Biomedical subjects
Publications and source records attributed to M Smith.
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The term, "chondrodysplasia punctata" (CDP) denotes a pattern of abnormal punctate calcification of dystrophic epiphyseal cartilage and certain other cartilaginous structures, such as the larynx. CDP occurs in a variety of genetic disorders associated with skeletal dwarfism and can also be caused by prenatal exposure to warfarin. Although the most studied clinical syndrome with CDP, rhizomelic chondrodysplasia punctata (RCDP), is known to be caused by several different abnormalities of plasmalogen biosynthesis, there are many other genetic disorders with CDP for which the biochemical cause is unknown. Because patients with Smith-Lemli-Opitz syndrome, a primary disorder of sterol biosynthesis, often have rhizomesomelic limb shortness and, less commonly, CDP, we assessed sterol levels and metabolism in patients with different clinical forms of CDP. By quantitative sterol analysis of a variety of tissues, we identified 5 patients with similar radiological findings and abnormally increased levels of 8-dehydrocholesterol and cholest-8(9)-en-3beta-ol, suggesting a deficiency of 3beta-hydroxysteroid-delta8,delta7-isomerase, a principal enzyme of cholesterol biosynthesis. Cultured cells available from one patient showed increased levels of the same two sterols, decreased synthesis of cholesterol, and a pattern of inhibition by triparanol and AY-9944 consistent with a deficiency of 3beta-hydroxysteroid-delta8,delta7-isomerase. Clinical diagnoses among the 5 patients included X-linked dominant Conradi-Hünermann-Happle syndrome and nonspecific lethal CDP. We conclude that abnormal cholesterol biosynthesis is a characteristic of some clinical syndromes with rhizomesomelic dwarfing and CDP.
OBJECTIVE: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine whose involvement in tumor necrosis factor alpha (TNFalpha) synthesis and T cell activation suggests a role in the pathogenesis of rheumatoid arthritis (RA). Antagonism of MIF is associated with marked inhibition of animal models of RA. Uniquely, MIF is inducible by low concentrations of glucocorticoids. We sought to investigate the expression of MIF in RA synovial tissue. METHODS: MIF was demonstrated in human RA synovium by immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). Regulation of MIF expression was investigated by treatment of cultured fibroblast-like synoviocytes (FLS) with interleukin-1beta (IL-1beta), TNFalpha, or interferon-gamma (IFNgamma), and dexamethasone (DEX). Mononuclear cell TNFalpha release after exposure to FLS-conditioned medium was measured by ELISA. RESULTS: MIF was present in RA synovial lining CD14+ macrophages and FLS. Constitutive MIF messenger RNA (mRNA) expression was demonstrated by RT-PCR of RNA from unstimulated cultured RA FLS, which also released abundant MIF. Serum, synovial fluid, and FLS intracellular MIF were significantly higher in RA patients than in controls. Synoviocyte MIF was not increased by IL-1beta, TNFalpha, or IFNgamma. In contrast, DEX 10(-7)M significantly reduced synoviocyte MIF, while DEX 10(-10)-10(-12)M induced a significant increase in MIF and MIF mRNA. Peripheral blood mononuclear cell TNFalpha release was induced by culture in RA FLS-conditioned medium, and this induction was significantly abrogated by monoclonal anti-MIF antibody, suggesting that MIF is an upstream regulator of TNFalpha release. CONCLUSION: These data represent the first demonstration of the cytokine MIF in human autoimmune disease and suggest MIF as a potential therapeutic target in RA.
OBJECTIVE: To develop, validate, and determine the measurement characteristics of a quantitative tool for assessing the severity of muscle involvement in children with idiopathic inflammatory myopathies. METHODS: The Childhood Myositis Assessment Scale (CMAS) was developed from 2 existing observational functional assessment tools to assess muscle function in the areas of strength and endurance across a wide range of ability and ages. The 14 ordinal items included were chosen to assess primarily axial and proximal muscle groups and are ranked with standard performance and scoring methods. Following the development of the CMAS, a training video and written instructions were developed and reviewed by the physicians participating in this study. Subsequently, utilizing a randomized block design, 12 physicians independently scored 10 children (9 with dermatomyositis, 1 with polymyositis; ages 4-15 years) twice in one day (morning and afternoon) on the CMAS. A pediatric physical therapist performed quantitative manual muscle strength testing (MMT) twice on each child (morning and afternoon), including the neck, trunk, and proximal and distal extremity muscle groups. RESULTS: The CMAS has a potential range of 0-51, with higher scores indicating greater muscle strength and endurance. The observed mean for the 10 patients was 36.4 (median 44, SD 14.1, observed range 5-51). The total score for the CMAS correlated with the physician's global assessment (by visual analog scale) of disease activity, the MMT score, serum creatine kinase level, and the Juvenile Arthritis Functional Assessment Report score. The score on the CMAS was not correlated with patient age. Interrater reliability (Kendall's coefficient of concordance) ranged from 0.77 to 1.0 for individual items (all P < 0.001), and overall, it was 0.95 (P < 0.001). Intrarater reliability for the individual physicians was measured by correlation of the CMAS scores for each patient on 2 separate evaluations and ranged from 0.97 to 0.99, with an overall correlation for all physicians of 0.98 (all P < 0.001). CONCLUSION: The CMAS demonstrated an acceptable range of observed scores, excellent convergent validity, and excellent inter- and intrarater reliability. The CMAS is validated to quantitatively assess muscle function in the areas of strength and endurance in children with idiopathic inflammatory myopathies. It can be used in routine clinical care as well as therapeutic trials.
