A novel human leucocyte antigen-DRB1 genotyping method based on multiplex primer extension reactions.

We have developed and validated a semi-automated fluorescent method of genotyping human leucocyte antigen (HLA)-DRB1 alleles, HLA-DRB1*01-16, by multiplex primer extension reactions. This method is based on the extension of a primer that anneals immediately adjacent to the single-nucleotide polymorphism with fluorescent dideoxynucleotide triphosphates (minisequencing), followed by analysis on an ABI Prism 3700 capillary electrophoresis instrument. The validity of the method was confirmed by genotyping 261 individuals using both this method and polymerase chain reaction with sequence-specific primer (PCR-SSP) or sequencing and by demonstrating Mendelian inheritance of HLA-DRB1 alleles in families. Our method provides a rapid means of performing high-throughput HLA-DRB1 genotyping using only two PCR reactions followed by four multiplex primer extension reactions and PCR-SSP for some allele groups. In this article, we describe the method and discuss its advantages and limitations.

The effect of transforming growth factor beta1 gene polymorphisms in ankylosing spondylitis.

OBJECTIVES: To determine whether genetic polymorphisms in or near the transforming growth factor beta1 (TGFB1) locus were associated with susceptibility to or severity of ankylosing spondylitis (AS). METHODS: Five intragenic single-nucleotide polymorphisms (SNP) and three microsatellite markers flanking the TGFB1 locus were genotyped. Seven hundred and sixty-two individuals from 184 multiplex families were genotyped for the microsatellite markers and two of the promoter SNPs. One thousand and two individuals from 212 English and 170 Finnish families with AS were genotyped for all five intragenic SNPs. A structured questionnaire was used to assess the age of symptom onset, disease duration and disease severity scores, including the BASDAI (Bath Ankylosing Spondylitis Disease Activity Index) and BASFI (Bath Ankylosing Spondylitis Functional Index). RESULTS: A weak association was noted between the rare TGFB1 +1632 T allele and AS in the Finnish population (P = 0.04) and in the combined data set (P = 0.03). No association was noted between any other SNPs or SNP haplotype and AS, even among those families with positive non-parametric linkage scores. The TGFB1 +1632 polymorphism was also associated with a younger age of symptom onset (English population, allele 2 associated with age of onset greater by 4.2 yr, P = 0.05; combined data set, allele 2 associated with age of onset greater by 3.2 yr, P = 0.02). A haplotype of coding region SNPs (TGFB1 +869/+915+1632 alleles 2/1/2) was associated with age of symptom onset in both the English parent-case trios and the combined data set (English data set, haplotype 2/1/2 associated with age of onset greater by 4.9 yr, P = 0.03; combined data set, haplotype 2/1/2 associated with greater age of onset by 4.2 yr, P = 0.006). Weak linkage with AS susceptibility was noted and the peak LOD score was 1.3 at distance 2 cM centromeric to the TGFB1 gene. No other linkage or association was found between quantitative traits and the markers. CONCLUSION: This study suggests that the polymorphisms within the TGFB1 gene play at most a small role in AS and that other genes encoded on chromosome 19 are involved in susceptibility to the disease.

Interleukin 10 polymorphisms in ankylosing spondylitis.

Genetic polymorphisms of the IL10 promoter region have been implicated in many autoimmune diseases, including seronegative spondyloarthropathies. We studied three SNPs (IL10-1087, -824, and -597) and two microsatellites (IL10R and IL10G) lying within the promoter region of IL10 for association with susceptibility to and clinical manifestations of ankylosing spondylitis (AS), a common form of spondyloarthritis. Four hundred and sixty-eight individuals from 182 Finnish families affected with AS were studied. No association between individual IL10 promoter region polymorphisms or marker haplotype was observed with susceptibility to AS, but weak association was noted between the IL10-597 and -824 SNPs and age of disease onset (P=0.01 for each SNP). The IL10.G4 allele was associated with BASFI (corrected for disease duration) (P=0.03). We conclude that IL10 promoter polymorphisms have no significant effect on susceptibility to AS, but may play a minor role in determining age of disease onset and disease severity.

