Selective gene expression using a DF3/MUC1 promoter in a human esophageal adenocarcinoma model.

The efficacy of replication-deficient adenoviral vectors in gene therapy is confined to the number of tumor cells the vector infects. To focus and enhance the therapeutic efficacy, we employed a conditionally replication-competent adenoviral vector with a tissue-specific promoter, DF3/MUC1, in a human esophageal adenocarcinoma model. Our results demonstrate that Ad.DF3.E1A.CMV.TNF (Ad.DF3.TNF) specifically replicates in Bic-1 (DF3-producing cells) and mediates an enhanced biologic effect due to increased TNF-alpha in the same DF3-producing cells. We also show that the increased TNF-alpha interacts with ionizing radiation to produce greater tumor regression and a greater delay in tumor regrowth in Bic-1 (DF3-producing cells) compared to Seg-1 (DF3 non-producers). Tumor cell targeting using conditionally replication-competent adenoviral vectors with tumor-specific promoters to drive viral replication and deliver TNF-alpha provides a novel approach to enhancing tumor radiosensitivity.

Antecedents and neonatal consequences of low Apgar scores in preterm newborns: a population study.

BACKGROUND: To examine the antenatal and early neonatal correlates of low Apgar scores (<3 and <6 at 1 and 5 minutes) in preterm newborns (23-34 weeks' gestation). OBJECTIVE: The use of Apgar scoring for premature newborns remains widespread, despite controversy regarding its reliability as a measure of morbidity and mortality in the neonatal period. DESIGN: A cohort of 852 preterm newborns born during a 34-month period between 1984 and 1987 was studied. Newborns were stratified into 2 groups by gestational age (23-28 weeks and 29-34 weeks), and data were analyzed, controlling for gestational age in single weeks. SETTING: Two academic and 1 community hospital, which together accounted for 83% of all preterm births in a tri-county area of central New Jersey during the study period. PATIENTS: All premature newborns (birth weight <2000 g and gestational age <35 weeks) born in the participating hospitals during the study period were evaluated. MAIN OUTCOME MEASURES: Antecedents included maternal illness during pregnancy, maternal complications of labor and delivery, and fetal heart rate abnormalities during labor and delivery. Consequences included delivery room resuscitation, abnormal physical findings, diagnoses, and therapeutic interventions in the first 6 to 8 hours of life. RESULTS: Premature newborns with low Apgar scores received more cardiopulmonary resuscitation in the delivery room and in the first 6 to 8 hours of neonatal intensive care. Mortality was significantly increased among newborns with low Apgar scores (54% vs. 26% in the 23- to 28-week stratum, 30% vs 6% in the 29- to 34-week stratum). Newborns with low Apgar scores in the 29- to 34-week stratum more often required intubation, positive pressure ventilation, and umbilical vessel catheterization. Newborns with low Apgar scores had higher rates of bradycardia, pneumothoraces, acidosis, and increased oxygen requirement during the first 6 to 8 hours of life. Maternal illness, complications of labor and delivery;, and fetal heart rate decelerations did not correlate with subsequent Apgar scores of newborns. The presence of severe bradycardia (<90/min) and fetal heart rate accelerations correlated with low Apgar scores in the 29- to 34-week group. CONCLUSION: Low Apgar scores are associated with increased neonatal morbidity and mortality in preterm newborns. Antenatal maternal history, and pregnancy complications are not clearly associated with low Apgar scores. Therefore, the Apgar score is a useful tool in assessing neonatal short-term prognosis and the need for intensive care among preterm newborns.

NM-3, an isocoumarin, increases the antitumor effects of radiotherapy without toxicity.

