Efficient recovery of HLA class I expression in human tumor cells after beta2-microglobulin gene transfer using adenoviral vector: implications for cancer immunotherapy.

Here we report a successful use of a non-replicating adenovirus expressing the wild-type human beta2m gene in recovery of normal human leucocyte antigen (HLA) class I expression in beta2m-null cancer cells. Total loss of HLA class I expression in these cell lines is caused by a mutation in beta2m gene and a loss of heterozygosity in chromosome 15 carrying another copy of that gene. Normal HLA class I expression on the tumour cell surface is critical for the successful outcome of cancer immunotherapy as T cells can only recognize tumour-derived peptides in a complex with self-HLA class I molecules. In this report we characterize the newly generated adenoviral vector AdCMVbeta2m and demonstrate an efficient beta2m gene transfer in tumour cell lines of different histological origin, including melanoma, prostate and colorectal carcinoma. The beta2m re-expression lasted for an extended period of time both in vitro and in vivo in human tumour xenograft transplants. We propose that in a subset of cancer patients with structural defect in beta2m gene or chromosome 15, the adenoviral-mediated recovery (or even increase) of HLA class I expression on tumour cells in combination with vaccination or adoptive T-cell therapy can provide a complementary approach to improve the clinical efficacy of cancer immunotherapy.

A recessive allele inhibiting saponin synthesis in two lines of Bolivian quinoa (Chenopodium quinoa Willd.).

Quinoa cultivars currently grown in North America and Europe require removal of bitter-tasting saponins from the grain prior to human consumption. This need for postharvest processing is a barrier to expanding production of the crop outside its Andean area of origin. Grain saponin content in quinoa shows continuous variation and is considered to be a quantitative trait. However, segregation for the presence or absence of grain saponin in F2 generations derived from crosses between high- and low-saponin parents indicates a major gene effect, with plants homozygous for a recessive allele spl having no detectable grain saponin. Variation in saponin levels among F2 plants with detectable grain saponin was consistent with polygenic inheritance. It appears that grain saponin level in quinoa is both qualitatively and quantitatively controlled, with saponin production requiring at least one dominant allele at the Sp locus and the amount of grain saponin being determined by an unknown number of additional quantitative loci. Introgression of sp1 into day-neutral lines will facilitate the development of short-season "sweet" quinoa cultivars which do not require postharvest processing to remove grain saponin.

Differential expression of C-protein isoforms in the developing heart of normal and cardiac lethal mutant axolotls (Ambystoma mexicanum).

Regulated assembly of contractile proteins into sarcomeric structures, such as A- and I-bands, is still currently being defined. The presence of distinct isoforms of several muscle proteins suggests a possible mechanism by which myocytes regulate assembly during myofibrillogenesis. Of several muscle isoforms located within the A-band, myosin binding proteins (MyBP) are reported to be involved in the regulation and stabilization of thick filaments during sarcomere assembly. The present confocal study characterizes the expression of one of these myosin binding proteins, C-protein (MyBP-C) in wild-type and cardiac lethal mutant embryos of the axolotl, Ambystoma mexicanum. C-protein isoforms are also detected in distinct temporal patterns in whole-mounted heart tubes and thoracic skeletal muscles. Confocal analysis of axolotl embryos shows both cardiac and skeletal muscles to regulate the expression of C-protein isoforms over a specific developmental window. Although the CPROAxslow isoform is present during the initial heartbeat stage, its expression is not retained in the adult heart. C-protein isoforms are simultaneously expressed in both cardiac and skeletal muscle during embryogenesis.

Expression of myosin heavy chain transcripts in normal and cardiac mutant Mexican axolotls.

