Purification and transcription repression by negative cofactor 2.
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Biochemistry↗
Biomedical subjects
S Gilfillan
Publications and source records attributed to S Gilfillan.
Long-term sequelae of HFE deletion in C57BL/6 x 129/O1a mice, an animal model for hereditary haemochromatosis.
BACKGROUND: HFE knockout mice (C57BL/6 x 129/Ola strain) mimic the functional aberrations of human hereditary haemochromatosis (HH) in short-term experiments. The present study investigates functional and morphological long-term changes. METHODS: HFE(o/o), HFE(+/o) and HFE(+/+) mice were maintained on iron-rich and control diets for 2 weeks, 3, 12 and 18 months. Light microscopic tissue iron distribution, pathomorphological alterations, tissue iron content and oxidative stress were analysed in liver, pancreas, spleen, gastrointestinal tract, kidneys and myocardium. Additionally, duodenal 59Fe absorption and 59Fe whole body loss were measured. RESULTS: Iron distribution between organs and microscopic iron deposition in the tissues resembled the patterns described in HH. After 3 months of iron-rich feeding duodenal 59Fe absorption decreased to approximately 15% of iron-adequate controls but remained about twice as high in HFE(o/o) as in HFE(+/+) mice. Hepatic iron concentrations reached only half the values known to induce hepatic fibrosis in rats and humans, while whole body 59Fe loss was about twice as high. Consequently no hepatic fibrosis developed, although massive hepatocellular iron deposition and indication for oxidative stress were observed. CONCLUSION: C57BL/6 x 129/O1a HFE(o/o) mice mimic HH iron distribution and the regulation of intestinal iron absorption after long-term feeding. However, characteristic morphological late changes in untreated HH are not modelled.
Animals↗
The long isoform of terminal deoxynucleotidyl transferase enters the nucleus and, rather than catalyzing nontemplated nucleotide addition, modulates the catalytic activity of the short isoform.
During variable/diversity/joining (V[D]J) recombination, the enzyme terminal deoxynucleotidyl transferase (Tdt) adds random nucleotides at the junctions of the rearranging gene segments, increasing diversity of the antibody (Ab) and T cell receptor repertoires. Two splice variants of Tdt have been described, but only one (short isoform of Tdt [TdtS]) has been convincingly demonstrated to catalyze nontemplated (N) addition in vitro. We have expressed each splice variant of Tdt in transgenic (Tg) mice and found that the TdtS transgene catalyzes N addition on the endogenous Tdt(-/)- background and in fetal liver, but that the long isoform of Tdt (TdtL) transgene does neither. In contrast to previous in vitro results, both TdtS and TdtL are translocated to the nucleus in our model. Furthermore, TdtL/TdtS double Tg mice exhibit less N addition in fetal liver than do TdtS Tg mice. Whereas the TdtS transgene was shown to have functional consequences on the antiphosphorylcholine (PC) B cell repertoire, TdtL Tg mice exhibit a normal PC response, and Tdt(-/)- mice actually exhibit an increase in the PC response and in TEPC 15 idiotype(+) Ab production. We conclude that TdtL localizes to the nucleus in vivo where it serves to modulate TdtS function.
Alternative Splicing↗
The nature of help-seeking during psychiatric emergency service visits by a patient and an accompanying adult.
Utilization rates for urban psychiatric emergency services remain high, and the decision to seek care in this setting is poorly understood. Three hundred individuals accompanying patients to a psychiatric emergency service were interviewed about their help seeking and choice of treatment setting. Twenty-three of the interviewees (7.7 percent) were caregivers accompanying patients with severe and persistent mental illness. They were significantly more likely than other interviewees to know the difference between psychiatric emergency services and services offered by other outpatient providers. More than half reported that the patient they accompanied was intermittently noncompliant, which required visiting either a walk-in service during a moment when the patient was cooperative or a facility equipped to provide involuntary treatment.
Adolescent↗
Terminal deoxynucleotidyl transferase and repertoire development.
In mice, the absence of terminal deoxynucleotidyl transferase (Tdt) expression during fetal and neonatal life provides a window in development where clones of lymphocytes are generated that provide protective immunity. Introducing premature Tdt activity interferes with the development of these clones and results in an impaired ability to make protective antibodies. Conversely, gene-targeted disruption of Tdt prevents N additions at all stages of T and B-lymphocyte development and promotes the development of fetal-like T and B-cell clones into adulthood, with accompanying alterations in repertoire. The alternative splice forms of Tdt may be necessary to provide regulatory mechanisms to restrict N addition to appropriate stages of the developmental pathways, the details of which are being revealed. The evidence continues to build that Tdt is a key player in influencing the outcome of V(D)J recombination during lymphocyte and repertoire development.
