Depletion of CD4+CD25+CD127lo regulatory T cells does not increase allergen-driven T cell activation.

BACKGROUND: It has been suggested that allergic diseases are caused by defective suppression of allergen-specific Th2 cells by CD4(+)CD25(+) regulatory T cells. However, such studies have been hampered by the difficulty in distinguishing regulatory T cells from CD25-expressing activated T cells. Recently, it was shown that conventional T cells expressed high levels of CD127, whereas regulatory T cells were CD127(lo), allowing discrimination between these distinct T cell subpopulations. OBJECTIVE: The aim of this study was to study whether the putative regulatory subset defined as CD4(+)CD25(+)CD127(lo) was involved in grass pollen-reactive T cell responses. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from allergic donors and non-atopic controls out of season. Grass pollen-induced cytokine production and proliferation were compared in cultures of undepleted cells and cells depleted of CD4(+)CD25(+), CD4(+)CD25(+)CD127(hi) or CD4(+)CD25(+)CD127(lo) T cells. RESULTS: Undepleted cell cultures from allergic patients showed significantly increased proliferation and Th2 cytokine production compared with non-atopic controls. Depletion of all CD25(+) T cells did not increase cytokine production or proliferation, and more importantly, no increase in Th2 cytokine production or proliferation was observed in cell cultures depleted of CD4(+)CD25(+)CD127(lo) cells (putative regulatory T cells) compared with undepleted PBMCs in both the allergic and the non-atopic group. CONCLUSION: Our study showed that T cells from grass pollen-allergic patients and non-atopic controls responded very differently to grass pollen extract, but this difference could not be explained by differences in regulatory T cell function. Further studies are needed to understand the importance of regulatory T cells in allergy.

Muramyldipeptide modulates CXCL-8 release of BEAS-2B cells via NOD2.

Chronic inflammation and acute exacerbations are pathophysiological features of chronic obstructive pulmonary disease (COPD). An impaired immune response to bacterial pathogens can contribute to both of them. Nucleotide oligomerization domain 2 (NOD2) is an intracellular receptor of innate immunity for muramyldipeptide (MDP). Mutations of the NOD2 gene followed by decreased recognition of MDP are associated with chronic intestinal inflammation and pulmonary complications of patients with allogenic stem cell transplant and sepsis. Our study provides evidence that NOD2, toll-like receptor 4 (TLR4) and the adapter protein receptor-interacting protein 2 (RIP2) are induced by tumor-necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the bronchial epithelial cell line BEAS-2B. We also demonstrate that lipopolysaccharide (LPS) can further increase NOD2 transcription in a TNF-alpha and IFN-gamma-induced activation state. In addition, we show that, while MDP fails to enhance CXCL-8 release from otherwise unstimulated BEAS-2B cells, a 12 h prestimulation period with TNF-alpha and IFN-gamma primes the cells for an additional increase of CXCL-8 secretion via induction of NOD2 and RIP2. LPS itself significantly augments CXCL-8 production and co-administration of MDP further increases cytokine secretion. Finally, overexpression of an SNP13 mutant decreased MDP-induced chemokine production in BEAS-2B cells compared with NOD2 wild type overexpression. Taken together, our work indicates that MDP and NOD2 play an important role for CXCL-8 release of BEAS-2B cells following LPS-challenge via synergistic interactions between MDP and LPS.

Reduction of endogenous transforming growth factors beta prevents ontogenetic neuron death.

We show that following immunoneutralization of endogenous transforming growth factors beta (TGF-beta) in the chick embryo, ontogenetic neuron death of ciliary, dorsal root and spinal motor neurons was largely prevented, and neuron losses following limb bud ablation were greatly reduced. Likewise, preventing TGF-beta signaling by treatment with a TbetaR-II fusion protein during the period of ontogenetic cell death in the ciliary ganglion rescued all neurons that normally die. TUNEL staining revealed decreased numbers of apoptotic cells following antibody treatment. Exogenous TGF-beta rescued the TGF-beta-deprived phenotype. We conclude that TGF-beta is critical in regulating ontogenetic neuron death as well as cell death following neuronal target deprivation.

Characterization of bone morphogenetic protein family members as neurotrophic factors for cultured sensory neurons.

