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1.
Tissue Antigens ; 81(3): 141-9, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23398507

ABSTRACT

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome; distinguishing the thousands of HLA alleles is challenging. Next generation sequencing of exonic amplicons with the 454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software provides high resolution, high throughput HLA genotyping for eight class I and class II loci. HLA typing of potential donors for unrelated bone marrow donor registries typically uses a subset of these loci at high sample throughput and low cost per sample. The Fluidigm Access Array System enables the incorporation of 48 different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples with up to 48 different primer pairs in a microfluidic device generating 2304 parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are used. During genomic PCR, in this 4-primer system, the outer set of primers containing the MID and the 454 adaptor sequences are incorporated into an amplicon generated by the inner HLA target-specific primers each containing a common sequence tag at the 5' end of the forward and reverse primers. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 Life Sciences GS FLX System, followed by genotyping with Conexio software. We have genotyped 192 samples with 100% concordance to known genotypes using 8 primer pairs (covering exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1, 3/4/5 and DQB1) and 96 MIDs in a single GS FLX run. An average of 166 reads per amplicon was obtained. We have also genotyped 96 samples at high resolution (14 primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1, 3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1), recovering an average of 173 sequence reads per amplicon.


Subject(s)
Gene Library , Genotyping Techniques/methods , High-Throughput Nucleotide Sequencing/methods , Histocompatibility Testing/methods , Microfluidics/methods , Sequence Analysis, DNA/methods , Cell Line , DNA Primers/metabolism , Humans , Polymerase Chain Reaction , Software
2.
Tissue Antigens ; 77(3): 206-17, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21299525

ABSTRACT

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.


Subject(s)
HLA Antigens/genetics , High-Throughput Nucleotide Sequencing/methods , Sequence Analysis, DNA/trends , Alleles , Base Sequence , Double-Blind Method , Family Characteristics , Genotype , HLA Antigens/analysis , Humans , Models, Biological , Molecular Sequence Data , Multicenter Studies as Topic , Sequence Analysis, DNA/methods , Software
3.
Arch Psychiatr Nurs ; 8(1): 14-21, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8203939

ABSTRACT

The deinstitutionalization movement has left a legacy of need for appropriate care for inpatients with chronic refractory mental illness. This article describes a nurse managed program for such patients based on the principles of a therapeutic milieu. The program was formally defined for a 6-month period with an effort to include outcome measures for program evaluation. Multidisciplinary treatment team planning with a psychiatric nursing clinical specialist as team leader provided a format for putting therapeutic concepts into practice. The nurse clinical specialist and two nurse practitioners served as case managers as well as primary care providers. Program evaluation showed that cost-effective quality care could be achieved with a reduction in polypharmacy and overall neuroleptic usage. The notion of a restraint appropriate environment, with the goal of minimizing restraint use, was introduced and put into practice.


Subject(s)
Inpatients , Managed Care Programs/organization & administration , Mental Disorders/therapy , Milieu Therapy/organization & administration , Patient Care Planning , Patient Care Team/organization & administration , Psychiatric Nursing/organization & administration , Adult , Aged , Aged, 80 and over , Chronic Disease , Cost-Benefit Analysis , Deinstitutionalization , Drug Utilization , Health Services Needs and Demand , Humans , Male , Middle Aged , Models, Nursing , Nurse Clinicians , Nurse Practitioners , Outcome Assessment, Health Care , Program Evaluation
4.
Microb Ecol ; 17(1): 105-10, 1989 Jan.
Article in English | MEDLINE | ID: mdl-24197127

ABSTRACT

Enzootic leprosy has been recognized in armadillos in Louisiana since 1975. Contact with armadillos is being assessed as a risk factor for leprosy in three white women, lifelong residents of separate rural areas in northern Louisiana, which is a region without endemic leprosy. None has had any known exposure to human leprosy. Each was aware of armadillos (Dasypus novemcinctus) near or under her home for decades. In considering Possible environmental sources forMycobacterium leprae, we observed that all three had earthworm growth areas for fishing bait where soil was kept moist near their homes. The worms attracted armadillos. Since armadillos subsist on worms, grubs, and insects and because of the common feature of a "worm farm" near each home, we reasoned that earthworms might containM. leprae and be part of a cycle involving the armadillo and human beings. Worms from each home worm farm were studied. One site was sampled twice at patient 1's home, five sites were sampled once at patient 2's home, and three sites were sampled once at patient 3's home. A sample consisted of 3-4 worms, which were washed, purged, fixed live in 10% formalin, embedded in paraffin, sectioned, and stained with the Fite stain. Each was sagittally sectioned and examined by three independent observers. No acid-fast bacilli or other acid-fast structures were identified. We conclude that it is unlikely that earthworms are an environmental source or reservoir ofM. leprae.