Animals↗
Toward a redefinition of psychiatric emergency.
OBJECTIVE: To compare three methods for rating legitimate use of psychiatric emergency services (PES) in order to develop criteria that can differentiate appropriate from inappropriate PES service requests. METHOD: Ratings of PES visits by treating physicians and ratings of the same visits made during review of medical records. STUDY DESIGN: Two previously used methods of identifying justified PES service use were compared with the treating physician's rating of the same: (1) hospitalization as visit outcome and (2) retrospective chart ratings of visit characteristics using traditional medico-surgical criteria for "emergent" illness episodes. DATA EXTRACTION METHODS: Data were extracted through use of a physician questionnaire, and medical and administrative record review. PRINCIPAL FINDINGS: Agreement between the methods ranged from 47.1 percent to 74.1 percent. A total of 21.7 percent of visits were rated as true health "emergencies" by the traditional definition, while 70.4 percent of visits were rated as "necessary" by treating physicians, and 21.0 percent resulted in hospitalization. Acuteness of behavioral dyscontrol and imminent dangerousness at the time of the visit were common characteristics of appropriate use by most combinations of the three methods of rating visits. CONCLUSIONS: The rating systems employed in similar recent studies produce widely varying percentages of visits so classified. However, it does appear likely that a minimum of 25-30 percent of visits are nonemergent and could be triaged to other, less costly treatment providers. Proposed criteria by which to identify "legitimate" psychiatric emergency room treatment requests includes only patient presentations with (a) acute behavioral dyscontrol or (b) imminent dangerousness to self or others.
Adolescent↗
Experimental hemochromatosis due to MHC class I HFE deficiency: immune status and iron metabolism.
The puzzling linkage between genetic hemochromatosis and histocompatibility loci became even more so when the gene involved, HFE, was identified. Indeed, within the well defined, mainly peptide-binding, MHC class I family of molecules, HFE seems to perform an unusual yet essential function. As yet, our understanding of HFE function in iron homeostasis is only partial; an even more open question is its possible role in the immune system. To advance on both of these avenues, we report the deletion of HFE alpha1 and alpha2 putative ligand binding domains in vivo. HFE-deficient animals were analyzed for a comprehensive set of metabolic and immune parameters. Faithfully mimicking human hemochromatosis, mice homozygous for this deletion develop iron overload, characterized by a higher plasma iron content and a raised transferrin saturation as well as an elevated hepatic iron load. The primary defect could, indeed, be traced to an augmented duodenal iron absorption. In parallel, measurement of the gut mucosal iron content as well as iron regulatory proteins allows a more informed evaluation of various hypotheses regarding the precise role of HFE in iron homeostasis. Finally, an extensive phenotyping of primary and secondary lymphoid organs including the gut provides no compelling evidence for an obvious immune-linked function for HFE.
Animals↗
A conserved sequence block in the murine and human TCR J alpha region: assessment of regulatory function in vivo.
Temporal control of rearrangement at the TCR alpha/delta locus is crucial for development of the gamma delta and alpha beta T cell lineages. Because the TCR delta locus is embedded within the alpha locus, rearrangement of any V alpha-J alpha excises the delta locus, precluding expression of a functional gamma delta TCR. Approximately 100 kb spanning the C delta-C alpha region has been sequenced from both human and mouse, and comparison has revealed an unexpectedly high degree of conservation between the two. Of interest in terms of regulation, several highly conserved sequence blocks (> 90% over > 50 bp) were identified that did not correspond to known regulatory elements such as the TCR alpha and delta enhancers or to coding regions. One of these blocks lying between J alpha 4 and J alpha 3, which appears to be conserved in other vertebrates, has been shown to augment TCR alpha enhancer function in vitro and differentially bind factors from nuclear extracts. To further assess a plausible regulatory role for this element, we have created mice in which this conserved sequence block is either deleted or replaced with a neomycin resistance gene driven by the phosphoglycerate kinase promoter (pgk-neor). Deletion of this conserved sequence block in vivo did have a local effect on J alpha usage, echoing the in vitro data. However, its replacement with pgk-neor had a much more dramatic, long range effect, perhaps underscoring the importance of maintaining overall structure at this locus.
