Protein-based vaccine candidate MecVax broadly protects against enterotoxigenic Escherichia coli intestinal colonization in a rabbit model.

There are no vaccines licensed against enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea and the most common cause of travelers' diarrhea. Multivalent vaccine candidate MecVax unprecedentedly targets two ETEC enterotoxins (heat-stable toxin, STa; heat-labile toxin, LT) and the seven most prevalent ETEC adhesins (colonization factor antigen, CFA/I, coli surface antigens, CS1-CS6) and has been demonstrated preclinically to protect against STa- and LT-mediated ETEC clinical diarrhea and prevent intestinal colonization from ETEC strain H10407 (CFA/I, STa, LT). However, it is unattested whether MecVax broadly protects against intestinal colonization from ETEC strains producing the other six adhesins (CS1-CS6) also targeted by this product. In this study, we immunized rabbits with MecVax and challenged them with heterogeneous ETEC strains that express CS1-CS6 adhesins to evaluate MecVax's efficacy against bacterial intestinal colonization, thus providing broad vaccine protection against ETEC infection. Data revealed that rabbits intramuscularly immunized with MecVax developed robust responses to both ETEC enterotoxins (STa, LT) and seven adhesins (CFA/I, CS1-CS6), and when challenged with ETEC isolates expressing CS1/CS3, CS2/CS3, CS4/CS6, CS5/CS6, or CS6 adhesin, the immunized rabbits prevented over two logs (>99%) of bacteria from colonization in small intestines. Additionally, compared to a CFA-toxoid fusion protein, which is another potential ETEC vaccine antigen to target two ETEC enterotoxins and the seven adhesins, MecVax exhibited better protection against ETEC intestinal colonization. These results, in conjunction with the protection data from early studies, evidenced that MecVax is broadly protective, validating MecVax's candidacy as an effective vaccine against ETEC-associated diarrhea and accelerating ETEC vaccine development.

Polyvalent Protein Adhesin MEFA-II Induces Functional Antibodies against Enterotoxigenic Escherichia coli (ETEC) Adhesins CS7, CS12, CS14, CS17, and CS21 and Heat-Stable Toxin (STa).

There are no licensed vaccines for enterotoxigenic Escherichia coli (ETEC), a common cause of children's diarrhea and travelers' diarrhea. ETEC strains producing enterotoxins (heat-labile toxin, LT; heat-stable toxin, STa) and adhesins CFA/I, CFA/II (CS1-CS3) or CFA/IV (CS4-CS6) attributed to a majority of ETEC-associated diarrheal cases, thus the two toxins (STa, LT) and the seven adhesins (CFA/I, CS1 to CS6) are historically the primary targets in ETEC vaccine development. Recent studies, however, revealed that ETEC strains with adhesins CS14, CS21, CS7, CS17, and CS12 are also prevalent and cause moderate-to-severe diarrhea; these adhesins are now considered antigen targets as well for ETEC vaccines. In this study, we applied the epitope- and structure-based multiepitope-fusion-antigen (MEFA) vaccinology platform and constructed a polyvalent protein to present immuno-dominant continuous B-cell epitopes of these five adhesins (also an STa toxoid); we then characterized this protein antigen's (termed as adhesin MEFA-II) broad immunogenicity and evaluated antibody functions against each targeted adhesin and STa toxin. Data showed that mice intramuscularly immunized with adhesin MEFA-II protein developed robust IgG to the targeted adhesins and toxin STa. Importantly, the antigen-derived antibodies significantly inhibited adherence of ETEC bacteria expressing adhesin CS7, CS12, CS14, CS17, or CS21 and reduced STa enterotoxicity. These results indicated that adhesin MEFA-II protein is broadly immunogenic and induces cross-functional antibodies, suggesting adhesin MEFA-II can be an effective ETEC vaccine antigen; if included in an ETEC vaccine candidate, adhesin MEFA-II can expand vaccine coverage and increase efficacy against ETEC-associated children's diarrhea and travelers' diarrhea. IMPORTANCE An effective vaccine is lacking against ETEC, a primary cause of children's diarrhea and traveler's diarrhea and a threat to global health. The key challenge in ETEC vaccine development is that ETEC bacteria express heterogeneous virulence determinants (>25 adhesins and two toxins). While the current strategy to target the seven most prevalent ETEC adhesins (CFA/I, CS1 to CS6) potentially lead to a vaccine against many clinical cases, the prevalence of ETEC strains shifts chronically and geographically, and ETEC expressing other adhesins, mainly CS7, CS12, CS14, CS17, and CS21, also cause moderate-to-severe diarrhea. However, it is impossible to develop an ETEC vaccine to target as many as 12 adhesins under conventional approaches. This study used a unique vaccinology platform to create a polyvalent antigen and demonstrated the antigen's broad immunogenicity and functions against the targeted ETEC adhesins, enabling the development of a broadly protective vaccine essentially against all of the important ETEC strains.

