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Biomedical subjects

A J Carmichael

Publications and source records attributed to A J Carmichael.

At least 37 records · Page 2Linked to original sources
PubMed2000

Determination of the isotopic composition of uranium in urine by inductively coupled plasma mass spectrometry.

A simple method based on inductively coupled plasma mass spectrometry (ICP-MS) was developed to identify exposure to depleted uranium by measuring the isotopic composition of uranium in urine. Exposure to depleted uranium results in a decreased percentage of 235U in urine samples causing measurements to vary between natural uranium's 0.72% and depleted uranium's 0.2%. Urine samples from a non-depleted uranium exposed group and a suspected depleted uranium exposed group were processed and analyzed by ICP-MS to determine whether depleted uranium was present in the urine. Sample preparation involved dry-ashing the urine at 450 degrees C followed by wet-ashing with a series of additions of concentrated nitric acid and 30% hydrogen peroxide. The ash from the urine was dissolved in 1 M nitric acid, and the intensity of 235U and 238U ions were measured by ICP-MS. After the samples were ashed, the ICP-MS measurements required less than 5 min. The 235U percentage in individuals from the depleted uranium exposed group with urine uranium concentrations greater than 150 ng L(-1) was between 0.20%-0.33%, correctly identifying depleted uranium exposure. Samples from the non-depleted uranium exposed individuals had urine uranium concentration less than 50 ng L(-1) and 235U percentages consistent with natural uranium (0.7%-1.0%). A minimum concentration of 14 ng L(-1) uranium was required to obtain sufficient 235U to allow calculating a valid isotopic ratio. Therefore, the percent 235U in urine samples measured by this method can be used to identify low-level exposure to depleted uranium.

Humans↗
PubMed1999

Human virus-specific CD8+ CTL clones revert from CD45ROhigh to CD45RAhigh in vivo: CD45RAhighCD8+ T cells comprise both naive and memory cells.

It has been generally believed that human CD8+ memory cells are principally found within the CD45ROhigh population. There are high frequencies of CD8+ memory CTL specific for the human CMV tegument phosphoprotein pp65 in PBMC of long-term virus carriers; the large population of memory CTL specific for a given pp65 peptide contains individual CTL clones that have greatly expanded. In this study, we found high frequencies of pp65 peptide-specific memory CTL precursors in the CD45ROhighCD45RA- population, but also appreciable frequencies in the CD45RAhigh subpopulation. Because the majority of CD8+ T cells in PBMC are CD45RAhigh, more of the total pp65-specific memory CTL pool is within the CD45RAhigh than in the CD45ROhigh compartment. Using clonotypic oligonucleotide probes to quantify the size of individual pp65-specific CTL clones in vivo, we found the CD45RAhigh population contributed 6- to 10-fold more than the CD45ROhigh population to the total virus-specific clone size in CD8+ cells. During primary CMV infection, an individual virus-specific CTL clone was initially CD45ROhigh, but after resolution of infection this clone was detected in both the CD45ROhigh and the CD45RAhigh populations. We conclude that CD45RA+ human CD8+ T cells do not solely comprise naive cells, but contain a very significant proportion of memory cells, which can revert from the CD45ROhigh to CD45RAhigh phenotype in vivo.

Carrier State↗
PubMed1999

Human CD28-CD8+ T cells contain greatly expanded functional virus-specific memory CTL clones.

At birth, almost all human peripheral blood CD8+ T cells express the costimulatory molecule CD28. With increasing age, the proportion of CD8+ T cells that lack CD28 increases. Because the Ag specificity of CD28-CD8+ T cells has not previously been defined, we studied the contribution of CD28-CD8+ T cells to the memory CD8+ CTL response against two human persistent viruses, human CMV (HCMV) and HIV. From PBMC of healthy virus carriers we generated multiple independent CTL clones specific for defined viral peptides and sequenced their TCR beta-chains. We designed clonotypic oligonucleotides complementary to each beta-chain hypervariable sequence and quantified the size of individual immunodominant CTL clones in PBMC. Some individual CTL clones were very large, comprising up to 3.1% of all CD8+ T cells in PBMC, and were generally maintained at a stable level for months. Individual virus-specific CTL clones were consistently more abundant in purified CD28- cells than in the CD8+ population as a whole. Because CD28-CD8+ cells as a population have been reported to proliferate poorly in response to mitogen, we studied the function of these virus-specific CD28- CTL clones by quantifying the frequency of peptide-specific CTL precursors using limiting dilution analysis. CD28-CD8+ T cells contained high frequencies of functional memory CTL precursors specific for peptides of HCMV or HIV, generally higher than in the CD8+ T cell population as a whole. We conclude that in asymptomatic HCMV and HIV infection, human CD28-CD8+ T cells contain high frequencies of functional virus-specific memory CTL clones.

