STAT3 activation is also impaired, particularly at nonsaturating G-CSF concentrations, in bone marrow cells from mice expressing a truncated G-CSF-R (gcsfr-triangle up715). These findings suggest that G-CSF-induced STAT3 activation during basal granulopoiesis (low G-CSF) and "emergency" granulopoiesis (high G-CSF) are differentially controlled. In addition, the data establish the importance of the G-CSF-R C-terminus in STAT3 activation in primary cells, which has implications for understanding why truncated G-CSF-R derived from SCN patients are defective in maturation signaling.     

An immune complex selective affinity column utilizing site-specific attachment of bovine conglutinin

Established leukemic cell lines have been useful models for studying the biology of leukemia. Analysis of the actions of differentiating agents on leukemic cell lines in vivo has been limited by an inability to unambiguously distinguish host hematopoietic elements from differentiated leukemic cells. In order to identify and quantify leukemic cells during in vivo studies, a derivative of the murine myelomonocytic leukemia cell line WEHI-3B D+, which stably expresses beta-galactosidase, was constructed utilizing retroviral vector gene transfer. This cell line, termed WEHI-3B D+/lacZ 2.8, demonstrated in vitro growth and differentiation properties similar to the parental cell line. WEHI-3B D+/lacZ 2.8 expressed high levels of beta-galactosidase following prolonged in vitro growth and following differentiation in suspension cultures and clonogenic assays. In vivo, WEHI-3B D+/lacZ 2.8 was leukemogenic and high level expression of beta-galactosidase was maintained. Quantification of tissue involvement with WEHI-3B D+/lacZ 2.8 leukemia was performed utilizing staining with the fluorogenic beta-galactosidase substrate fluorescein di-beta-galactoside and fluorescence-activated cell sorting analysis. In vivo differentiation efficiency following granulocyte colony-stimulating factor (G-CSF) administration was determined using a simultaneous nuclear and cytoplasmic staining procedure. Results indicate that treatment of mice inoculated with WEHI-3B D+/lacZ 2.8 cells with G-CSF administration causes detectable but limited differentiation.     

Isolation and characterization of pectin methylesterase from papaya

To estimate energy expenditure (EE) in elderly subjects, more age-specific data are required on energy costs of standardized activities. EE was assessed by using indirect calorimetry in 28 women aged 72 +/- 4 y (mean +/- SD) and in 29 middle-aged women (42 +/- 1 y) at rest (resting metabolic rate; RMR) and during sitting, sitting with standardized arm activity, and walking on a treadmill at 3 km/h. RMR and EE during sitting, and sitting with standardized arm activity did not differ significantly between the groups, although EE expressed as a ratio of arm activity to RMR (physical activity ratio, PAR) tended to be higher in the elderly subjects. Walking EE was significantly higher in the elderly women (16.4 +/- 4.0 kJ/min) than in the middle-aged women (12.7 +/- 2.3 kJ/min), also when expressed as PAR. It is suggested that elderly women walk less efficiently. Because PARs are frequently used to estimate daily EE, it is important to note that additional age-specific data might be required.     

VH gene family utilization of anti-acetylcholine receptor antibodies in experimental autoimmune myasthenia gravis

The immunoglobulin heavy chain (VH) gene family usage in experimental autoimmune myasthenia gravis (EAMG) model was investigated by RNA slot blot hybridization using VH gene family specific probes. Anti-acetylcholine receptor (AChR) monoclonal antibodies (mAbs) isolated from susceptible C57BL/6 and resistant BALB/c mice were found to be encoded by VH genes from at least six different families. The Vgam3.8 family was overrepresented in alpha-bungarotoxin blocking mAbs. Expression of cross-reactive idiotypes by anti-AChR mAbs was irrespective of the VH gene family usage. Strain dependent differences in susceptibility for EAMG were not reflected in an aberrant VH gene family usage of anti-AChR mAbs.