Ameliorative effects of supplemental folinic acid on Lamotrigine-induced fetal malformations in the mouse.

Data from our previous work indicate that Lamotrigine (LTG) is teratogenic in the mouse. In the present study, we attempted to determine the possible protective effects of exogenous folate on LTG-induced fetal anomalies in TO mouse. Experiment I entailed administering 4 mg/kg of folinic acid (FA) and (25 mg/kg) of LTG intraperitoneally three times on gestation day (GD) 8 to a group of mice; other groups were a group that received similar volumes of saline, a group that received LTG and Saline, a group that received FA and saline. Experiment 2 involved administering groups of mice with daily 3 doses FA (or proportionate volume of saline) on GD 5 through 10 and either 3 doses of saline on GD8, or 3 doses of LTG on GD8. Maternal plasma concentrations of FA, vitamin B12 and homocysteine were determined an hour after the last injection from one-half of all animals. The other half were allowed to go to term (GD18) when they were euthanized and their fetuses were examined for visceral and skeletal malformations. A high incidence of resorption, abortion, embryolethality, congenital malformations, and intrauterine growth restriction (IUGR), was observed in the LTG-treated group. Folic acid and B12 levels were decreased and homocysteine concentration increased significantly in LTG groups. Mice receiving LTG with FA had normal levels of folate, Vitamin B12 and homocysteine levels, and the fetuses had fewer birth defects similar to the controls which were given saline only. Supplemental FA ameliorated to a great extent the LTG-induced embryonic resorption and malformations and restored the FA status.

Acute cadmium exposure causes systemic and thromboembolic events in mice.

Cadmium (Cd), an environmental and industrial pollutant, poses a potential threat and affects many systems in human and animals. Although several reports on Cd toxicity were presented, the acute effect of Cd on systemic and thrombotic events was not reported so far. Cd (2.284 mg/kg) or saline (control) was injected intraperitoneally (ip), and the systemic parameters were assessed in mice. Compared to control group, acute intraperitoneal injection of Cd, in mice showed significant quickening of platelet aggregation (P<0.001) leading to pial cerebral thrombosis. Likewise, Cd exposure caused a significant increase in white blood cell numbers (P<0.05) indicating the occurrence of systemic inflammation. Also, alanine aminotransferase (ALT) (P<0.05) and creatinine (P<0.01) levels were both significantly increased. Interestingly, the superoxide dismutase activity was significantly decreased in Cd treated group compared to control group (P<0.001), suggesting the occurrence of oxidative stress. We conclude that the Cd exposure in mice causes acute thromboembolic events, oxidative stress and alter liver and kidney functions.

Heart rate, body temperature and physical activity are variously affected during insulin treatment in alloxan-induced type 1 diabetic rat.

Diabetes mellitus is associated with a variety of cardiovascular complications including impaired cardiac muscle function. The effects of insulin treatment on heart rate, body temperature and physical activity in the alloxan (ALX)-induced diabetic rat were investigated using in vivo biotelemetry techniques. The electrocardiogram, physical activity and body temperature were recorded in vivo with a biotelemetry system for 10 days before ALX treatment, for 20 days following administration of ALX (120 mg/kg) and thereafter, for 15 days whilst rats received daily insulin. Heart rate declined rapidly after administration of ALX. Pre-ALX heart rate was 321+/-9 beats per minute, falling to 285+/-12 beats per minute 15-20 days after ALX and recovering to 331+/-10 beats per minute 5-10 days after commencement of insulin. Heart rate variability declined and PQ, QRS and QT intervals were prolonged after administration of ALX. Physical activity and body temperature declined after administration of ALX. Pre-ALX body temperature was 37.6+/-0.1 °C, falling to 37.3+/-0.1 °C 15-20 days after ALX and recovering to 37.8+/-0.1 °C 5-10 days after commencement insulin. ALX-induced diabetes is associated with disturbances in heart rhythm, physical activity and body temperature that are variously affected during insulin treatment.

Vigabatrin (VGB) administered during late gestation lowers maternal folate concentration and causes pregnancy loss, fetal growth restriction and skeletal hypoplasia in the mouse.