Animals↗
Alpha beta/gamma delta lineage commitment in the thymus of normal and genetically manipulated mice.
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Animals↗
Selection and function of CD4+ T lymphocytes in transgenic mice expressing mutant MHC class II molecules deficient in their interaction with CD4.
Interactions of the T cell coreceptors, CD4 and CD8, with MHC molecules participate in regulating thymocyte development and T lymphocyte activation and differentiation to memory T cells. However, the exact roles of these interactions in normal T cell development and function remain unclear. CD4 interacts with class II MHC7 molecules via several noncontiguous regions in both the class II MHC alpha- and beta-chains. We have introduced a double mutation that disrupts interaction with CD4 into the I-A(beta)k gene and used this construct to generate transgenic mice expressing only mutant class II MHC. Although CD4+ thymocytes matured to the single-positive stage in these mice, their frequency was reduced by threefold compared with that of wild-type transgenics. Positive selection of CD4+ T cells in the mutant transgenic mice may have been mediated by TCRs with a higher than usual affinity for class II MHC/Ag complexes. In A(beta)k mutant transgenics, peripheral CD4+ lymphocytes promoted B cell differentiation to plasma cells. These CD4+ T cells also secreted IFN-gamma in response to various stimuli (e.g., protein Ag, bacterial superantigen, and alloantigen), but were deficient in IL-2 secretion. Interactions between CD4 and class II MHC molecules appeared to regulate lymphokine production, with a strong bias toward IFN-gamma and against IL-2 in the absence of these interactions. Our results have implications for the manipulation of T cell-dependent immune responses.
Amino Acid Substitution↗
Terminal deoxynucleotidyl transferase deficiency reduces the incidence of autoimmune nephritis in (New Zealand Black x New Zealand White)F1 mice.
Terminal deoxynucleotidyl transferase (TdT) enzyme activity in lymphocytes generates diversity in the Ag receptor repertoires by adding template-independent N nucleotides and disrupting homology-directed rearrangements. The importance of this diversity in vivo and the significance of the suppression of TdT during fetal life remain uncertain. Previous studies have shown that in TdT knockout mice (TdT(0)) 1) the T cell repertoire is less peptide oriented; and 2) natural autoantibody, particularly anti-DNA autoantibodies, are less polyreactive, and their mean affinities are reduced. Consequently, the suppression of TdT during early T/B cell ontogeny may participate in controlling autoimmunity. To study the impact of TdT suppression in autoimmune-prone mice, we introduced the TdT null mutation into the (NZB x NZW)F1 (B/W) mouse strain. We show that TdT deficiency significantly reduces the incidence of autoimmune nephritis and prolongs survival compared with those in control mice. Surprisingly, the long-term survivor TdT(0) mice produced amounts of anti-ADN and anti-histone autoantibodies similar to those of their TdT+ littermates. However, these TdT(0) mice showed no evidence of renal inflammation, and the immune deposits were restricted to the mesangium, whereas basal membrane deposits were clearly correlated with overt renal disease. The present study supports the idea that the absence of TdT enzyme activity in lymphocytes protects mice against autoimmunity and could offer a therapeutic approach to autoimmune diseases. Moreover, our results may help to unravel the mechanisms of lupus nephritis.
Animals↗
A comparison of psychotic and nonpsychotic substance users in the psychiatric emergency room.
Current illicit drug and alcohol users were identified by laboratory evaluation of urine samples from nonpsychotic patients without a primary clinical diagnosis of a substance use disorder seen in a psychiatric emergency room. Urine screens revealed that 32 of 93 nonpsychotic patients (34 percent) had used a substance just before visiting the emergency room. Compared with nonusers, users were more often Caucasian females with adjustment disorders who admitted their previous substance use. The prevalence of concurrent use among nonpsychotic patients was higher than among psychotic patients. Nonpsychotic and psychotic users differed in gender, marital status, level of suicidality, self-report of use, the clinician's suspicion of use, use of seclusion during the visit, admitting status, level of care, and disposition.
Adjustment Disorders↗
Autoantibodies in mice lacking terminal deoxynucleotidyl transferase: evidence for a role of N region addition in the polyreactivity and in the affinities of anti-DNA antibodies.
