Biosynthetic and synthetic AChR sequences to study T cells in myasthenia gravis.
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Autoantigens↗
Biomedical subjects
J Howard
Publications and source records attributed to J Howard.
Acetylcholine receptor-specific CD4+ T cells in myasthenia gravis patients have individual, but restricted TCR V beta usage.
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CD4-Positive T-Lymphocytes↗
Processivity of the motor protein kinesin requires two heads.
A single kinesin molecule can move for hundreds of steps along a microtubule without dissociating. One hypothesis to account for this processive movement is that the binding of kinesin's two heads is coordinated so that at least one head is always bound to the microtubule. To test this hypothesis, the motility of a full-length single-headed kinesin heterodimer was examined in the in vitro microtubule gliding assay. As the surface density of single-headed kinesin was lowered, there was a steep fall both in the rate at which microtubules landed and moved over the surface, and in the distance that microtubules moved, indicating that individual single-headed kinesin motors are not processive and that some four to six single-headed kinesin molecules are necessary and sufficient to move a microtubule continuously. At high ATP concentration, individual single-headed kinesin molecules detached from microtubules very slowly (at a rate less than one per second), 100-fold slower than the detachment during two-headed motility. This slow detachment directly supports a coordinated, hand-over-hand model in which the rapid detachment of one head in the dimer is contingent on the binding of the second head.
Animals↗
Inhibition of domestic cat spermatozoa acrosome reaction and zona pellucida penetration by tyrosine kinase inhibitors.
Spermatozoa from teratospermic domestic cats (> 60% morphologically abnormal spermatozoa per ejaculate) consistently exhibit lower levels of oocyte penetration in vitro than their normospermic (< 40% abnormal spermatozoa per ejaculate) counterparts. This could be caused by structural abnormalities or intracellular defects resulting in disruption of normal cellular functions. Spermatozoa from teratospermic cats also are compromised in the ability to capacitate and undergo the acrosome reaction (AR) in vitro. Further, we recently identified two tyrosine phosphorylated proteins (95- and 160-kDa) localized over the acrosome region in domestic cat spermatozoa. Phosphorylation of these proteins is reduced in teratospermic compared with normospermic ejaculates. To begin to understand the relationship between tyrosine phosphorylation and sperm function, we examined the effects of two protein tyrosine kinase inhibitors (tyrphostin RG-50864 and genistein) on (1) sperm motility; (2) protein tyrosine phosphorylation; (3) the ionophore A23187-induced AR; (4) the spontaneous and zona pellucida (ZP)-induced AR, and (5) the ability of spermatozoa from normospermic cats to penetrate conspecific ZP-intact oocytes. Over a wide range of concentrations, neither inhibitor affected sperm percentage motility during incubation (P > 0.05). Preincubation with either inhibitor reduced tyrosine phosphorylation of both (95- and 160-kDa) sperm proteins. Although both inhibitors blocked the ZP-induced AR, neither influenced the spontaneous AR nor the A23187-induced AR, suggesting that tyrosine phosphorylation may be involved in physiologic AR. No differences (P > 0.05) were observed in the ability of control or inhibitor-treated spermatozoa to bind to or penetrate the outer ZP layer. However, percentages of oocytes with treated spermatozoa in the inner ZP (tyrphostin, 8.7%; genistein, 20.4%) and perivitelline space (tyrphostin, 0%; genistein, 2.3%) were less (P < 0.001) than untreated controls (inner ZP, 62.7%; perivitelline space, 10.2%). These results (1) demonstrate that ZP-induced acrosomal exocytosis in domestic cat spermatozoa is regulated via a tyrosine kinase-dependent pathway and (2) suggest that defects in these signaling pathways may represent one of the causes for compromised sperm function in teratospermic males.
Acrosome↗
Molecular mimicry of the inflammation modulatory proteins (IMPs) of poxviruses: evasion of the inflammatory response to preserve viral habitat.
