Initiating joint attention use in infants at high-risk for autism spectrum disorder.

BACKGROUND: Impairment in initiating joint attention (IJA) is associated with autism spectrum disorder (ASD) in children, although it is unclear when impairments arise. Due to the early development of IJA use and late diagnosis of ASD, groups at high-risk of ASD, such as infants with an older sibling with ASD (ASIBs) and infants with fragile X syndrome (FXS), provide opportunities to study early IJA behaviours for children who are later diagnosed with ASD. This study analysed these two groups to determine if IJA use differed compared with typically developing (TD) peers at 12 months and whether IJA was associated with later ASD outcomes. METHOD: An experimental attention task was used to analyse IJA gaze shifts and gestures in the high-risk groups. Clinical best estimate diagnoses were given to each participant to compare IJA behaviours to ASD severity. RESULTS: No differences in the frequency of IJA gaze shifts and gestures were found between 12-month-old ASIBs and TD controls, but infants with FXS demonstrated a significantly reduced range of IJA gaze shifts relative to TD controls. Additionally, ASD outcomes at 24 months were related to IJA use for infants with FXS at 12 months, but not infant ASIBs, although these findings were explained by differences in nonverbal cognitive development. CONCLUSIONS: Although previous studies have reported delays in IJA use in children with FXS and ASIBs at ages 21 and 14 months, respectively, our results suggest IJA behaviours for these high-risk groups are not distinct from TD children at 12 months. When differences were found at 12 months, they were explained by nonverbal cognitive development, particularly for infants with FXS. Differences in IJA use at 12 months in this study were too small to serve as a potential indicator of later ASD.

Molecular heterogeneity of a lymphocyte glycoprotein in immunodeficient patients.

Previous evaluation of lymphocytes taken from patients with Wiskott-Aldrich syndrome (WAS) and other X-linked immunodeficiencies has revealed deficiencies of a lymphocyte sialoglycoprotein with a relative molecular mass of 115 kD (designated gpL-115) found in normal lymphocytes. The development of monoclonal antibodies to gpL-115 has permitted the detection of molecular heterogeneity in gpL-115 from the lymphocytes of immunodeficient patients. When lymphocytes from normal individuals were analyzed by immunoblotting, gpL-115 with only a single molecular species (115 kD) was detected. Lymphocytes from 17 immunodeficient patients were analyzed after overnight incubation. Two patients had no gpL-115 with an Mr of 115 kD, but gpL-115 with an Mr of either 95 or 135 kD was detected. Nine patients had gpL-115 with Mr equally of 95 and 115 Kd. Other patients exhibited gpL-115 with combinations of 95, 115, and 135 kD. The heterogeneity of the degraded gpL-115 suggests that WAS and other X-linked immunodeficiencies are due to a series of abnormalities, all of which involve gpL-115, and may explain the clinical heterogeneity of the diseases.

Identification of receptor regulatory proteins, membrane glycoproteins, and functional characteristics of adenylate cyclase in vesicles derived from the human neutrophil.

Human neutrophils were disrupted by brief sonication under conditions which preserve the hormone sensitivity of adenylate cyclase and yield minimal granule lysis. Fractions enriched in adenylate cyclase were analysed for hormonal and guanine nucleotide regulation of the enzyme as well as structural proteins. Adenylate cyclase was activated by PGE1 and isoproterenol in a GTP-dependent fashion, while f-met-leu-phe and C5a gave no stimulation. Cholera toxin treatment, which specifically modifies cyclase-related GTP-binding proteins, caused a dose-dependent enhancement of GTP activation, in which GTP alone activated maximally and PGE1 was without further effect. The following proteins were detected in the cyclase-containing vesicles: a 42 K mol. wt protein labeled selectively by cholera toxin; protein subunits observed in SDS gels at 214, 165, 105 and 47 K, of which the 47 K band was the most prominent and comigrated with actin; prominent lectin-binding activities at 165 K (concanavalin A and wheat germ agglutinin) as well as at 100 K (wheat germ agglutinin); and a set of proteins and lectin-binding activities in fractions containing beta-glucuronidase activity distinct from adenylate cyclase containing vesicles. The identification of receptor-controlled cyclase, GTP-binding regulatory proteins, cytoskeletal elements and unique lectin-binding activities in a single vesicle preparation should contribute to an understanding of receptor-mediated control of neutrophil function.

A method for the preparation of mononuclear cells devoid of platelet contamination and its application to the evaluation of putative alpha-receptors in normal and asthmatic subjects.