5.
Arch Intern Med ; 148(9): 1987-92, 1988 Sep.
Article in English | MEDLINE | ID: mdl-3046539

ABSTRACT

Northern Louisiana has been essentially free of indigenous leprosy, and now it is not. Six new cases of leprosy have been diagnosed: three in 1986, the other three in 1985, 1983, and 1982, respectively. The patients had been lifelong residents of six scattered rural parishes. Leprosy had never been reported from five of them. No patient had had contact with human leprosy. The patients were white; four were women; the mean +/- SD age at onset was 60.3 +/- 16.4 years (age range, 31 to 80 years); and the mean +/- SD interval to diagnosis was 1.2 +/- 1.4 years. One patient had Hodgkin's disease at the age of 25 years and leprosy at the age of 31 years; another patient had cervical carcinoma. All rural northern Louisiana residents coexist with armadillos (Dasypus novemcinctus), some of which are infected with Mycobacterium leprae, the significance of which is unknown. Hypothetically, exposure to an unknown human case, reactivation of "asymptomatic" leprosy through immunosenescence or immunosuppression, or infection from an environmental source might have occurred. Because the patients lacked contact, travel, residence, and exposure risk factors, the origin of leprosy in the new indigenous cases is noteworthy and is not understood.


Subject(s)
Leprosy/epidemiology , Adult , Age Factors , Aged , Aged, 80 and over , Animals , Armadillos/microbiology , Female , Humans , Leprosy/etiology , Leprosy/immunology , Leprosy/transmission , Leprosy/veterinary , Louisiana , Male , Middle Aged , Mycobacterium leprae/immunology , Risk Factors , Rural Population , Space-Time Clustering , Zoonoses
6.
J Anat ; 155: 195-202, 1987 Dec.
Article in English | MEDLINE | ID: mdl-3332273

ABSTRACT

A stereotaxic atlas has been prepared for the medulla oblongata of the adult goat's brain using the technique described previously (Tindal et al. 1968). The atlas consists of transverse stereotaxic planes passing caudally at 1 mm intervals from posterior 10 mm (P10) at the level of the junction between brainstem and cerebellum to posterior 20 mm (P20) at the level of the obex.


Subject(s)
Anatomy, Artistic , Brain Mapping , Goats/anatomy & histology , Medulla Oblongata/anatomy & histology , Animals , Male , Stereotaxic Techniques
7.
Rev Infect Dis ; 9(3): 562-77, 1987.
Article in English | MEDLINE | ID: mdl-3299637

ABSTRACT

Leprosy has been considered to occur only after exposure to a human case. However, evidence has been accumulating that this conventional view is wrong and that an environmental nonhuman source is critical to some human infections with Mycobacterium leprae. Observations, some of which date back to the nineteenth century, support soil, vegetation, water, arthropods, and armadillos (Dasypus novemcinctus) as environmental sources of leprosy. Disparate clinical, epidemiologic, and microbiologic evidence has been critically reviewed in light of the fact that 50%-70% of sporadic cases of leprosy in well-studied populations occur in persons who have had no known contact with human leprosy. Historical data and current information alike substantiate the concept of nonhuman sources of the disease; recent observations with monoclonal antibody have shown that phenolic glycolipid-I antigen, which is unique to the M. leprae cell wall, is present in soil. In the absence of a technique for in vitro cultivation, indirect methods and the body of observations reviewed here persuasively favor but do not prove the existence of environmental nonhuman sources of M. leprae.


Subject(s)
Disease Reservoirs , Leprosy/transmission , Animals , Armadillos/microbiology , Arthropod Vectors , Arthropods/microbiology , Female , Humans , Leprosy/veterinary , Male , Mycobacterium leprae/isolation & purification , Plants/microbiology , Soil Microbiology , Water Microbiology
8.
J Endocrinol ; 109(3): 405-9, 1986 Jun.
Article in English | MEDLINE | ID: mdl-3525729

ABSTRACT

Transection of the ventral central grey and surrounding midbrain tegmentum in anaesthetized lactating rabbits caused repeated milk ejections which, on comparison with the effects of i.v. infusions of synthetic oxytocin, synthetic arginine-vasopressin or a mixture of the two, were attributed to continuous release of 1.25-2.5 mu. oxytocin/min, although it is not known whether lesser amounts of vasopressin might also have been released. It is suggested that the ascending midbrain reticular formation, which is known to project rostrally through this region, controls the central inhibition of oxytocin release via the previously described septo-hippocampo-subicular route to the hypothalamus.