Microorganisms encode numerous immunomodulators that resemble, in structure and function, molecules captured over the millennia from their hosts [G. J. Kotwal J. Leukoc. Biol. 62, 415-429]. The vaccinia virus complement control protein (VCP) was the first soluble microbial protein to have a postulated role in the immunomodulation and evasion of host defense [G. J. Kotwal and B. Moss Nature 355, 176-179]. Purified bioactive VCP has been shown to bind to C3 and C4, block the complement cascade at multiple sites [G. J. Kotwal et al. Science 250, 827-830; R. Mckenzie, G. J. Kotwal et al. J. Infect. Dis. 166, 1245-1250] and exhibit a greater potency than the human complement 4b binding protein, C4b-BP [G. J. Kotwal, Am. Biotech. Lab. 9, 76]. The importance of this protein to poxviruses was further demonstrated in rabbits and guinea pigs through the use of recombinant virus lacking an intact DNA coding for VCP [Isaacs, G. J. Kotwal, and B. Moss Proc. Natl. Acad. Sci. 89, 628-672]. Studies in mice have shown that the homolog of VCP in cowpox virus (CPV), referred to as the inflammation modulatory protein (IMP) can, in a mouse model, significantly diminish the specific footpad swelling response [C. G. Miller, S. N. Shchelkunov, and G. J. Kotwal Virol. 229, 126-133]. To determine the precise cellular changes at the site of infection, BALB/c mice were subcutaneously injected (in the backs) with CPV or a recombinant virus lacking IMP, CPV-IMP. Differences in histology were observed by staining the adjoining skin tissue sections with hematoxylin & eosin or by removal of the connective tissue and staining with May-Grunwald-Geimsa. All mice that were injected with the CPV-IMP experienced severe tissue destruction and formation of nodular lesions compared with the mice injected with CPV. Microscopic examination indicated significantly greater cellular infiltration and destruction of skeletal muscle cells in the sections of connective tissue and adjoining skin tissue, respectively, of the mice injected with the CPV-IMP [G. J. Kotwal et al. Mol. Cell. Biochem. in press]. Thus IMP preserves the tissue at the site of infection (viral habitat). In this review, we present evidence for molecular mimicry and evolutionary relationship to other homologs of IMP and discuss their relationships with other IMPs such as the poxviral chemokine and cytokine receptor-like proteins.
Amino Acid Sequence↗
Memory, simple and complex language, and the temporal lobe.
Nineteen patients with intractable temporal lobe epilepsy who underwent anterior temporal lobectomy were given a highly specific memory battery (23 tests) pre- and post- (1 week; 1, 2, and 6 months; 1 and 2 years) resection. Sixteen of 23 tests revealed that memory performance of temporal lobe epilepsy patients was worse than normal controls prior to surgery (p < .001), while the most profound differences were seen in the remembering and generation of inferences from connected discourse. Almost no differences were observed in delayed nonmatching to sample tasks (recognition without language task). MRI results revealed that anterior, middle, and posterior hippocampal abnormality was extensive in 12 of 19 patients, and 12 also showed medial temporal lobe abnormalities and volume loss. Hippocampal damage was negatively correlated with extended delay memory performance for connected discourse: worse performance was associated with greater damage. Few differences in less complex memory performance were observed pre-postsurgery. While ordinary recognition functions were preserved, results demonstrated that dominant medial temporal lobe structures appeared heavily involved in language-generated memory, and hippocampus is heavily implicated in both simple and complex language.
Adult↗
Response of CD4+ T cells from myasthenic patients and healthy subjects of biosynthetic and synthetic sequences of the nicotinic acetylcholine receptor.
We investigated the suitability of pools of overlapping synthetic peptides spanning the complete alpha 1 subunit sequence of the human muscle acetylcholine receptor (AChR) (alpha 1 pool) or the extracellular domain (residues 1-218, alpha 11-218 pool), and of biosynthetic alpha 1 constructs from E. coli, as stimulants of human CD4+ cells from myasthenia gravis (MG) patients and healthy subjects. A construct corresponding to residues alpha 11-209 was obtained as solubilized inclusion bodies (ib alpha 11-209), or purified by SDS gel electrophoresis (pur alpha 11-209). A second construct included the extracellular, cytoplasmic and carboxylterminal domains plus histidine residues, and was obtained as inclusion bodies (ib alpha 1NoTrans) or purified by gel permeation and histidine tag affinity chromatography (pur alpha 1NoTrans). A biosynthetic extracellular domain of the neuronal AChR alpha 7 subunit (ib alpha 71-206) isolated from E. coli as inclusion bodies served as control for bacterial contaminants. We used ib alpha 11-209, pur alpha 11-209 and peptide