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Pacific Northwest National Laboratory reports on the detection of 39Ar at the location of an underground nuclear explosion on the Nevada Nuclear Security Site. The presence of 39Ar was not anticipated at the outset of the experimental campaign but results from this work demonstrated that it is present, along with 37Ar and 85Kr in the subsurface at the site of an underground nuclear explosion. Our analysis showed that by using state-of-the-art technology optimized for radioargon measurements, it was difficult to distinguish 39Ar from the fission product 85Kr. Proportional counters are currently used for high-sensitivity measurement of 37Ar and 39Ar. Physical and chemical separation processes are used to separate argon from air or soil gas, yielding pure argon with contaminant gases reduced to the parts-per-million level or below. However, even with purification at these levels, the beta decay signature of 85Kr can be mistaken for that of 39Ar, and the presence of either isotope increases the measurement background level for the measurement of 37Ar. Measured values for the 39Ar measured at the site ranged from 36,000 milli- Becquerel/standard-cubic-meter-of-air (mBq/SCM) for shallow bore holes to 997,000 mBq/SCM from the rubble chimney from the underground nuclear explosion.
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Argón/análisis , Armas Nucleares , Monitoreo de Radiación , Contaminantes Radiactivos/análisis , NevadaRESUMEN
Low-background lead for radiation measurement shielding is often assayed for 210Pb to ensure acceptable backgrounds. Samples of lead assayed with a germanium spectrometer calibrated for bremsstrahlung-based assay of 210Pb provide a view into the 210Pb content of commercial lead in the U.S. (other than stockpiled Doe Run lead). Results suggest that the loss of lead smelting in the U.S. has eliminated the traditional supply of "low background" lead (~30Bqkg-1), and indicate current commercial supplies contain roughly an order of magnitude higher 210Pb levels.
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The Ultra-Low Background Liquid Scintillation Counter developed by Pacific Northwest National Laboratory will expand the application of liquid scintillation counting by enabling lower detection limits and smaller sample volumes. By reducing the overall count rate of the background environment approximately 2 orders of magnitude below that of commercially available systems, backgrounds on the order of tens of counts per day over an energy range of ~3-3600keV can be realized. Initial test results of the ULB LSC show promising results for ultra-low background detection with liquid scintillation counting.
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Pacific Northwest National Laboratory has recently opened a shallow underground laboratory intended for measurement of low-concentration levels of radioactive isotopes in samples collected from the environment. The development of a low-background liquid scintillation counter is currently underway to further augment the measurement capabilities within this underground laboratory. Liquid scintillation counting is especially useful for measuring charged particle (e.g., ß and α) emitting isotopes with no (or very weak) gamma-ray yields. The combination of high-efficiency detection of charged particle emission in a liquid scintillation cocktail coupled with the low-background environment of an appropriately designed shield located in a clean underground laboratory provides the opportunity for increased-sensitivity measurements of a range of isotopes. To take advantage of the 35m-water-equivalent overburden of the underground laboratory, a series of simulations have evaluated the scintillation counter's shield design requirements to assess the possible background rate achievable. This report presents the design and background evaluation for a shallow underground, low background liquid scintillation counter design for sample measurements.
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'Calciphylaxis', a calcification syndrome associated with ischaemic cutaneous necrosis, is acquired naturally in humans in disease states. It is a life and limb-threatening complication, usually observed in patients with renal disease and secondary hyperparathyroidism, but known to occur in the absence of renal or parathyroid disease. The reported mortality rate, which ranges from 60-80%, relates to wound infection, sepsis and organ failure. It is a small-vessel vasculopathy, which is estimated to occur in about 4% of haemodialysis patients. Clinically, violaceous, reticulate areas of cutaneous necrosis and eschar may be evident, particularly in the extremities. In addition to the clinical picture, a raised calcium phosphorous product, an elevated parathyroid hormone level, radiographic evidence of vessel and soft-tissue calcification and the finding of mural calcification affecting small arteries and arterioles on histopathology help to confirm the diagnosis of this entity which generally has a poor prognosis. A high index of suspicion and an active multidisciplinary management approach, with rigorous attention to wound care and prevention of sepsis, are vital in the management of these patients. In this overview, we discuss the pathophysiology, clinical features and associations, risk factors, diagnosis and management issues relating to calciphylaxis.