The sexually dimorphic number of cells expressing arginine vasopressin (AVP) in the bed nucleus of the stria terminalis and the density of AVP fibers within the lateral septum appear to be organized by pre- and postnatal androgens. Social recognition behaviors are also sexually dimorphic and AVP-dependent. Whereas AVP antagonists prevent males from recognizing familiar intruders by olfactory investigation of the anal-genital area, they have no effect in females. To test the hypothesis that the male's dependency upon AVP to form social recognition memories begins prior to birth, we compared the effectiveness of an AVP antagonist to block social recognition in control males and females with that seen in male offspring whose mothers were treated prenatally with an androgen antagonist (flutamide). In an initial study we showed that while sexual experience may enhance social recognition in males, virgin males exhibit the ability to recognize conspecifics and are sensitive to the memory blocking actions of AVP antagonists. In a second experiment, pregnant rats were treated daily for the last 10 days of gestation with either flutamide (10 mg) or control vehicle. Within 12 h of birth, male offspring from flutamide litters were injected with either testosterone proprionate (50 microg TP) or vehicle control. AVP-antagonist treatment in adults eliminated the ability of control males to recognize familiar juvenile intruders, but had no effect on males exposed prenatally to flutamide, regardless of whether these males were treated with TP or vehicle on day 1 of life. These data support the hypothesis that the development of the male's dependency upon AVP to express social recognition memories begins with the organizational actions of prenatal androgens.
Full-length human gelatinase B (FLGelB) and its C-terminal truncated form (dGelB) were expressed in Pichia pastoris strain GS115, using the Saccharomyces cerevisiae Mat alpha signal peptide. In both cases, a high level of the secreted protein could be detected by SDS-PAGE. The truncated gene was also expressed using the human gelatinase B native signal peptide. Secretion using the Mat alpha signal peptide was significantly greater than that from the native signal peptide. The recombinant products were purified and characterized biochemically. The recombinant proteins, FLGelB and dGelB, were found to have similar biochemical properties and activity to that of the human gelatinase B native protein.
BACKGROUND: Some benefits of laparoscopic (LC) and minilaparotomy (MC) cholecystectomy may reflect attenuation of the acute phase response. The authors examined components of this response. METHODS: Patients were randomized to LC (n = 11) or MC (n = 11). C-reactive protein (CRP), alpha-1-antitrypsin (AAT), retinol-binding protein (RBP), transferrin, and albumin were measured preoperatively and on postoperative days 1, 2, 4, and 7. Interleukin-1 receptor antagonist (IL-1ra), IL-6, and tumor necrosis factor (TNF-alpha) were measured more frequently perioperatively. Peak expiratory flow rate, forced expiratory volume in 1 second, and forced vital capacity were measured daily. RESULTS: The IL-6 increase was more persistent and marked in the MC patients from hour 8 to day 7 postoperatively (P < 0.05). Alterations in CRP, AAT, and albumin were similar. Postoperative deficits of respiratory function correlated with the magnitude of acute phase protein alteration. CONCLUSIONS: Minimal access surgery induces an acute phase response that is less prominent after a laparoscopic technique.
