A comparison of antibody testing, permeability testing, and zonulin levels with small-bowel biopsy in celiac disease patients on a gluten-free diet.

Active celiac disease is associated with positive endomysial (EMA) and tissue transglutaminase (TTG) antibodies, elevated zonulin levels, and increased intestinal permeability. There is little known about what happens to these immunologic and structural abnormalities in patients on a gluten-free diet and their correlation with small-bowel biopsy changes. Adult patients previously diagnosed with celiac disease and on a gluten-free diet for greater than 1 year were considered for the study. All patients underwent the following: measurement of EMA and TTG antibodies, serum zonulin levels, intestinal permeability (IP) testing with lactulose/mannitol ratios, food diary analysis for gluten ingestion and small- bowel biopsy. A total of 21 patients on a gluten-free diet for a mean of 9.7 years completed the study. There were ten patients who had normalization of intestinal biopsies, IP and TTG, and EM antibodies. Six patients had Marsh type 2 or 3 lesions and all had either abnormal IP (5/6) or TTG antibody (4/6). In patients with Marsh type 3 lesions, there was a correlation between IP and zonulin levels. A subgroup of patients with celiac disease on a gluten-free diet has complete normalization of intestinal biopsies, intestinal permeability defects, and antibody levels. Patients with Marsh type 3 lesions have abnormal TTG antibodies and intestinal permeability with zonulin levels that correlate with IP. These abnormalities may be due to continued gluten ingestion. Further study is needed to determine the clinical utility of TTG antibodies and IP testing in following patients with celiac disease.

Electron microscope autoradiography of erythroid cells using radioactive iron.

The "circle analysis" method of Williams (1969) and a new improved method employing hypothetical grains described in the previous paper have been used to analyze the distribution of autoradiographic grains over erythroid bone marrow cells labeled with radioactive iron, (55)Fe. The resolution obtainable with this isotope was determined by measuring the distribution of grains about a thin line source. This distribution was also used in calculation of the circle size for the Williams's analysis and the distances of hypothetical grains for the new method. The new method provides estimates for the amount of activity in the regions of condensed and extended nuclear chromatin and for the concentration of isotope at the junction between these two areas. The possible significance of activity in this junctional region is discussed.

A new method for analyzing electron microscope autoradiographs using hypothetical grain distributions.

A new method for the analysis of electron microscope autoradiographs is described which has two advantages over other methods. This method provides estimates for the activity in different regions of the section and takes into account the actual geometrical shape and position of different regions within the section. Use is made of the measured distribution of grains about a thin line source to construct a hypothetical distribution of autoradiographic grains which is then compared with the actual distribution of grains. Different activities are then ascribed to various regions until a satisfactory agreement with the autoradiographic grains is obtained using the chi(2) significance. In the next paper, an example of the use of this method is given for the distribution of grains in the nucleus of erythroid bone marrow cells labeled with radioactive iron, (55)Fe.

Separation of A- versus C-nociceptive inputs into spinal-brainstem circuits.

This study was designed to determine the organization of nociceptive inputs with different behavioral significance into spinal-brainstem circuits in the rat. Induction of Fos protein was used to localize spinal dorsal horn and hypothalamic neurons activated by noxious heating of the hind paw dorsum at rates known to preferentially activate C- or A-heat nociceptors. This was combined with retrograde transport of cholera toxin subunit B from the dorsolateral/lateral- (DL/L-) or the ventrolateral- (VL-) periaqueductal gray (PAG) in order to map the organization of A- and C-fiber input to spinal-brainstem circuits. The majority of dorsal horn heat-activated neurons were located in laminae I and II. A significantly larger proportion of C-fiber-activated neurons projected to the VL-PAG (P<0.05) compared with its DL/L-sector. In contrast, there was no columnar separation in the projections of A-fiber-activated neurons. However, a significantly greater proportion of A-fiber-activated neurons (P<0.05) were retrogradely labeled from the DL/L-PAG, when compared with C-fiber-activated neurons. A large proportion (25-50%) of A- and C-fiber-activated neurons in the lateral spinal nucleus projected to the PAG. A-fiber-activated neurons were found throughout the rostral hypothalamus but those projecting to the PAG were focused in the lateral area of the anterior hypothalamus (LAAH), from where approximately 20% projected to the VL-PAG, which was significantly more than to the DL/L PAG (P<0.05). We hypothesize that the organization of A- versus C-fiber inputs to the PAG enables the coordination of coping strategies appropriate to meet the demands imposed by these different noxious stimuli. Hypothalamic-PAG projections activated by A-fiber inputs did not reflect this level of organization and we suggest that this may relate to their role in thermoregulation as opposed to autonomic responses to particular nociceptive inputs.

