Refined characterization and reference values of the pediatric T- and B-cell compartments.

Work in the past years has led to a refined phenotypical description of functionally distinct T- and B-cell subsets. Since both lymphocyte compartments are established and undergo dramatic changes during childhood, redefined pediatric reference values of both compartments are needed. In a cohort of 145 healthy children, aged 0-18 years, the relative and absolute numbers of the various T- and B-cell subsets were determined. In addition, we found that besides thymic output, naive (CD27(+)CD45RO(-)) T-cell proliferation contributed significantly to the establishment of the naive T-cell compartment. At birth, regulatory (CD25(+)CD127(-)CD4(+)) T cells (Tregs) mainly had a naive (CD27(+)CD45RO(-)) phenotype whereas 'memory or effector-like' (CD45RO(+)) Tregs accumulated slowly during childhood. Besides the CD27(+)IgM(+)IgD(+) memory B-cell population, the recently identified CD27(-)IgG(+) and CD27(-)IgA(+) memory B-cell populations were already present at birth. These data provide reference values of the T- and B-cell compartments during childhood for studies of immunological disorders or immune reconstitution in children.

Effect of catheter tip length and position on lesion volume in temperature controlled RF ablation in canine tricuspid valve annulus.

BACKGROUND: Radiofrequency (RF) energy has been delivered to the tricuspid valve annulus (TVA) in humans with both 4 mm and 8 mm long catheter tip electrodes to treat atrial flutter. However, lesion volume with temperature controlled RF delivery systems has not been previously characterized. METHODS: In 10 anesthetized canines, a single pulse of temperature controlled RF energy at a 70 degrees C set point, 30 second duration was delivered with either a 7 Fr/4 mm tip or a 7 Fr/8 mm tip electrode in a position both anterolateral and posteroseptal to the tricuspid valve annulus (TVA). Surface echocardiogram was obtained prior and after ablation. The animals were sacrificed after ablation and the lesions underwent gross and histological examination. RESULTS: Lesion size, tip temperature and power were related to tip electrode surface area (SA). Eight mm tips (SA = 59 mm2) tended to create significantly larger lesions than 4 mm tips (SA = 29 mm2). Median lesion volume was 22 vs. 1.5 mm3, respectively. Eight mm tips were also associated with higher power requirements and lower temperatures than 4 mm tips. Posteroseptal TVA lesions tended to be larger than anterolateral lesions. No significant complications were noted. CONCLUSIONS: Using temperature controlled RF ablation, large lesions may be safely created on the canine TVA using 7 Fr catheters with 8 mm long tips.

Segment by segment linear ablation with an articulated bilimb catheter: initial experimental results.

Transcatheter radiofrequency (RF) ablation of atrial fibrillation or flutter requires the creation of linear lesions. However, conventional catheters are not predictably effective because of poor endocardial contact, and limited lesion size and penetration. The purpose of the study was to assess in the right atrium, the efficacy and safety of a new catheter designed to create long myocardial tissue lesions using RF energy. The main characteristics of this 8 Fr deflectable RF ablation catheter were: (1) a perpendicularly contacting articulated bilimb electrodes ensuring stable and firm endocardial firm contact; and (2) an irrigated planar interface. Three different electrode prototypes were tested. Fourteen anesthetized sheep weighing 61 +/- 7 kg underwent RF ablation in the right atrium using three incremental power levels (25 to 45, 50, 55 to 75 W) with the aim of creating fully transmural (FT) lesions, defined as continuous and complete epicardial and endocardial lesion imprints. The animals were euthanized 1 hour later for macroscopic and histologic examinations. Forty-three of the 80 right atrial lesions created, in smooth as well as in trabeculated areas, were FT. The percentage of FT lesions increased with the applied power from 37% with 25-45 W, to 49% with 50 W, and up to 86% for 55-75 W. In all but two cases, histologic examination showed no discontinuity between FT lesions produced by both limbs resulting in 19 coalescent linear lesions with an average size of 25 x 6 x 4 mm (length x width x depth). In situ bipolar electrograms after ablation in the FT group showed split potentials and/or a marked decrease in amplitude from 2.85 +/- 1.79 to 0.33 +/- 0.14 mV (P = 0.001) accompanied by near complete disappearance of unipolar electrograms. There was no perforation due to RF ablation. In conclusion, continuous and FT lesions can be achieved in various areas of sheep atria, including the trabecular right atrium, with a perpendicularly contacting bilimb electrode catheter. It represents a promising tool for catheter ablation of atrial fibrillation and flutter.

Temperature controlled RF ablation in canine ventricle and coronary sinus using 7 Fr or 5 Fr ablation electrodes.