pools to propagate CD4+ lines from two MG patients. The lines obtained using pur alpha 11-209 and the peptide pools recognized the peptide pools and alpha 1 constructs tested well, but ib alpha 71-206 poorly or not at all. These lines recognized peptides known to form CD4+ epitopes in these patients. The ib alpha 11-209 lines recognized ib alpha 11-209 and ib alpha 71-206 strongly, but recognized poorly pur alpha 11-209 and the alpha 11-218 pool. We propagated T-cell lines from a healthy subject using pur alpha 11-209 and ib alpha 11-209. The pur alpha 11-209 line recognized pur alpha 11-209 and the alpha 11-218 pool, but not ib alpha 11-209 or ib alpha 71-206. The ib alpha 11-209 line recognized ib alpha 11-209 and ib alpha 71-206, but not pur alpha 11-209 or the alpha 11-218 pool. We tested blood CD4+ cells from six MG patients and eight healthy subjects with ib alpha 11-209, pur alpha 11-209, the alpha 11-218 pool and--in the healthy subjects--also ib alpha 71-206, ib alpha 1NoTrans and pur alpha 1NoTrans. In both populations, the alpha 11-218 pool elicited low and sporadic responses, while the constructs elicited clear responses that were frequently higher for ib alpha 11-209 than pur alpha 11-209. The responses to ib alpha 71-206 were strong and comparable to those to ib alpha 11-209, ib alpha 1NoTrans, and pur alpha 1NoTrans. These results indicate that even purified constructs from E. coli contain bacterial contaminants recognized by CD4+ cells. They should not be used to test unselected blood CD4+ cells, because they may evoke strong CD4+ responses to the bacterial antigens. Purified recombinant sequences may be suitable for propagation of CD4+ cell lines, if the specificity of the lines can be verified using different antigen preparations. Short synthetic peptide sequences can be safely used for propagation of specific CD4+ cells. Although they are poor stimulants for unselected blood CD4+ cells, the low responses they elicit are probably due to these cells.
Amino Acid Sequence↗
Neoral cyclosporine as maintenance immunosuppression in pediatric small bowel transplantation.
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Administration, Oral↗
Summary of questions and answers on chickenpox for primary healthcare personnel. Prepared by the UK Advisory Group on Chickenpox.
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Adult↗
Bach Flower Remedies: a personal commentary on the work of Dr Edward Bach.
During my nursing career, I remember constantly being aware of patients who needed some emotional help--those who were frightened about a forthcoming operation, those who were shocked and despairing after being given their diagnosis and prognosis, others who were down in the dumps because they couldn't go home as soon as they had hoped. There were also the 'ward clowns' who tried to make everyone laugh with their good humour and little pranks, yet felt no less anxious, worried or depressed than anyone else. Patients seem to fit into categories: the nervous ones, the depressives, the jovial types, the moaners, those who demand attention and those who shun it. I feel sure that every nurse has noticed the different 'types' of people who fill hospital beds-ordinary people who seem to take on a new persona as soon as they get into their pyjamas and become a 'patient'. Somehow, their identity gets folded up and put away in their locker along with their outdoor clothes and other reminders of the outside world.
History, 20th Century↗
Surveillance of non-fatal workplace assault injuries, using police and employers' reports.
Although the majority of work-related homicides are routinely reported in the United States, information on non-fatal events is less complete. Comprehensive surveillance of non-fatal events depends on understanding reporting trends to different agencies. This study characterizes workplace assaults reported to police and through employers in eight southern California cities. Employers' reports filed from October 1, 1994, through January 31, 1995, and police reports filed from June 1, 1994, through March 31, 1995, that involved a non-fatal workplace assault injury were included. Reports from police and employers were linked, and annualized rates combining non-duplicative reports were calculated and event characteristics compared. The combined annualized rate of workplace assault injury for the eight cities was 184.6 per 100,000 workers, which was almost twice the rate found in either reporting source individually. Police reports differed from employers' reports by industry and occupation of victim but not type of event or weapons used. Examination of multiple reporting sources for non-fatal workplace assault injuries is essential to identifying the magnitude of these events. Understanding trends in reporting is important for the effective design of prevention programs.
California↗
T-cell recognition of muscle acetylcholine receptor subunits in generalized and ocular myasthenia gravis.