Methodological issues in longitudinal studies: vestibular schwannoma growth rates in neurofibromatosis 2.

Four longitudinal studies of vestibular schwannoma (VS) growth rates in neurofibromatosis 2 (NF2) have yielded very different results on the relationship of VS growth rates to age. The studies had different patient eligibility criteria, indices of VS growth rates, VS volumetric methods, and sample sizes. We reanalysed data from two of the longitudinal studies and used data from the population based United Kingdom NF2 Registry to determine the most likely reason for the different results and the actual relationship of VS growth rates to age. We found that the eligibility criterion in one study caused selection bias for slower growing VS. The proper interpretation of the results from the four studies is that VS growth rates in NF2 are highly variable but tend to decrease with increasing age. Clinical trials for VS in NF2 should focus on younger patients because VS growth rates tend to decrease with increasing age, and because faster growing VS are more likely to be excised with increasing age than slower growing VS.

The location of constitutional neurofibromatosis 2 (NF2) splice site mutations is associated with the severity of NF2.

Neurofibromatosis 2 (NF2) patients with constitutional splice site NF2 mutations have greater variability in disease severity than NF2 patients with other types of mutations; the cause of this variability is unknown. We evaluated genotype-phenotype correlations, with particular focus on the location of splice site mutations, using mutation and clinical information on 831 patients from 528 NF2 families with identified constitutional NF2 mutations. The clinical characteristics examined were age at onset of symptoms of NF2 and number of intracranial meningiomas, which are the primary indices of the severity of NF2. Two regression models were used to analyse genotype-phenotype correlations. People with splice site mutations in exons 1-5 had more severe disease than those with splice site mutations in exons 11-15. This result is compatible with studies showing that exons 2 and 3 are required for self-association of the amino terminal of the NF2 protein in vitro, and that deletions of exons 2 and 3 in transgenic and knockout mouse models of NF2 cause a high prevalence of Schwann cell derived tumours.

Carotid body tumors in humans: genetics and epidemiology.

Genetic factors in the etiology of carotid body tumors (CBT) were sought in a medical record review of 222 histologically diagnosed cases at 12 U.S. medical centers. Patients in the series, which had a marked female predominance (146 females:76 males), usually developed tumors between the fourth and seventh decades of life (mean, 44.7 yr). In 16 patients who also had other extra-adrenal paragangliomas, suggesting a multiple primary tumor syndrome, CBT were diagnosed significantly earlier (mean, 35.4 yr; P less than 0.01). The occurrence of thyroid cancer in 5 other patients appeared to be excessive. Familial CBT was recognized in 16 patients from 13 affected families, including 9 newly ascertained kindreds. Compared with non-familial lesions, familial CBT tended to develop bilaterally (38% vs. 8% unilaterally) and at slightly earlier ages (41.6 vs. 44.9 yr). CBT was reported to occur in an autosomal dominant pattern in some families and within sibships in others; relatives were not examined for confirmation. The familial findings are generally consistent with a two-step mutation model of the development of hereditary and nonhereditary CBT; apparent deviations from the model might be clarified with additional data on this rare neoplasm.

Primitive roles for inhibitory interneurons in developing frog spinal cord.

Understanding the neuronal networks in the mammal spinal cord is hampered by the diversity of neurons and their connections. The simpler networks in developing lower vertebrates may offer insights into basic organization. To investigate the function of spinal inhibitory interneurons in Xenopus tadpoles, paired whole-cell recordings were used. We show directly that one class of interneuron, with distinctive anatomy, produces glycinergic, negative feedback inhibition that can limit firing in motoneurons and interneurons of the central pattern generator during swimming. These same neurons also produce inhibitory gating of sensory pathways during swimming. This discovery raises the possibility that some classes of interneuron, with distinct functions later in development, may differentiate from an earlier class in which these functions are shared. Preliminary evidence suggests that these inhibitory interneurons express the transcription factor engrailed, supporting a probable homology with interneurons in developing zebrafish that also express engrailed and have very similar anatomy and functions.