The bone morphogenetic proteins have been implicated in several inductive processes throughout vertebrate development including nervous system patterning. Recently, these proteins have also emerged as candidates for regulating survival of mesencephalic dopaminergic and sympathetic neurons. Interestingly, we have found that several bone morphogenetic proteins can be detected in developing embryonic day 14 rat dorsal root ganglia by means of reverse transcription-polymerase chain reaction and immunocytochemistry. To further elucidate their potential role during the period of ontogenetic neuron death, serum-free cultures of dorsal root sensory neurons from developing chick and rat embryos were treated with distinct bone morphogenetic proteins with or without simultaneous addition of other "established" neurotrophic factors. Our results show that bone morphogenetic proteins exert survival promoting effects on their own, and that they can positively modulate the effects of neurotrophins on sensory neurons. In particular, growth/differentiation factor-5, bone morphogenetic protein-2, -4, -7 and -12 significantly increased the survival promoting effects of neurotrophin-3 and nerve growth factor on cultured dorsal root ganglion neurons. These results fit well into the current concept that neurotrophic factors may act synergistically in ensuring neuronal survival. Moreover, these data suggest potential instructive interactions of bone morphogentic proteins and neurotrophins during sensory neuron development. Finally, the documented neurotrophic capacity of bone morphogenetic protein family members may have potential relevance for the treatment of peripheral neuropathies.

Heparin-binding epidermal growth factor-like growth factor: a component in chromaffin granules which promotes the survival of nigrostriatal dopaminergic neurones in vitro and in vivo.

Chromaffin cells can restore function to the damaged nigrostriatal dopaminergic system in animal models of Parkinson's disease. It has been reported that a protein which is released from chromaffin granules can promote the survival of dopaminergic neurones in vitro and protect them against N-methylpyridinium ion toxicity. This neurotrophic effect has been found to be mediated by astroglial cells and blocked by inhibitors of the epidermal growth factor (EGF) receptor signal transduction pathway. Here we report the identification of bovine heparin-binding EGF-like growth factor (HB-EGF) in chromaffin granules and the cloning of the respective cDNA from bovine-derived adrenal gland. Protein extracts from bovine chromaffin granules were found to promote the survival of embryonic dopaminergic neurones in culture, to the same extent as recombinant human HB-EGF. Furthermore, the neurotrophic action of the chromaffin granule extract could be abolished by antiserum to recombinant human HB-EGF. We also show that intracerebral injection of recombinant human HB-EGF protected the nigrostriatal dopaminergic system in an in vivo adult rat model of Parkinson's disease. Intracerebral administration of this protein at the same time as a 6-hydroxydopamine lesion of the medial forebrain bundle was found to spare dopamine levels in the striatum and tyrosine hydroxylase-immunopositive neurones in the midbrain. This study has found that the main component in chromaffin granules responsible for their neurotrophic effect on dopaminergic neurones is HB-EGF. Furthermore, HB-EGF has significant protective effects on nigrostriatal dopaminergic neurones in vivo, making it a potential candidate for use in the treatment of Parkinson's disease.

Findings in children exposed in utero to phenytoin and carbamazepine monotherapy: independent effects of epilepsy and medications.

Our objective was to evaluate the patterns of malformations in children exposed in utero to phenytoin (DPH) and carbamazepine (CBZ) monotherapy, and to compare them prospectively with matched mother-child pairs exposed to nonteratogens, and to separate the effects of antiepileptic drugs (AEDs) from those of epilepsy by collecting groups of untreated epileptics and those treated with DPH and CBZ for conditions other than epilepsy. This was a prospective, controlled, and blinded observational study. Thirty-six mother-child pairs exposed to CBZ monotherapy, 34 pairs exposed to DPH monotherapy, and 9 nonmedicated epileptic women and their children were compared with matched mother-child pairs exposed to nonteratogens. The control mothers were matched for maternal age, time of consultation, obstetric history, and socioeconomic status (SES). One main outcome measures a "blinded" morphological assessment of the offspring. We found that minor anomalies were significantly more common among children of epileptics on either drug (P = 0.01) and among DPH-treated nonepileptic offspring (P = 0.03). Among epileptics, the relative risk for minor anomalies following DPH (2.1) was similar to that after exposure to either DPH (P = 0.006) or CBZ (P = 0.01). Increased rates of hypertelorism were detected among DPH-exposed offspring. High forehead, frontal bossing, malar hypoplasia, epicanthus and micrognathia were associated with untreated epilepsy, as well as with DPH and CBZ treatment.