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Phytosterol esters (PE) are intended for use as a novel food ingredient, primarily in margarines and spreads as a functional component with plasma cholesterol lowering activity. Phytosterols and their esters are present naturally in vegetable oils and on average people consume 200 mg/day, but their consumption at this level is not sufficient to lower plasma cholesterol levels. Therefore, through the incorporation of PE into margarines/spreads, the intake can be increased by approximately 10-fold by consuming the PE-containing margarine/spread at normal intake levels. As part of an extensive programme of safety evaluation studies a subchronic rat toxicity study has been conducted in which groups of Alpk:AP(f)SD (Wistar derived) rats (20 males and 20 females/group) were fed diets containing PE at levels of 0, 0.16, 1.6, 3.2 and 8.1% (w/w) in the diet for 90 days. Throughout the study, clinical observations, body weights, and food and water consumption were measured. At the end of the study the rats were subjected to a full post-mortem examination, cardiac blood samples were taken for clinical pathology, selected organs were weighed, and a full tissue list was taken for subsequent histological examination. There were no treatment-related changes that were considered to be of toxicological significance. Therefore, a nominal PE concentration of 8.1% was considered to be the no-observed-adverse- effect level (NOAEL) following daily oral administration to rats for 90 days. This was equivalent to a dose of 6.6 g/kg body weight/day PE or 4.1 g/kg/day phytosterol.
More than half of all cancer patients now use some form of complementary/alternative medicine, yet the majority of these patients do not disclose this use to their physicians. Health care practitioners need to educate themselves about the complementary/alternative medicine products their patients are using. Eight herbal products (astragalus, essiac, Asian ginseng, Siberian ginseng, green tea, garlic, Hoxsey formula and iscador) commonly used by cancer patients are reviewed here and a list of recommended reference texts is provided. In addition, health care providers are encouraged to initiate discussions about complementary/alternative products and therapies with their patients so that they may help them make safe and informed decisions about these products. Not knowing what patients are taking is definitely a less desirable option.
The homeobox gene Hb9, like its close relative MNR2, is expressed selectively by motor neurons (MNs) in the developing vertebrate CNS. In embryonic chick spinal cord, the ectopic expression of MNR2 or Hb9 is sufficient to trigger MN differentiation and to repress the differentiation of an adjacent population of V2 interneurons. Here, we provide genetic evidence that Hb9 has an essential role in MN differentiation. In mice lacking Hb9 function, MNs are generated on schedule and in normal numbers but transiently acquire molecular features of V2 interneurons. The aberrant specification of MN identity is associated with defects in the migration of MNs, the emergence of the subtype identities of MNs, and the projection of motor axons. These findings show that HB9 has an essential function in consolidating the identity of postmitotic MNs.
Malnutrition among renal patients has been widely documented and is associated with increased morbidity and mortality. Advances in dialysis technology and transplantation have helped to increase patient long-term survival, and as more elderly patients commence dialysis programs, the problem of malnutrition is escalating. Hospitalized renal patients are at a greater risk, as dietary intakes may be reduced for a number of reasons. A multidisciplinary team decided to review the existing cook-chill plated meal system and all food provision to the renal ward. The aim of this review was to assess the nutritional intake of renal inpatients and to gauge patients' and relatives' attitudes towards hospital food provision. From these results, we hoped to go on and implement some changes to help improve the situation. Results showed that 34% of the patients ate half or less of the hospital food provided, and 80% of patients surveyed relied on food brought in by relatives and friends. Actual dietary intakes were compared to Dietary Reference Values (DRVs; Department of Health [DoH], UK, 1995). One hundred percent of the patients did not achieve the DRVs for energy, iron, potassium, zinc, folate, B6, and riboflavin. Sixty-six percent of the patients did not achieve the DRV for protein. These results were discussed by the multidisciplinary group, and it was decided to trial a cook-chill bulk trolley to replace the existing plated meal system. Unfortunately, to implement a bulk trolley system, the ward needs someone to serve the food. This could be the job of a "feeding assistant" or "ward hostess." A bid has been put forward to the hospital Trust Board to obtain funding for these "feeding assistants, " and the bulk trolley can be acquired from existing funds. It is hoped that the creation of these new posts will go some of the way towards improving the patients' dietary intake while in the hospital.
Previous in vitro studies indicated that mutation of both K234 and K236 to arginine, glutamine, or glutamic acid impaired the ability of the voltage-dependent anion channel (VDAC1) to insert into the outer membrane of the mitochondria (Smith et al. 1995). These same mutants were expressed in a strain of Saccharomyces cerevisiae with a disruption in the VDAC1 gene. The mutant VDAC1 forms were found in the mitochondria suggesting that they were correctly sorted to the outer membrane. However, only very small amounts of the mutants were inserted into the mitochondrial membranes. Mitochondria isolated from the strains expressing the mutants were capable of catalyzing the translocation of both wild-type VDAC1 and pre-alcohol dehydrogenase III indicating that the translocation apparatus was functional. These results confirm the previously drawn conclusion that K234 and K236 are part of a membrane insertion motif. The failure of the mutant VDAC1 forms to insert did not cause VDAC1 precursors to accumulate in the soluble cell cytoplasm or in the microsomal fraction. The apparent lack of a "precursor pool" suggested that a post-transcriptional control mechanism might limit the amounts of VDAC1 precursors in the cell. Such a control mechanism is consistent with the observation that the amount of VDAC1 was very similar after epichromosomal (gene in a 2u plasmid controlled by a Gal1 promoter) and chromosomal expression (endogenous gene controlled by the endogenous promoter).