OBJECTIVES: Our purpose was to identify the muscle acetylcholine receptor (AChR) subunits recognized by autoimmune CD4+ T cells in myasthenia gravis (MG) and determine whether they differ in generalized (gMG) and ocular MG (oMG), and as gMG progresses. METHODS: We tested the proliferative response of blood CD4+ cells from 25 patients with gMG and four patients with oMG to synthetic peptides spanning the sequence of each subunit of human muscle AChR. We also investigated the antisubunit response of Th1 cells (a CD4+ subset frequently involved in autoimmune phenomena) using an enzyme-linked immunospot (ELISPOT) assay of antigen-induced secretion of interferon-gamma by individual CD4 cells. RESULTS: In gMG patients both the total CD4+ population and the Th1 subset recognized all AChR subunits to comparable extents. oMG patients recognized the AChR epsilon subunit minimally, and other subunits consistently and more strongly. gMG patients whose disease had lasted less than 5 years had lower antisubunit responses, and several of them did not recognize some AChR subunits; patients whose disease had lasted for 5 or more years had higher antisubunit responses and always responded to all AChR subunits. CONCLUSIONS: CD4+ and Thl responses in MG involve the entire AChR molecule. This likely results from spreading of the CD4+ sensitization to increasingly larger parts of the AChR as the disease progresses. The differential recognition of AChR subunits in oMG might be related to the preferential involvement of extrinsic ocular muscles, which express AChR containing the gamma subunit.
Adult↗
Coroners' records of rural and non-rural cases of youth suicide in New South Wales.
OBJECTIVE: The aim of this study is to compare the frequency of certain putative risk factors for youth suicide in New South Wales (especially use of alcohol, social class, unemployment, and internal migration) in metropolitan and rural settings. METHOD: A review of 137 files for 10-19-year-old subjects judged by the Coroner to have committed suicide in 1988-1990 was carried out. RESULTS: One hundred and fifteen males and 21 females were identified (one subjects sex was unavailable). The male-female ratio was higher in rural (13.0) areas than non-rural (4.9 chi 2 = 12.14, p < 0.01). Of 27 subjects migrating within Australia, most migrated in a rural direction, and most to rural shires. Unemployment was somewhat more common among rural (38.5%) than non-rural (28.9%) subjects (chi 2 = 0.75, p = 0.39). Eleven of 50 non-rural parents of the deceased, but none of the 11 rural parents, were ranked as being in social classes 2 or 3. Alcohol consumption appeared more common in rural shires (44%) than metropolitan areas (32.9%), but this was not statistically significant. Medical services were less utilised prior to death in rural (15%) than non-rural (25%) areas (chi 2 = 1.69, p = 0.19), and a psychiatric diagnosis was recorded more commonly in non-rural areas. CONCLUSIONS: Incomplete coronial file data and relatively small numbers limit this study's conclusions. Male suicides, principally by firearms, predominated in rural areas. Youth firearm access remains highly relevant to rural communities. Possible trends among rural subjects toward rural migration, higher unemployment, lower social class and lower medical attendance may point to resource deprivation among this group; these matters require further investigation.
Adolescent↗
Restricted HIV-1 infection of human astrocytes: potential role of nef in the regulation of virus replication.
A small percentage of astrocytes are consistently infected in vivo by HIV-1 and may contribute to neuropathogenesis despite a non-productive infection. Overexpression of the nef gene product has been associated with their infection both in vivo and in vitro. We examined the role of the nef gene during HIV replication in astrocytes (U251MG cells) following transfection with pNL4-3 proviral plasmid or isogenic strains containing a deletion or point mutation in the nef gene (pNL4-3deltaNef; pNL4-3-nef-stop). We were able to initiate virus replication which peaked at 5 days post-transfection and became non-productive after 21 days. Nef protein expression by wild type pNL4-3 was observed at low levels compared to control HeLa cells at peak virus replication. At later time points after development of a non-productive infection, viral antigen and Nef protein was not detectable however virus was readily recovered by co-culture with CD4+T-cells. Interestingly, virus production was significantly enhanced by a 222 base pair deletion in the nef reading frame. This was not observed with a frame shifting point mutation in nef, indicating a suppressive effect of nef on virus production in astrocytes. The enhanced virus production from nef-deleted pNL4-3 in U251MG cells was not reversed by co-expression of Nef from a second Nef-expressing plasmid, and in fact Nef expression in trans had a further positive effect on virus production. This suggested opposing effects of the Nef protein and elements contained within the nef sequence on virus production in astrocytes. Despite the low expression of Nef by U251MG astrocytes, relatively high amounts of multiply spliced 2 kb mRNA were present compared to HeLa cells. These data demonstrate that an acute low-level infection of astrocytes rapidly becomes a non-productive infection and this process is assisted by sequences in nef. The low level Nef protein expression, despite high levels of mRNA, suggests a block in translation of multiply spliced HIV mRNA in astrocytes, or a translational control mechanism not yet characterised.