Vigabatrin (VGB) has several therapeutic advantages over older antiepileptic drugs (AED), but there is a lack of information about its potential reproductive toxicologic effects. Our aim was to evaluate the consequences of VGB administered during late gestation on fetal growth and development in the mouse. Based on the results of our previous study, we administered groups of mice a single dose of 450 mg/kg VGB on one of gestation days (GD) 15, 16 or 17. Fetuses were collected on GD 18. VGB groups had a significant incidence of fetal death, abortion, intrauterine growth restriction (IUGR), and hypoplasia of the axial skeleton, metacarpals, metatarsal and phalanges. Abortion was characterized by visible hemorrhagic expulsion of the embryos with their membranes. Maternal plasma folate (FA) and vitamin B12 concentrations were found to be markedly reduced within 12h of VGB treatment. Mice were supplemented with FA from GD 12 through GD 17 with or without a single dose of VGB on GD 15. This group had no abortions. Their fetuses had better body weight and lower frequency of IUGR than those of the non-supplemented VGB group. These data suggest that reductions in maternal FA and vitamin B12 concentrations play an important role in fetal loss, IUGR and skeletal hypoplasia induced by VGB during late gestation in the mouse. In view of the finding that a significant maternal toxicity is associated with this dose regimen, additional groups of mice were treated with 350 mg/kg VGB during embryogenesis and late gestation. This treatment was found to be maternally nontoxic. However, this low dose also resulted in significant fetal loss and IUGR when treatment occurred during late gestation. These data support the hypothesis that late gestation is particularly susceptible to VGB-induced fetal loss and IUGR in the mouse.

Efficacy of eight experimental bispyridinium oximes against paraoxon-induced mortality: comparison with the conventional oximes pralidoxime and obidoxime.

Recently, several experimental K-oximes with two functional aldoxime groups have been synthesized that show excellent in vitro efficacy in protecting acetylcholinesterase (AChE) from inhibition by a broad variety of organophosphorus compounds (OPCs). However, oximes themselves are also AChE inhibitors, albeit at higher concentrations, which is a major cause of their toxicity and may be a dose-limiting factor in oxime therapy. To assess the efficacy of the experimental K-oximes in vivo, the extent of oxime-conferred protection from mortality induced by paraoxon was quantified. Rats received paraoxon in a dosage of 1, 5, or 10 mumol, and immediately thereafter intraperitoneal injections of the respective oxime at a dosage of half the LD(01). The relative risk of death (RR) over time was estimated by Cox survival analysis for treatment with experimental K-oximes (K-53, K-74, K-75, K-107, K-108, and K-113), with the clinically available oximes pralidoxime (2-PAM) and obidoxime, and with the well-characterized K-oximes K-27 and K-48, comparing results with the no-treatment group. Best protection was conferred by K-27, reducing the RR to 20% of controls (P

Altered gene expression may underlie prolonged duration of the QT interval and ventricular action potential in streptozotocin-induced diabetic rat heart.

Ventricular electrical conduction has been investigated in the streptozotocin (STZ)-induced diabetic rat. Diabetes was induced with a single injection of STZ (60 mg/kg bodyweight, ip). The ECG was measured continuously, in vivo, using a biotelemetry system. Left ventricular action potentials were recorded with an extracellular suction electrode. Expression of mRNA transcripts for selected ion transport proteins was measured in left ventricle with real-time RT-PCR. At 10 weeks after STZ treatment, in vivo heart rate (HR) was reduced (267 +/- 3 vs. 329 +/- 5 BPM), QRS complex duration and QT interval were prolonged in diabetic rats compared to controls. In vitro spontaneous HR was reduced and paced heart action potential repolarization was prolonged in diabetic rats compared to controls. The mRNA expression for Kcnd2 (I (to) channel) and Kcne2 (I (kr) channel) was significantly reduced in diabetic rats compared to controls. Altered gene expression and, in particular, genes that encode K(+) channel proteins may underlie delayed propagation of electrical activity in the ventricular myocardium of STZ-induced diabetic rat.

Effect of intrathecal pralidoxime administration upon survival of rats exposed to the organophosphate paraoxon.