Maintaining the cornea and the general physiological environment in visual neurophysiology experiments.

Neurophysiologists have been investigating the responses of neurons in the visual system for the past half-century using monkeys and cats that are anesthetized and paralyzed, with the non-blinking eyelids open for prolonged periods of time. Impermeable plastic contact lenses have been used to prevent dehydration of the corneal epithelium, which would otherwise occur in minutes. Unfortunately, such lenses rapidly introduce a variety of abnormal states that lead to clouding of the cornea, degradation of the retinal image, and premature termination of the experiment. To extend the viability of such preparations, a new protocol for maintenance of corneal health has been developed. The protocol uses rigid gas permeable contact lenses designed to maximize gas transmission, rigorous sterile methods, and a variety of methods for sustaining and monitoring the overall physiology of the animal. The effectiveness of the protocol was evaluated clinically by ophthalmoscopy before, during, and after the experiments, which lasted 8-10 days. Histopathology and quantitative histology were performed on the corneas following the experiment. Our observations showed that this protocol permits continuous contact lens wear without adversely affecting the corneas. Thus, it is possible to collect data 24 h each day, for the entire duration of the experiment.

Motion direction signals in the primary visual cortex of cat and monkey.

When an image feature moves with sufficient speed it should become smeared across space, due to temporal integration in the visual system, effectively creating a spatial motion pattern that is oriented in the direction of the motion. Recent psychophysical evidence shows that such "motion streak signals" exist in the human visual system. In this study, we report neurophysiological evidence that these motion streak signals also exist in the primary visual cortex of cat and monkey. Single neuron responses were recorded for two kinds of moving stimuli: single spots presented at different velocities and drifting plaid patterns presented at different spatial and temporal frequencies. Measurements were made for motion perpendicular to the spatial orientation of the receptive field ("perpendicular motion") and for motion parallel to the spatial orientation of the receptive field ("parallel motion"). For moving spot stimuli, as the speed increases, the ratio of the responses to parallel versus perpendicular motion increases, and above some critical speed, the response to parallel motion exceeds the response to perpendicular motion. For moving plaid patterns, the average temporal tuning function is approximately the same for both parallel motion and perpendicular motion; in contrast, the spatial tuning function is quite different for parallel motion and perpendicular motion (band pass for the former and low pass for the latter). In general, the responses to spots and plaids are consistent with the conventional model of cortical neurons with one rather surprising exception: Many cortical neurons appear to be direction selective for parallel motion. We propose a simple explanation for "parallel motion direction selectivity" and discuss its implications for the motion streak hypothesis. Taken as a whole, we find that the measured response properties of cortical neurons to moving spot and plaid patterns agree with the recent psychophysics and support the hypothesis that motion streak signals are present in V1.

Projections of the median raphe nucleus in the rat.

No previous report in any species has examined comprehensively the projections of the median raphe (MR) nucleus with modern tracing techniques. The present report represents an in depth analysis of the projections of MR by use of the anterograde anatomical tracer Phaseolus vulgaris-leucoagglutinin. MR fibers descend along the midline within the brainstem and mainly ascend within the medial forebrain bundle in the forebrain. MR fibers distribute densely to the following brainstem/forebrain sites: caudal raphe nuclei, laterodorsal tegmental nucleus, dorsal raphe nucleus, interpeduncular nucleus, medial mammillary body, supramammillary nucleus, posterior nucleus and perifornical region of the hypothalamus, midline and intralaminar nuclei of thalamus, dopamine-containing cell region of medial zona incerta, lateral habenula, horizontal and vertical limbs of the diagonal band nuclei, medial septum, and hippocampal formation. Virtually all of these structures lie on or close to the midline, indicating that the MR represents a midline/para-midline system of projections. Overall, MR projections to the cortex are light. MR projects moderately to the perirhinal, entorhinal and frontal cortices, but sparingly to remaining regions of cortex. A comparison of MR with dorsal raphe (DR) projections (Vertes RP. 1991. J Comp Neurol 313:643-668) shows that these two major serotonin-containing cell groups of the midbrain distribute to essentially nonoverlapping regions of the forebrain; that is, the MR and DR project to complementary sites in the forebrain. A direct role for the MR in the desynchronization of the electroencephalographic activity of the hippocampus and its possible consequences for memory-associated functions of the hippocampus is discussed.