In this study, we have cloned a 1.0 kb myosin heavy chain (MHC) cDNA by screening an axolotl heart cDNA library with the monoclonal antibody MF20 against a light meromyosin (LMM) region of MHC. The nucleotide sequence analysis shows 85-86% homology at the amino acid and 78-81% homology at the nucleic acid level with MHC from other vertebrates. Phylogenetic analyses suggest that axolotl beta-MHC forms a cluster with the myosin II group of vertebrate striated muscles. Within the myosin II cluster, axolotl beta-MHC forms a distinct subclade from avian MHC and is instead closer to mammalian MHC. RT-PCR analyses show that transcripts of beta-MHC are present at stage 2 and the onset of the MHC gene expression is at stage 8-10 (gastrulation). Expression increases with embryonic development and reaches a maximum at stage 20. Beyond stage 35, the heart-beat initiation stage, the expression level of beta-MHC is higher in cardiac muscle than in skeletal muscle. We could not detect significant differences in the levels of expression of MHC transcripts in normal and cardiac lethal mutant (c/c) axolotls (Ambystoma mexicanum).

Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl, Ambystoma mexicanum.

Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and alpha-actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, local- izes only in skeletal muscle of the axolotl. This slow axolotl isoform (Axslow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C315 antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Axcard2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.

Cloning, sequencing and expression of an isoform of cardiac C-protein from the Mexican axolotl (Ambystoma mexicanum).

C-protein, a myosin binding protein, is thought to regulate and stabilize thick filaments during assembly of sarcomeric A-bands. Multiple isoforms of C-protein have been characterized in avian and mammalian systems. We now report the isolation and the nucleic acid sequence of a partial C-protein cDNA clone from an axolotl heart cDNA expression library in lambda gt11. The clone was isolated by screening the library with a heterologous monoclonal anti-C-protein antibody (MF1). Sequence comparison revealed that CPROAxocard1 has an average sequence identity of 62-68% at the nuclei acid and 72-78% at the amino acid levels respectively to human and chicken sequences. We could not detect any significant differences at the levels of expression of the cardiac isoform of C-protein (CPROAxocard1) in normal and non-beating heart tissues of the double-recessive cardiac lethal mutant (c/c) axolotl, Ambystoma mexicanum. This is the first report of a C-protein sequence from an amphibian species.

Use of nasal continuous positive airway pressure as treatment of childhood obstructive sleep apnea.

OBJECTIVE: To determine the safety and efficacy of nasal continuous positive airway pressure (CPAP) for obstructive sleep apnea (OSA) during childhood and the effects of growth and maturation on CPAP requirements. DESIGN: Retrospective study with use of a written questionnaire administered to pediatric practitioners treating sleep disorders. SETTING: Nine academic pediatric sleep disorders centers. RESULTS: Data were obtained for 94 patients. Three percent of patients receiving CPAP were less than 1 year, 29% were 1 to 5 years, 36% were 6 to 12 years, and 32% were 13 to 19 years of age; 64% were boys. The longest duration of CPAP use was 4 years. Indications for CPAP included OSA associated with obesity (27%), craniofacial anomalies (25%), idiopathic OSA persisting after adenoidectomy and tonsillectomy (17%), and trisomy 21 (13%). Continuous positive airway pressure was effective in 81 patients (86%), in one patient it was unsuccessful, and in 12 patients compliance was inadequate. The median pressure required was 8 cm H2O (range, 4 to 20 cm H2O); pressure requirements were independent of age or diagnosis. Twenty-two percent of patients eventually required a modification of CPAP levels. Complications of CPAP were minor. Sixty-four percent of centers reported difficulty in obtaining funding for CPAP. CONCLUSIONS: Continuous positive airway pressure is safe, effective, and well tolerated by children and adolescents with OSA. Experience in infants is limited. As pressure requirements change with patient growth, we recommend that CPAP requirements be regularly reevaluated over time. The marked center-to-center variability in CPAP use suggests that specific indications for this therapy require clarification.

Incidence of sudden infant death syndrome in infants with sickle cell trait.

The significantly higher incidence of both sickle cell trait (SCT) and sudden infant death syndrome (SIDS) in the black population suggests that SCT and SIDS may be epidemiologically related. To study this possibility, we identified, for the period of February 1990 to February 1992, all infants with SCT born in Los Angeles County whose disease was diagnosed through the California Newborn Screening Program. We matched these infants with all confirmed cases of SIDS in Los Angeles County from February 1990 to March 1993. Three cases of SCT among 589 infants confirmed to have had SIDS were identified. The incidence of SIDS was 1.25/1000 live births for the general population versus 0.58/1000 cases for the SCT group. This finding remained unchanged when rates were adjusted for ethnicity. We conclude that infants born with SCT are not at increased risk of dying of SIDS.

