Population studies of the fragile X: a molecular approach.

The fragile X mutation can now be recognised by a variety of molecular techniques. We report a pilot screening survey of a population of children with mental impairment in which we used Southern blotting methods to detect the fragile X mutation, augmented by cytogenetic studies on children whose phenotype suggested a possible chromosome abnormality. There were 873 children with special educational needs in our survey and 310 fulfilled our criteria for testing. A sample was obtained from 254, of whom four were found to have a full fra(X) mutation (delta L) and none to have a premutation. The number of CGG repeats in our population of X chromosomes was measured by PCR analysis and the genotype at the closely linked polymorphic locus FRAXAC1 established. The distribution of CGG repeat numbers was very similar to that of the control population reported by Fu et al and the distribution of FRAXAC1 alleles almost identical to that of the control population reported by Richards et al. Among the non-fragile X chromosomes, we found a very significant correlation between the size of the CGG repeat and the FRAXAC1 genotype. There was a dearth of A and D genotypes in subjects with a small number of CGG repeats and an excess of the A genotype in those with a large number of CGG repeats. These observations are considered in the light of the reported disequilibrium between the A (and possibly also the D) genotype and the fra(X) mutation.     

Reduced cell proliferation in fetal lung after maternal administration of pilocarpine. A scintillation autoradiographic study

Fetuses were obtained on the 28th gestational day from pregnant New Zealand white rabbits treated daily, on the 24th through the 27th gestational day, with pilocarpine HCl, 5 mg/kg in saline, or saline alone. Lung fragments from these fetuses were incubated for two hours in medium containing ³H-thymidine. Scintillation autoradiography of 1-μm-thick sections of these fetal lungs revealed that the lung tissue from pilocarpine-treated fetuses had significantly lower labelled cell indices for both alveolar epithelial cells and interstitial cells. These results indicate that pilocarpine treatment promotes differentiation of immature cells in the fetal lung at the expense of cell proliferation.     

Immunologic Memory Induced by a Glycoconjugate Vaccine in a Murine Adoptive Lymphocyte Transfer Model

We have developed an adoptive cell transfer model in mice to study the ability of a glycoprotein conjugate vaccine to induce immunologic memory for the polysaccharide moiety. We used type III capsular polysaccharide from the clinically relevant pathogen group B streptococci conjugated to tetanus toxoid (GBSIII-TT) as our model vaccine. GBS are a major cause of neonatal infections in humans, and type-specific antibodies to the capsular polysaccharide protect against invasive disease. Adoptive transfer of splenocytes from mice immunized with the GBSIII-TT conjugate vaccine conferred anti-polysaccharide immunologic memory to naive recipient mice. The transfer of memory occurred in a dose-dependent manner. The observed anamnestic immune response was characterized by (i) more rapid kinetics, (ii) isotype switching from immunoglobulin M (IgM) to IgG, and (iii) 10-fold-higher levels of type III-specific IgG antibody than for the primary response in animals with cells transferred from placebo-immunized mice. The adoptive cell transfer model described in this paper can be used for at least two purposes: (i) to evaluate conjugate vaccines with different physicochemical properties for their ability to induce immunologic memory and (ii) to study the cellular interactions required for an immune response to these molecules.     

The radial depth-dose distribution of a 188W/188Re beta line source measured with novel,ultra-thin TLDs in a PMMA phantom: comparison with Monte Carlo simulations

The radial depth-dose distribution of a prototype 188W/188Re beta particle line source of known activity has been measured in a PMMA phantom, using a novel, ultra-thin type of LiF:Mg,Cu,P thermoluminescent detector (TLD). The measured radial dose function of this intravascular brachytherapy source agrees well with MCNP4C Monte Carlo simulations, which indicate that 188Re accounts for > or = 99% of the dose between 1 mm and 5 mm radial distance from the source axis. The TLDs were calibrated using a 90Sr/90Y beta secondary standard. Several correction factors are calculated using analytical and Monte Carlo methods. An analysis of the measurement uncertainty is made. Since it is partly determined by components of uncertainty arising from random effects, repeated measurements yield a lower uncertainty. The expanded uncertainty in the absolute dose at 2 mm radial distance equals 11%, 10%, 9% and 8% for 1, 2, 3 and 5 measurements, respectively. After a correction for source non-uniformity, the measured dose rate per unit source activity at 2 mm radial distance equals (1.53 +/- 0.16) Gy min(-1) GBq(-1) (2sigma), in agreement with the value of (1.45 +/- 0.01) Gy min(-1) GBq(-1) (2sigma) predicted by the MCNP4C simulations.     

Rapid neurofibrillary tangle formation after localized gene transfer of mutated tau

Neurofibrillary pathology was produced in the brains of adult rats after localized gene transfer of human tau carrying the P301L mutation, which is associated with frontotemporal dementia with parkinsonism. Within 1 month of in situ transfection of the basal forebrain region of normal rats, tau-immunoreactive and argyrophilic neuronal lesions formed. The fibrillar lesions had features of neurofibrillary tangles and tau immunoreactivity at light and electron microscopic levels. In addition to neurofibrillary tangles, other tau pathology, including pretangles and neuropil threads, was abundant and widespread. Tau gene transfer to the hippocampal region of amyloid-depositing transgenic mice produced pretangles and threads, as well as intensely tau-immunoreactive neurites in amyloid plaques. The ability to produce neurofibrillary pathology in adult rodents makes this a useful method to study tau-related neurodegeneration.     