Time-dependent effects of acute ethanol administration on regional cerebral blood flow in the rat.

The present study investigated the role of the postinjection interval in determining the functional consequences of acute ethanol administration in the CNS. Regional cerebral blood flow (RCBF) was determined by the [14C]iodoantipyrine method in 33 brain structures of ethanol-naive Sprague-Dawley rats. In the first experiment, changes in RCBF were assessed 5 and 15 min after a 0.8 g/kg (i.p.) dose of ethanol or water. Five minutes after treatment, rates of RCBF were increased in the motor cortex, agranular insular cortex, and the olfactory tubercle compared to water controls. No significant differences compared to control were found at the 15-min time point, despite the continued presence of ethanol in the blood. Experiment 2 tested whether blood ethanol level was the sole determinant of this response to ethanol by comparing animals with the same blood ethanol level at the 5- and 15-min time points. Greater rates of RCBF were found at 5 min postinjection compared to 15 min, in the motor cortex, agranular insular cortex, caudate/putamen, cerebellum, and the lateral septum. These data demonstrate that the rates of cerebral blood flow are increased in regionally discrete portions of the rat brain shortly after ethanol administration. Furthermore, blood ethanol level is not the exclusive factor governing this functional response.

Distribution, quantification, and morphological characteristics of serotonin-immunoreactive cells of the supralemniscal nucleus (B9) and pontomesencephalic reticular formation in the rat.

In their initial report on the rat, Dahlstrom and Fuxe ([1964] Acta Physiol. Scand. 62:1-55) identified nine brainstem serotonin-containing cell groups, which they termed B1-B9. B9 has received considerably less attention than other serotonergic nuclei (B1-B8) due in part to the fact that its precise location and extent have not been well documented in subprimates. B9 (supralemniscal nucleus; SLN) has been viewed as a minor serotonergic cell group. In addition, 5-hydroxytryptamine (5-HT)-containing cells have been shown to be only sparsely distributed throughout the pontomesencephalic reticular formation (PMRF). By using 5-HT immunohistochemical techniques, we examined the distribution and morphological characteristics of SLN and PMRF 5-HT neurons of the pontomesencephalic tegmentum. We showed that 5-HT cells of both SLN and the PMRF extend rostrocaudally from the rostral midbrain to the midpons. 5-HT SLN cells are located within or dorsal to the medial lemniscus (ML); those of the PMRF are widely distributed throughout the PMRF. The mean numbers of 5-HT containing cells in the SLN, PMRF, dorsal raphe, and median raphe nuclei were 4,571, 1,948, 15,191, and 4,114, respectively. The SLN (B9) contains more 5-HT neurons than any serotonergic group other than the dorsal raphe nucleus. The dendrites of both SLN and PMRF 5-HT cells are primarily oriented mediolaterally and generally extend for long distances (75-300 microns), running perpendicular to the fibers of the ML (SLN) or, to those coursing through the brainstem (PMRF). The present anatomical delineation of SLN and PMRF shows that they are major 5-HT-containing cell groups in the rat and provides the foundation for the further examination of their properties and functions.

Descending projections of the posterior nucleus of the hypothalamus: Phaseolus vulgaris leucoagglutinin analysis in the rat.