The therapeutic results of systemic administration of pralidoxime (2-PAM) in the treatment of poisoning with organophosphate-type cholinesterase inhibitors are disappointing. It has been hypothesized that this is due to poor entry of 2-PAM into the brain. To test if survival rates can be improved by direct administration of 2-PAM into the cerebrospinal fluid (CSF), the effect of intrathecal 2-PAM injections upon mortality after paraoxon intoxication was examined. Eight groups of rats (n=30 each) were examined, all of which received paraoxon (1 micromol=272 microg, 3 micromol=816 microg, or 5 micromol=1.36 mg) intraperitoneally (i.p.). One group received no further treatment; the other groups were given 50 micromol (=8.63 mg) 2-PAM i.p., 5 micromol (=863 microg) 2-PAM intrathecally and pentobarbital/lidocaine in various combinations. Results were compared with the no treatment group and the control groups that did not receive any paraoxon injections, but were given intrathecal injections of saline or 2-PAM. The relative risk of death was estimated by Cox survival analysis. Mortality was lowest after treatment with a combination of both i.p. and intrathecal 2-PAM plus pentobarbital, and with 2-PAM i.p. alone plus pentobarbital. Both treatments were significantly better than 2-PAM i.p. alone (p

Chronic effects of type 2 diabetes mellitus on cardiac muscle contraction in the Goto-Kakizaki rat.

Type 2 diabetes mellitus accounts for more than 90% of all cases of diabetes mellitus, and cardiovascular complications are the major cause of mortality and death in diabetic patients. The chronic effects of type 2 diabetes mellitus on heart function have been investigated in the Goto-Kakizaki (GK) rat. Experiments were performed in GK rats and age-matched Wistar control rats at 18 months of age. The progressive effects of diabetes on glucose metabolism were monitored periodically by application of the glucose tolerance test. Ventricular action potentials were measured in isolated, perfused heart. Shortening and intracellular Ca(2+) were measured in electrically stimulated ventricular myocytes. The GK rats displayed mild fasting hyperglycaemia and progressively worsening glucose tolerance. At 18 months of age and 180 min after intraperitoneal injection of glucose (2 g (kg body weight)(-1)), blood glucose was 436 +/- 47 mg dl(-1) in GK rats compared with 153 +/- 18 mg dl(-1) in control animals. Heart weight to body weight ratio was significantly increased in GK rats (4.10 +/- 0.09 mg g(-1), n = 5) compared with control animals (3.36 +/- 0.22 mg g(-1), n = 4). Spontaneous heart rate was slightly reduced in GK rats compared with control rats. Although the amplitude of shortening was not altered, the amplitude of the Ca(2+) transient was significantly increased in myocytes from GK rats (0.78 +/- 0.11 ratio units) compared with control rats (0.50 +/- 0.06 ratio units). Despite progressively worsening glucose metabolism, at 18 months of age the contractile function of the heart appears to be well preserved.

Five oximes (K-27, K-48, obidoxime, HI-6 and trimedoxime) in comparison with pralidoxime: survival in rats exposed to methyl-paraoxon.

There is a clear need for broad-spectrum cholinesterase reactivators (active against a multitude of organophosphorus ester enzyme inhibitors) with a higher efficacy than pralidoxime. The purpose of the study was to quantify in vivo the extent of oxime-conferred protection, using methyl-paraoxon [dimethyl p-nitrophenyl phosphate; (methyl-POX)] as a cholinesterase inhibitor. There were seven groups of six rats in each cycle of the experiment. Group 1 (G1) received 2 micromol methyl-POX ( approximately LD(50)), the other groups (G2-7) received 2 micromol methyl-POX + one of the six reactivators. The animals were monitored for 48 h and the time of mortality was recorded. The procedure was repeated six times. All substances were applied i.p. The experiments were repeated using 3 and 5 micromol methyl-POX. Mortality data were compared and hazards ratios (relative risks) ranked using the Cox proportional hazards model with methyl-POX dose and group (reactivator) as time-independent covariables. The relative risk of death estimated by Cox analysis (95% CI) in oxime-treated animals when compared with untreated animals, adjusted for methyl-POX dose (high/low) was K-27, 0.58 (0.42-0.80); K-48, 0.60 (0.43-0.83); trimedoxime, 0.76 (0.55-1.04); pralidoxime, 0.88 (0.65-1.20); obidoxime, 0.93 (0.68-1.26); HI-6, 0.96 (0.71-1.31). Only K-27 and K-48 provided statistically significant protection in rats exposed to methyl-POX. Despite the lower inhibitory potency (higher IC(50)) of methyl-POX compared with POX (ratio 4:1), the ability of oxime reactivators to protect from methyl-POX induced mortality was reduced compared with protection from POX (ethyl-analog).