Recurrent apparent life-threatening events during infancy: a manifestation of inborn errors of metabolism.

To determine how often inborn errors of metabolism may cause unexplained apnea or recurrent apparent life-threatening events in infants, we retrospectively reviewed the records of 166 infants who were referred for apnea evaluation. A metabolic disorder was identified in 7 infants (4.2%), all of whom had recurrent apparent life-threatening events.

Prevalence of medium-chain acyl-coenzyme A dehydrogenase deficiency in the sudden infant death syndrome.

Disorders of fatty acid beta-oxidation have been suggested as playing a significant role in the sudden infant death syndrome (SIDS). To elucidate the role of medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency in SIDS, we identified all cases of SIDS occurring in Los Angeles County between January 1986 through December 1991. A total of 1304 SIDS deaths were identified; tissue samples were collected in 1236 cases (94.8%). Extraction of DNA was successful in 1224 tissue samples (93.9%), which were examined for the presence of the G985 mutation, identified as occurring in more than 88% of affected cases of MCAD deficiency. Three heterozygotes and no homozygotes were identified; this incidence does not differ from that reported in the general population. Review of the pathologic specimens from the identified heterozygotes and from 18 ethnic-, age-, and sex-matched control subjects revealed significant fatty infiltration of all organs examined in one of the three heterozygotes and in none of the control subjects. We conclude that MCAD deficiency does not play a significant role in the causation of SIDS.

Use of enzyme immunoassays to compare the effect and assess the dosage regimens of three Brazilian Bothrops antivenoms. The Butantan Institute Antivenom Study Group (BIASG).

The effect of the three main Brazilian polyspecific antivenoms on venom clearance was assessed in 118 moderately envenomed victims of bites by Bothrops species (mainly B. jararaca) in Sao Paulo State, Brazil. Serum samples taken from patients at intervals during their stay in the hospital and at followup approximately four weeks later were tested by enzyme immunoassay for the presence of whole venom and therapeutic antivenom. Results indicated that in patients treated with the standard regimen of either four (40 ml) or eight (80 ml) ampules of each antivenom, venom was cleared from the circulation within four days of antivenom administration. However, high concentrations of antivenom persisted for approximately 10 days and remained detectable until 30-50 days after administration. This suggests that patients may be being treated with excessive amounts of antivenom in Brazil. This practice increases the national cost of antivenom therapy and may contribute to the high frequency of antivenom reactions. Clinically, there was no obvious difference in the efficacy between the three antivenoms.

Responses to hypoxia and hypercapnia in infants of substance-abusing mothers.

Because infants of substance-abusing mothers (ISAM) have an increased risk of sudden infant death syndrome and have abnormal sleeping ventilatory patterns, we studied the effects of mild hypoxia during quiet sleep on ventilatory pattern, heart rate, and arousal in 23 healthy ISAM (mean +/- SEM: 9.0 +/- 0.49 weeks of age) and 15 healthy, similarly aged, control infants. Hypercapnic challenges were performed in six ISAM and eight control subjects. Hypoxic arousal responses were elicited by rapidly decreasing inspired oxygen tension to 80 mm Hg for 3 minutes or until arousal occurred. Failure to arouse to hypoxia occurred in the majority of infants in both groups. All infants had a fall in end-tidal carbon dioxide tension during hypoxia, suggesting that each had a hypoxic ventilatory response. However, the fall in end-tidal carbon dioxide tension was significantly less in the ISAM (mean +/- SEM: -4.0 +/- 0.3 vs -8.0 +/- 1.0 mm Hg), suggesting blunted ventilatory responses to hypoxia. Periodic breathing occurred during 9.5% of hypoxic challenges in control infants compared with 37% in ISAM (p = 0.056). Heart rates were significantly higher in the ISAM before, during, and after hypoxic challenges. Hypercapnic challenges (inspired carbon dioxide tension of 60 mm Hg for a maximum of 3 minutes) resulted in arousal in all infants; however, ISAM required a significantly longer exposure to hypercapnia before arousal (mean +/- SEM; 116 +/- 7.8 vs 79 +/- 13.9 seconds; p < 0.02). We conclude that ISAM have an impaired repertoire of protective responses to hypoxia and hypercapnia during sleep, and that this may play a role in their increased risk for sudden infant death syndrome.