A meiotic mutant affecting recombination in female Drosophila melanogaster.

mei-S282 is a female meiotic mutant isolated from a natural population of Drosophila melanogaster. It is a recessive mutation located at approximately map position 5 on the third chromosome which has two major effects. It causes a nonuniform decrease in recombination which is most drastic in distal chromosome regions and nondisjunction of all chromosome pairs is elevated at the first meiotic division. Nondisjunctional events are positively correlated; furthermore, nondisjoining chromosomes, themselves nonrecombinant, are preferentially recovered from cells in which nonhomologs are preferentially recovered from cells in which nonhomologs are also non-recombinant.-It is concluded that mei-S282 is a defect which occurs early in meiosis I prior to the time of exchange. In the mutant, the frequency of no-exchange tetrads for each of the major chromosomes is increased-and in cells which contain two or more no-exchange tetrads, an interaction between these chromosomes leads to correlated nondisjunction. mei-S282(+) then, is an exchange precondition necessary for the normal frequency and distribution of exchanges.

The genetic identification of a heterochromatic segment on the X chromosome of Drosophila melanogaster.

An autosomal euchromatic maternal-effect mutant, abo (= abnormal oocyte), interacts with, or regulates the activity of, the heterochromatin of the sex chromosomes of Drosophila melanogaster. It is shown that this interaction or regulation with the X chromosome involves a specific heterochromatic locus or small region that maps to the distal penultimate one-eighth of the basal X-chromosome heterochromatic segment.

Segmental aneuploidy and the genetic gross structure of the Drosophila genome.

By combining elements of two Y-autosome translocations with displaced autosomal breakpoints, it is possible to produce zygotes heterozygous for a deficiency for the region between the breakpoints, and also, as a complementary product, zygotes carrying a duplication for precisely the same region. A set of Y-autosome translocations with appropriately positioned breakpoints, therefore, can in principle be used to generate a non-overlapping set of deficiencies and duplications for the entire autosomal complement.-Using this method, we have succeeded in examining segmental aneuploids for 85% of chromosomes 2 and 3 in order to assess the effects of aneuploidy and to determine the number and location of dosage-sensitive loci in the Drosophila genome (Figure 5). Combining our data with previously reported results on the synthesis of Drosophila aneuploids (see Lindsley and Grell 1968), the following generalities emerge.-1. The X chromosome contains no triplo-lethal loci, few or no haplo-lethal loci, at least seven Minute loci, one hyperploid-sensitive locus, and one locus that is both triplo-abnormal and haplo-abnormal. 2. Chromosome 2 contains no triplo-lethal loci, few or no haplo-lethal loci, at least 17 Minute loci, and at least four other haplo-abnormal loci. 3. Chromosome 3 contains one triplo-lethal locus that is also haplo-lethal, few or no other haplo-lethal loci, at least 16 Minute loci, and at least six other haplo-abnormal loci. 4. Chromosome 4 contains no triplo-lethal loci, no haplo-lethal loci, one Minute locus, and no other haplo-abnormal loci.-Thus, the Drosophila genome contains 57 loci, aneuploidy for which leads to a recognizable effect on the organism: one of these is triplo-lethal and haplo-lethal, one is triplo-abnormal and haplo-abnormal, one is hyperploid-sensitive, ten are haplo-abnormal, 41 are Minutes, and three are either haplo-lethals or Minutes. Because of the paucity of aneuploid-lethal loci, it may be concluded that the deleterious effects of aneuploidy are mostly the consequence of the additive effects of genes that are slightly sensitive to abnormal dosage. Moreover, except for the single triplo-lethal locus, the effects of hyperploidy are much less pronounced than those of the corresponding hypoploidy.

Multiple schwannomas and meningiomas associated with irradiation in childhood.