Amino Acid Sequence↗
PubMed1999

Large clonal expansions of human virus-specific memory cytotoxic T lymphocytes within the CD57+ CD28- CD8+ T-cell population.

The proportion of human peripheral blood CD8+ T cells that are CD57+ CD28- is low at birth but increases with age and in individuals infected with human cytomegalovirus (HCMV) or human immunodeficiency virus (HIV). These CD57+ CD28- CD8+ T cells contain large oligoclonal T-cell expansions whose antigen specificity is unknown. We identified clonal expansions of virus-specific memory cytotoxic T-lymphocyte precursors (CTLp) in both healthy carriers of HCMV and in asymptomatic HIV-infected subjects. In each subject, from the T-cell receptor (TCR) beta-chain hypervariable sequence of each immunodominant CTL clone, we designed complementary oligonucleotide probes to quantify the size and phenotypic segregation of individual virus-specific CTL clones in highly purified populations of peripheral blood CD8+ T cells. We found large clonal expansions of virus-specific CTL clonotypes in CD57+ CD28- CD8+ T cells. Using limiting dilution analysis, we found functional peptide-specific CTLp at high frequency in CD57+ CD28- cells. Thus, memory CTL specific for persistent viruses account for many oligoclonal expansions within CD57+ CD28- CD8+ T cells.

CD28 Antigens↗
PubMed1999

ATP2A2 mutations in Darier's disease: variant cutaneous phenotypes are associated with missense mutations, but neuropsychiatric features are independent of mutation class.

Darier's disease (DD) is an autosomal dominant skin disorder characterized clinically by multiple keratotic papules, and histologically by focal loss of adhesion between epidermal cells (acantholysis) and by abnormal keratinization. Variant forms of cutaneous phenotype, sometimes familial, have been described. Associated neuropsychiatric features, including mental handicap, schizophrenia, bipolar disorder and epilepsy, have also been reported. The cause of DD was shown recently to be mutation in the ATP2A2 gene at 12q24.1, which encodes the sarco-endoplasmic reticulum calcium ATPase type 2 (SERCA2). Here, we show that while both common isoforms of SERCA2 are expressed in the cytoplasm of cultured keratinocytes and fibroblasts, in adult skin sections only the longer isoform, SERCA2b, was expressed abundantly in epidermal structures. Extended mutation analysis in European DD patients using single-strand conformation polymorphism and/or direct sequencing identified 40 different patient-specific mutations in 47 families. The majority (23/40) were likely to result in nonsense-mediated RNA decay. The remaining 17 were missense mutations distributed throughout the protein and were associated significantly with atypical clinical features. The clearest association was with the familial haemorrhagic variant where all four families tested had a missense mutation. Three of the families (one Scottish family and two unrelated Italian families) exhibited the same N767S substitution in the M5 transmembrane domain, and a fourth family, from Sweden, had a C268F substitution in the M3 transmembrane domain. Neuropsychiatric features did not appear to be associated with a specific class of mutation and may be an intrinsic, but inconsistent, effect of defective ATP2A2 expression.

Calcium-Transporting ATPases↗
PubMed1999

The memory cytotoxic T-lymphocyte (CTL) response to human cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone extensive expansion in vivo.

Human cytomegalovirus (HCMV)-specific CD8(+) cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vbeta segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo.

Amino Acid Sequence↗
PubMed1999

Response of normal human keratinocytes to sulfur mustard (HD): cytokine release using a non-enzymatic detachment procedure.

Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by sulfur mustard (HD). This study describes responses of normal human epidermal keratinocyte (NHEK) cells to 2,2'-dichlorodiethyl sulfide, sulfur mustard (HD), defined by interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) release. A new method for detaching cell to cell adhesion between keratinocytes has been applied. This method permits the characterization of endogenous fluid from cellular content that could be applied for the development of therapeutic intervention. NHEK (typical average cell density 4.4 x 10(6) cells/mL) were exposed to HD (100 and 300 microM) in keratinocyte growth medium (KGM) for 24 h at 37 C in humidified air. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 microM of HD. Exposure to 100 microM HD increased release of cytokines. IL-1beta (exposed: 1.41 x 10(-5) pg/ cell+/-1.60 x 10(-6) pg/cell: control 7.10 x 10(-6) pg/ cell+/-1.20 x 10(-6) pg/cell), TNF-alpha (exposed: 1.06 x 10(5) pg/cell+/-7.3 x 10(-7)pg/cell; control: 4.04 x 10(-6)+/-2.80 x 10(-7) pg/cell) and IL-8 (exposed: 3.71 x 10(-5) pg/ cell+/- 3.26 x 10(-6) pg/cell; control: 2.99 x 10(-6) pg/cell+/-8.80 x 10(-7) pg/cell) were significantly enhanced when NHEK cells were detached from culture flasks by non-enzymatic procedures. Cell suspensions of NHEK released low amounts of IL-6 when exposed to 100 microM for 24 h (exposed: 1.47 x 10(-6)+/-1.60 x 10(-7) pg/cell; control: 1.28 x 10(-6)+/-8.40 x 10(-8) pg/cell). However, cell suspensions of NHEK increased levels of IL-6 after exposure to 300 microM HD (4.67 x 10(-5) pg/cell+/-3.90 x 10(-6) pg/cell; control: 3.99 x 10(-6) pg/cell+/-5.50 x 10(-7) pg/cell). The amount of IL-8 and TNF-alpha present in cell suspensions increased up to 59-fold and fourfold, respectively, above control levels when NHEK cells were exposed to 300 microM HD. Exposure of NHEK to 300 microM HD had a highly variable effect on the release of IL-1beta, where sometimes the secretion of IL-1beta increased above baseline level and other times decreased in cell suspensions. Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 microM and significantly increased levels of IL-6 were observed. IL-6 was released in a concentration-dependent manner, 3.6-fold up to 8.4-fold, respectively, in supernatant. These pro-inflammatory mediators IL-1beta, IL-8, TNF-alpha and IL-6 may play an important role in HD injury. The present findings suggest that cytokine changes detected could be used as potential biomarkers of cutaneous vesicant injury.

Cell Adhesion↗
PubMed1997

Allergic contact dermatitis to bisphenol-A-glycidyldimethacrylate (BIS-GMA) dental resin associated with sensitivity to epoxy resin.

A patient presented with recurrent facial dermatitis associated with dental work. Dermatology referral identified the cause as allergic contact dermatitis to the epoxy acrylate BIS-GMA. Occupationally-induced allergic contact dermatitis to epoxy resin was also demonstrated. A structurally distinct aliphatic acrylate was successfully substituted. Contact sensitivity to BIS-GMA is reviewed and the potential for epoxy sensitive patients to cross-react to BIS-GMA is discussed.

Bisphenol A-Glycidyl Methacrylate↗
PubMed1997

Trichloroethylene radicals generated by ionizing radiation. An EPR/spin trapping study.

Trichloroethylene (TCE) was exposed in the presence of the spin trap N-tert-butyl-alpha-phenyl nitrone (PBN, 0.1 M) to ionizing radiation from two different sources in an attempt to determine the origin of the spin-trapped radicals generating the EPR spectra in precision cut liver slices. TCE samples were irradiated with 18 MeV electrons to a total dose of 1000 Gy in a linear accelerator (LINAC) or exposed to 60Co gamma-rays to total doses of 100 Gy and 1000 Gy. The results show that three PBN adducts were generated during the LINAC radiations. Two of these spin adducts correspond to the addition of carbon-centered radicals to PBN, and the third adduct is consistent with a decomposition product of PBN. The predominant carbon-entered radical yields a PBN adduct that is more stable, persists for over 24 h and has identical hyperfine coupling constants (aN = 1.61 mT, aH beta = 0.325 mT) to the PBN adduct obtained when precision-cut liver slices were exposed to TCE. Gamma radiation (100 Gy) of TCE yields PBN adducts with lower primary nitrogen hyperfine coupling constants (aN = 1.45 mT and aN = 1.54 mT). The results (gamma-radiation) suggest that the carbon-centered radical is formed on a single TCE carbon that is different than the predominant radical formed during LINAC radiations. This difference is confirmed by experiments using 13C-TCE. The results further suggest that, during gamma-radiation of TCE, the radicals are formed by dechlorination at the TCE carbon containing two chlorine atoms. The results obtained during LINAC radiations suggest that the predominant radical is formed by dechlorination at the TCE carbon containing a single chlorine and a single proton. In addition, it is possible that this radical is the initial TCE radical formed during exposure of liver slices to TCE.