We examined the effects of a new antiangiogenic isocoumarin, NM-3, as a radiation modifier in vitro and in vivo. The present studies demonstrate that NM-3 is cytotoxic to human umbilical vein endothelial cells (HUVECs) but not to Lewis lung carcinoma (LLC) cells nor Seg-1, esophageal adenocarcinoma cells, in clonogenic survival assays. When HUVEC cultures are treated with NM-3 combined with ionizing radiation (IR), additive cytotoxicity is observed. In addition, the combination of NM-3 and IR inhibits HUVEC migration to a greater extent than either treatment alone. The effects of treatment with NM-3 and IR were also evaluated in tumor model systems. C57BL/6 female mice bearing LLC tumors were given injections for 4 consecutive days with NM-3 (25 mg/kg/day) and treated with IR (20 Gy) for 2 consecutive days. Combined treatment with NM-3 and IR significantly reduced mean tumor volume compared with either treatment alone. An increase in local tumor control was also observed in LLC tumors in mice receiving NM-3/IR therapy. When athymic nude mice bearing Seg-1 tumor xenografts were treated with NM-3 (100 mg/kg/day for 4 days) and 20 Gy (four 5 Gy fractions), significant tumor regression was observed after combined treatment (NM-3 and IR) compared with IR alone. Importantly, no increase in systemic or local tissue toxicity was observed after combined treatment (NM-3 and IR) when compared with IR alone. The bioavailability and nontoxic profile of NM-3 suggests that the efficacy of this agent should be tested in clinical radiotherapy.

Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation.

The family of vascular endothelial growth factor (VEGF) proteins include potent and specific mitogens for vascular endothelial cells that function in the lation of angiogenesis Inhibition of VEGF-induced angiogenesis either by neutralizing antibodies or dominant-negative soluble receptor, blocks the growth of primary and metastatic experimental tumors Here we report that VEGF expression is induced in Lewis lung carcinomas (LLCs) both in vitro and vivo after exposure to ionizing radiation (IR) and in human tumor cell lines (Seg-1 esophageal adenocarcinoma, SQ20B squamous cell carcinoma, T98 and U87 glioblastomas, and U1 melanoma) in vitro. The biological significance of IR-induced VEGF production is supported by our finding that treatment of tumor-bearing mice (LLC, Seg-1, SQ20B, and U87) with a neutralizing antibody to VEGF-165 before irradiation is associated with a greater than additive antitumor effect. In vitro, the addition of VEGF decreases IR-induced killing of human umbilical vein endothelial cells, and the anti-VEGF treatment potentiates IR-induced lethality of human umbilical vein endothelial cells. Neither recombinant VEGF protein nor neutralizing antibody to VEGF affects the radiosensitivity of tumor cells These findings support a model in which induction of VEGF by IR contributes to the protection of tumor blood vessels from radiation-mediated cytotoxicity and thereby to tumor radioresistance.

Phase I evaluation of humanized OKT3: toxicity and immunomodulatory effects of hOKT3gamma4.

Murine anti-CD3 (OKT3, Muromonab-CD3) is a potent human T-lymphocyte mitogen. A previous clinical Phase I trial examined OKT3 as an immunomodulator for the treatment of cancer. However, the murine monoclonal antibody triggered a potent humoral response that neutralized the antibody activity during subsequent administration. Thus, a "humanized" form of OKT3 (hOKT3gamma4) was developed to minimize immunogenicity. The genetically engineered human anti-CD3 retained its binding activity and effectively activated T cells in vitro. Therefore, we evaluated the safety and activity of hOKT3gamma4 in a Phase I clinical trial. hOKT3gamma4 was administered as a 10-min i.v. infusion every 2 weeks for three injections (one course of therapy). Six dose levels ranging from 50 to 1600 microg/injection were evaluated. Headache and fever were common, transient toxicities but were not dose limiting. The dose-limiting toxicities were rigors and dyspnea at the 1600-microg dose level, which defined 800 microg as the maximally tolerated dose in this trial. A dose-dependent in vivo T-lymphocyte activation was produced by this treatment, and the most significant T-lymphocyte activation occurred in patients treated at the two highest dose levels (800 and 1600 microg). Persistent CD3 modulation occurred after administration of 1600 microg of hOKT3gamma4. Anti-idiotypic antibodies were detected in only 6 of 24 patients after multiple injections and were not associated with attenuation of T-lymphocyte activation. Malignant ascites resolved in three patients, one each with peritoneal mesothelioma, pancreatic adenocarcinoma, and ovarian adenocarcinoma. hOKT3gamma4 can induce T-lymphocyte activation in patients with cancer, and the immunogenicity of the "humanized" antibody is sufficiently reduced relative to its murine "parent" to permit immunostimulation by repetitive i.v. administration. The therapeutic potential of biweekly i.v. hOKT3gamma4 at a dose of 800 microg should be further evaluated.

Developmental implications of head growth following intracranial hemorrhage.