Receptor studies of human mononuclear leukocytes (MNLs) are complicated by the presence of contaminating platelets which have common receptors. A method was devised to produce MNLs free of platelets (less than 1%) and consists of sequential Ficoll-Hypaque gradients, a BSA gradient and a washing step. Lack of platelet contamination was confirmed by the following criteria: (a) microscopic evaluation using fluorescent dyes showed less than 1% platelets; (b) PGE1 stimulation of the leukocyte membrane adenylate cyclase required addition of exogenous GTP while the platelet cyclase did not; (c) immunoblots of the cells and membranes using antibodies strongly reactive against platelet membranes showed no reactivity against MNL membranes; (d) [3H]yohimbine showed no binding in MNL membranes under conditions where substantial binding to platelets was detected. MNLs were viable as judged by dye exclusion. PHA stimulation of lymphocytes was unimpaired. Plasma membranes of MNLs were prepared by brief sonication and fractionation on a sucrose step gradient. Binding studies using 3H-DHE, an alpha-receptor ligand, revealed no binding in MNLs from normal subjects (n = 6). By contrast, studies on cells from subjects with mild asthma with medication appropriately withheld (n = 8) showed low levels of binding (60-300 fmol/10(6) cells). The subtype and functionality of the putative alpha-receptors are being further evaluated.

Distribution of prostatic acid phosphatase isoenzymes in normal and cancerous states.

We have studied the IEF (isoelectric focusing) profiles and the sedimentation characteristics of intracellular and secretory prostatic acid phosphatase (PAP) in normal and cancerous states. IEF studies show a similar relative distribution of tartrate inhibitable pI 4.9 (approximately 80%) and 5.6 (approximately 20%) forms of this enzyme in normal as well as cancerous prostate. The same IEF profile is obtained regardless of whether an enzymatic or RIA method is utilized for detection of PAP. Of these two isoenzymes, only the form of pI 4.9 predominates in prostatic and seminal fluids and in Stage IV serum. Sedimentation analysis shows that the purified enzyme is exceptionally stable since it retains an S020,w value of 5.7 at low concentrations (ng/ml). While only the 5.7S form is observed in normal and cancerous tissues as well as in prostatic fluid, analysis of Stage IV serum reveals an additional form at 8.7S. Control experiments suggest that the 8.7S form is not induced by non-specific association with normal serum proteins or by the inhibitor tartrate. Our results suggest that: (a) of the two major isoenzymes in tissue, only the pI 4.9 isoenzyme predominates in secretion, (b) this relationship of intracellular to secretory forms is unaltered in the transition from normal to cancerous tissue, and (c) the utility of PAP as a tumor marker is derived at least in part by the intrinsic stability of the 5.7S form. The significance of the 8.7S form is unknown at the present time, but it does not distort the clinical (RIA) measurement of PAP in serum.

Ligand requirements for the relaxation of adenylate cyclase from activated and inhibited states.

The turkey erythrocyte adenylate cyclase system binds tightly the inhibitory nucleotide GDP, and a pretreatment step with isoproterenol and GMP is required to restore activation. Under identical pretreatment conditions, the release of labeled nucleotide is complete within 1 min whereas the restoration of activation by Gpp(NH)p requires 15 min. A study of the ligand requirements of the slow step shows the following: (a) The role of GMP is that of an obligatory allosteric regulator. (b) Cholera toxin modification of the system abolishes the requirement for GMP with a considerable enhancement in the reaction rate. (c) GMP is without effect on the relaxation process with the activator Gpp(NH)p as the resident nucleotide. In sharp contrast, ethylenediamine-tetraacetic acid (without effect in a GDP-occupied complex) markedly potentiates alterations from the Gpp(NH)p-occupied state. (d) Formation of a GDP/guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) hybrid leads to the suppression of both F- and Gpp(NH)p activation. F- activation is restored by isoproterenol alone, while GMP is still required to restore Gpp(NH)p activation. The results suggest that covalent modification or nucleotide analogue occupancy of the regulatory complex can modify the allosteric role for GMP, with consequences for the rate of the slow step.

PPD-induced monocyte chemotactic factor production by human milk cells.

The functional capability of antigen-stimulated breast milk cells to produce an immunologic mediator was examined. Colostrum and comparison peripheral blood samples were obtained from ten women, two to four days postpartum, and supernatants from PPD-stimulated mononuclear cell cultures were assayed for the lymphokine, monocyte chemotactic factor. Five of the ten women studied had a history of a positive tuberculin skin test and one had received BCG immunization. Peripheral blood lymphocyte cultures and colostrum cell cultures from four of these six women produced monocyte chemotactic factor. These results demonstrated the functional capability of antigen-stimulated colostral cells to produce immunologic mediators.

IgD in human colostrum.