Craniofacial variability index: a simple measure of normal and abnormal variation in the head and face.

We propose a numerical means of increasing the objectivity of describing, characterizing, and evaluating craniofacial morphology, including dysmorphology. A craniofacial variability index (CVI) can be created for an individual by obtaining a series of anthropometric measurements of the head and face, converting each measurement value to a standardized z-score, and then calculating the standard deviation for the whole set of these z-score values. This value is lowest for those faces in which all of the z-scores are in the same direction and of relatively the same magnitude and highest for faces with pronounced difference in direction and size of the individual z-scores. The CVI has a range of values with an approximately normal distribution in a reference population of 1,312 individuals. Examination of a small sample of individuals with known craniofacial syndromes indicates that the CVI in such cases consistently falls outside of the normal range of the index, and its value is highest in individuals with the greatest subjective degree of facial involvement. Finally, the CVI is robust: age, sex, size of the individual, and changes in the number of variables used in its calculation have little impact on its distribution. When used in conjunction with traditional clinical assessment, the CVI has a number of potential clinical applications including initial diagnostic screening, demonstrating age-related changes in postnatal development of patients with facial syndromes, and pre- and post-surgical assessments of individuals with craniofacial anomalies.

Anthropometric craniofacial pattern profiles in Down syndrome.

A series of 21 anthropometric craniofacial measurements was performed on 199 individuals with Down syndrome (DS), age 6 months to 61 years. These were compared to age and sex-matched normal standards, and Z score pattern profiles were constructed. These profiles confirmed brachycephaly and reduced ear length. With increasing age, maxillary growth was reduced in comparison to mandibular growth. Clinically, this was manifested by a change in facial shape from the characteristic round face of infancy to an oval shape in later life. Stepwise forward discriminant function analysis identified a subset of three variables (ear length, maxillary arc, and upper facial depth) which could accurately classify greater than 99% of the individuals in the combined sample of affected and unaffected individuals. Of the subjects with DS, 96.8% were classified correctly. These findings demonstrate the usefulness of anthropometric craniofacial pattern profiles in defining abnormal facial dimensions in particular syndromes and documenting the changes that occur with age. The technique should facilitate syndrome recognition, identification of carriers, and comparisons between syndromes.

A comprehensive scoring system for evaluating Noonan syndrome.

A multidisciplinary team assessed 23 patients with various manifestations of the Noonan syndrome, including pulmonary valve stenosis (with leaflet dysplasia), "typical" facial appearance (including hypertelorism, epicanthic folds, flat nasal bridge, and apparently low-set ears), short stature, and mental retardation. Seven patients had a family history of the syndrome. A comprehensive scoring system was devised on the basis of frequency and severity of manifestations and results of invasive and noninvasive tests in these patients and those reported in the literature. The scoring system was condensed into a score card for clinical use and validated by "blind" application to patients with isolated pulmonary valve stenosis or suspected Noonan syndrome. Use of a scoring system to diagnose a syndrome for which there is no specific diagnostic test facilitates accuracy and decreases observer bias. In the case of unusual congenital disorders it is particularly valuable for a pediatrician in general practice.

Genetic latent structure analysis of dysmorphology in attention deficit disorder.

Dysmorphology--in the form of minor physical anomalies--has been frequently reported in children with attention deficit disorder (ADD). The authors report an overrepresentation of minor physical anomalies in both ADD probands and their first-degree relatives. Further, ADD probands who are not dysmorphic have non-ADD relatives who are dysmorphic; this familial pattern suggests that a single underlying factor may influence transmission of both traits. A genetic latent structure model was fit to these data to describe the factor's mode of transmission. In this analysis, an autosomal dominant model emerged. Successfully fitting this model is not equivalent to testing the validity of the model itself. Meaningful tests of the model will require larger samples than available at present, and would benefit from diagnostic refinement of the ADD and dysmorphic phenotypes.

Characterization of growth/differentiation factor 5 (GDF-5) as a neurotrophic factor for cultured neurons from chicken dorsal root ganglia.

