Biomedical subjects
M Howard
Publications and source records attributed to M Howard.
The role of autoantigens in autoimmune disease.
Many autoantigens have been identified in human patients and in rodent models. In numerous experimental settings, these autoantigens or related autoreactive lymphocytes can transfer autoimmunity. Although autoreactivity spreads to new epitopes during the course of disease, single-epitope-specific therapies show considerable efficacy in multi-epitope-induced models of autoimmunity. These observations may indicate that epitope-specific therapies operate at the level of regulating mechanisms of immune tolerance rather than exerting a direct effect on autoreactive T lymphocytes.
The left atrial appendage: our most lethal human attachment! Surgical implications.
OBJECTIVES: To prevent death from atrial fibrillation, a cardiac disease which kills by producing emboli. Atrial fibrillation causes about 25% of strokes and increases stroke rate by five times. Over 90% of these embolic strokes are from clots originating in the left atrial appendage. This study addresses the surgical feasibility of removing the appendage to prevent future deaths in two subcategories of patients. (1) Prophylactic removal during open-heart surgery to study its safety. Theoretically, as these patients age and some develop atrial fibrillation, protection from embolic strokes would already be present. (2) Therapeutic removal in chronic atrial fibrillation patients by means of a thorascopic approach. Its technical feasibility is demonstrated. Its actual stroke prevention potential awaits large studies. METHODS: Appendectomy has been evaluated three ways. (1) Experimentally, thorascopic appendage removal was performed on 20 goats with endoscopic approach. Late studies showed a cleanly healed atrial closure after stapling, and no puckering of tissue as seen with the purse-string approach. (2) Safety of human appendectomy was demonstrated in 437 patients (1995-1997). Routine appendectomy was performed during open-heart surgery. Forty-three appendages were stapled, 391 sutured off. (3) Thorascopic appendectomy in seven patients with chronic atrial fibrillation has been successfully accomplished as an isolated surgical procedure. Stapling or suture closure provides a much cleaner, non-puckered suture line than a purse string. RESULTS: In prophylactic removal, no acute bleeding occurred. No late problems have been identified. Endoscopic removal of the appendage has been successful in seven atrial fibrillation patients. CONCLUSIONS: The left atrial appendage is a lethal source of emboli in atrial fibrillation patients. As patients age and often develop atrial fibrillation, prophylactic appendage removal whenever the chest is open is suggested as a method to prevent future strokes. In chronic atrial fibrillation patients, appendectomy can be done with a mini-thorascopic approach. Further studies are planned to demonstrate the effectiveness of appendectomy in preventing strokes in the chronic fibrillating patients.
Chemokines in autoimmune diseases.
Autoimmune diseases like multiple sclerosis (MS) and insulin-dependent diabetes (IDD) are believed to be mediated by pathogenic CD4+ autoreactive T cells which mediate selective destruction of specific host cells. Interrupting the trafficking of such T cells from host circulation to the sites of pathology, such as the central nervous system in the case of MS and the pancreas in the case of IDD, potentially offers a novel opportunity for therapeutic intervention in these diseases. The following summarizes our evolving thoughts on the role of the chemokine network in MS and IDD, and focuses on the chemokine receptor CXCR3 as a potential target for impeding T-cell-mediated destruction in these disease settings.
Multiple myeloma and Kaposi's sarcoma: what is the association?
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Cyclin D2 is essential for BCR-mediated proliferation and CD5 B cell development.
Progression into G(1) in B lymphocytes is regulated by cyclins D2 and D3, components of the cell cycle machinery currently believed to have overlapping and potentially redundant roles in cell cycle control. To study the specific role of cyclin D2 in B lymphocyte proliferation, we examined B cells from cyclin D2(-/-) mice and demonstrate a specific requirement for cyclin D2 in BCR- but not CD40- or lipopolysaccharide-induced proliferation. Furthermore, conventional B cell development proceeds normally in the mutant mice; however, the CD5 B cell compartment is dramatically reduced, suggesting that cyclin D2 is important in CD5 B cell development as well as antigen-dependent B cell clonal expansion.
Psychopathology, adversity, and service utilization of young refugees.
OBJECTIVE: To investigate the psychopathology, social impairment, adversities, and service utilization of refugee families and their children seeking help at a child and adolescent psychiatry clinic in London. METHOD: A retrospective case-control study of 30 refugee children and families individually matched with nonrefugee immigrant families and white British families. Case note review was carried out to obtain data on diagnosis, social adjustment, past adversity, exposure to violence, current socioeconomic circumstances, and use of the child and adolescent psychiatric service. RESULTS: Refugee children tended to have disorders with a psychosocial etiology rather than neurobiological disorders. Refugees had similar levels of social impairment compared with the other groups. Refugees were much more isolated and disadvantaged and had different referral pathways but were not more likely to drop out of treatment prematurely. CONCLUSIONS: Refugee children and families had been exposed to high levels of adversity. The ability of community agencies to refer families who could use treatment has significant resource implications.