No previous report in any species has systematically examined the descending projections of the posterior nucleus of the hypothalamus (PH). The present report describes the descending projections of the PH in the rat by using the anterograde anatomical tracer, Phaseolus vulgaris leucoagglutinin. PH fibers mainly descend to the brainstem through two routes: dorsally, within the central tegmental tract, and ventromedially, within the mammillo-tegmental tract and its caudal extension, ventral reticulo-tegmental tracts. PH fibers were found to distribute densely to several nuclei of the brainstem. They are (from rostral to caudal) 1) lateral/ ventrolateral regions of the diencephalo-mesopontine periaqueductal gray (PAG); 2) the peripeduncular nucleus; 3) discrete nuclei of pontomesencephalic central gray (dorsal raphe nucleus, laterodorsal tegmental nucleus, and Barrington's nucleus); 4) the longitudinal extent of the central core of the mesencephalic through meduallary reticular formation (RF); 5) the ventromedial medulla (nucleus gigantocellularis pars alpha, nucleus raphe magnus, and nucleus raphe pallidus); 6) the ventrolateral medulla (nucleus reticularis parvocellularis and the rostral ventrolateral medullary region); and 7) the inferior olivary nucleus. PH fibers originating from the caudal PH distribute much more heavily than those from the rostral PH to the lower brainstem. The PH has been linked to the control of several important functions, including respiration, cardiovascular activity, locomotion, antinociception, and arousal/wakefulness. It is likely that descending PH projections, particularly those to the PAG, the pontomesencephalic RF, Barrington's nucleus, and parts of the ventromedial and ventrolateral medulla, serve a role in a PH modulation of complex behaviors involving integration of respiratory, visceromotor, and somatomotor activity.

Genetic control of cytokines. Cytokine gene polymorphisms in alopecia areata.

It is likely that alopecia areata is a multifactorial disease determined by a combination of genetic and environmental factors. The interaction of susceptibility genes with environmental factors gives rise to the disease phenotype, and then genetic modifying factors determine the extent of the inflammatory response and thereby the clinical outcome. Cytokines regulate the inflammatory response. Polymorphisms in these genes may therefore determine the amount of a cytokine that is produced in response to an environmental trigger such as a bacterial or viral infection.

Promoter region polymorphism in the human TNF-alpha gene is not associated with lichen sclerosus.

The pathogenesis of lichen sclerosus remains unknown. However, it has been frequently associated clinically with autoimmunity. The MHC haplotype A1, B8, DR3 is associated with many autoimmune conditions and has also been associated with the uncommon allele of the tumour necrosis factor (TNF-alpha) promoter polymorphism. This allele is also associated with higher production of TNF in vivo and in vitro, and thus it has been speculated that it is the TNF-alpha gene which underlies the genetic association of many diseases with the autoimmune haplotype. There have been many reports of HLA associations with lichen scleroses, but these have not been concordant. We therefore decided to analyse the TNF-alpha polymorphism in patients with lichen scleroses to determine if TNF-alpha was likely to play a role in susceptibility or severity of lichen scleroses. No association between alleles of the TNF-alpha polymorphism and lichen scleroses was found.

Effective cryopreservation and long-term storage of primary human hepatocytes with recovery of viability, differentiation, and replicative potential.

Despite reports of successful cryopreservation of primary human hepatocytes, existing methods do not produce sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocellular transplantation. We now describe a protocol for efficient cryopreservation of primary human hepatocytes using University of Wisconsin (UW) solution, fetal bovine serum, and dimethyl sulfoxide (DMSO). This method provides > 90% viability of differentiated, primary human hepatocytes 8 mo after cryopreservation as measured by trypan blue exclusion, preserves hepatocyte morphology, liver-specific gene expression (alpha 1 antitrypsin), and replication. The effectiveness of UW solution as a cryopreservative agent suggests that metabolic as well as ultrastructural factors may be important in the effective cryopreservation of primary human hepatocytes. The present method represents an effective protocol for cryopreserving differentiated primary human hepatocytes for research. This method may allow characterization and banking of human hepatocytes for clinical applications, including hepatocellular transplantation and hepatic assist devices.

Ascending projections of the posterior nucleus of the hypothalamus: PHA-L analysis in the rat.