Hypercapnic and hypoxic ventilatory and cardiac responses in school-aged siblings of sudden infant death syndrome victims.

Siblings of sudden infant death syndrome (SIDS) victims have been shown to have abnormal ventilatory patterns and altered responses to respiratory stimuli during infancy. To evaluate whether these abnormalities persist, we studied ventilatory responses in 20 older SIDS siblings (9.8 +/- 0.9 (mean +/- SEM) years of age) and 20 control subjects (10.2 +/- 0.9 years of age). To evaluate hypercapnic ventilatory responses, we had subjects rebreathe 5% carbon dioxide and 95% oxygen until end-tidal carbon dioxide tension reached 65 mm Hg. Instantaneous minute ventilation, mean inspiratory flow, and respiratory rate were calculated breath by breath. Hypercapnic responses did not differ between SIDS siblings (2.08 +/- 0.14 L/min per mm Hg) and control subjects (1.90 +/- 0.10 L/min per mm Hg; not significant). To assess hypoxic ventilatory responses, we asked subjects to rebreathe 13% oxygen and 7% carbon dioxide, with the balance nitrogen, at mixed-venous end-tidal carbon dioxide tension, until arterial oxygen saturation by pulse oximetry fell to 75%. No differences in hypoxic ventilatory responses were found between the SIDS siblings (-1.39 +/- 0.15 L/min/% saturation) and the control subjects (-1.22 +/- 0.17 L/min/% saturation; not significant). The mean inspiratory flow, tidal volume, respiratory rate, and heart rate responses to hypercapnia and hypoxia were also similar in the two groups. We conclude that there is no difference in hypercapnic and hypoxic ventilatory and cardiac responses, as assessed by rebreathing techniques, between school-aged SIDS siblings and control subjects. We speculate that in SIDS siblings the control of breathing is immature during infancy and that they achieve maturity of control and resolution of breathing abnormalities with time.

Sudden infant death syndrome in infants of substance-abusing mothers.

A population-based study was performed to determine whether substance abuse during the perinatal period may be a risk factor for sudden infant death syndrome (SIDS). The incidence of SIDS was studied in 2143 infants of substance-abusing mothers (ISAM) born in Los Angeles County during 1986 and 1987 who were reported to the Los Angeles County Department of Health Services because of a history of drug exposure or positive urine test results in the mother, infant, or both. By comparing the ISAM birth reports with records of autopsy-proven SIDS in Los Angeles County, we found 19 SIDS cases in the population of 2143 ISAM, a SIDS rate of 8.87 cases per 1000 ISAM (95% confidence interval 5.3 to 13.8). This was significantly higher than the SIDS rate for the non-ISAM general population: 396 SIDS deaths among 325,372 live births, an incidence rate of 1.22 cases per 1000 births, p less than 0.00001. The age of ISAM at death was 99 +/- 63 (mean +/- SD) days compared with 91 +/- 52 days for the non-ISAM population (not significant). The incidence of SIDS was significantly greater in male infants, during the winter months, in black infants, and in non-Hispanic white infants in the non-ISAM population. Such differences were not observed in the ISAM group. A greater incidence of symptomatic apnea was reported before SIDS for the ISAM than for the non-ISAM population (22% vs 5.4%, p = 0.022). We conclude that ISAM have a higher incidence of SIDS than the non-ISAM general population. However, it was not possible to separate maternal substance abuse from other confounding variables that may also have had an impact on SIDS risk in the ISAM group.

Evaluation of epiglottoplasty as treatment for severe laryngomalacia.