Effect of maternal exposure to homocystine on sodium valproate-induced neural tube defects in the mouse embryos.

BACKGROUND: Neural tube defects (NTD) are mainly of multifactorial origin. Maternal treatment with valproic acid (VPA) during pregnancy induces NTD in susceptible fetuses. Elevated levels of homocysteine are observed in pregnancies with NTD. The mechanism by which homocysteine might cause NTD is unknown. AIM OF THE STUDY: The aim of this study was to determine if homocystine would augment VPA-induced exencephaly in an experimental model. METHODS: Groups of mice were injected (IP) on gestational day 8 (GD) with a single dose of 75 mg/kg of L: -Homocystine (HC) or a proportionate volume of saline, followed by a single dose of 600 mg/kg of VPA or an equal volume of saline. In a second experiment, mice were treated with a daily dose of 75 mg/kg of HC or an equal volume of saline (IP) from GD 5 and continued through GD 10. These animals had a single exposure to 600 mg/kg of VPA or saline (IP) on GD 8. All animals were killed by cervical dislocation on GD 18. Plasma homocysteine, folate and vitamin B12 were determined on GD 8 and GD 10 from single and multiple dose groups of mice, respectively, from additional experiments. RESULTS: The VPA and HC+VPA induced significantly higher rates of embryonic resorption and intrauterine growth retardation (IUGR) than HC or saline alone. HC + VPA groups had significantly more numerous fetuses with severe IUGR than HC alone or VPA alone groups. Both single and multiple doses of HC augmented VPA-induced reduction in fetal body weight. Successive doses of HC did not augment the rate of IUGR more significantly than a single dose of HC. Incidence of exencephaly was significantly enhanced in the HC + VPA groups compared to that in the HC or VPA alone groups. HC alone was not teratogenic. Plasma homocysteine levels increased several fold both in HC and HC + VPA groups and the increase was not particularly more marked in multiple dose groups than in the single dose groups. VPA did not elevate homocysteine concentration. Both FA and vitamin B12 concentrations were reduced by VPA, HC and HC + VPA, but HC and VPA when combined did not produce an additive effect on vitamin levels. CONCLUSION: These data indicate that HC and VPA interact in neurulation stage embryos, affect fundamental processes of closure of the neural tube and lead to enhanced incidence of NTD.

Effects of insulin treatment on heart rhythm, body temperature and physical activity in streptozotocin-induced diabetic rat.

1. Streptozotocin (STZ)-induced diabetic cardiomyopathy is frequently associated with depressed diastolic/systolic function and altered heart rhythm. 2. The effects of insulin treatment on heart rhythm, body temperature and physical activity in STZ-induced diabetic rats were investigated using biotelemetry techniques. 3. Transmitter devices were surgically implanted in the peritoneal cavity of young adult male Wistar rats. Electrodes from the transmitter were arranged in Einthoven bipolar - Lead II configuration. Electrocardiogram, physical activity and body temperature data were recorded with a telemetry system for 10 days before STZ treatment, for 20 days following administration of STZ (60 mg/kg) and thereafter, for 30 days while rats received daily insulin. 4. Heart rate, physical activity and body temperature declined rapidly 3-5 days after administration of STZ. Pre-STZ heart rate was 362 +/- 7 b.p.m., falling to 266 +/- 12 b.p.m. 5-15 days after STZ with significant recovery to 303 +/- 14 b.p.m. 10-20 days after commencement of insulin. Pre-STZ body temperature was 37.5 +/- 0.1C, falling to 37.2 +/- 0.2C 5-15 days after STZ with significant recovery to 37.5 +/- 0.1C 10-20 days after commencement of insulin. Physical activity and heart rate variability were also reduced after STZ but there was no significant recovery during insulin replacement. 5. Defective autonomic regulation and/or mechanisms of control that are intrinsic to the heart may underlie disturbances in heart rhythm in the STZ-induced diabetic rat.

Tiapride pre-treatment in acute exposure to paraoxon: comparison of effects of administration at different points-in-time in rats.