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Calcifilaxia , Calcifilaxia/diagnóstico , Calcifilaxia/etiología , Calcifilaxia/fisiopatología , Calcifilaxia/terapia , Diagnóstico Diferencial , Humanos , Hiperparatiroidismo Secundario/complicaciones , Fallo Renal Crónico/complicaciones , Pronóstico , Factores de RiesgoRESUMEN
BACKGROUND: Erythrokeratodermia variabilis (EKV) is an autosomal dominant or recessive genodermatosis characterized by the coexistence of randomly occurring, transient, erythematous patches and hyperkeratosis of the skin. The disorder has been mapped to chromosome 1p35.1 but is genetically heterogeneous. EKV may be caused by pathogenic mutations in one of two neighbouring connexin genes, GJB3 and GJB4, encoding the gap junction proteins Cx31 and Cx30.3, respectively. Twelve distinct mutations identified to date cluster either at the cytoplasmic amino-terminus or in the four transmembrane domains. OBJECTIVES: To report a large family with EKV and an unrelated sporadic case. METHODS: DNA amplification and mutation analysis, followed by denaturing high-performance liquid chromatography to confirm the segregation of the mutations in the two families with EKV. RESULTS: A novel, recurrent GJB3 mutation (625C-->T; L209F) was identified in the family with EKV and in the unrelated sporadic case. CONCLUSIONS: This mutation is the first to affect a conserved residue in the cytoplasmic carboxy-terminus of any connexin gene with a cutaneous phenotype, emphasizing its structural and/or functional importance.
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Conexinas/genética , Eritema/genética , Hiperqueratosis Epidermolítica/genética , Mutación Puntual , Enfermedades Cutáneas Genéticas/genética , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , Femenino , Genes Dominantes , Genes Recesivos , Genotipo , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Alineación de SecuenciaRESUMEN
A major challenge to the concept of gene therapy for dominant disorders is the silencing or repairing of the mutant allele. Supplementation therapy is an alternative approach that aims to bypass the defective gene by inducing the expression of another gene, with similar function but not susceptible to the disrupting effect of the mutant one. Epidermolysis bullosa simplex (EBS) is a genetic skin fragility disorder caused by mutations in the genes for keratins K5 or K14, the intermediate filaments present in the basal cells of the epidermis. Keratin diseases are nearly all dominant in their inheritance. In cultured keratinocytes, mutant keratin renders cells more sensitive to a variety of stress stimuli such as osmotic shock, heat shock or scratch wounding. Using a 'severe' disease cell culture model system, we demonstrate reversion towards wild-type responses to stress after transfection with human desmin, an intermediate filament protein normally expressed in muscle cells. Such a supplementation therapy approach could be widely applicable to patients with related individual mutations and would avoid some of the financial obstacles to gene therapy for rare diseases.