Comparison of direct immunofluorescence and direct immunoperoxidase procedures for detection of herpes simplex virus antigen in lesion specimens

Direct immunofluorescence and direct immunoperoxidase staining were equally sensitive and specific for detection of herpes simplex virus antigen in lesion specimens, and each method showed 82% agreement with virus isolation results.     

Electron microscopic demonstration of horseradish peroxidase-tetramethylbenzidine reaction product as a method for identifying sensory nerve fibers in the rat tooth pulp

The purpose of the present investigation was to determine if the horseradish peroxidase could be used as a method for labeling sensory nerve fibers (specifically, tooth pulp afferents) for detailed ultrastructural analyses. HRP injected into the trigeminal ganglion of adult rats was taken up by ganglion cell bodies and transported anterogradely to their peripheral endings in the dental tissues. Following perfusion-fixation, the teeth were decalcified in EDTA, sectioned, reacted for HRP activity according to the tetramethylbenzidine (TMB) technique, and processed for electron microscopy. The HRP-TMB reaction product was clearly visible within most of the axons in the dental pulp, appearing as conspicuous, rectangular shaped aggregates of fine rods or needles.     

Processing of radical prostatectomy specimens for correlation of data from histopathological, molecular biological, and radiological studies: a new whole organ technique

AIMS: To develop a method of processing non-formalin fixed prostate specimens removed at radical prostatectomy to obtain fresh tissue for research and for correlating diagnostic and molecular results with preoperative imaging. METHODS/RESULTS: The method involves a prostate slicing apparatus comprising a tissue slicer with a series of juxtaposed planar stainless steel blades linked to a support, and a cradle adapted to grip the tissue sample and receive the blades. The fresh prostate gland is held in the cradle and the blades are moved through the cradle slits to produce multiple 4 mm slices of the gland in a plane perpendicular to its posterior surface. One of the resulting slices is preserved in RNAlater. The areas comprising tumour and normal glands within this preserved slice can be identified by matching it to the haematoxylin and eosin stained sections of the adjacent slices that are formalin fixed and paraffin wax embedded. Intact RNA can be extracted from the identified tumour and normal glands within the RNAlater preserved slice. Preoperative imaging studies are acquired with the angulation of axial images chosen to be similar to the slicing axis, such that stained sections from the formalin fixed, paraffin wax embedded slices match their counterparts on imaging. CONCLUSIONS: A novel method of sampling fresh prostate removed at radical prostatectomy that allows tissue samples to be used both for diagnosis and molecular analysis is described. This method also allows the integration of preoperative imaging data with histopathological and molecular data obtained from the prostate tissue slices.     

Lymphopenia in acute nitrofurantoin pleuropulmonary reactions

Each of 5 patients with acute nitrofurantoin pleuropulmonary reactions had profound lymphopenia and 4 had eosinophilia developing early in the clinical course after the drug was withdrawn. The 2 patients tested had only one third of the normal numbers of E rosettes (T lymphocytes) in the peripheral blood during recovery. Lymphoblastic transformation tests with purified nitrofurantoin were done in 3 patients and all of them were negative; responses to phytohemagglutinin, concanavalin A, and pokeweed were decreased but still normal. The diagnosis of various nitrofurantoin hypersensitivity reactions relies on clinical data. The mechanisms of these reactions presently remain unclear.     

Differential effects of upper gastrointestinal fill on milk ingestion and nipple attachment in the suckling rat

Milk intake and nipple attachment behaviors were studied in the natural suckling situation after gastrointestinal preloading. Rat pups, deprived of their dam for 9 hr at 1, 10, or 20 days of age, were preloaded by gavage with volumes ranging from 2 to 16% of their body weight and returned to suckle. Preloads of artificial bitch's milk decreased the intake of mother's milk in a volume-related manner at all ages. At 1 and 10 days 4% preloads decreased milk intake without affecting attachment behaviors; larger prelods of 8 and 16% decreased intake and reduced the incidence of attachment. At 20 days of age small and large preloads decreased both incidence of attachment and milk intake. Preloads up to 8% of body weight had no effect on latency to attach at any age. Complete subdiaphragmatic bilateral vagotomy increased milk intake of 7–9–day-old pups fed automatically through an anterior mouth cannula in a nonsuckling situation. Vagotomy combined with spinal cordotomy (T2-T3) resulted in a synergistic hyperphagia and massive distension of the upper GI tract. The results indicate that suckling rats can control their intake of mother's milk while remaining attached to a nipple as early as 1 day of age. The suppression of ingestion in response to GI filling appears to be mediated by visceral afferent activity. Conversely, attachment behaviors are less affected by GI fill. This suggests that ingestive behaviors and attachment behaviors have different controls during the 1st 10 days of postnatal development.