INTRODUCTION: Accidental and suicidal exposures to organophosphorus compounds (OPC) are frequent. The inhibition of esterases by OPC leads to an endogenous ACh poisoning. Recently, the FDA approved, based on animal experiments, for military combat medical use oral pyridostigmine (PSTG) for pre-exposure treatment of soman; the concept is to block the cholinesterase reversibly using the carbamate pyridostigmine in order to deny access to the active site of the enzyme to the irreversible inhibitor (OPC) on subsequent exposure. We have shown previously that tiapride (TIA) is in vitro a weak inhibitor of AChE. We also have shown recently that in rats coadministration of TIA with the organophosphate paraoxon significantly decreases mortality without having an impact on red blood cell cholinesterase (RBC-AChE) activity. PURPOSE OF THE STUDY: To establish in a prospective, non-blinded study in a rat model of acute high dose OPC (paraoxon; POX) exposure the ideal point in time for TIA pre-treatment administration and to correlate it with measured TIA plasma levels. MATERIAL AND METHODS: There were six groups of rats in each cycle of the experiment and each group contained six rats. The procedure was repeated twelve times (cycles) (n = 72 for each arm; half male and half female). All substances were applied ip. All groups (1-6) received 1 microMol POX ( approximately LD(75)); groups 1-5 also received 50 microMol TIA at different points in time. Group 1 (G(1)): TIA 120 min before POX Group 2 (G(2)): TIA 90 min before POX, Group 3 (G(3)): TIA 60 min before POX, Group 4 (G(4)): TIA 30 min before POX, Group 5 (G(5)): TIA & POX simultaneously, Group 6 (G(6)): POX only. The animals were monitored for 48 hours and mortality/survival times were recorded at 30 min, 1, 2, 3, 4, 24 and 48 h. AChE activities were determined at 30 min, 24 and 48 h in surviving animals. Statistical analysis was performed on the mortality data, cumulative survival times and enzyme activity data. Mortality data was compared using Kaplan-Meier plots. Cumulative survival times and enzyme activites were compared using the Mann-Whitney rank order test. No Bonferroni correction for multiple comparisons was applied and an alpha < or= 0.05 was considered significant. RESULTS: Mortality is statistically significantly reduced by TIA pre-treatment at all points-in-time. Highest protection is achieved if TIA is given 90 to 0 min before OPC exposure. The reduction in mortality is not correlated to TIA plasma levels (C (max) approximately 120 min post ip-administration). TIA pre-treatment is not affecting AChE activity regardless of the timing of administration. CONCLUSION: The lack of correlation between TIA plasma levels and degree of mortality reduction as well as the lack of protective effect on enzyme activity seem to indicate that the site of action of TIA is not the blood. While our hypothesis that TIA would protect AChE in a pyridostigmine-like manner (via protection of the enzyme) could not be confirmed, the reduction in mortality with TIA pre-treatment is nevertheless of potential interest.

Long-term effects of streptozotocin-induced diabetes on the electrocardiogram, physical activity and body temperature in rats.

In vivo biotelemetry studies have demonstrated that short-term streptozotocin (STZ)-induced diabetes is associated with a reduction in heart rate (HR) and heart rate variability (HRV) and prolongation of QT and QRS intervals. This study investigates the long-term effects of STZ-induced diabetes on the electrocardiogram (ECG), physical activity and body temperature. Transmitter devices were surgically implanted in the peritoneal cavity of young adult male Wistar rats. Electrodes from the transmitter were arranged in Einthoven bipolar lead II configuration. ECG, physical activity and body temperature data were continuously recorded with a telemetry system before and following the administration of STZ (60 mg kg(-1)) for a period of 22 weeks. HR, physical activity and body temperature declined rapidly 3-5 days after the administration of STZ. The effects became conspicuous with time reaching a new steady state approximately 1-2 weeks after STZ treatment. HR at 4 weeks was 268 +/- 5 beats min(-1) in diabetic rats compared to 347 +/- 12 beats min(-1) in age-matched controls. HRV at 4 weeks was also significantly reduced after STZ treatment (18 +/- 3 beats min(-1)) compared to controls (33 +/- 3 beats min(-1)). HR and HRV were not additionally altered in either diabetic rats (266 +/- 5 and 20 +/- 4 beats min(-1)) or age-matched controls (316 +/- 6 and 25 +/- 4 beats min(-1)) at 22 weeks. Reduced physical activity and/or body temperature may partly underlie the reductions in HR and HRV. In addition, the increased power spectral low frequency/high frequency ratio from 4 weeks after STZ treatment may indicate an accompanying disturbance in sympathovagal balance.

Effect of maternal administration of vigabatrin during late gestation on fetoplacental amino acid profile in the mouse.