AIDS Dementia Complex↗
Molecular motors: structural adaptations to cellular functions.
Molecular motors are protein machines whose directed movement along cytoskeletal filaments is driven by ATP hydrolysis. Eukaryotic cells contain motors that help to transport organelles to their correct cellular locations and to establish and alter cellular morphology during cell locomotion and division. The best-studied motors, myosin from skeletal muscle and conventional kinesin from brain, are remarkably similar in structure, yet have very different functions. These differences can be understood in terms of the 'duty ratio', the fraction of the time that a motor is attached to its filament. Differences in duty ratio can explain the diversity of structures, speeds and oligomerization states of members of the large kinesin, myosin and dynein families of motors.
Animals↗
Healing of laser incisions in rat dermis: comparisons of the carbon dioxide laser under manual and computer control and the scalpel.
BACKGROUND AND OBJECTIVE: The Computer-Assisted Surgical Techniques (CAST) program was researched to decrease lateral tissue damage and improve wound healing subsequent to laser incision. CAST differs from the traditional laser because it makes the incision in a discontinuous manner, allowing tissue to cool during the incision process. STUDY DESIGN/MATERIAL AND METHODS: The transient temperature changes in the tissue adjacent to the incision were measured with a thermocouple in a rat model. The subsequent wound healing was studied with histology and tensiometry. RESULTS: The thermal measurements demonstrated that all CAST settings were cooler than the continuous mode of laser incision. However, histology and tensiometric studies showed mixed results. CONCLUSION: This research demonstrates that CAST can be used in future surgical applications with no delay in wound healing as compared to the manually controlled laser. However, this study also finds no decrease in the wound healing time when using the CAST program.
Animals↗
TCR V beta usage by acetylcholine receptor-specific CD4+ T cells in myasthenia gravis.
In myasthenia gravis the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. AChR epitopes recognized by CD4+ T cells in myasthenic patients have been identified. AChR-specific CD4+ cell lines can be propagated by stimulation of blood lymphocytes with synthetic or biosynthetic AChR sequences. We analysed, using a semi-quantitative PCR assay, the T cell receptor (TCR) V beta usage of 16 anti-AChR polyclonal CD4+ T cell lines of known epitope specificity, propagated from myasthenic patients using pools of overlapping peptides corresponding to the sequence of an AChR subunit, or individual synthetic AChR sequences. Twelve lines had been propagated for less than 2 months, four lines for 3.5-5 months. Most lines had limited V beta usage, but in most cases different V beta regions were used for different epitopes in the same patient, and for the same epitope in different patients. In a few patients, the same V beta regions were used for recognition of different epitopes. The V beta 4 and V beta 6 regions were used most frequently. These findings suggest that the potentially autoimmune T cells that survive clonal deletion have a limited TCR repertoire. Although the present data do allow conclusions on the role of a superantigen in triggering the anti-AChR autoimmune response, the finding that different V beta regions were used in different patients does not support an important role of a superantigen in the maintenance of the CD4+ response in myasthenia gravis.
Amino Acid Sequence↗
Suicide among young rural Australians 1964-1993: a comparison with metropolitan trends.
The study tested hypotheses that from 1964 to 1993: (1) suicide rates among Australian 15- to 24-year-old males rose more sharply in rural than metropolitan areas; (2) firearm suicide rates among 15- to 24-year-old males, declining throughout Australia recently, rose continuously in rural areas; (3) suicide rates among 15- to 24-year-old females did not change significantly in either metropolitan or rural areas. Suicides of those aged 10-24 years recorded by the Australian Bureau of Statistics (ABS) were classified according to the subject's residential grouping. Rates were calculated using ABS population data corresponding to these groupings. Results were analysed using log-linear analysis and chi-square statistics. The results supported the first two hypotheses, but not the third. Suicide rates for 15- to 24-year-old males rose by a factor of 2.2 in metropolitan areas, by 4-fold in towns with populations between 4,000 and 25,000, and by 12-fold in towns with populations less than 4,000. Male firearm suicide rates continued to rise in rural areas, and the greatest proportion of deaths in those locations were by firearms, though male hanging rates increased most in recent years in all locations. Female youth suicide rates did not change overall, but in towns with populations less than 4,000, they increased 4.5-fold. Possible explanations for this epidemic, which are mostly speculative and require confirmation, are discussed.
Adolescent↗