Surface critical behavior in systems with nonequilibrium phase transitions
We study the surface critical behavior of branching-annihilating random walks with an even number of offspring (BARW) and directed percolation (DP) using a variety of theoretical techniques. Above the upper critical dimensions d(c), with d(c)=4 (DP) and d(c)=2 (BARW), we use mean field-theory to analyze the surface phase diagrams using the standard classification into ordinary, special, surface, and extraordinary transitions. For the case of BARW, at or below the upper critical dimension d</=d(c), we use field theoretic methods to study the effects of fluctuations. As in the bulk, the field-theory suffers from technical difficulties associated with the presence of a second critical dimension. However, we are still able to analyze the phase diagrams for BARW in d=1 and 2, which turn out to be very different from their mean field analog. Furthermore, for the case of BARW only (and not for DP), we find two independent surface beta(1) exponents in d=1, arising from two distinct definitions of the order parameter. Using an exact duality transformation on a lattice BARW model in d=1, we uncover a relationship between these two surface beta(1) exponents at the ordinary and special transitions. Many of our predictions are supported using Monte Carlo simulations of two different models belonging to the BARW universality class.
A new visual indicator of chlamydial cervicitis?
OBJECTIVES: To determine the usefulness of endocervical discharge opacity as a risk indicator for chlamydial infection in relation to two acknowledged visual indicators--yellow endocervical discharge and easily induced mucosal bleeding of the cervix. METHODS: Women from two family planning clinics, a therapeutic abortion clinic, and a university student health clinic (n = 1418 total) consented to a pelvic examination and chlamydia testing, and completed a questionnaire on socio-demographics, sexual behaviour, medical history, and symptoms. A case of chlamydia was defined as positive by culture or blocked enzyme immunoassay in an endocervical swab. RESULTS: The prevalence of chlamydial infection in the clinics was 6.3%. All three of the visual indicators--yellow endocervical discharge, easily induced bleeding, and opaque cervical discharge--were statistically significantly and independently associated with chlamydial infection (odds ratios 2.8, 2.3, and 2.9 respectively), independent of clinic type. Adjustment for the other visual indicators made little difference to the odds ratios. CONCLUSION: Opacity of endocervical discharge was at least as important as the other two commonly acknowledged indicators of chlamydial cervicitis--yellow endocervical discharge and easily induced mucosal bleeding of the cervix.
Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells.
Channel gating of the cystic fibrosis transmembrane conductance regulator (CFTR) is activated in response to cAMP stimulation. In addition, CFTR activation may also involve rapid insertion of a subapical pool of CFTR into the plasma membrane (PM). However, this issue has been controversial, in part because of the difficulty in distinguishing cell surface vs. intracellular CFTR. Recently, a fully functional, epitope-tagged form of CFTR (M2-901/CFTR) that can be detected immunologically in nonpermeabilized cells was characterized (Howard M, Duvall MD, Devor DC, Dong J-Y, Henze K, and Frizzell RA. Am J Physiol Cell Physiol 269: C1565-C1576, 1995; and Schultz BD, Takahashi A, Liu C, Frizzell RA, and Howard M. Am J Physiol Cell Physiol 273: C2080-C2089, 1997). We have developed replication-defective recombinant adenoviruses that express M2-901/CFTR and used them to probe cell surface CFTR in forskolin (FSK)-stimulated polarized Madin-Darby canine kidney (MDCK) cells. Virally expressed M2-901/CFTR was functional and was readily detected on the apical surface of FSK-stimulated polarized MDCK cells. Interestingly, at low multiplicity of infection, we observed FSK-stimulated insertion of M2901/CFTR into the apical PM, whereas at higher M2-901/CFTR expression levels, no increase in surface expression was detected using indirect immunofluorescence. Immunoelectron microscopy of unstimulated and FSK-stimulated cells confirmed the M2-901/CFTR redistribution to the PM upon FSK stimulation and demonstrates that the apically inserted M2-901/CFTR originates from a population of subapical vesicles. Our observations may reconcile previous conflicting reports regarding the effect of cAMP stimulation on CFTR trafficking.
Characterization of mucins from cultured normal human tracheobronchial epithelial cells.
Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentiate into a mucociliary epithelium over a 2- to 3-wk period and express increasing mRNA levels of the airway mucin genes MUC5AC and MUC5B as the cultures age; the levels of MUC2 mRNA were very low throughout the study. Using specific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase in these two mucins in the intracellular and apically secreted pools as a function of time. A low level of MUC2 mucin was detected, which did not change with time. The intracellular and apically secreted mucins isolated from day 14 and day 21 cultures by density gradient centrifugation were similar in density to those previously isolated from human respiratory mucus secretions. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-associated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associated and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was of similar charge density to its in vivo counterpart; however, MUC5B was more homogeneous than that found in vivo. Finally, evidence is presented for an intracellular NH(2)-terminal cleavage of the MUC5B mucins. These studies indicate that the mucins produced by cultured NHTBE cells are similar to those found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.