The objectives were to determine if vigabatrin in late gestation affects fetal growth and causes alterations in amino acid concentrations in the mouse. A single dose of 450 mg/kg VGB in saline or a proportionate volume of saline was administered intraperitoneally (IP) to Theiler outbred (TO) mice on gestation day 15 and maternal plasma, placentae and fetuses were collected at different time intervals after treatment. VGB attained peak concentration in the maternal plasma and the fetus at 2 h after treatment and in the placenta at 4 h. At 12 h significantly lower concentrations of several amino acid including methionine were found in the placentae and fetuses in the treated group. After 24 h, no difference was seen in the plasma amino acid concentrations but in the placentae and fetuses a significant decrease occurred in some amino acids in the treated group. At 48 and 72 h, a generalized increase in most amino acid levels occurred in the fetus and placenta but not in maternal plasma of the treated group although the fetal and placental weights were significantly reduced. VGB during late gestation causes fetal growth retardation accompanied by an initial disruption of amino acid supply followed by an increase in amino acid concentrations for the next 2 days. This increase did not help restore growth suggesting that early fetal period is particularly vulnerable to VGB-induced intrauterine growth retardation in the mouse.

Effect of maternal diabetes and ethanol interactions on embryo development in the mouse.

The aim of this study was to determine the possible fetal effects of interaction between maternal diabetes and acute doses of alcohol. Pregnant TO mice were made diabetic by a single injection of streptozotocin (STZ) on gestation day (GD) 2. Single dose of 0.003 or 0.03 ml/g body weight of fresh ethanol (25% v/v of absolute alcohol in normal saline) was injected into groups of diabetic and nondiabetic animals on GD 7 or 8. One group of diabetic animals had a daily dose of 6-8 IU of insulin subcutaneously. Fetuses were collected on GD 18. There was a significant increase in the incidence of implantation failure in the diabetes plus ethanol groups and insulin control group. Ethanol injection on GD 7 accentuated diabetes-related embryonic resorption and intrauterine growth retardation (IUGR). This effect was less marked in the diabetic group treated with ethanol on GD 8. Diabetes alone produced a greater incidence of IUGR than ethanol alone. Midfacial hypoplasia and minor anomalies were found more frequently in the combination treatment groups. Holoprosencephaly and thymus hypoplasia observed in diabetic groups were found to be reduced in frequency in the diabetes plus ethanol groups, suggesting an antagonistic type of ethanol-diabetes interaction, stage-dependently. Since severely malformed embryos are known to be resorbed/killed in utero in mice, this reduction might reflect the magnitude of early death of severely malformed embryos. These data suggest that the interaction effects are possibly related to alterations in fundamental developmental processes of early embryos.

Amelioration of sodium valproate-induced neural tube defects in mouse fetuses by maternal folic acid supplementation during gestation.

Infants of epileptic women treated with valproic acid (VPA) during pregnancy have a higher risk of developing spina bifida than those of the general population. VPA induces exencephaly in experimental animal embryos. But the pathogenetic mechanism remains rather elusive. Antiepileptic drugs (AED) in general accentuate pregnancy-imposed fall in maternal folate levels. Periconceptional folic acid supplementation is reported to protect embryos from developing neural tube defects (NTD). Conflicting results have been reported by experimental studies that attempted to alleviate VPA-induced NTD by folic acid. Our objectives were to determine the critical developmental stages and an effective dose of folic acid for the prevention of VPA-induced exencephaly in mouse fetuses. A single teratogenic dose of 400 mg/kg of VPA was administered to TO mice on gestation day (GD) 7 or 8. It was followed by (1) a single dose of 12 mg/kg of FA (folinic acid) or (2) 3 doses of FA 4 mg/kg each. In experiment (3), FA (4 mg/kg) was administered thrice daily starting on GD 5 and continued through GD 10. These animals received VPA on GD 7 or 8. VPA and B12 concentrations were determined by radioimmunoassay. The single heavy dose of FA had no rescue effect on NTD. Three divided doses of FA on GD 7 and continuous dosing of FA from GD 5 through GD 10 substantially reduced the VPA-induced exencephaly in the fetuses. In the later experiments, the neural folds elevated faster than the non-supplemented group. VPA considerably reduced maternal plasma folate and B12 concentrations. The heavy dose of FA only moderately improved vitamin levels. Three divided doses of FA elevated the vitamin levels slightly better but it was the prolonged dosing of FA that was associated with sustained elevation of plasma levels higher than the control levels and acceleration of neural tube closure thus accounting for the pronounced protection against VPA-induced NTD development. These data suggest that plasma levels of FA and B12 have to be kept substantially elevated and maintained high throughout organogenesis period to protect embryos against VPA-induced NTD in this mouse model.