OBJECTIVE: To determine the pattern of neural tumors (schwannomas, vestibular schwannomas [acoustic neuromas], and meningiomas) that developed in 3013 people who received radiation treatment with x-ray beam therapy for benign conditions of the head and neck area before their 16th birthday. METHODS: The surgical and pathology reports and pathology slides were reviewed for all neural tumors in the cohort. Patients with more than 1 neural tumor were compared with those with 1 neural tumor and those with no neural tumors. RESULTS: There were 7 patients with multiple neural tumors and 63 with single neural tumors. The distribution of tumors in these 2 groups differed. The group with multiple tumors had more spinal nerve root schwannomas, while the group with single tumors had more cranial nerve schwannomas. Six of the 7 patients did not meet the diagnostic criteria for neurofibromatosis type 2. CONCLUSIONS: Our findings suggest that host factors that increase susceptibility to radiation may be involved in the development of the multiple neural tumors. Clinically, patients with multiple neural tumors who do not meet the diagnostic criteria for neurofibromatosis type 2 should be questioned about radiation exposure. If exposure is confirmed, then screening for other radiation-related tumors should be initiated.

Sites of [3H]taurine Uptake in the Rat Substantia Nigra in Relation to the Release of Taurine from the Striatonigral Pathway.

The autoradiographic localization of radiolabelled taurine taken up in the rat substantia nigra in vivo together with conditions of release of the [3H]taurine taken up into brain slices were studied to determine whether they are consistent with the hypothesis that taurine may act as a neurotransmitter in the striatonigral pathway. At the light microscopic level the main cellular elements that became radiolabelled following the injection of [3H]taurine into the substantia nigra could be identified as glial cells. Electron microscope autoradiography confirmed that a subpopulation of glial cells including astrocytes, pericytes, and oligodendrocytes were radiolabelled and that neuronal perikarya were not radiolabelled. In addition, axonal elements including both terminal and preterminal boutons were found to have silver grains overlying them and were thus considered to be radiolabelled. This was supported by a quantitative analysis of the distribution of the silver grains; whereas glial elements had a significantly higher number of grains associated with them than with any other structure, axonal elements had a significantly greater number of grains than dendritic structures. Release of the preloaded [3H]taurine from superfused slices of substantia nigra occurred in response to veratridine, was calcium-dependent and was sensitive to inhibition by high magnesium concentrations or tetrodotoxin. Following the destruction of neurons in the striatum by ibotenic acid injections, although the weight of the ipsilateral substantia nigra was reduced, the uptake of [3H]taurine was not altered. In contrast to this, the veratridine-stimulated release was markedly attenuated, implying that the destruction of striatal neurons causes the loss of sites in the substantia nigra from which exogenous taurine is released. These results add further support to previous suggestions that taurine might act as a neurotransmitter or neuromodulator in the striatonigral pathway.

In vitro radiosensitivity of fibroblasts from thyroid and skin cancer patients treated with X-rays for tinea capitis.

To investigate the hypothesis that persons who developed thyroid or skin cancer subsequent to scalp irradiation for tinea capitis are particularly sensitive to radiation, possibly because of a high frequency of ataxia-telangiectasia, we used an in vitro cell survival assay to evaluate radiosensitivity of their fibroblast cell strains. Study subjects were selected from a cohort of 10,834 Israelis irradiated during childhood for tinea capitis. Skin fibroblasts were obtained from thyroid and skin cancer patients (cases) as well as a sample of subjects who did not have cancer (controls). Fibroblasts were cultured and then loss of colony-forming ability as a result of acute X-irradiation was evaluated. Comparison of survival curve parameters (mean inverse of the slope and the dose needed to reduce colony survival to 10%) between 12 thyroid cancer and 12 control strains showed no differences (P > 0.5). A slightly increased radiation sensitivity of the skin cancer cases compared with their controls was observed. Although based on few subjects (14 cases and 11 controls), the findings were similar whether the mean inverse of the slope (P = 0.06) or the dose needed to reduce colony survival to 10% (P = 0.05) was evaluated. However, because of the small size of the study and potential errors inherent in survival assays, our finding that cell strains derived from patients who developed skin cancer exhibit enhanced radiosensitivity should be viewed as preliminary and interpreted cautiously.