The safety and lesion volume of temperature controlled radiofrequency ablation (TCRFA) in the right ventricle (RV), left ventricle (LV), and coronary sinus (CS) comparing long 5 Fr to standard tip electrodes have not been previously reported. In 10 canines, TCRFA was delivered at a 70 degrees C set point for 30 seconds. Lateral and septal RV lesions were made with either a 5 Fr/5 mm or 7 Fr/4 mm tip. Lateral and septal LV lesions were made with either a 5 Fr/7 mm or 7 Fr/4 mm tip. Proximal and distal CS lesions were made with either a 7 Fr/4 mm, 5 Fr/5 mm or 5 Fr/7 mm tip. Gross and histologic examination of the lesions was completed. Lesion size, tip temperature and power required are related to electrode surface area (SA) when ablating in the RV, LV or CS. 5 Fr/7 mm tips (SA = 36 mm2) tended to create larger lesions than 7 Fr/4 mm tips (SA = 29 mm2) in the LV. 7 Fr4 mm tips tended to create larger lesions than 5 Fr/5 mm tips (SA = 26 mm2) in the RV. 7 Fr/4 LV lesions exceeded 7 Fr/4 mm RV lesions due to thicker LV walls. In the CS, 5 Fr/7 mm tips tended to create the largest lesions. In the RV, LV and CS, tips with larger SA tended to have lower temperatures and require higher power. No high temperature or high impedance shut-downs were observed. In conclusion, varying 5 Fr tip length can safely produce larger or smaller lesions compared to those created with 7 Fr/4 mm tips.

Interactions among genes regulating ovule development in Arabidopsis thaliana.

The INNER NO OUTER (INO) and AINTEGUMENTA (ANT) genes are essential for ovule integument development in Arabidopsis thaliana. Ovules of ino mutants initiate two integument primordia, but the outer integument primordium forms on the opposite side of the ovule from the normal location and undergoes no further development. The inner integument appears to develop normally, resulting in erect, unitegmic ovules that resemble those of gymnosperms. ino plants are partially fertile and produce seeds with altered surface topography, demonstrating a lineage dependence in development of the testa. ant mutations affect initiation of both integuments. The strongest of five new ant alleles we have isolated produces ovules that lack integuments and fail to complete megasporogenesis. ant mutations also affect flower development, resulting in narrow petals and the absence of one or both lateral stamens. Characterization of double mutants between ant, ino and other mutations affecting ovule development has enabled the construction of a model for genetic control of ovule development. This model proposes parallel independent regulatory pathways for a number of aspects of this process, a dependence on the presence of an inner integument for development of the embryo sac, and the existence of additional genes regulating ovule development.

A comparison of bronchial drainage treatments in cystic fibrosis.

We compared standard chest physical therapy and postural drainage (CPT/PD) with a new airway clearance therapy called high-frequency chest wall oscillation (HFCWO) in a group of stable cystic fibrosis (CF) patients. In this crossover trial, 29 CF patients (15 males, 14 females), aged 7-47 years that met the inclusion criteria were randomly assigned to alternate CPT/PD and HFCWO, on a daily basis, over a 4 day period. Each patient received 2 days of each form of therapy; treatment frequency and the length of treatment were the same for both techniques. Expectorated secretions were collected during each 30 minute therapy session and for 15 minutes following treatment. The wet and dry weights of collected secretions were determined gravimetrically, and the therapy methods were compared. Significantly more sputum was expectorated during HFCWO than during CPT/PD as determined by both the wet (P < 0.001) and the dry (P < 0.01) measurements. This study suggests that HFCWO is at least as effective as manual CPT/PD in clearing secretions from the airways in patients with cystic fibrosis.

A national survey of cigarette smoking in fourth-form school children in New Zealand.

AIMS: To determine the prevalence of cigarette smoking in 14 and 15 year old school children in New Zealand and to examine associated risk factors. METHODS: Nationwide cross-sectional survey of fourth-form school children in New Zealand by means of an anonymous self administered questionnaire in November 1992. RESULTS: Questionnaires from 14,097 fourteen and fifteen year-olds were analysed. 65.6% had tried smoking, and 36.1% regarded themselves as smokers. Females and Maori had significantly higher prevalence rates. Of Maori females 44.6% were current smokers (more than one per month) compared to 24.0% for the whole group, and 33.0% were daily smokers. Pacific Island students, who have similar socioeconomic disadvantage to Maori, have a lower relative risk of smoking (RR) 0.79, (95% confidence interval (CI) 0.68, 0.91), than Europeans. Major independent risk factors were identified and population attributable risk was calculated for parental smoking (22.9%), poor knowledge of adverse health effects (7.3%) and watching televised sports (13.4%). These three modifiable factors accounted for 36.1% of the total smoking prevalence in these children. CONCLUSION: The continued high prevalence of smoking in New Zealand children, especially in Maori and in females, prove current public health measures to be inadequate. Our results suggest that strategies aimed at decreasing parental smoking, improving student knowledge of adverse health effects and preventing tobacco sponsorship of television sports could greatly decrease the smoking prevalence in this age group.