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Desmina/genética , Epidermólisis Ampollosa Simple/terapia , Terapia Genética/métodos , Queratinocitos/fisiología , Queratinas/genética , Movimiento Celular/fisiología , Células Cultivadas , Citoesqueleto/fisiología , Epidermólisis Ampollosa Simple/genética , Genes Dominantes/genética , Calor , Humanos , Queratina-14 , Queratina-5 , Músculo Esquelético/fisiopatología , Mutación , Ósmosis/fisiología , Transfección , Cicatrización de Heridas/genéticaRESUMEN
BACKGROUND: Epidermolysis bullosa simplex (EBS) is an inherited skin fragility disorder caused by mutations in keratin intermediate filament proteins. While discoveries of these mutations have increased understanding of the role of keratins and other intermediate filaments in epithelial tissues, progress towards the development of therapy for these disorders is much slower. OBJECTIVES: Cell culture model systems that display these structural defects are needed for analysis of the cellular consequences of the mutations and to enable possible therapeutic strategies to be developed. Our aim was to generate immortalized cell lines as such model systems for the study of EBS. METHODS: We generated a series of stable cell lines expressing EBS-associated keratin mutations, by immortalizing keratinocytes from EBS-affected skin biopsies with either simian virus 40 (SV40) T antigen or human papillomavirus 16 (HPV16) E6/E7, and assessed their keratin expression (by immunofluorescence), proliferation rates and migratory behaviour (in outgrowth and scratch wound assays). RESULTS: Clonal immortalized keratinocyte cell lines KEB-1, KEB-2, KEB-3 (using SV40 T antigen) and KEB-4, KEB-7 and NEB-1 (using HPV16 E6/E7) were established. These include two lines from a single individual with Weber-Cockayne EBS (i.e. KEB-3 and KEB-4, mutation K14 V270M), and three cell lines from a second family, two from siblings carrying the same mutation (KEB-1, KEB-2 lines from Dowling-Meara EBS, mutation K5 E475G) and one from an unaffected relative (NEB-1). The sixth cell line (KEB-7), with a previously unreported severe mutation (K14 R125P), was the only one to show keratin aggregates in resting conditions. Despite variations in the immortalization procedure, there was no significant difference between cell lines in keratin expression, outgrowth capabilities or response to transient heat shock. However, cell migration, as measured by speed of scratch wound closure, was significantly faster in cells with severe EBS mutations. CONCLUSIONS: These cell lines provide useful culture systems in which to assess aspects of EBS-induced cell changes. The faster migration after scratch wounding of the EBS keratinocytes may be a consequence of the known upregulation of stress-activated kinase pathways in these cells.
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Línea Celular/metabolismo , Epidermólisis Ampollosa Simple/patología , Queratinas/genética , Mutación , Cicatrización de Heridas/genética , División Celular/genética , Línea Celular/patología , Movimiento Celular/genética , Transformación Celular Viral , Preescolar , Análisis Mutacional de ADN/métodos , Epidermólisis Ampollosa Simple/genética , Epidermólisis Ampollosa Simple/metabolismo , Calor , Humanos , Filamentos Intermedios/genética , Queratinocitos/patología , Queratinas/metabolismo , Papillomaviridae , Virus 40 de los SimiosRESUMEN
Keratins are intermediate filament proteins whose expression in epithelial tissues is closely linked to their differentiated state. The greatest complexity of this expression is seen in the epidermis and associated structures. The critical basal (proliferative) cell layer expresses the major keratin pair, K5 and K14, but it also expresses an additional type I keratin, K15, about which far less is known. We have compared the expression of K15 with K14 in normal, pathological, and tissue culture contexts; distinct differences in their expression patterns have been observed that imply different regulation and function for these two genes. K15 appears to be preferentially expressed in stable or slowly turning over basal cells. In steady-state epidermis, K15 is present in higher amounts in basal cells of thin skin but in lower amounts in the rapidly turning over thick plantar skin. Although remaining high in basal cell carcinomas (noninvasive) it is suppressed in squamous cell carcinomas (which frequently metastasize). Wounding-stimulated epidermis loses K15 expression, whereas K14 is unchanged. In cultured keratinocytes, K15 levels are suppressed until the culture stratifies, whereas K14 is constitutively expressed throughout. Therefore, unlike K14, which appears to be a fundamental component of all keratinocytes, K15 expression appears to be more tightly coupled to a mature basal keratinocyte phenotype.
