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Biomedical subjects

R A Clark

Publications and source records attributed to R A Clark.

At least 73 records · Page 4Linked to original sources
PubMed1999

Fibrin microbeads (FMB) as biodegradable carriers for culturing cells and for accelerating wound healing.

We have developed biodegradable fibrin-derived microbeads as potent cell carriers. The fibrin-derived microbeads, 50-200 microm in diameter, were tested for their attachment to a wide range of cell types. Fibrin-derived microbeads were shown to be greatly haptotactic to cells (such as endothelial cells, smooth muscle cells and fibroblasts), which respond to fibrinogen in contrast to keratinocytes and different cell lines derived from leukocytic lineage. The cells on fibrin-derived microbeads could be maintained for more than 10 d and achieved a high density. 31P-nuclear magnetic resonance was employed to monitor phosphate metabolism in cells, with densities on the order of 100 million cells per g of fibrin-derived microbeads. The 31P-nuclear magnetic resonance adenosine triphosphate and phosphocreatine signals, equivalent to the signal obtained with perfused normal skin, indicated that metabolism of cells on fibrin-derived microbeads was responsive to oxygenation and nutrients. Light, fluorescent, and confocal laser microscopy revealed that the porous fibrin-derived microbeads accommodate up to 200-300 cells due to their high surface area which minimized contact inhibition. Cells could degrade the fibrin-derived microbeads and be transferred to seed culture flasks without trypsinization. In a pig skin wound healing model, fibrin-derived microbeads + fibroblasts were transplanted into full thickness punch wounds. This procedure was compared with other treatment modalities, such as the addition of human platelet-derived growth factor BB or fibrin-derived microbeads alone. By the third day after wounding, only the wounds in which fibroblasts on fibrin-derived microbeads were added showed significant formation of granulation tissue. Based on the above, we project many uses of our novel fibrin-derived microbead technology for cell culturing, wound healing and tissue engineering.

Animals↗
PubMed1999

Fibrin and collagen differentially regulate human dermal microvascular endothelial cell integrins: stabilization of alphav/beta3 mRNA by fibrin1.

Integrin alphavbeta3 is specifically but transiently expressed on the tips of capillary sprouts as they invade the fibrin clot during angiogenesis of cutaneous wound repair. Specific blocking of alphavbeta3 function inhibits granulation tissue formation in cutaneous wounds. The mechanisms of regulation of alphavbeta3 expression on human dermal microvascular endothelial cells, however, have not been fully delineated. As alphavbeta3 was highly expressed on capillary sprouts in 5 d wounds rich in fibrin, but was almost undetectable on blood vessels in 7 d wounds rich in collagen, we hypothesized that the extracellular matrix environment could regulate human dermal micro- vascular endothelial cell alphavbeta3 expression. To address this, human dermal microvascular endothelial cells were cultured on surfaces coated with collagen, fibronectin, and gelatin, and mRNA levels of integrin alphav/beta3 were determined. Compared with human dermal microvascular endothelial cells on collagen, mRNA levels of alphav/beta3 were higher in human dermal microvascular endothelial cells on fibronectin and on gelatin. To simulate the in vivo environment better, human dermal microvascular endothelial cells cultured on collagen were overlaid by fibrin or collagen gels prior to assessment of alphav/beta3 mRNA levels. alphav/beta3 mRNA levels were higher in human dermal microvascular endothelial cells surrounded by a three-dimensional fibrin gel compared with a collagen gel, whether angiogenic factors were present or absent. As modulation of mRNA stability is a potential regulatory mechanism for integrin expression, integrin subunit mRNA stability was assessed. beta3 mRNA decayed much faster than alphav, alpha2, and beta1 mRNA. Three-dimensional fibrin gels enhanced alphav/beta3 mRNA stability compared with collagen gels. We propose that the provisional matrix molecules in the wound clot regulate angiogenesis associated with cutaneous wound repair through their modulation of integrin receptor expression.

Collagen↗
PubMed1999

Activation of the neutrophil respiratory burst oxidase.