Six patients with severe laryngomalacia underwent epiglottoplasty. Four of these patients had life-threatening episodes of airway obstruction before surgery; of these, two had required tracheal intubation and one had required cardiopulmonary resuscitation. Two patients had failure to thrive and two had cor pulmonale. Patients had required a mean of two hospitalizations related to upper airway obstruction. We performed polysomnography during a daytime nap, both before and after epiglottoplasty, in all patients. Respiratory effort, arterial oxygen saturation, and end-tidal carbon dioxide pressure were monitored with continuous electrocardiograms and electrooculograms. All patients had abnormal polysomnograms preoperatively. Six patients had obstructive apnea, four had hypoxemia (arterial oxygen saturation less than 90% while breathing room air), and four had hypoventilation (end-tidal carbon dioxide pressure greater than 45 mm Hg) before epiglottoplasty. Mean age (+/- SEM) at epiglottoplasty was 10.3 +/- 5.3 months. No patients had surgical complications. An endotracheal tube was in place for 25 +/- 7 hours postoperatively, and patients were discharged 4 +/- 1 days postoperatively. Polysomnography performed 2.8 +/- 1.0 months after surgery showed that all patients had improved. Two patients had residual, mild episodes of obstructive apnea, and one patient had mild hypoventilation and desaturation. No patient had further life-threatening events or required further hospitalizations after epiglottoplasty. We conclude that epiglottoplasty is an effective and safe treatment for a selected group of patients with severe laryngomalacia.

Abnormal control of ventilation in adolescents with myelodysplasia.

Infants with myelomeningocele have abnormalities in ventilatory control. To determine whether these persist into later life, we studied 14 patients with myelomeningocele and Arnold-Chiari malformation (age 18.0 +/- 0.8 (SE) years), and compared them with 14 control subjects (age 24.0 +/- 0.9 years). Pulmonary function and ventilatory muscle strength did not differ between patients with myelomeningocele and control subjects. Hypercapnic ventilatory responses were significantly lower in the group with myelomeningocele (1.98 L/min/mm Hg) compared with control values (3.33 L/min/mm Hg; p less than 0.01). Hypoxic ventilatory responses (-1.4 L/min/%oxygen saturation of hemoglobin in arterial blood) were not significantly different from control values (-2.14 L/min/%oxygen saturation). In control subjects the hypercapnic and hypoxic ventilatory responses were highly correlated with each other within subjects (r = 0.84; p less than 0.002) but not in those with myelomeningocele (r = 0.34; not significant). We concluded that adolescents and young adults with myelomeningocele have abnormalities in control of ventilation during sleep and wakefulness. We speculate that the Arnold-Chiari malformation interferes with central chemosensitivity (hypercapnic ventilatory response) and central integration of chemoreceptor output.

Abnormal ventilatory patterns during sleep in infants with myelomeningocele.

Some infants with myelomeningocele, hydrocephalus, and Arnold-Chiari malformation have symptomatic apnea or hypoventilation. The incidence of abnormalities of the ventilatory pattern during sleep in asymptomatic infants with myelomeningocele is not known. Therefore we performed overnight pneumograms (recordings of ventilatory pattern and electrocardiogram) in 18 asymptomatic infants with myelomeningocele and compared them with pneumograms from 64 control infants. Infants with myelomeningocele had longer total sleep time (596 +/- 16 minutes vs 536 +/- 10 minutes, P less than 0.005), longer episodes of longest apnea (12.6 +/- 0.8 seconds vs 8.1 +/- 0.3 seconds, P less than 0.001), greater total duration of apnea greater than or equal to 6 seconds as percent total sleep time (1.02% +/- 0.18% vs 0.23% +/- 0.03%, P less than 0.001), and lower mean heart rates (120 +/- 5 vs 145 +/- 5, P less than 0.001) than did control infants. No abnormal bradycardia was observed in either group. Thirteen (72%) of 18 infants with myelomeningocele had abnormal pneumograms, compared with 4 (6%) of 64 control infants (P less than 0.0005). We conclude that asymptomatic infants with myelomeningocele have a high incidence of ventilatory pattern abnormalities during sleep.