Subject(s)
Lactation , Mesencephalon/physiology , Milk Ejection , Oxytocin/metabolism , Animals , Arginine Vasopressin/pharmacology , Female , Lactation/drug effects , Mesencephalon/surgery , Milk Ejection/drug effects , Oxytocin/pharmacology , Pregnancy , Rabbits , Stereotaxic Techniques
9.
J Endocrinol ; 104(1): 159-63, 1985 Jan.
Article in English | MEDLINE | ID: mdl-3881551

ABSTRACT

Six rumen-fistulated, overnight-fasted, female British Saanen goats were used. In preliminary experiments on two of the goats, evidence was obtained that inflation of a balloon with water in the cranial sac of the rumen was accompanied by inhibition of GH release. In a definitive series of experiments on the other four goats (one goat on 1 day = one experiment), a total of 96 experiments were carried out, 48 of which were balloon inflations with 2.5 litres water at 37 degrees C in the cranial sac of the rumen for 40 min and 48 were control experiments on alternate days. It was found that the mean plasma GH concentration of the samples taken at the end of the 40-min inflation period was significantly (P less than 0.05) lower than the mean value of the samples taken at the start of inflation. Mean control values at the start and finish of inflation were not significantly different. There were no significant differences between mean plasma insulin concentrations at the start and finish of inflation in experimental and control groups. It is concluded that distension of the cranial rumen in the goat is a signal which can inhibit the release of GH.


Subject(s)
Growth Hormone/metabolism , Rumen/physiology , Animals , Eating , Female , Goats , Growth Hormone/blood , Insulin/blood , Pressure
10.
Biochem J ; 218(2): 573-81, 1984 Mar 01.
Article in English | MEDLINE | ID: mdl-6370242

ABSTRACT

Bovine growth hormone (somatotropin) was extracted from anterior pituitaries and fractionated into four protein peaks (A-D) by chromatography on DEAE-Sephacel. Analysis by high-pressure liquid chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the homogeneity of the material increased from fraction A through to D. The properties of the fractions were examined in the following manner: immunological activity (radioimmunoassays for ruminant growth hormone and prolactin); growth-promoting activity (rat tibia test); lipolytic activity (release of glycerol from rat epididymal fat in the presence of dexamethasone); diabetogenic activity (rate of glucose transport in epididymal fat of hypophysectomized rats and intravenous insulin-tolerance tests in goats). None of the fractions contained immunoreactive prolactin and all were equally lipolytic. Although fraction A contained a small quantity of immunoreactive growth hormone it had no growth-promoting or diabetogenic activities. Both fractions B and C were diabetogenic and contained high concentrations of immunoreactive growth hormone, consistent with their growth-promoting activity. Although the growth-promoting activity of fraction D was higher than that of the other three fractions, it was not diabetogenic and was only weakly immunoreactive. These results for bovine growth hormone support the contention that growth hormone, as commonly extracted, is a mixture of different molecular forms and that these different metabolic properties of the hormone might be explained in terms of this heterogeneity.


Subject(s)
Growth Hormone , Pituitary Gland, Anterior/analysis , Animals , Biological Transport/drug effects , Cattle , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Glucose/metabolism , Goats , Growth Hormone/immunology , Growth Hormone/pharmacology , Insulin/metabolism , Lipolysis/drug effects , Radioimmunoassay , Rats
11.
J Endocrinol ; 100(1): 125-9, 1984 Jan.
Article in English | MEDLINE | ID: mdl-6361194

ABSTRACT

Removal of discrete parts of the forebrain of the anaesthetized lactating rabbit by suction caused repeated milk-ejection responses. These were associated with transection or removal of the subiculum, which represents the major efferent pathway from the hippocampal formation to the diencephalon. It is suggested that the hippocampus may exert a tonic, inhibitory influence over oxytocin release, that this inhibition may represent a cascade of events which originates in the brainstem and culminates in the subiculum and that removal of this inhibition may lead to release of oxytocin.


Subject(s)
Hippocampus/physiology , Lactation , Milk Ejection , Neural Inhibition , Telencephalon/physiology , Afferent Pathways/physiology , Animals , Diencephalon/physiology , Efferent Pathways/physiology , Female , Pregnancy , Rabbits , Stereotaxic Techniques
12.
Horm Metab Res ; 14(8): 425-9, 1982 Aug.
Article in English | MEDLINE | ID: mdl-6751977