The neutrophil respiratory burst oxidase is a multicomponent activatable enzyme comprising one of the major phagocyte antimicrobial systems. In the genetic disorder chronic granulomatous disease, absent oxidase function is associated with recurrent, severe, and often life-threatening infections. The components of the oxidase system include both membrane-bound and soluble cytosolic proteins. A primary feature of stimulus-dependent activation is the translocation of a complex of cytosolic factors to the membrane, where they associate with a flavocytochrome enzyme. Interactions among the various oxidase components occur through a number of specific regions, including SH3 domains and proline-rich motifs. The fully assembled complex functions as an electron transport system, moving electrons from cytosolic NADPH to molecular oxygen to form superoxide, which, along with subsequent reactive products, exerts microbicidal and cytotoxic activities.

Cytosol↗
PubMed1999

Localization of intracellular Ca2+ stores in HeLa cells during infection with Chlamydia trachomatis.

Chlamydia trachomatis elementary bodies (EBs) enter epithelial cells within membrane-bound endosomes that aggregate with each other in a calcium-regulated process, but avoid fusion with lysosomes. Annexin III but not I translocates to chlamydial aggregates and inclusions. In this study, we localize the intracellular Ca2+ stores during the course of infection by analyzing the distribution of three intracellular Ca2+ store proteins: calreticulin, type-1 inositol-1,4, 5-trisphosphate receptor (IP3-R), and Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase type 2 (SERCA2) in HeLa cells infected with C. trachomatis serovar L2. In uninfected cells, immunofluorescence staining of the proteins showed a fine granular distributed pattern for all three proteins. After infection with C. trachomatis, calreticulin was found at the periphery of chlamydial aggregates and inclusions from 3 to 48 hours post-infection. In infected cells, SERCA2 was intimately associated with chlamydial inclusions after 3 and 24 hours, but not after 48 hours. Moreover, IP3-R was translocated to and colocalized with EB aggregates and chlamydial inclusions and had a distribution very similar to that of SERCA 2. After 24 hours incubation with chlamydiae, there was a local accumulation of [Ca2+]i (105+/-17 nM) in the proximity of chlamydial inclusions, compared to 50+/-13 nM in other parts of the cell cytoplasm. In the absence of extracellular Ca2+, this local accumulation of Ca2+ increased to 295+/-50 nM after adding 50 microM ATP, and to a similar extent after adding 100 nM thapsigargin (Tg). These data indicate that during infection of HeLa cells with chlamydiae, intracellular Ca2+ stores are redistributed, causing local accumulation of Ca2+ in the vicinity of chlamydial inclusions. These changes may trigger the association of certain proteins such as annexins with chlamydia-containing vesicles, and thereby regulation of membrane-membrane interaction during endosome aggregation and inclusion formation.

Adenosine Triphosphate↗
PubMed1999

Characteristics of HIV-infected men with low serum testosterone levels.

Low levels of serum testosterone may have negative implications on morbidity in HIV-infected men. The purpose of this study was to determine demographic and clinical characteristics that predict low serum testosterone among men attending our HIV clinic. A cross-sectional study of 587 HIV-positive male patients who presented at the Louisiana State University HIV Outpatient (HOP) Clinic between August 1997 and January 1999 was conducted. Demographic and clinical characteristics were collected and analysed. Of the 587 men studied, 119 (20.3%) had a serum testosterone level below 400 ng/dl. Significantly more men with low serum testosterone levels had a presence of opportunistic infection (especially HIV wasting syndrome, oesophageal candidiasis, or dementia), CD4+ cell counts below 200 cells/mm3, or were taking megestrol acetate. Early detection of low serum testosterone will allow for expedient testosterone supplementation therapy, which could improve morbidity and quality of life for HIV-infected men.

AIDS Dementia Complex↗
PubMed1999

Acute infection with Trichophyton rubrum associated with flares of atopic dermatitis.