Cobalt Radioisotopes↗
PubMed1997

Cutis marmorata telangiectatica congenita with hypoplasia of the right iliac and femoral veins.

We describe a 37-year-old woman with an extensive congenital reticulate vascular naevus affecting predominantly the right side of the body associated with a naevus anaemicus. Investigation of persistent ulceration and scarring of the lower legs revealed right-sided hypoplasia of the hemipelvis and underlying ipsilateral hypoplasia of the deep veins of the pelvis and leg. This association has not previously been reported and its implications for morbidity and management are discussed.

Adult↗
PubMed1996

The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL.

Cytotoxic T lymphocytes (CTL) appear to play an important role in the control of human cytomegalovirus (HCMV) in the normal virus carrier: previous studies have identified peripheral blood CD8+ CTL specific for the HCMV major immediate-early gene product (IE1) and more recently, by bulk culture and cloning techniques, have identified CTL specific for a structural gene product, the lower matrix protein pp65. In order to determine the relative contributions of CTL which recognize the HCMV proteins IE1, pp65, and glycoprotein B (gB) to the total HCMV-specific CTL response, we have used a limiting-dilution analysis system to quantify HCMV-specific CTL precursors with different specificities, allowing the antigenic specificity of multiple short-term CTL clones to be assessed, in a group of six healthy seropositive donors. All donors showed high frequencies of HCMV-specific major histocompatibility complex-restricted CTL precursors. There was a very high frequency of CTL specific for pp65 (lower matrix protein); IE1-specific CTL were also detectable at lower frequencies in three of five donors, while CTL directed to gB were undetectable. A pp65 gene deletion mutant of HCMV was then used to estimate the contribution of pp65-specific CTL to the total HCMV-specific CTL response; this showed that between 70 and 90% of all CTL recognizing HCMV-infected cells were pp65 specific. Analysis of the peptide specificity of pp65-specific CTL showed that some donors have a highly focused response recognizing a single peptide; the T-cell receptor Vbeta gene usage in these two donors was shown to be remarkably restricted, with over half of the responding CD8+ T cells utilizing a single Vbeta gene rearrangement. Other subjects recognized multiple pp65 peptides: nine new pp65 CTL peptide epitopes were defined, and for five of these the HLA-presenting allele has been identified. All four of the HLA A2 donors tested in this study recognized the same peptide. This apparent domination of the CTL response to HCMV during persistent infection by a single structural protein, irrespective of major histocompatibility complex haplotype, is not clearly described for other persistent virus infections, and the mechanism requires further investigation.

Amino Acid Sequence↗
PubMed1996

Trichloroethylene metabolism in vitro: an EPR/SPIN trapping study.

Trichloroethylene (TCE) was hypothesized to produce free radicals which could be detected using electron paramagnetic resonance spectroscopy with the spin trap, PBN (alpha-phenyl tert-butyl nitrone). The free radicals detected following incubation of precision cut liver slices in media containing 10 mM PBN had hyperfine coupling constants aN = 1.61 mT and aH = 0.325 mT. There was a linear increase in free radicals detected in the bathing media when the headspace TCE concentration was increased from 2500-10000 p.p.m. The levels of conjugated dienes measured in the slices incubated in PBN supplemented media were less than slices exposed to TCE in incubation media without PBN. The PBN trap may act as a scavenger preventing the propagation of free radicals and inhibiting lipid peroxidation. The experiments suggest that free radical formation by TCE leads to a concomitant increase in conjugated dienes in liver slices which may contribute to the pathological changes which occur in liver following TCE exposure.

Animals↗