Presymptomatic diagnosis for neurofibromatosis 2 with chromosome 22 markers.

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder characterized by multiple tumors of the central nervous system, predominantly bilateral vestibular schwannomas. The gene for NF2 is located in the chromosomal region 22q12 between the loci D22S1 and D22S28. We have performed genetic linkage analysis on 13 NF2 families with a total of nine polymorphic DNA markers, including five which we have recently mapped to this region. Two loci, D22S32 and NEFH, are linked to the NF2 locus at 0% recombination (lod scores of 6.03 and 4.28, respectively). By multipoint linkage analysis, we assign the NF2 gene to an interval of 7 cM, between the loci D22S212 and D22S28. We have used this set of nine markers to construct chromosome 22 haplotypes for the 82 at-risk individuals in this pedigree set. It has been possible to determine, with a high degree of certainty, the carrier status of 70 (85%) of these at-risk individuals. Risk prediction was possible in every case where DNA was available from both parents. Fifty-three of the 70 (76%) informative individuals were assigned decreased risks of being carriers. The use of chromosome 22 probes for risk assessment should result in a greatly reduced number of individuals who require periodic screening for NF2.

Serotonergic transmission in the periaqueductal gray matter in relation to aversive behaviour: morphological evidence for direct modulatory effects on identified output neurons.

Intracellular recordings were made from 21 cells in the dorsolateral periaqueductal gray matter in coronal midbrain slices. In the majority (n = 20) bath application of 5-hydroxytryptamine (30 or 150 mM) evoked either hyperpolarizing (n = 11) or depolarizing (n = 9) responses. Reconstructions of 11 neurons in the dorsolateral periaqueductal gray matter after filling with biocytin revealed a population of output neurons whose axons followed a dorsolateral trajectory towards the perimeter of the ipsilateral periaqueductal gray matter. In seven cells, the axon could be followed into the adjacent mesencephalic reticular formation. At the light microscopic level, immunostaining for 5-hydroxytryptamine revealed immunoreactive processes throughout the dorsolateral periaqueductal gray matter but no labelled somata or dendrites. Close associations (i.e. no discernible gap) were observed between serotonergic profiles and the somata and dendrites of biocytin-filled cells. At the ultrastructural level, serial sections through 21 appositions on to biocytin-filled dendrites in three slices revealed 19 true appositions (i.e. having closely parallel plasma membranes with no intervening glial cell profiles) with the biocytin-filled dendrite. Only four of the appositions (21%) showed evidence of synaptic specializations which included aggregations of synaptic vesicles, and some thickening of the apposing membrane. The dense reaction product in the biocytin-filled cells precluded identification of the ultrastructure of postsynaptic elements. However, examination of contacts between 5-hydroxytryptamine-immunoreactive profiles and unlabelled elements in material taken from the contralateral side of the periaqueductal gray matter (i.e. no biocytin present) or in material taken from perfusion-fixed whole brain, in which ultrastructural preservation was superior compared with slices, revealed a similar incidence (21% and 23%, respectively) of synaptic specializations. The data indicate that serotonergic transmission on to output neurons in the dorsolateral periaqueductal gray matter is largely mediated by non-junctional contacts, suggesting that the actions of 5-hydroxytryptamine on these cells are mediated predominantly by volume rather than wiring transmission.

A simplified method of "hypothetical grain" analysis of electron microscope autoradiographs.

An improved method for the analysis of high resolution electron microscope autoradiographs is described which is simple and quick to perform. The method uses a transparent screen superimposed over the autoradiograph prints to provide information on the distribution of grains over neighboring structures, produced by radioactive disintegrations occurring in different regions of the section. This "cross-scatter" information is used to estimate the radioactivity in different structures without the need to make any assumptions about the size, shape and arrangement of the structures within the autoradiographs being analyzed. The method allows activity in membranes and other linear structures to be determined and also the accuracy attributable to the activity values obtained.

Isotope decay range distribution curves for use in the analysis of electron microscope autoradiographs.