Trichophyton rubrum has been implicated as a potential trigger in flares of atopic dermatitis. We describe a patient with atopic dermatitis who presented with a history of multiple flares and concurrent acute tinea pedis and onychomycosis. Symptoms of atopic dermatitis and culture-positive acute infection with T. rubrum resolved during each flare using systemic antifungals. Flares of atopic dermatitis may be triggered by acute T. rubrum infections. Antifungal therapy should be considered in these patients.

Acute Disease↗
PubMed1998

Th1/Th2 cytokine expression in saliva of HIV-positive and HIV-negative individuals: a pilot study in HIV-positive individuals with oropharyngeal candidiasis.

Current data suggest that T-helper (Th)2-type cytokine responses are often associated with progression to AIDS in HIV-positive individuals. Similarly, Th2-type cytokines are associated with susceptibility to mucosal candidiasis, of which oropharyngeal candidiasis (OPC) is one of the most common opportunistic infections in HIV-positive individuals. Although little information is available on host defense mechanisms at the level of the oral mucosa, recent studies suggest that local cell-mediated immunity (CMI) is equally or more important than that in the periphery for host defense against mucosal Candida albicans infections. This study investigated the potential presence of oral-associated CMI through the expression of Th1/Th2-type cytokines in saliva of immunocompetent and immunocompromised individuals with and without OPC. Results showed a constitutive mixed Th1/Th2 cytokine expression (Th0) in whole saliva of healthy HIV-negative individuals. In contrast, HIV-positive individuals had a dominant Th2-type salivary cytokine profile (interleukin-4 [IL-4], IL-10) (IL-2, interferon-y [IFN-gamma], IL-12) that seemingly resulted from a lack of Th1-type cytokines rather than enhanced Th2-type cytokines. Moreover, pilot analyses of those with OPC showed evidence for a more profound salivary Th2-type profile. Both HIV-positive and HIV-negative patients, irrespective of CD4 counts, had some level of positive in vitro systemic lymphocyte proliferative responses to C albicans antigens. These results suggest that the Th1/Th2 cytokine dichotomy in HIV disease is detectable in situ in oral secretions and may be a useful indicator of oral-associated CMI to better understand resistance/susceptibility of HIV-positive individuals to oral opportunistic infections, including OPC.

AIDS-Related Opportunistic Infections↗
PubMed1998

Effect of asthma and its treatment on growth: four year follow up of cohort of children from general practices in Tayside, Scotland.

OBJECTIVE: To investigate whether asthma or its treatment impairs children's growth, after allowing for socioeconomic group. DESIGN: 4 year follow up of a cohort of children aged 1-15. SETTING: 12 general practices in the Tayside region of Scotland. SUBJECTS: 3347 children with asthma or features suggestive of asthma registered with the general practices. MAIN OUTCOME MEASURES: Height and weight standard deviation scores. RESULTS: Children who lived in areas of social deprivation (assessed by postcode) had lower height and weight than their contemporaries (mean standard deviation score -0.26 (SD 1.02) and -0.18 (1.15) respectively, P < 0.001 for both). Children who were receiving > or = 400 micrograms daily of inhaled steroids and who were attending both hospital and general practice for asthma care had lower height and weight than average, independent of the effect of deprivation (mean standard deviation score -0.62 (1.01), P = 0.002, for height and -0.58 (0.94), P = 0.005, for weight). Children receiving high doses of inhaled corticosteroids also showed lower growth rates (mean change in standard deviation score -0.19 (0.51), P = 0.003). However, no other children with asthma showed growth impairment. CONCLUSION: Most children with asthma were of normal height and weight and had normal growth rates. However, children receiving high doses of inhaled steroids and requiring both general practice and hospital services had a significant reduction in their stature. This effect was independent from but smaller than the effect of socioeconomic group on stature.

Adolescent↗
PubMed1998

A three-dimensional collagen lattice activates NF-kappaB in human fibroblasts: role in integrin alpha2 gene expression and tissue remodeling.