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Diferenciación Celular , Queratinocitos/metabolismo , Queratinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular , Línea Celular , Cartilla de ADN , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Inmunohistoquímica , Queratinocitos/citología , RatonesRESUMEN
Mutations in keratin genes give rise to a number of inherited skin fragility disorders, demonstrating that the intermediate filament cytoskeleton has an essential function in maintaining the structural integrity of epidermis and its appendages. Epidermolysis bullosa simplex (EBS) is an autosomal dominant disorder caused by mutations in keratins K5 or K14, which are expressed in the basal layer of stratified epithelia. Using a keratinocyte cell line established from an EBS patient, we investigated whether the muscle-specific intermediate filament protein desmin would be able to functionally complement a mutant keratin 14 in cultured keratinocytes. We show that in stably transfected EBS cells, desmin forms an extended keratin-independent cytoskeleton. Immunogold-EM analysis demonstrated that in the presence of numerous keratin filaments attached to desmosomes, desmin could nevertheless interact with desmosomes in the same cell, indicating the dynamic nature of the filament-desmosome association. When desmin-transfected cells were subjected to heat shock, the mutant keratin filaments showed a transient collapse while desmin filaments were maintained. Thus the defective keratin filaments and the wild-type desmin filaments appear to coexist in cells without interference. Expression of a type III intermediate filament protein like desmin may offer a strategy for the treatment of patients suffering from epidermal keratin mutations.
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Citoesqueleto de Actina/fisiología , Desmina/genética , Queratinocitos/fisiología , Queratinas/genética , Citoesqueleto de Actina/química , Animales , Células Cultivadas , Cricetinae , Proteínas del Citoesqueleto/análisis , Desmina/análisis , Desmoplaquinas , Epidermólisis Ampollosa Simple/genética , Epidermólisis Ampollosa Simple/terapia , Técnica del Anticuerpo Fluorescente , Expresión Génica/fisiología , Terapia Genética , Humanos , Inmunohistoquímica , Queratina-14 , Queratinocitos/química , Queratinocitos/ultraestructura , Queratinas/análisis , Microscopía Electrónica , Receptor Cross-Talk/fisiología , Piel/citología , Temperatura , TransfecciónRESUMEN
Bullous congenital ichthyosiform erythroderma (BCIE) is a dominantly inherited keratinizing disorder characterized by erythroderma and blistering in neonates and generalized epidermolytic hyperkeratosis (EH) in adulthood. Previously, it has been shown that BCIE can be caused by mutations in either of the genes encoding K1 or K10, the keratins predominantly expressed in suprabasal layers of the epidermis. Using direct sequencing of genomic PCR fragments, we have analyzed 4 British families with BCIE, all of whom were found to carry mutations in K10. In 1 family, the affected person was found to have an unusual dinucleotide transversion mutation, 2138CC-->AA, causing two amino acid substitutions, D155E and R156S, also in the 1A domain of the K10 polypeptide. In 2 further kindreds, the previously reported "hotspot" mutations 2139C-->T and 2140G-->A were found. These mutations predict amino acid substitutions in the helix 1A domain of K10, designated R156C and R156H respectively. The proband in the fourth family was found to carry a novel mutation 4724T-->C, predicting the amino acid change L452P in the helix 2B domain of K10. All mutations were confirmed in the affected persons and were excluded from a population of 50 normal, unrelated individuals by restriction enzyme analysis. The location of these mutations in the highly conserved helix boundary motif sequences of K10 are consistent with previously reported dominant negative mutations in K10 and other keratins. Despite the unusual nature of two of these mutations, in particular the double missense mutation, the phenotypes of the affected individuals in these 4 families were entirely typical of BCIE.
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Eritrodermia Ictiosiforme Congénita/genética , Queratinas/genética , Mutación Missense , Sustitución de Aminoácidos , Automatización , Secuencia de Bases , Exones , Femenino , Tamización de Portadores Genéticos , Humanos , Masculino , LinajeRESUMEN
Point mutations in the keratin intermediate filament genes for keratin 5 or keratin 14 are known to cause hereditary skin blistering disorders such as epidermolysis bullosa simplex, in which epidermal keratinocytes are extremely fragile and the skin blisters on mild trauma. We show that in 2 phenotypically diverse cases of epidermolysis bullosa simplex, the keratin mutations result in a thermoinstability of the intermediate filament cytoskeleton which can be reproducibly demonstrated even in the presence of tissue culture-induced keratins and in conditions where filament fragility is not otherwise obvious. SV40-T antigen and HPV16 (E6--E7) immortalised keratinocyte cell lines were examined, established from control and epidermolysis bullosa simplex-affected individuals with either severe (Dowling-Meara) or mild (Weber-Cockayne) forms of the disease. In standard tissue culture conditions no significant and consistent abnormality of the keratin cytoskeleton could be demonstrated. However after thermal stress a reduced stability of the keratin filaments was demonstrable in the epidermolysis bullosa simplex cell lines, with filaments breaking into aggregates similar to those seen in skin from EBS patients. These aggregates were maximal at 15 minutes after heat shock and the filament network structure was substantially reversed by 60 minutes. Differences were also seen in the cells during respreading after replating: cells containing mutant keratins were slower to respread than controls and fine aggregates were seen at the cell margins in the Dowling-Meara derived cell line. Such delays in restoring the normal intermediate filament network after physiological processes involving cytoskeleton remodelling may render the cells vulnerable to cytolysis in vivo if physically challenged during this time window. The steady reduction in the mitotic index of the epidermis during the first few years of life could then explain the clinical improvement which is frequently observed in growing children.