Analysis of autoradiographs at the electron microscope level requires special procedures, since the size of the radioactive structures visualized are comparable to the range of the radioactive decay particles emitted. Quantitative analysis in these circumstances requires that the sizes, shapes, and juxtaposition of the various structures be taken into account in relation to the range distribution of silver grains, produced by the decays, from a point source for the particular isotope and autoradiographic conditions employed. We present the distribution of silver grains about a point source for the four electron capture isotopes 51Cr, 55Fe, 111In, and 125I. Thin radioactive line sources were constructed and the distribution of autoradiographic grains measured. The grain distributions are discussed in relation to the number of particles per disintegration and their energy and range. A simple calculation enables these line source distributions to be converted into point source distributions, which can then be used for whichever method of quantitative analysis is considered appropriate for a particular problem. An outline is given of some of the more important aspects of various methods of analysis.

Effects of exogenous progesterone and oestradiol on prostaglandin F and 13,14-dihydro-15-oxo prostaglandin F2alpha concentrations in uteri and plasma of ovariectomized ewes.

PIP: The effects of progesterone and estradiol administration on plasma and uterine concentrations of prostaglandin F2alpha (PGF2a) and 13,14-dihydro-15-oxo PGF (PGFM) were studied in ovariectomized ewes. Treatment with 30 mcg/day estradiol for 9 days did not alter the concentration of PGF2a in uterine caruncles and intercaruncular tissue, the release of PGF2a or PGFM from these tissues in vitro, or the concentrations of PGF2a in the utero-ovarian vein or PGFM in the jugular vein. Nonetheless, there was an accumulation of estradiol in uterine tissue. Treatment with 20 mg/day progesterone for 9 days resulted in a significant (p less than .01) increase in PGF2a concentration in the uterine caruncles, a significant (p less than .01) increase in the release of PGF2a from caruncles incubated in the presence of arachidonic acid, and an increase in the concentration of PGFM in the jugular vein. The administration of estradiol to animals pretreated with progesterone further increased the PGF content of uterine caruncles, the release of PGF2a into the utero-ovarian vein, and the concentration of PGFM in the jugular vein. The amount of PGF2a in the caruncles was significantly (p less than .05) greater than that in the intercaruncular area, and the amount of PGF2a and PGFM released in vitro was also greater in the caruncles. Animals treated with progesterone plus estradiol showed a good correlation between PGF2a concentrations in blood samples obtained at the same time from the right and left utero-ovarian vains, and all animals showed a high correlation between utero-ovarian PGF2a and peripheral PGFM concentrations. Lipid droplets were also more predominant in the caruncular epithelium of progesterone-treated animals than in other groups. The results demonstrate the necessary presence of progesterone for the activation of prostaglandin synthetase activity and the promotion of PGF2a production.^ieng

Photoperiodic control of the development of the LHRH neurosecretory system of European starlings (Sturnus vulgaris) during puberty and the onset of photorefractoriness.

The development of the reproductive system was studied in juvenile starlings during the acquisition of photosensitivity, the attainment of sexual maturation after photostimulation and the subsequent onset of photorefractoriness, using immunohistochemistry for LHRH and radioimmunoassay measurements of hypothalamic, pituitary and plasma hormone concentrations. The first stage of sexual development induced by exposure of photorefractory immature starlings to short days (8 h light:16 h darkness; 8L:16D) was characterized by a decrease in pituitary prolactin content within 1 week and an increase in hypothalamic LHRH content, in the size of the LHRH perikarya and in the intensity of immunostaining in the median eminence in 4-6 weeks. Sexual maturation occurring after exposure to long days (18L:6D) was associated with further increases in LHRH content and cell size, and increases in LH and prolactin concentrations. During testicular regression, LHRH perikarya were reduced in size and staining intensity but LHRH immunostaining in the median eminence and content in the hypothalamus remained high until gonadal regression was almost complete. Prolactin levels were maximal during testicular regression. These results suggest that gonadal regression is initiated by a reduction in LHRH synthesis and possibly, in addition, an external inhibitory influence on LHRH release. Hypothalamic LHRH content eventually declined and LHRH immunostaining in the median eminence was much reduced in fully photorefractory starlings maintained under long days.