Normal adult human dermal fibroblasts grown in a three-dimensional collagen lattice increase mRNA level of collagen receptor integrin subunit alpha2 (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239- 249.) and DNA binding activity of a nuclear transcription factor, NF-kappaB (Xu, J., and R.A.F. Clark. 1997. J. Cell Biol. 136:473-483.). Here we present evidence that the collagen lattice induced the nuclear translocation of p50, one member of NF-kappaB family, and the degradation of an NF-kappaB inhibitor protein, IkappaB-alpha. The inhibition of NF-kappaB activity by SN50, a peptide inhibitor targeted at nuclear translocation of NF-kappaB, significantly reduced the induction of integrin alpha2 mRNA and protein by the collagen lattice. A region located between -549 and -351 bp in the promoter of integrin alpha2 gene conferred the inducibility by three-dimensional collagen lattice. The presence of either SN50 or IkappaB-alpha32, 36, a stable mutant of IkappaB-alpha, abrogated this inducibility, indicating that the activation of integrin alpha2 gene expression was possibly mediated by NF-kappaB through this region. Although there were three DNA-protein binding complexes forming in this region that are sensitive to the inhibition of NF-kappaB nuclear translocation, NF-kappaB was not directly present in the binding complexes. Therefore, an indirect regulatory mechanism by NF-kappaB in integrin alpha2 gene expression induced by three-dimensional collagen lattice is suggested. The involvement of NF-kappaB in reorganization and contraction of three-dimensional collagen lattice, a process that requires the presence of abundant integrin alpha2beta1, was also examined. The inhibition of NF-kappaB activity by SN50 greatly blocked the contraction, suggesting its critical role in not only the induction of integrin alpha2 gene expression by three-dimensional collagen lattice, but also alpha2beta1-mediated tissue-remodeling process.

Adult↗
PubMed1998

L-Selectin ligands that are O-glycoprotease resistant and distinct from MECA-79 antigen are sufficient for tethering and rolling of lymphocytes on human high endothelial venules.

During the process of lymphocyte recirculation, lymphocytes bind via L-selectin to sulfated sialyl-Lewisx (sLex)-containing carbohydrate ligands expressed on the surface of high endothelial venules (HEV). We have examined the expression of sLex on HEV using a panel of mAbs specific for sLex and sLex-related structures, and have examined the function of different sLex-bearing structures using an in vitro assay of lymphocyte rolling on HEV. We report that three sLex mAbs, 2F3, 2H5, and CSLEX-1, previously noted to bind with high affinity to glycolipid-linked sLex, vary in their ability to stain HEV in different lymphoid tissues and bind differentially to O-linked versus N-linked sLex on glycoproteins. Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant. Treatment of tissue sections with O-sialoglycoprotease under conditions that removed the vast majority of MECA-79 staining, only partially reduced staining with the 2F3 and 2H5 mAbs. Using a novel rolling assay in which cells bind under flow to HEV of frozen tissue sections, we demonstrate that a pool of O-sialoglycoprotease-resistant molecules is present on HEV that is sufficient for attachment and rolling of lymphocytes via L-selectin. This interaction is not inhibited by the mAb MECA-79. Furthermore, MECA-79 mAb blocks binding to untreated sections by only 30%, whereas the sLex mAb 2H5 blocks binding by approximately 60% and a combination of MECA-79 and 2H5 mAb blocks binding by 75%. We conclude that a pool of O-glycoprotease-resistant sLex-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb. We postulate that these ligands may participate in lymphocyte binding to HEV.

Antibodies, Monoclonal↗
PubMed1998

Digital mammography: hybrid four-channel wavelet transform for microcalcification segmentation.