A prospective pilot study was performed in order to assess the safety of treating recurrent malignant gliomas (MGs) with locally infused autologous tumor infiltrating lymphocytes (TILs) and recombinant interleukin-2 (rIL-2). Six patients were entered between June 27, 1994 and June 2, 1995 and followed until July 1, 1998. At surgery an Ommaya reservoir was placed for later infusion of TILs and rIL-2. Following surgery, autologous TILs were expanded in vitro in the presence of rIL-2 and infused on treatment days 1 and 14, with concurrent rIL-2 infusions performed three times each week for one month. Following completion of immunotherapy all patients were offered chemotherapy. Phenotypic analysis demonstrated TILs to be T-lymphocytes (87-99% CD3+). Of these, 4 of 6 cases (67%) phenotyped as cytotoxic/suppressor T-lymphocytes (CD8+) and 2 of 6 cases (33%) phenotyped as helper/inducer T-lymphocytes (CD4+). TILs demonstrated limited selective cytotoxicity, with dose dependent cytotoxicity against autologous tumor, allogenic tumor and long term MG cell lines. There were no significant (Grade 3 or 4) complications. One patient developed transient low grade fevers, and 2 developed asymptomatic hydrocephalus. All patients developed transient and asymptomatic cerebral swelling, noted on the immediate post-treatment imaging studies. At three and six month follow-up, 3 patients responded with partial response, 2 demonstrated stable disease and 1 patient progressed. At long term follow-up, 1 patient had a complete response (45 month follow-up), 2 had a partial response (48 and 47 month follow-up) and 3 patients expired as a result of progressive disease (at 12, 12 and 18 months following immunotherapy). A relationship between subsequent chemotherapy or extent of resection to outcome was not apparent but could not be excluded. This pilot study demonstrated that locally infused autologous TILs and rIL-2 could be delivered without serious toxicity. Further studies are indicated to determine the safety and long term efficacy of TIL immunotherapy.
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X-linked dominant Conradi-Hünermann syndrome (CDPX2; MIM 302960) is one of a group of disorders with aberrant punctate calcification in cartilage, or chondrodysplasia punctata (CDP). This is most prominent around the vertebral column, pelvis and long bones in CPDX2. Additionally, CDPX2 patients may have asymmetric rhizomesomelia, sectorial cataracts, patchy alopecia, ichthyosis and atrophoderma. The phenotype in CDPX2 females ranges from stillborn to mildly affected individuals identified in adulthood. CDPX2 is presumed lethal in males, although a few affected males have been reported. We found increased 8(9)-cholestenol and 8-dehydrocholesterol in tissue samples from seven female probands with CDPX2 (ref. 4). This pattern of accumulated cholesterol intermediates suggested a deficiency of 3beta-hydroxysteroid-delta8,delta7-isomerase (sterol-delta8-isomerase), which catalyses an intermediate step in the conversion of lanosterol to cholesterol. A candidate gene encoding a sterol-delta8-isomerase (EBP) has been identified and mapped to Xp11.22-p11.23 (refs 5,6). Using SSCP analysis and sequencing of genomic DNA, we found EBP mutations in all probands. We confirmed the functional significance of two missense alleles by expressing them in a sterol-delta8-isomerase-deficient yeast strain. Our results indicate that defects in sterol-delta8-isomerase cause CDPX2 and suggest a role for sterols in bone development.
Darier disease (DD) is an autosomal-dominant skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. Recently we constructed a 2.4-Mb, P1-derived artificial chromosome contig spanning the DD candidate region on chromosome 12q23-24.1. After screening several genes that mapped to this region, we identified mutations in the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca2(+)-ATPase type 2 isoform (SERCA2) and is highly expressed in keratinocytes. Thirteen mutations were identified, including frameshift deletions, in-frame deletions or insertions, splice-site mutations and non-conservative missense mutations in functional domains. Our results demonstrate that mutations in ATP2A2 cause DD and disclose a role for this pump in a Ca(2+)-signalling pathway regulating cell-to-cell adhesion and differentiation of the epidermis.