With the exception of a report by R.B. Veazey, D.G. Amaral, and W.M. Cowan (1982, J. Comp. Neurol. 207:135-156) that examined the projections of the posterior hypothalamic area in the monkey by using the autoradiographic technique, the ascending projections of the posterior nucleus (PH) of the hypothalamus have not been systematically examined in any species. The present report describes the ascending projections of PH in the rat by using the anterograde anatomical tracer, Phaseolus vulgaris-leucoagglutinin (PHA-L). The major ascending route for PH fibers is the medial forebrain bundle. PH fibers project densely to several subcortical and cortical sites. The subcortical sites are the subthalamus/hypothalamus (zona incerta, the supramammillary nucleus, lateral, perifornical, dorsal, and anterior nuclei/areas), the thalamus (lateroposterior, laterodorsal, parafascicular, reuniens, paraventricular, central medial, paracentral, central lateral and intermediodorsal nuclei), the amygdala (central, lateral, and medial nuclei), the septal area (bed nucleus of stria terminalis, medial and lateral septum), and the basal forebrain (horizontal/vertical limbs of diagonal band nuclei and lateral preoptic area). The cortical sites are the perirhinal, insular, frontal (lateral agranular), prelimbic, and infralimbic cortices. The diversity of PH projections to subcortical and cortical "limbic-related" sites and to several structures with direct input to the hippocampus (supramammillary nucleus, reuniens, paraventricular and laterodorsal nuclei of the thalamus, medial and lateral septum, and perirhinal cortex) suggest that the PH may serve a critical role in various components of emotional behavior, including mnemonic processes associated with significant emotional events.

Analysis of time courses of metabolic precursors and products in heterogeneous rat brain tissue: limitations of kinetic modeling for predictions of intracompartmental concentrations from total tissue activity.

The efficacy of various kinetic models to predict time courses of total radioactivity and levels of precursor and metabolic products was evaluated in heterogeneous samples of freeze-blown brain of rats administered [14C]deoxyglucose ([14C]DG). Two kinetic models designed for homogeneous tissues, i.e., a no-product-loss, three-rate-constant (3K) model and a first-order-product-loss, four-rate-constant (4K) model, and a third kinetic model designed for heterogeneous tissues without product loss [Tissue Heterogeneity (TH) Model] were examined. In the 45-min interval following a pulse of [14C]DG, the fit of the TH Model to total tissue radioactivity was not statistically significantly better than that of the 3K Model, yet the TH Model described the time courses of [14C]DG and its metabolites more accurately. The TH- and 4K-Model-predicted time courses of [14C]DG and its metabolites were similar. Whole-brain glucose utilization (CMRglc) calculated with the TH or 3K Model, approximately 75 mumol 100 g-1 min-1, was similar to values previously determined by model-independent techniques, whereas CMRglc calculated with the 4K Model was 44% higher. In a separate group of rats administered a programmed infusion to attain a constant arterial concentration of [14C]DG that minimizes effects of tissue heterogeneity as well as any product loss, CMRglc calculated with all three models was 79 mumol 100 g-1 min-1 at 45 min after initiation of the infusion. Statistical comparisons of goodness of fit of total tissue radioactivity were, therefore, not indicative of which models best describe the tissue precursor and product pools or which models provide the most accurate rates of glucose utilization.

Comparative genetic association of human leukocyte antigen class II and tumor necrosis factor-alpha with dermatitis herpetiformis.

Dermatitis herpetiformis is a chronic subepidermal vesicular autoimmune skin disease characterized by a strong association with the human leukocyte antigen A1-B8-DR3-DQ2 haplotype. Although the strongest major histocompatibility complex association has been shown to be with the DQw2 (DQB1*0201/DQA1*0501) heterodimer, recent evidence has suggested that there may be up to three susceptibility loci within the major histocompatibility complex. Tumor necrosis factor-alpha (TNF-alpha) is a cytokine with a broad range of proinflammatory, immunomodulating, and catabolic activities. We have recently described the first known polymorphism in the human TNF-alpha gene, which is biallelic and lies in the promoter region. The rare allele, TNF2, is in strong linkage disequilibrium with the human leukocyte antigen A1-B8-DR3-DQ2 haplotype. We therefore examined TNF-alpha genotypes in patients with dermatitis herpetiformis and controls and compared the association with that of the class II alleles. Although TNF2 is strongly associated with dermatitis herpetiformis, this was weaker than the association with the class II loci, with DQw2 (DQB1*0201/DQA1*0501) showing the strongest disease association. Of the four patients negative for this marker, only one carried the TNF2 allele. These results indicate that TNF2 is not a major disease susceptibility marker, although our results do not exclude a minor role.