RATIONALE AND OBJECTIVES: The authors evaluated an algorithm for the automatic segmentation of microcalcification clusters (MCCs) at digital mammography. Two- and four-channel wavelet transforms were evaluated to determine whether sensitivity in the detection of MCCs can be improved and if the selective reconstruction of the higher-order M2 subimages allows better preservation of the segmented MCCs, which is required for their classification. MATERIALS AND METHODS: The hybrid method involved the use of a nonlinear filter for image noise suppression coupled with wavelet transforms for image decomposition and an adaptive method for selective subimage reconstruction as a basis for segmentation of MCCs. The two- and four-channel wavelet transforms were implemented with different filter bank structures (i.e., polyphase quadrature mirror filters [QMFs], tree structure, and lattice structure) to determine if their computational efficiency can be improved while retaining properties such as near-perfect reconstruction. The hybrid wavelet transforms were applied to a common image database of biopsy-proved MCCs (100 images, 105-micron resolution, 12 bits deep; 52 cases with at least one MCC of varying subtlety [46 malignant and six benign cases] and eight normal cases). RESULTS: The two- and four-channel wavelet transforms yielded sensitivities of 93% and 94% and false-positive (PP) detection rates of 1.58 and 1.35 MCCs per image, respectively. The lattice structure provided greater than fivefold improvement in computational speed compared to the polyphase QMF structure, particularly for the higher order of channels (M = 4). CONCLUSION: The four-channel wavelet transform provided better sensitivity and FP detection rates and greater image detail preservation for the segmented MCCs.

Algorithms↗
PubMed1998

Simulation model of mammographic calcifications based on the American College of Radiology Breast Imaging Reporting and Data System, or BIRADS.

RATIONALE AND OBJECTIVES: The authors developed and evaluated a method for the simulation of calcification clusters based on the guidelines of the Breast Imaging Reporting and Data System of the American College of Radiology. They aimed to reproduce accurately the relative and absolute size, shape, location, number, and intensity of real calcifications associated with both benign and malignant disease. MATERIALS AND METHODS: Thirty calcification clusters were simulated by using the proposed model and were superimposed on real, negative mammograms digitized at 30 microns and 16 bits per pixel. The accuracy of the simulation was evaluated by three radiologists in a blinded study. RESULTS: No statistically significant difference was observed in the observers' evaluation of the simulated clusters and the real clusters. The observers' classification of the cluster types seemed to be a good approximation of the intended types from the simulation design. CONCLUSION: This model can provide simulated calcification clusters with well-defined morphologic, distributional, and contrast characteristics for a variety of applications in digital mammography.

Breast Neoplasms↗
PubMed1998

Heterotopic muscle pulleys or oblique muscle dysfunction?

INTRODUCTION: The description of connective tissue sleeves that function as pulleys for the rectus extraocular muscles (EOMs) suggests that abnormalities of EOM pulley position might provide a mechanical basis for some forms of incomitant strabismus. Pulleys determine the paths and thus the pulling directions of EOMs. METHODS: High-resolution magnetic resonance images spanning the orbits were obtained in primary position, upgaze, and downgaze for each subject. Paths of the EOMs were measured with reference to the orbital center and permitted inference of pulley locations. RESULTS: Data from 18 orbits of orthotropic subjects defined means and SDs of normal EOM pulley coordinates. Eight patients, aged 17 to 60 years, had heterotopic EOM pulleys, defined as displaced at least 2 SDs from normal. We found one to eight heterotopic pulleys (considering both orbits) in each of four patients who had been diagnosed with marked superior oblique (SO) overaction and mild to marked inferior oblique (IO) underaction. Each patient had superior mislocation of at least one lateral rectus pulley by 1.8 to 4.9 mm. Three patients diagnosed with mild to moderate IO overaction and mild to moderate SO underaction in only one orbit had one to three heterotopic EOM pulleys. Each of those patients had at least one lateral rectus pulley inferiorly dislocated by 1.9 to 4.9 mm. The final patient, who was diagnosed with mild IO underaction and normal SO function bilaterally, had bilateral superior mislocation of the medial rectus pulleys by greater than 2 mm. Computer simulations using the Orbit program (Eidactics, San Francisco) incorporating individually measured pulley positions reproduced the clinical patterns of incomitant strabismus in all cases without postulating abnormalities of oblique muscle innervation or contractility. CONCLUSION: Heterotopic EOM pulleys can cause patterns of incomitant strabismus that have been attributed to oblique muscle dysfunction. Even isolated mislocations of less than 2 mm, coupled with smaller mislocations of the other pulleys, can produce the clinical appearance of bilateral oblique dysfunction. Pulley heterotopy should be considered in the differential diagnosis of incomitant strabismus and oblique dysfunction.