Expression of HAND gene products may be sufficient for the differentiation of avian neural crest-derived cells into catecholaminergic neurons in culture.
Members of the basic helix-loop-helix family of DNA binding proteins have important roles in the development of subpopulations of neural crest-derived neurons. We have cloned the chicken homologues of dHAND (HAND2) and eHAND (HAND1), basic helix-loop-helix DNA binding proteins whose neuronal expression is restricted to sympathetic and enteric neural crest-derived ganglia. Transcripts encoding dHAND and eHAND are expressed in sympathetic ganglia beginning at Hamburger and Hamilton stage 17-18. Antisense blockade of transcripts encoding HAND genes in neural crest-derived cells in vitro results in a significant reduction in neurogenesis. Differentiation of catecholaminergic neurons is also reduced by 52% if the expression of transcripts encoding dHAND and eHAND is reduced using antisense oligonucleotide blockade. The effect on neurogenesis and phenotypic expression of neural crest-derived neurons is specific; blockade of HAND gene expression has no apparent influence on the differentiation in vitro of neural tube-derived neurons. Use of a replication-competent avian retrovirus to constitutively express HAND genes in neural crest-derived cells in vitro, under nonpermissive growth conditions in medium supplemented with 2% chick embryo extract (CEE), induced precocious catecholaminergic differentiation. Constitutive expression of HAND gene products resulted in a significant increase in catecholaminergic differentiation of cells grown in medium supplemented with 10% CEE, a permissive growth condition for catecholaminergic development. These results suggest that the expression by neural crest cells of dHAND and eHAND may be both sufficient and necessary for catecholaminergic phenotypic expression.
Physical characterization of a low-charge glycoform of the MUC5B mucin comprising the gel-phase of an asthmatic respiratory mucous plug.
We have previously noted that sequential extraction of an asthmatic mucous exudate with 6 M guanidinium chloride yielded a fraction of the mucins that were most resistant to solubilization and of high Mr [Sheehan, Richardson, Fung, Howard and Thornton (1995) Am. J. Respir. Cell Mol. Biol. 13, 748-756]. Here we show that this mucin fraction is dominated (at least 96% of the total) by the low-charge glycoform of the MUC5B gene product. Seen in the electron microscope the mucins appeared mainly as compact 'island' structures composed of linear threads often emanating from globular 'nodes' rather than the discrete linear threads more typical of mucins that we have previously described. The effect of reducing agents was as expected for other gel-forming mucins, i.e. reduced subunits or monomers of Mr 3x10(6)) were produced within 15 min of treatment. Kinetic experiments on the cleavage of the intact mucins with the proteinase trypsin indicated two clear regimes of fragmentation. An initial rapid cleavage generated mucins ranging from Mr=4x10(6) to 30x10(6) that in the electron microscope appeared as polydisperse threads (500-3000 nm in length), similar to normal and other respiratory mucins that we have previously characterized. A subsequent slower fragmentation over many hours yielded a major fragment of Mr 3x10(6) and length 200-600 nm, very similar in size and Mr to the subunits obtained by reduction. The results suggest that the MUC5B mucin is assembled, first into polydisperse linear threads, which are then linked together via a protein-mediated process. This might involve part of the mucin polypeptide or an as yet unidentified protein(s). The high proteinase susceptibility of the linkage suggests that it might be a point of control for mucin size and thus mucus rheology.
A conformationally-constrained MHC class II I-Ag7-derived peptide protects NOD mice from the development of diabetes.
Allele-specific peptide vaccination against disease-associated MHC class II molecules is a promising new strategy for modulating self-antigen presentation to autoreactive T cells in autoimmune diseases. To evaluate the potential of this approach for treatment of insulin-dependent diabetes mellitus (IDDM), we have designed a cyclic peptide vaccine, DiavaX, from the third hypervariable region of the beta-chain of the NOD mouse MHC class II I-Ag7. NOD mice were treated at 5 and 9 weeks of age with 100 microg DiavaX emulsified in alum, a control peptide in alum, or alum alone. At the end of the study, 87% of alum treated mice had developed diabetes, compared with only 28% of DiavaX-treated mice. None of the control peptides, including a linear I-Ag7, a scrambled cyclic I-Ag7, or an analogous cyclic I-Aspeptide, reduced the incidence of diabetes, demonstrating that the protective effect of DiavaX is conformationally dependent and both allele- and sequence-specific. DiavaX treatment did not cause any general immune suppression, but did induce peptide-specific antibodies and memory T cells. DiavaX-induced protection from diabetes was associated with the maintenance of a non-destructive islet-associated autoimmune response. These data indicate that a conformationally constrained peptide from the disease-associated MHC represents a potential vaccine candidate for the prevention of clinical IDDM.