The generation of terminal deoxynucleotidyl transferase knockout mice (TdT0) has demonstrated that TdT is the only major activity involved in N region addition. This enzyme generates diversity by adding random nucleotides at the V-D-J junctions and by disrupting the formation of repetitive "homology-directed" junctions. Several studies have demonstrated that the Ig heavy chain third complementarity-determining region (H-CDR3) and the N region play a critical role: 1) in distinguishing between polyreactive and monospecific combining sites in natural and Ag-induced Abs; and 2) in the specificity and polyreactivity of natural autoantibodies (autoAbs) and in particular of anti-DNA Abs. To examine the impact of the lack of TdT on the natural autoAb repertoire in adult mice, we have stimulated TdT0 and TdT+ littermates with LPS. Serum studies demonstrate that TdT is not critical for the generation of B cells expressing autoAbs including anti-DNA Abs and rheumatoid factors. However, the generation of a large collection of hybridomas indicates that the frequencies of these cells are reduced in TdT0 mice mainly due to a lower incidence of polyreactivity; also, the lack of N region diversity seems to negatively affect the affinity of anti-DNA Abs. The physiologic relevance of these data is discussed.
Amino Acid Sequence↗
Substance use among patients with a psychotic disorder in a psychiatric emergency room.
OBJECTIVE: This study used laboratory tests to identify current drug and alcohol users among psychotic patients seeking treatment in an urban psychiatric emergency room. Rates of clinician-suspected use and self-reported use were compared, as were treatment and disposition of users and nonusers. METHODS: Logistic regression modeling was used to identify factors that differentiated current substance users from nonusers in a sample of 112 psychotic patients. RESULTS: Laboratory analyses revealed that 24 of the 112 psychotic patients (21 percent) had used alcohol or an illegal substance before visiting the emergency room. Younger age, male gender, African-American ethnicity, clinician-suspected substance use, and presentation in the emergency room between 7 p.m. and 7 a.m. were associated with a higher likelihood of positive results on the urine test. Only five of the patients who had positive results (21 percent) self-reported substance use. Clinicians suspected that 59 patients (53 percent) were under the influence; however, only 17 of those suspected (29 percent) had positive laboratory results. Patients with positive laboratory results required more intense care in the psychiatric emergency room and were more often hospitalized. CONCLUSIONS: Some demographic and clinical factors were associated with concurrent substance use among psychotic patients in the emergency room. Clinicians' suspicions of use in this sample of psychotic patients lacked specificity due to the fact that potential use was suspected in a large number of cases for which laboratory results were negative. In contrast, self-reported use was uncommon among patients with positive results. Because neither clinician judgment nor patient self-report meaningfully predicts current substance use, routine urine drug screens may be appropriate.
Adult↗
Somatic hypermutation occurs in B cells of terminal deoxynucleotidyl transferase-, CD23-, interleukin-4-, IgD- and CD30-deficient mouse mutants.
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Animals↗
Efficient immune responses in mice lacking N-region diversity.
Mice with a null mutation in the terminal deoxynucleotidyl transferase (TdT) gene harbor immunoglobulin and T cell receptor repertoires essentially devoid of N-region diversity. Consequently, the CDR3 loops important for antigen recognition are shorter and considerably less diverse than those of wild-type controls. We find surprisingly normal immune responses in TdT0 mice, as regards both efficiency and specificity. This provokes a reconsideration of the assumption that N-region diversity is required for an effective T and B cell repertoire.
Amino Acid Sequence↗
Mice lacking terminal deoxynucleotidyl transferase: adult mice with a fetal antigen receptor repertoire.
TdT knock-out mice have established the role of this enzyme in vivo: TdT mediates the transition from the relatively limited fetal to the highly diverse adult antigen receptor repertoire by adding template independent "N" nucleotides and disrupting homology-directed recombination. Lacking this source of diversity, TdT degree mice harbor essentially fetal antigen receptor repertoires. In alpha beta TCRs, the TdT null mutation affects the length and diversity of the CDR3 loops thought to be important in "directing" MHC/peptide recognition. N- CDR3 loops appear to wield less influence than do their N+ counterparts--positive selection is more efficient in the TdT degree animals and the peripheral repertiore is more polyreactive and less peptide-oriented than is the N+ repertoire. However, this loss of specificity does not markedly diminish the response to specific peptides. Overall, mice harboring essentially fetal repertoires are robust and effectively respond to a wide variety of challenges to the immune system.
Animals↗