ABSTRACT

It was confirmed that, as in overnight-fasted castrate male sheep, plasma growth hormone (GH) levels also fell in overnight-fasted castrate male goats shortly after they were given hay the following morning and that GH levels could also fall in anticipation of being fed. A further observation was that plasma GH levels usually remained low for 2--3 hours if the goats were allowed to continue eating but that if the hay was removed after only one hour of eating the plasma GH level rose 20--50 min later. Although initial attempts to determine the role of visceral stimuli in the regulation of GH release in castrate male goats by bilateral cooling of exteriorized vagi were inconclusive because of side-effects, they drew attention to the role of rumen distension and activation of stretch receptors in the modulation of GH release. This was investigated by artificial distension of the cranial sac of the rumen with a balloon in a castrate male, overnight-fasted goat prepared with a rumen fistula. Inflation of the balloon with warm water was followed by a fall, and deflation of the balloon by a rise, in plasma GH levels. It is concluded that rumen distension and hence activation of ruminal stretch receptors is a sufficient stimulus to explain the immediate inhibition of GH release by feeding in the goat. As a result of these, and other, observations during this work, it is also suggested that unrestrained release of GH, and hence large oscillations in plasma GH level, may occur in the goat unless it is suppressed by any of several factors, which include activation of rumen stretch receptors by feeding or inflation, anticipation of being fed, stress and metabolic satiety.


Subject(s)
Cold Temperature , Food , Growth Hormone/blood , Rumen/physiology , Vagus Nerve/physiology , Animals , Goats , Insulin/blood , Male , Time Factors
13.
J Endocrinol ; 86(3): 525-31, 1980 Sep.
Article in English | MEDLINE | ID: mdl-7000954

ABSTRACT

Discrete, transverse stereotaxic cuts in th forebrain of the anesthetized lactating rabbit caused spontaneous milk-ejection responses repeated at intervals of 1-5 min. The region of the brain in which cuts were effective included the diagonal band of Broca, the entire rostrocaudal extent of the dorsal medial septum, part of the cingulum and part of the hippocampal fimbria. It is proposed that severance of a septo-hippocampal patway frees the hippocampus from inhibition and allows oxytocin release to occur. It remains to be determined whether the hippocampus itself is directly responsible for periodic activation of neurosecretory cells.


Subject(s)
Diencephalon/physiology , Lactation , Milk Ejection , Neural Inhibition , Telencephalon/physiology , Afferent Pathways/physiology , Animals , Female , Mammary Glands, Animal/physiology , Oxytocin/pharmacology , Pregnancy , Pressure , Rabbits , Stereotaxic Techniques
14.
J Endocrinol ; 76(2): 333-46, 1978 Feb.
Article in English | MEDLINE | ID: mdl-564386

ABSTRACT

Recording electrodes were implanted in contact with the dura mater overlying the parietal cortex of six female goats, four of which were lactating. After recovery from surgery and complete familiarization with the housing conditions, the personnel and the recording technique, each goat was observed continuously for 24 h with simultaneous recording of the cortical electroencephalogram (EEG). Remote blood sampling was carried out every 30 min without disturbing the animal. Apart from the release of growth hormone (GH) associated with morning milking in two of the goats, there was no consistent relationship between the apparently spontaneous, episodic release of GH and behvaiour, stages of sleep, cortical EEG, air temperature, time of day or night, obvious environmental stimuli which arose from the normal husbandry routine, or the levels of porlactin, insulin, glucose or free fatty acids in the blood. There was also no relationship between the release of prolactin and the stages of sleep.


Subject(s)
Behavior, Animal/physiology , Goats/physiology , Growth Hormone/metabolism , Lactation , Animals , Blood Glucose/metabolism , Electroencephalography , Fatty Acids, Nonesterified/blood , Female , Growth Hormone/blood , Insulin/blood , Pregnancy , Prolactin/blood , Sleep Stages/physiology , Temperature
15.
J Endocrinol ; 70(1): 19-23, 1976 Jul.
Article in English | MEDLINE | ID: mdl-778321

ABSTRACT

Placental lactogen has been detected in the field vole, Microtus agrestis, and the bank vole, Clethrionomys glareolus, using a co-culture technique. In field voles this activity could be detected from about day 8 of pregnancy to shortly before term, and stimulated both mouse and vole mammary gland to secrete in vitro. Partial immunological cross-reaction was detected in a radioimmunoassay system between rat prolactin and either extracts of vole pituitaries or media on which vole pituitaries had been cultured; vole placental lactogen showed no cross-reaction with rat prolactin. One site of origin for this hormone is probably the trophoblastic giant cells.


Subject(s)
Arvicolinae/physiology , Placental Lactogen/analysis , Rodentia/physiology , Animals , Cross Reactions , Female , Mammary Glands, Animal/metabolism , Pituitary Gland, Anterior/physiology , Placenta/physiology , Pregnancy , Prolactin/immunology , Radioimmunoassay , Stimulation, Chemical
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