Growth/differentiation factor-5 (GDF-5), a morphogenetic protein, has previously been shown to act as a neurotrophic factor for midbrain dopaminergic neurons. To further elucidate the neurotrophic potential of GDF-5, serum free cultures of dorsal root ganglionic (DRG) neurons from developing chick embryos were treated with GDF-5 with or without the simultaneous addition of other trophic factors. Our results show that GDF-5 has a minor promoting effect on its own, but it can enhance the survival promoting effect of neurotrophin-3 (NT-3) and nerve growth factor (NGF) on cultured DRG neurons. Our finding fits well into the concept that neurotrophic factors may act synergistically in ensuring survival of different neuronal populations. The capacity of GDF-5 to reduce the requirement of a subpopulation of sensory neurons for NT-3 may have implications for the treatment of peripheral neuropathies.

Natural accommodation in the growing chicken.

A new technique, Infrared Photoretinoscopy, has been employed for recording natural accommodation in the chicken. The illumination of the pupil by the fundus reflection of infrared light provided by high output light emitting diodes (I.R. LED's) was monitored on a video screen. The defocus of the eye could be calculated by evaluating the fraction of the pupil which was illuminated. It was found that: in the chick the full range of accommodation (about 17D) is present in the first day after hatching, accommodation acts completely independently in both eyes, the "near pupillary response" is weaker in younger chicks, the pupil constriction in response to light starts at higher luminance in the younger chicks the developmental decrease of the f/number is not sufficient to explain the change of the pupil reaction to light. Problems resulting from the use of drugs in order to measure the refractive state using normal retinoscopy are discussed.

The role of the benzodiazepine-GABA system in the memory processes of the day-old chick.

This series of experiments investigated the effect of the benzodiazepine diazepam on memory formation in day-old chicks trained on a single-trial, passive-avoidance task. The findings indicate that diazepam has a dose-specific and time-dependent effect on memory processes. A 0.125-mg/kg dose of diazepam administered immediately after training led to amnesia in these subjects only after 30 min following learning. Pretreatment with bicuculline and flumazenil were effective in ameliorating the memory deficits caused by diazepam, and consolidated memory function in saline-treated controls following strong and weak aversant training. These findings suggest that benzodiazepine effects on memory are mediated by their effects on arousal, possibly by the release of noradrenaline, which is critical to the establishment of long-term memory.

External septorhinoplasty in children: Outcome and effect on growth of septal excision and reimplantation.

Outcome and effect on nasal growth of external septorhinoplasty was evaluated in 32 children. All had septal disease anterior to the nasal spine. In all cases, the cartilaginous septum was totally excised, refashioned, and then reinserted. Sixteen children with follow-up for more than 2 years were identified. Nine children had preoperative and postoperative nasal airflow studies and demonstrated a reduction in total nasal airway resistance, from a mean untreated value of 6.1 cm of water per centimeter per second preoperatively to a mean of 2.5 cm of water per centimeter per second postoperatively. IN 10 of these 16 children, six postoperative anthropometric measures and one index were determined, and these measurements were within the range of age- and sex-specific normative data from the Craniofacial Measurements Laboratory at the Hospital for Sick Children, Toronto, Ontario.

Nasal growth after external septoplasty in children.

OBJECTIVE: To assess the impact of external septoplasty surgery on nasal growth in children. DESIGN: Twelve anthropometric measurements (9 linear and 3 angular) were obtained in patients who previously underwent external septoplasty surgery for severe nasal obstruction caused by septal deviation anterior to the nasal spine. Surgery consisted of excision, refashioning, and reinsertion of the quadrilateral cartilage. From these 12 measurements, 5 proportional indexes were calculated, and then all measurements and proportions were compared with previously published norms. Follow-up measurements were taken at least 2 years after surgery (average, 3.4 years). SETTING: The Hospital for Sick Children, Toronto, Ontario, a tertiary care children's hospital. PARTICIPANTS: Twenty-eight patients who underwent external septoplasty surgery between the ages of 6 and 15 years. RESULTS: The principal measurements of the face and nose were within 1 SD of the normative mean for the majority of those in the study group. This was true for nasal height, nasal tip protrusion, nasal width, columella width, columella length, upper face height, face height, face width, inclination of the upper face, inclination of the nasal dorsum, and inclination of the columella. Values for 4 of the 5 proportional indexes were also overwhelmingly in the normal range. Twenty-nine percent of nasal dorsum measurements and 57% of nasal dorsum indexes were more than 2 SDs from the mean, indicating a predominance of short nasal dorsums. CONCLUSIONS: External septoplasty does not affect most aspects of nasal and facial growth, but it may negatively influence growth of the nasal dorsum. Prospective studies are needed to clarify this issue.