Adenosine 5'-triphosphate and neuropeptide Y are co-transmitters in conjunction with noradrenaline in the human saphenous vein.
1. Human saphenous veins were used to assess the cooperative participation of adenosine 5-triphosphate (ATP), neuropeptide Y (NPY), and noradrenaline (NA) in the vasomotor responses elicited following electrical depolarization of the perivascular nerve terminals. Rings from recently dissected human biopsies were mounted to record isometric muscular contractions; the motor activity elicited in the circular muscle layer following electrical depolarization (2.5-20 Hz, 50 V, 0.5 msec) were recorded. 2. Incubation of the biopsies with either 100 nM tetrodotoxin (TTX) or 1 microM guanethidine abolished the vasomotor response elicited by electrical nerve depolarization. The independent application of either ATP or NA to vein rings induced concentration-dependent contractions. 3. Tissue incubation with 30 microM suramin or 10 nM prazosin produced 10 fold rightward displacements of the alpha,beta-methylene ATP and NA concentration-response curves respectively. NPY contracted a limited number of biopsies, the vasoconstriction elicited was completely blocked by 1 microM BIBP 3226. A 5 min incubation of the biopsies with 10-100 nM NPY synergized, in a concentration-dependent fashion, both the ATP and the ATP analogue-induced contractions. Likewise, tissue preincubation with 10 nM NPY potentiated the vasomotor responses evoked with 20-60 nM NA. 4. Neither suramin, BIBP 3226, nor prazosin was individually able to significantly modify the derived frequency-tension curves. In contrast, the co-application of 30 microM suramin and 10 nM prazosin or 30 microM suramin and 1 microM BIBP 3226, elicited a significant (P<0.01) downward displacement of the respective frequency-tension curves. 5. The simultaneous application of the three antagonists-30 microM suramin, 1 microM BIBP 3226 and 10 nM prazosin-caused a significantly greater displacement of the frequency-tension curve than that achieved in experiments using two of these antagonists. 6. Electrically-evoked vasomotor activity is blocked to a larger extent by tissue incubation with 2.5 microM chloroethylclonidine and 30 microM suramin rather than with 10 nM 5 methyl urapidil and 30 microM suramin. As a result, the alpha1-adrenoceptor involved in the vasomotor activity has tentatively been associated with the alpha1B adrenoceptor family subtype. 7. Results support the physiological role of ATP in sympathetic neurotransmission. The present results are consistent with the working hypothesis that human sympathetic vasomotor reflexes involve the coordinated motor action of ATP, NPY, and NA acting on vascular smooth muscle cells. The present results support the concept of sympathetic co-transmission in the human saphenous vein.
Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product.
The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the individual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin.
[Minimally invasive coronary artery surgery].
BACKGROUND: There is a growing interest to perform a left internal mammary artery (LIMA) graft to the left anterior descending coronary artery (LAD) on a beating heart through a minimally invasive access to the chest cavity. AIM: To report the experience with minimally invasive coronary artery surgery. PATIENTS AND METHODS: Analysis of 11 patients aged 48 to 79 years old with single vessel disease that, between 1996 and 1997, had a LIMA graft to the LAD performed through a minimally invasive left anterior mediastinotomy, without cardiopulmonary bypass. A 6 to 10 cm left parasternal incision was done. The LIMA to the LAD anastomosis was done after pharmacological heart rate and blood pressure control and a period of ischemic pre conditioning. Graft patency was confirmed intraoperatively by standard Doppler techniques. Patients were followed for a mean of 11.6 months (7-15 months). RESULTS: All patients were extubated in the operating room and transferred out of the intensive care unit on the next morning. Seven patients were discharged on the third postoperative day. Duplex scanning confirmed graft patency in all patients before discharge; in two patients, it was confirmed additionally by arteriography. There was no hospital mortality, no perioperative myocardial infarction and no bleeding problems. After follow up, ten patients were free of angina, in functional class I and pleased with the surgical and cosmetic results. One patient developed atypical angina on the seventh postoperative month and a selective arteriography confirmed stenosis of the anastomosis. A successful angioplasty of the original LAD lesion was carried out. CONCLUSIONS: A minimally invasive left anterior mediastinotomy is a good surgical access to perform a successful LIMA to LAD graft without cardiopulmonary bypass, allowing a shorter hospital stay and earlier postoperative recovery. However, a larger experience and a longer follow up is required to define its role in the treatment of coronary artery disease.