The Arabidopsis SUPERMAN Gene Mediates Asymmetric Growth of the Outer Integument of Ovules.

Arabidopsis superman (sup, also referred to as floral mutant10) mutants have previously been shown to have flowers with supernumerary stamens and reduced carpels as a result of ectopic expression of the floral homeotic gene APETALA3 (AP3). Here, we report that sup mutations also cause specific alterations in ovule development. Growth of the outer integument of wild-type ovules occurs almost exclusively on the abaxial side of the ovule, resulting in a bilaterally symmetrical hoodlike structure. In contrast, the outer integument of sup mutant ovules grows equally on all sides of the ovule, resulting in a nearly radially symmetrical tubular shape. Thus, one role of SUP is to suppress growth of the outer integument on the adaxial side of the ovule. Genetic analyses showed that the effects of sup mutations on ovule development are independent of the presence or absence of AP3 activity. Thus, SUP acts through different mechanisms in its early role in ensuring proper determination of carpel identity and in its later role in asymmetric suppression of outer integument growth.

Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases).

Using an in situ phosphorylation assay with zucchini (Cucurbita pepo L. cv Dark Green) seedling tissue, we have identified numerous polypeptides that are capable of acting as protein kinases. Total protein preparations from different organs contain different kinase profiles, but all are within the range of 55 to 70 kD. At least four kinases are associated with highly purified plasma membranes from etiolated zucchini hypocotyls. The major phosphorylated polypeptides from plasma membranes range in apparent molecular mass from 58 to 68 kD. The plasma membrane kinases are activated by micromolar concentrations of calcium and phosphorylate serine, and, to a lesser extent, threonine residues. These characteristics are similar to those of a soluble calcium-dependent protein kinase that has been purified to homogeneity from soybean suspension cultures. Three of the zucchini plasma membrane kinases share antigenic epitopes with the soluble soybean kinase. The presence of kinase activity at different apparent molecular masses may be indicative of separate kinases with similar characteristics. The zucchini hypocotyl protein kinases are not removed from plasma membrane vesicles by 0.5 M NaCl/5 mM ethylenediaminetetraacetate or by detergent concentrations below the critical micelle concentration of two types of detergent. This indicates that the plasma membrane protein kinases are tightly associated with the membrane in zucchini seedlings.

The altered gravitropic response of the lazy-2 mutant of tomato is phytochrome regulated.

Shoots of the lazy-2 (lz-2) gravitropic mutant of tomato (Lycopersicon esculentum Mill.) have a normal gravitropic response when grown in the dark, but grow downward in response to gravity when grown in the light. Experiments were undertaken to investigate the nature of the light induction of the downward growth of lz-2 shoots. Red light was effective at causing downward growth of hypocotyls of lz-2 seedlings, whereas treatment with blue light did not alter the dark-grown (wild-type) gravity response. Downward growth of lz-2 seedlings is greatest 16 h after a 1-h red light irradiation, after which the seedlings begin to revert to the dark-grown phenotype. lz-2 seedlings irradiated with a far-red light pulse immediately after a red light pulse exhibited no downward growth. However, continuous red or far-red light both resulted in downward growth of lz-2 seedlings. Thus, the light induction of downward growth of lz-2 appears to involve the photoreceptor phytochrome. Fluence-response experiments indicate that the induction of downward growth of lz-2 by red light is a low-fluence phytochrome response, with a possible high-irradiance response component.

In vitro resistance of the brown Norway rat acute myelocytic leukemia (BNML) to lymphokine-activated killer activity.