A study of anthropometric measures before and after external septoplasty in children: a preliminary study.

OBJECTIVE: To test the hypothesis that surgery on the growing nasal septum does not adversely affect nasal and midfacial dimensions. DESIGN: Paired study. SETTING: Tertiary care center. PARTICIPANTS: Children treated consecutively during a 4-year period; all had significant nasal obstruction and cosmetic disfigurement secondary to skeletal septal deformities. INTERVENTION: Nasal septal surgery (using an external approach), in which the quadrilateral cartilage was removed, remodeled, and reinserted as a free graft. OUTCOME MEASURES: Anthropometric linear measurements and indexes of the face and nose preoperatively and postoperatively; nasal dorsum length, nasal height, nasal dorsum index, nasal tip protrusion, columellar length, facial height, face width, upper face height, facial index, nose-upper face height index, and columellar length-nasal tip protrusion index. Continuous measurements were transformed into ordered categories with reference to normative data. Data were analyzed using Wilcoxon signed rank sum test (alpha level of.05) and by applying the Bonferroni adjustment for multiple testing. RESULTS: Twenty-six children were studied (12 females and 14 males); age at surgery ranged from 4.5 to 15.5 years (mean age, 9.5 years); average age at postoperative measurement, 12.5 years; mean follow-up, 3.1 years. Only nasal dorsum length (P =.007) and nasal tip protrusion (P =.04) were decreased by a statistically significant level before the Bonferroni adjustment. The change was not considered clinically significant. Thus, relative to age-appropriate norms, the dimensions of the nose and midface and their proportionality did not change after surgery. CONCLUSIONS: Appropriate nasal septal surgery involving excision and subsequent reinsertion of a remodeled segment of the quadrilateral cartilage has no deleterious effects on development of the nose and midface. We question the absolute dogma that nasal surgery in children must always be avoided.

Phase composition analysis of hydrous aluminium oxides by thermal analysis and infrared spectrometry.

A general method for determination of the phase composition of hydrous aluminium oxides by thermal analysis and infrared spectrometry, and determination of the transformation temperature of mixtures of Al(OH)(3) and AlOOH into alpha-Al(2)O(3) are described.

Occurrence of anaerobic bacterial, clostridial, and Clostridium perfringens spores in raw goose livers from a poultry processing plant in Hungary.

Anaerobic bacterial, clostridial, and Clostridium perfringens spores were enumerated in raw goose liver samples taken after evisceration of the birds (EB) in the slaughterhouse and after removal of blood vessels from the liver (RBVL) in the cannery. The samples taken after RBVL had significantly higher (P < 0.05) spore counts than did those taken after EB, indicating contamination of livers during processing. The number of C. perfringens spores was one log cycle higher in the samples taken after RBVL than in those taken after EB (P < 0.05). The confirmation of C. perfringens according to the profiles of Rapid ID 32 A tests was carried out by means of the ATB Plus computer program. With an identification percentage of 99.9 and a T-value of 0.65, the suspect colonies proved to be C. perfringens. Therefore, the importance of an appropriate cleaning and sanitation program and of personnel hygiene should be emphasized in the industry.

Anthropometry of the normal and defective ear.

Surface measurements of the ear are needed to assess damage in patients with disfigurement or defects of the ears and face. Population norms are useful in calculating the amount of tissue needed to rebuild the ear to adequate size and natural position. Anthropometry proved useful in defining grades of severe, moderate, and mild microtia in 73 patients with various facial syndromes. The division into grades was based on the amount of tissue lost and the degree of asymmetry in the position of the ears. Within each grade the size and position of the ears varied greatly. In almost one-third, the nonoperated microtic ears were symmetrically located, promising the best aesthetic results with the least demanding surgical procedures. In slightly over one-third, the microtic ears were associated with marked horizontal and vertical asymmetries. In cases of horizontal and vertical dislocation exceeding 20 mm, surgical correction of the defective facial framework should precede the building up of a new ear. Data on growth and age of maturation of the ears in the normal population can be useful in choosing the optimal time for ear reconstruction.