Simultaneous colostrum (C) and plasma samples (P) from 14 women, 1 to 5 days postpartum, were examined. Total IgD and specific IgD antibodies to beta-lactoglobulin, bovine serum albumin, Bermuda grass, and alpha-gliadin were measured by solid phase radioimmunoassay. The geometric mean concentrations of IgD were 35.8 (range 2.2-410) micrograms/dl for colostrum and 591.3 (range 72-4100) micrograms/dl for plasma. Six subjects had a specific IgD antibody C/P ratio more than 10-fold greater than the total IgD C/P ratio, suggesting enhancement of antibody to a specific antigen in the mammary gland. All six had C/P ratios suggestive of local enhancement of IgD antibody to Bermuda grass, and two met this criterion for enhancement of IgD antibodies to beta-lactoglobulin, bovine serum albumin, or alpha-gliadin. Specimens for these studies were obtained during the peak grass pollen season. Seventeen additional subjects were studied to compare total IgD in colostrum and plasma with total IgG and serum albumin. The mean C/P ratio for IgD (0.055 +/- 0.015) exceeded the C/P ratio for total IgG (0.015 +/- 0.003) or total albumin (0.020 +/- 0.002). For 14 of 17 subjects the colostrum/plasma ratio for IgD exceeded the C/P ratio for albumin or IgG. Data were transformed logarithmically and correlation coefficients calculated. For albumin versus IgG in colostrum, there was a strong correlation, r = 0.865, p = 0.001. This was different from albumin versus IgD, r = 0.489, p = 0.046 and from IgD versus IgG, r = 0.556, p = 0.020. These analyses support a different mechanism of entry of IgD into milk compared to IgG or albumin.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfer of tuberculin immunity from mother to infant.

The purpose of our study was to investigate transfer of tuberculin immunity from mother to infant via breast milk by studying newborn lymphocyte blastogenesis induced by purified protein derivative antigen at 1-5 days of age, 4-6 wk of age, and 3 months of age. Our study consisted of four mother-infant groups: breast-feeding and bottle-feeding infants of tuberculin-positive and tuberculin-negative mothers. A difference in the groups was found only at 4-6 wk of age where 17% (4/23) of breast-feeding infants and 13% (2/15) of bottle-feeding infants of tuberculin positive mothers had lymphocyte blastogenesis to purified protein derivative. None of the infants of tuberculin negative mothers had purified protein derivative-induced blastogenesis. Analysis of covariance with tests for equality of slopes showed that the responses of tuberculin-positive mothers were significantly different from the responses of tuberculin-negative mothers (p less than 0.05). These studies suggest transplacental transfer of tuberculin immunity evident at 4-6 wk of age which wanes by 3 months of age. We could not find evidence of transfer via human milk.

The beta-adrenergic receptor in the human neutrophil plasma membrane: receptor-cyclase uncoupling is associated with amplified GTP activation.

We compared the properties of the adenylate cyclase system in plasma membranes derived from gas cavitation and sonication of human neutrophils. In membranes prepared from cavitated cells, cyclase was stimulated by fluoride ion and Gpp(NH)p. Stimulation by isoproterenol (and PGE1) was absent, although the beta-receptor was present as judged by 125I-HYP binding. Two unusual characteristics of this cyclase system are a) substantial activation of cyclase by GTP in the absence of hormone, and b) reduction in the regulation of the beta-receptor by GTP. By contrast, vesicles isolated from sonicated cells displayed normal GTP-dependent activation by isoproterenol (as well as PGE1) and GTP regulation of the beta-receptor, while hormone-independent stimulation by GTP was negligible. Cholera toxin-catalyzed ADP ribosylation revealed a prominent band at 42K in both membranes, and a minor band at 35K, although the degree of labeling of this protein was lower in the "cavitated" vesicles. Our results suggest that the mode of cell lysis and membrane preparation influence receptor-cyclase interactions and that receptor-cyclase uncoupling is associated with amplified GTP activation and altered receptor regulation by GTP.

Limited T-cell receptor beta-chain diversity of a T-helper cell type 1-like response to Mycobacterium leprae.

Delayed-type hypersensitivity (DTH) is the standard measure of T-cell responsiveness to infectious organisms. For leprosy, the Mitsuda reaction, a local immune response to cutaneous challenge with Mycobacterium leprae, is considered to represent a measure of DTH against the pathogen. We analyzed the diversity of the T-cell receptor beta-chain repertoire in Mitsuda reactions to determine the breadth of the mycobacterial antigens involved. The polymerase chain reaction was used to compare V beta usage in the Mitsuda reaction T-cell lines established and unstimulated peripheral blood. These molecular analyses revealed a skewed T-cell receptor V beta gene usage in the Mitsuda reaction and in T-cell lines from lesions. To examine the reactivity of T cells from these lesions, T-cell lines were tested against the available native and recombinant antigens of M. leprae. T-cell lines derived from Mitsuda reactions responded more strongly to the 10-kDa M. leprae antigen, a homolog of GroES in Escherichia coli, than to other M. leprae proteins. T-cell lines were also shown to proliferate strongly in response to the 17- and 3-kDa proteins. The pattern of the lymphokine mRNA of these cells was reminiscent of the pattern of murine TH1 cells, positive for interleukin-2 and gamma interferon and weakly positive for interleukin-4. These data indicate that a limited array of T cells, perhaps recognizing stress proteins, secrete a type 1 lymphokine profile in the DTH response to mycobacteria.