This study examined the association between head size at birth, discharge, and 1 year and developmental outcome at 1 year in preterm infants, with and without intracranial hemorrhages (ICH) or associated periventricular echodensities (PVE). The data indicated that most sick preterm infants with small heads at discharge achieved appropriate head sizes at 1 year. Analyses of the 1-year mental and motor performances of 125 subjects revealed that for subjects who did not develop ICH, appropriate head sizes at birth and discharge were associated with good developmental outcome, whereas infants with small heads (< two standard deviations below the mean for age) before hospital discharge were more likely to show poorer developmental outcome at 1 year. For subjects with ICH, birth and discharge head circumference were not predictive of 1-year developmental status; however, normal head size at 1 year was associated with better outcome. This was true for children with transient PVE as well. However, persistent periventricular echodensities were associated with both mental and motor deficits at 1 year, regardless of head growth.

The apgar score and its components in the preterm infant.

OBJECTIVE: The Apgar score is well-characterized in full-term infants but not in premature infants. The objective of this study was to assess the Apgar score in preterm infants with respect to the relationships between the 1- and 5-minute scores, the correlation of the Apgar score with pH and with other variables, and the relationship among the individual Apgar components. METHODOLOGY: We recorded Apgar scores at 1 and 5 minutes in a population-based cohort of preterm infants (n = 1105) with birth weight <2000 g, from three intensive care nurseries in central New Jersey. Linear correlation analysis was used to examine the relationship between 1- and 5-minute Apgar scores and between the individual components of the Apgar score. Multiple regression analysis was used to explore the relationship between various perinatal characteristics and the Apgar score, and between pH and Apgar score. Stepwise logistic regression analysis was used to assess the determinants of mortality. RESULTS: The 1-minute Apgar score median (25%, 75%) was 6(4,8) and correlated with the 5-minute score of 8(7,9) at r = .78. Slight but significant differences were seen between male (n = 557) and female (n = 508) infants in the 1-minute (6[4,8] and 7[4,8]) Apgar scores. One- and 5-minute scores of white infants (7[4,8] and 8[7,9]; n = 713) were significantly higher than those of black infants (5[3,7] and 8[6,9]; n = 280). Birth weight and gestational age were both linearly related to both Apgar scores. Low Apgar score (<3 at 1 minute and <6 at 5 minutes) was significantly associated with birth weight, gestational age and mode of delivery. Low arterial blood pH (<7.01) at birth was significantly related to low Apgar score. One hundred fifty-nine infants died; these infants were significantly smaller (983 +/- 382 vs 1462 +/- 369 g), less mature (27 vs 31 weeks), had lower arterial blood pH (7.20 +/- 0.18 vs 7.31 +/- 0.11), had lower 1- (3[2,6] vs 7[4,8]) and 5-minute Apgar scores (6[4,8] vs 8[7,9]), and a greater incidence of low Apgar score (32% vs 6%) than did survivors. CONCLUSIONS: Among the components of the Apgar score, respiratory effort, muscle tone, and reflex activity correlated well with one another; heart rate correlated less well; and color the least. Our data confirms the limited use of the Apgar score in preterm infants and demonstrates the different responses of the Apgar score's components.

CD28/B7 costimulation regulates autoimmune diabetes induced with multiple low doses of streptozotocin.

Insulin-dependent diabetes mellitus is believed to occur as a result of a T cell-mediated destruction of the islets of Langerhans. The factors that regulate the T cell responses, in particular the costimulatory signals required for the T cell activation, which result in islet cell destruction, are still unclear. CD28/B7 interactions have been shown to be important in the regulation of T cell immune responses. We, therefore, have examined the role of CD28/B7 interactions in a model of insulin-dependent diabetes mellitus in which T cell-dependent insulitis and hyperglycemia occur over a brief period, following multiple low doses of streptozotocin (multidose streptozotocin (STZ)-induced diabetes mellitus). Expression of CD28 was necessary for diabetes because CD28 -/- C57BL/KsJ animals developed neither hyperglycemia nor insulitis, and did not express IFN-gamma mRNA following STZ, unlike CD28 +/- C57BL/KsJ mice. The expression of B7-1 (CD80) and B7-2 (CD86) molecules was closely regulated during development of the disease. Expression of both CD80 and CD86 increased on cells in pancreatic lymph nodes in STZ-treated C57BL/KsJ mice. Expression of only CD86 increased on islet cells in diabetic mice. In wild-type animals, treatment with mAb against CD86 prevented, whereas treatment with mAb against CD80 exacerbated, insulitis and hyperglycemia, indicating that mAbs against these molecules differentially affect development of disease. We conclude that CD28 signal transduction is required for development of diabetes in multidose STZ-induced diabetes mellitus. CD80 and CD86 molecules, the CD28/CTLA4 ligands, may have different roles in regulation of the disease and affect T cell function at steps beyond differentiation into mature phenotypes.