Role of the cerebellar fastigial nucleus in the physiological regulation of cerebral blood flow.

Local cerebral blood flow (ICBF) was measured with [14C]iodoantipyrine in conscious, unrestrained rats during electrical stimulation of the fastigial nucleus (FN). Electrode position in the FN was determined by blood pressure (MABP) responses to stimulation under anesthesia. In nine rats in which MABP responses had been variable under anesthesia, bipolar stimulation (50 Hz, 0.5 ms, 1 s on/1 s off) with currents of 30-100 microA after recovery from anesthesia produced stereotypic behavior but little effect on MABP and ICBF. In seven other conscious rats currents could be raised to 75-200 microA without inducing seizures, resulting in sustained MABP elevations during the ICBF measurement and significantly increased ICBF in the sensory-motor (+45%), parietal (+31%), and frontal cortices (+56%) and the caudate-putamen (+27%) above control values (n = 9). Glucose utilization, measured with [14C]deoxyglucose, in rats similarly stimulated was significantly increased in six structures, including some of the above, indicating increases in ICBF due to metabolic activation. Unilateral or bilateral electrolytic lesions of the FN, placed 6-7 days before ICBF measurement, had negligible effects on resting ICBF and on autoregulation in conscious rats. These results fail to support a specific role for the FN in physiological regulation of cerebral blood flow in unanesthetized rats.

Overexpression of human methylmalonyl CoA mutase in mice after in vivo gene transfer with asialoglycoprotein/polylysine/DNA complexes.

Methylmalonic acidemia resulting from genetic deficiency of methylmalonyl CoA mutase (MCM) is an often fatal metabolic disease. Somatic gene therapy for this disorder may require gene replacement in the liver. We describe overexpression of MCM in the liver of mice after in vivo gene delivery using asialoglycoprotein/polylysine/DNA (ASO/PL/DNA) targeted delivery to the liver of plasmids expressing recombinant MCM. After intravenous administration of the ASO/PL/DNA complex, the vector sequences are cleared from the blood with t1/2 = 2.5 min and > 95% of the vector is taken up by the liver. Vector sequences are cleared from the liver with t1/2 = 1.0-1.3 hr. MCM enzyme activity in the liver increases to levels 30-40% over baseline 6-24 hr after injection. No acute or chronic toxicity was observed. This net level of expression is likely to be therapeutic for MCM if the complex could be administered repetitively to treat acute episodes of life-threatening acidosis or establish a steady-state level of MCM activity. Repetitive administration of the ASO/PL/DNA complexes in mice was associated with formation of antibodies against asialo-orosomucoid and the asialo-orosomucoid complex but not against DNA.

Clustering of mutations in methylmalonyl CoA mutase associated with mut- methylmalonic acidemia.

Mutations have been described in human methylmalonyl CoA mutase (MCM) that exhibit partial defects in enzyme activity, including cobalamin-dependent (i.e., mut-) or interallelic complementation. This work describes mutations in cells from four patients, three of whom exhibit a cobalamin-dependent phenotype and all four of whom exhibit interallelic complementation. Four novel mutations (R694W, G648D, G630E, and G626C) are identified that cluster near the carboxyl terminus of the protein, a region close to another mut- mutation (G717V). Each of these mutations was shown to express a phenotype congruent with that of the parental cell line, after transfection into mut0 fibroblasts, and each exhibits interallelic complementation in cotransfection assays with clones bearing a R93H mutation. The activity of mutant enzymes expressed in Saccharomyces cerevisiae parallels the residual activity of the parental cell lines and exhibits novel sensitivities to pH and salt. The clustering of these mutations identifies a region of MCM that most likely represents the cobalamin-binding domain. The location of this domain, as well as the pattern of sequence preservation between the homologous human and Probiono-bacterium shermanii enzymes, suggests a mechanism for interallelic complementation in which the cobalamin-binding defect is complemented in trans from the heterologous subunits of the dimer.