Experimental studies on reproductive toxicologic effects of lamotrigine in mice.

BACKGROUND: Virtually all antiepileptic drugs (AED) tested so far have been found to be teratogenic. The second generation AED possess a number of therapeutic advantages over the older ones. There are, however, very little data on their effects on embryonic development. A recent report suggests that lamotrigine (LTG) can be teratogenic to human fetuses. With only a few cases of prenatal exposure to LTG in the record, however, it has not been possible to establish a recognizable pattern of malformations in the infants of LTG-treated mothers. OBJECTIVES: The objectives of the present study were to evaluate the reproductive toxic effects of LTG . RESULTS: Single (50-200 mg/kg) or multiple doses (25, 50, 75 mg/kg) of LTG were administered by intraperitoneal (i.p.) injection (note that the therapeutic administration is oral) to groups of TO mice on gestation day (GD) 7 or 8. Fetuses were collected on GD 18. Maternal toxic effects including a dose-related mortality, a high incidence of abortion, embryo lethality, congenital malformations and intrauterine growth retardation (IUGR) were observed in the LTG-treated group. Administration of LTG in multiple low doses resulted in a better maternal survival and increased incidence of embryonic resorption and malformations with increasing dose; IUGR was significant but not dose-dependent. The malformations characteristic of the LTG multiple low dose group fetuses included maxillary-mandibular hypoplasia, exencephaly, cleft palate, median facial cleft, urogenital anomalies and varying degrees of caudal regression. Skeletal malformations and developmental delay of the skeleton were observed both in single and multiple dose groups. CONCLUSIONS: The results of this study indicate that LTG administered i.p. at high doses can induce intrauterine growth retardation and at low multiple doses causes a dose-dependent increase in embryonic resorption, craniofacial and caudal malformations as well as maternal toxicity in the mouse. Previous studies in other laboratories have used oral route of exposure and concluded that there are no teratogenic effects of LTG at dose levels that are not maternally toxic.

Intrauterine growth retardation in experimental diabetes: possible role of the placenta.

Fetal growth disorders are common in pregnancy complicated by diabetes. Whereas macrosomia often occurs in infants of diabetic women, growth retardation is almost a rule in spontaneous and experimental diabetes in animals. However, it is not clear when during development growth inhibition starts and how placental pathology might affect fetal growth in maternal diabetes. In this study pregnant Wistar rats were injected (ip) with a single dose of 50 mg/kg of streptozotocin (STZ) on gestation day (GD) 2 and a blood glucose level of 200 mg/dl or more determined 24 hrs later indicated diabetes. The controls were non-treated, buffer treated or, following confirmation of diabetes, injected with a single dose of 2--6 IU of insulin (Novo Ultralente) once daily. Fetuses and placentae were collected from GD 14--20. Intrauterine growth retardation (IUGR) in STZ group was significant as early as GD 15 and persisted to GD 20. Insulin produced a significant recovery in fetal weight gain. The placentas of STZ-treated group were significantly heavier than those of the control groups. The reduction in cord length of the STZ group became apparent on GD 16 and remained so to term. The placenta of GD 14 STZ group had a thicker decidua basalis and dilated maternal sinusoids. By GD 16, the decidua basalis contained glycogen-containing decidual cells and scattered glycogen cells confirmed by Best's carmine with or without diastase. The glycogen cells of the basal zone were more abundant, and had degenerated in some sites leaving behind cysts with eosinophilic mass. The giant cells had proliferated enormously. The labyrinthine zone appeared spongy with persistent fetal mesenchyme, peri-vascular fibrosis, and enhanced placental barrier. The trophoblasts of the labyrinths also contained traces of glycogen unlike the controls. By GD 18, the decidua basalis of the STZ group was thinner than that of the controls and contained necrotic giant cells and lymphocytic aggregations. In the basal zone, the giant cells had proliferated further; more glycogen cells had degenerated. Perivascular fibrosis was still extensive in the labyrinthine zone. Bloodless maternal sinusoids, extensive vacuolization, degeneration of glycogen islands and formation of cysts characterized the labyrinthine zone. These changes varied in intensity from one area to another in the same placenta and between placentas of the same and of different litters. The development of the upper and lower jaws, elevation and fusion of palatal shelves, reduction of physiological umbilical hernia, descent of the testes, fusion of the urethral folds and separation of digits of the paws were significantly delayed in the STZ group. The consistent association of placental pathology with fetal growth retardation is suggestive of an alteration in placental function possibly contributing to IUGR in STZ-induced diabetes in rats.