CD28/B7 regulation of Th1 and Th2 subsets in the development of autoimmune diabetes.

CD28 ligation delivers a costimulatory signal important in T cell activation. This study demonstrates that the disruption of the CD28/B7 pathway early in the nonobese diabetic mouse strain, using CD28-/- and CTLA41g transgenic mice, promoted the development and progression of spontaneous autoimmune diabetes. Functional analyses of T cells isolated from CD28-deficient mice demonstrated that the GAD-specific T cells produced enhanced Th1-type cytokines (IL-2 and IFN gamma) and diminished Th2-type cytokine, IL-4. Moreover, there was a significant decrease in serum levels of anti-GAD antibodies of the IgG1 isotype consistent with a profound suppression of Th2-type responses in these animals. Thus, the early differentiation of naive diabetogenic T cells into the Th2 subset is dependent upon CD28 signaling and extends our understanding of the importance of Th1/Th2 balance in the regulation of this spontaneous autoimmune disease.

Blood pressure ranges in premature infants: II. The first week of life.

OBJECTIVE: To examine the arterial blood pressure in the first week of life in a healthy premature population. DESIGN: Population-based cohort study. SETTING: Three intensive care nurseries in central New Jersey. PATIENTS: Premature infants with birth weights less than 2000 g. MAIN OUTCOME MEASURES: We documented daily maximum and minimum systolic and maximum and minimun diastolic blood pressures during the first 7 days of life. To examine the effects on the ranges of blood pressure, we identified four groups of infants: (1) healthy infants without any of the major risk factors (n = 193); (2) infants who were mechanically ventilated but free of any of the other conditions (n = 225); (3) infants with histories of maternal hypertension or preeclampsia (n = 38) and (4) infants with low Apgar scores (less than 3 at 1 minute and less than 6 at 5 minutes) regardless of the presence of other conditions (n = 86). RESULTS: Blood pressure increased steadily in the first week of life in all four groups. There was no relationship between any of the four blood pressure variables, or trends in blood pressure over time, with birth weight, gender, or race. Regression equations (based on all infants with available data) for blood pressure ranges by day of life revealed that the maximum systolic blood pressure increased by 2.6 mm Hg/d, the minimum systolic blood pressure increased by 1.8 mm Hg/d,the maximum diastolic blood pressure increased by 2.0 mm HHg/d, and the minimum diastolic blood pressure increased by 1.3 Hg/d. CONCLUSIONS: Infants with birth asphyxia and ventilated infants had significantly lower systolic and diastolic blood pressures than healthy infants.

Blood pressure ranges in premature infants. I. The first hours of life.

We studied blood pressure in the first hours of life in a cohort of 1105 preterm infants weighing 501 to 2000 gm; these infants represented 83% of all births at these weights that resulted in admission to three intensive care nurseries during a 34-month period between 1984 and 1987. To assess the effects of specific risk factors, we identified 244 healthy infants, 164 infants who received mechanical ventilation but had no other conditions, 47 infants whose only risk factor was the presence of hypertension or preeclampsia in the mother, and 86 infants with depressed Apgar scores regardless of the presence of the other conditions. We documented each infant's minimum and maximum systolic (Smin, Smax) and diastolic (Dmin, Dmax) pressures during the first 3 to 6 hours of life. In the healthy group, Smin was 47 mmHg; Smax, 59 mmHg; Dmin, 24 mmHg; and Dmax, 35 mmHg. In the ventilation group, Smin was 41 mmHg; Smax, 57 mmHg; Dmin, 22 mmHg; and Dmax, 35 mmHg. The Smin and Dmin values were both significantly lower in infants who received mechanical ventilation than in healthy infants (p < 0.01). In the maternal hypertension group, Smin was 49 mmHg; Smax, 59 mmHg; Dmin, 25 mmHg; and Dmax, 34 mmHg. Only the Smin value was significantly higher than in healthy infants. In the group with low Apgar scores, Smin was 33 mmHg; Smax, 51 mmHg; Dmin, 19 Hg; and Dmax, 34 mmHg. Thus all these values were significantly lower than in all the other groups (p < 0.05). Of infants with low Apgar scores, 20% to 50% had values below the 5th percentile for healthy infants. Birth weight and gestational age correlated with blood pressure limits only in the infants with low Apgar scores. We conclude that in healthy premature infants the limits of systolic and diastolic blood pressure are independent of birth weight and gestational age. Infants with low Apgar scores tend to have lower pressures, and infants whose mothers have hypertension have higher pressures than infants in the healthy cohort.