In in vivo allogeneic bone marrow transplantation studies with the Brown Norway (BN) rat as recipient and the WAG/Rij rat as allogeneic donor a significant graft-versus-leukemia (GVL) effect is observed. Studies were performed to investigate whether lymphokine-activated killer (LAK) cells play a role in this GVL effect. Splenocytes from WAG/Rij and BN rats were activated in vitro by recombinant human interleukin-2 (rhIL-2) for 5-6 days. The cytolytic activity of these LAK cells was tested on four rat solid tumor cell lines, i.e. an ureter carcinoma, a rhabdomyosarcoma, and two lung tumors, and on leukemic cells derived from the BN rat acute myelocytic leukemia (BNML) and the WAG/Rij acute lymphocytic leukemia (L4415). The panel of target cells also included the murine cell lines P815 and YAC. Both WAG/Rij and BN LAK cells were not capable of lysing the leukemic cells in contrast to significant cytolytic activity on the rat solid tumor cell lines and P815 and YAC. BNML cells showed to be resistant to lysis by human NK cells. Phenotypical analysis of the rat LAK population revealed a decrease in the CD4/CD8 ratio compared to the unstimulated splenocyte population. Rat LAK cells displayed no antibody-dependent cellular cytotoxicity (ADCC) on the leukemic cells, whereas IL-2-stimulated human peripheral blood cells showed moderate ADCC activity on the leukemic cells. To investigate whether cytokines play a role in lysis of leukemic target cells, graded numbers of LAK cells and leukemic cells were co-cultivated for seven days in an agar-based colony culture system. This resulted in moderate suppression of leukemic colony formation. From the current in vitro studies it appears that the graft-versus-leukemia observed in in vivo allogeneic bone marrow transplantation studies is probably not due to a direct leukemic cell kill by LAK cells.

Alternative RNA splicing that is spatially regulated: generation of transcripts from the Antennapedia gene of Drosophila melanogaster with different protein-coding regions.

We have shown previously that transcription of the Drosophila homeotic gene Antennapedia results in four major RNA species which differ in long 5'- and 3'-untranslated sequences. The protein-coding portion of these transcripts, however, is located in exons common to all. Using RNase protection assays and further cDNA clone isolation, we have now detected two alternative splicing events between exons of this region. These result in four RNA variations which, if translated, would encode a family of Antennapedia proteins. By analyzing transcripts from various developmental stages and isolated tissues, we show that alternative splicing is under strict temporal and spatial regulation. For example, while similar patterns of splicing were found for all wild-type thoracic imaginal disks examined, these differed distinctly from the patterns observed in neural tissues. Our results suggest that individual RNAs may be associated with different biological roles, and provide molecular evidence that the Antennapedia gene is involved in multiple functions.

Congenital muscular torticollis: results of conservative management with long-term follow-up in 85 cases.

A retrospective review of 277 patients with congenital muscular torticollis seen between 1970 and 1982 was conducted. In 85 cases this was supplemented by questionnaires and recent photographs, permitting a two- to 13-year follow-up. The first visit for 81.6% of patients was before six months of age. All were enrolled in a specific physical therapy program at the time of the first visit, unless they presented with severe torticollis after 12 months of age. Torticollis was mild to moderately severe in 90.6% of cases. Sternomastoid fibrotic nodules were present in 38.6%, more frequently in the more severe cases. Hip dysplasia increased in direct relation to severity and occurred in 10.5% of cases. At 12 months the torticollis had been conservatively resolved in nearly 70% of patients regardless of severity and presence or absence of focal fibrosis. Tenotomies were indicated in only ten children, eight of whom had first been seen after 12 months of age. Long-term sequelae were mild and consisted of craniofacial asymmetry, intermittent head tilt, and mild scoliosis. Developmental asymmetry or high tone due to limited mobility in the cervical spine were noted in 25.3% of infants initially and tended to subside with appropriate therapy. However, 11.8% of patients with long-term follow-up showed persistent functional asymmetry of the involved body side despite mild or moderate severity, early diagnosis, and complete resolution of the torticollis. Long-term observations indicate that congenital torticollis rarely requires surgical treatment.

Radiosensitivity of cells in recurrent experimental tumours and the effectiveness of tumour retreatment.

Two experimental tumour models, a rat rhabdomyosarcoma (R-1) and a rat urether carcinoma (RUC-2) have been employed to evaluate the X-ray sensitivity of tumours recurrent after primary treatments with various doses of X-rays and to correlate changes in volume responses with the cellular radiosensitivity. The responsiveness of R-1 tumours, assessed from the volume reduction as a function of the time after treatment, was less for recurrent tumours, but their growth delay was slightly increased, while the X-ray sensitivity of the tumour cells, assessed by cell survival, was equal to that of the controls. For RUC-2 tumours, however, the reduction in volume after irradiation of the recurrent tumour was larger than after primary treatment, the growth delay was increased, but cell survival curves were not significantly different from those of the controls. It is concluded that differences in volume responses between untreated tumours and recurrent tumours are largely determined by a tumour bed effect (TBE) and that changes in cellular radiosensitivity in these tumours do not play a significant part.