Adolescent↗
PubMed1998

Genealogy of the CCR5 locus and chemokine system gene variants associated with altered rates of HIV-1 disease progression.

Allelic variants for the HIV-1 co-receptors chemokine receptor 5 (CCR5) and CCR2, as well as the ligand for the co-receptor CXCR4, stromal-derived factor (SDF-1), have been associated with a delay in disease progression. We began this study to test whether polymorphisms in the CCR5 regulatory regions influence the course of HIV-1 disease, as well as to examine the role of the previously identified allelic variants in 1,090 HIV-1 infected individuals. Here we describe the evolutionary relationships between the phenotypically important CCR5 alleles, define precisely the CCR5 regulatory sequences that are linked to the CCR5-delta32 and CCR2-641 polymorphisms, and identify genotypes associated with altered rates of HIV-1 disease progression. The disease-retarding effects of the CCR2-641 allele were found in African Americans but not in Caucasians, and the SDF1-3'A/3'A genotype was associated with an accelerated progression to death. In contrast, the CCR5-delta32 allele and a CCR5 promoter mutation with which it is tightly linked were associated with limited disease-retarding effects. Collectively, these findings draw attention to a complex array of genetic determinants in the HIV-host interplay.

Adolescent↗
PubMed1998

Identification of lectin-purified neural glycoproteins, GPs 180, 116, and 110, with NMDA and AMPA receptor subunits: conservation of glycosylation at the synapse.

The postsynaptic apparatus is associated with a number of glycoproteins with apparent molecular masses of 180, 116, and 110 kDa, which are highly concentrated in and may be uniquely associated with this structure. These glycoproteins, purified by concanavalin A lectin-affinity chromatography, showed immunoreactivity in the present study with subunit-specific antibodies to glutamate receptors as follows: GP 180, NMDA receptor subunits NR2A/NR2B; GP 116, NMDA receptor NR1 (1a); and GP 110, pan-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (pan-AMPA) receptors. Sensitivities to the glycosidases peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H on both western blots and silver-stained gels suggested that the glutamate receptors were at least major constituents of the glycoprotein bands. Similar detailed glycosylation was observed for all three glycoproteins, with neutral oligosaccharides being dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for approximately 50% of the neutral sugars, with Man 5 (at almost 20% of the neutral sugars) always the major glycan. Other abundant neutral oligosaccharides were of the complex type. Similar sensitivities to peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H were observed for cell line-expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly processed oligosaccharides will be expressed. This may be indicative of glycosylation sites in these receptors that are inaccessible to the later processing enzymes and favours the oligomannosidic class of glycans in functional roles.

Amidohydrolases↗
PubMed1998

Infected cardiac myxoma. Case report and literature review.

We present a case of a left atrial myxoma infected with Porphyromonas asaccharolytica in a 55-year-old man, successfully treated with surgical excision and a brief course of antibiotic therapy. Infected cardiac myxomas are extremely rare, with only 39 cases previously reported. They can be difficult to diagnose due to their protean clinical manifestations, which can often be seen in uninfected myxomas as well. We suggest that blood cultures and careful pathologic examination be performed in all cases of cardiac myxoma with constitutional symptoms. However, fever and elevated sedimentation rate are significantly more common in infected tumors. Organisms responsible are similar in distribution to those causing bacterial endocarditis. Emboli, though frequent, may not be more common in infected than uninfected myxomas. Case reports have become more common since the development of better diagnostic techniques. Echocardiography, especially by the transesophageal approach, is the diagnostic procedure of choice, and sensitivity approaches 100%. Surgical excision is curative and generally has low morbidity and mortality.

Bacteroidaceae Infections↗