Neurodevelopmental outcome of infants with apnea of infancy.

The neurodevelopmental outcome of premature infants with persistent apnea of prematurity (AOP) is reported. Sixty premature infants (birthweight [BW], 1469 +/- 533 gm; gestational age [GA , 31 +/- 3 weeks) with AOP were compared to 47 control infants (BW, 1586 +/- 581 gm; GA, 31 +/- 3 weeks) matched for gestational age and degree of neonatal illness. The infants were enrolled in a multidisciplinary follow-up program, and outcome data between 12 and 24 months are reported. Assessments included the Bayley Scores of Infant Development, neurologic examinations, speech and hearing examinations. There were no significant differences in the cognitive outcome between the premature infants with AOP and the premature control group. In 50 of 60 infants the Bayley Mental Developmental Index was in the normal range (112 +/- 18) as was 39 of 47 of the control group (112 +/- 13). Delays in motor development were seen in both premature groups, although a greater percentage of premature infants with persistent apnea had mild motor delays than did control infants. There was a comparable incidence of cerebral palsy (8% vs 11%), speech delays (20% vs 23%), retinopathy (8% vs 13%), and esotropia (7% vs 4%) between the infants with AOP and the premature control infants. The presence of persistent neonatal apnea without additional adverse perinatal events did not appear to be associated with a higher incidence of significant developmental problems.

Cerebral blood flow measurements in the newborn dog.

Current methods of cerebral blood flow (CBF) determination provide only discrete, episodic data and are limited in assessing dynamic changes in cerebral circulation. We adapted a venous outflow technique employing cannulation of the superior sagittal sinus in newborn puppies to measure changes in CBF rapidly and sequentially during ventilatory maneuvers. CBF velocity (CBFV) was measured simultaneously with Doppler ultrasound of the anterior cerebral artery. Relationships between flow and velocity were determined during hypocarbia, hypercarbia, hypoxia and asphyxia. During hyperventilation, CBF decreased by a mean of 14%, and CBFV by 13%, whereas maximal CO2 inhalation increased CBF by 59% and CBFV by 110%. Although CBFV exhibited the same directional changes as shown by the area under the velocity curve, the magnitude of change was not proportional to flow. CBFV indicated trends in the direction of changes in flow in individual animals, but did not correlate with flow between animals. A fluctuating unstable pattern of CBFV, previously associated with neonatal intracranial hemorrhage, was demonstrated with hyperventilation and hypocarbia. During asphyxia, CBFV ceased 2-5 min prior to CBF, presumably reflecting continued brain perfusion from vessels other than anterior cerebral artery.

Neurodevelopmental outcome in infants with apnea.

The neurodevelopmental outcome of term infants with acute life-threatening events (ALTE) and preterm infants with persistent apnea of prematurity were compared to matched control populations. No differences in developmental outcomes were seen between the study and control populations.

Visitation to a neonatal intensive care unit.

Preterm newborns may experience extended periods of hospitalization which disrupt the normal early contact between the newborn and its family. Variations in the frequency of visits to 164 preterm neonates in a neonatal intensive care unit were examined in relation to infant and family status variables and compliance with follow-up appointments at 3 months postterm. The mean number of visitors decreased from day 2 to day 12 of hospitalization and then remained stable through day 21. There was a corresponding increase in the number of days with no visitors through day 12, and then stabilization. Neonates who had intraventricular hemorrhages, whose parents did not live together, and who were not firstborn had the most days with no visitors. While the mother was hospitalized herself, her condition was the only variable related to percentage of no-visitor days. The sicker the mother, the more likely the newborn had no visitors. The greater the number of days with no visitors, the poorer the likelihood that the infant was brought to a 3-month follow-up clinic appointment.