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Citoesqueleto/metabolismo , Epidermólisis Ampollosa Simple/patología , Queratinas/metabolismo , Línea Celular/citología , Movimiento Celular/fisiología , Citoesqueleto/ultraestructura , Técnica del Anticuerpo Fluorescente , Expresión Génica/fisiología , Calor , Humanos , Queratinocitos/citología , Queratinocitos/ultraestructura , Queratinas/genética , Microscopía Electrónica , Mutación/fisiologíaRESUMEN
Pachyonychia congenita (PC) is a group of autosomal dominant disorders characterized by dystrophic nails and other ectodermal aberrations. A gene for Jackson-Lawler PC was recently mapped to the type I keratin cluster on 17q. Here, we show that a heterozygous missense mutation in the helix initiation motif of K17 (Asn92Asp) co-segregates with the disease in this kindred. We also show that Jadassohn-Lewandowsky PC is caused by a heterozygous missense mutation in the helix initiation peptide of K16 (Leu130Pro). The known expression patterns of these keratins in epidermal structures correlates with the specific abnormalities observed in each form of PC.
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Displasia Ectodérmica/genética , Queratinas/genética , Mutación , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , Cartilla de ADN/genética , Displasia Ectodérmica/clasificación , Displasia Ectodérmica/patología , Femenino , Genes Dominantes , Genotipo , Heterocigoto , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
Ichthyosis bullosa of Siemens (IBS) is a congenital bullous ichthyosis without erythroderma. In contrast to bullous congenital ichthyosiform erythroderma (BCIE), there is a relatively mild involvement of the skin and epidermolytic hyperkeratosis (EHK) is restricted to the upper suprabasal layers of the epidermis. Tonofilament aggregation was observed by EM in suprabasal cells from affected patients in the two families under study, indicative of a keratin abnormality. Keratin 2e is a differentiation specific type II keratin expressed suprabasally in the epidermis. Part of the K2e gene was amplified by polymerase chain reaction using genomic DNA from affected and unaffected individuals from two IBS families. Direct sequencing of polymerase chain reaction products revealed a point mutation in the highly conserved helix termination motif, producing the protein sequence change LLEGEE-LLEGKE. This mutation was found in all affected members of a five-generation kindred and also in a sporadic case in a second unrelated family. No mutation was seen in unaffected individuals. The mutation destroys a MnlI restriction site, which allowed exclusion of the mutation from a population of 50 unaffected unrelated individuals by restriction fragment analysis of K2e PCR products. This is the sixth keratin gene found to be involved in an inherited epidermal disorder.
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Ictiosis/genética , Ictiosis/patología , Queratinas/genética , Secuencia de Bases , Niño , Diagnóstico Diferencial , Femenino , Heterocigoto , Humanos , Hiperqueratosis Epidermolítica/patología , Filamentos Intermedios/ultraestructura , Queratina-2 , Microscopía Electrónica , Sondas Moleculares/genética , Datos de Secuencia Molecular , Linaje , Mutación Puntual , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Bullous congenital ichthyosiform erythroderma is a human hereditary skin disorder in which suprabasal keratinocytes rupture. Recent reports have implicated keratins K1 and K10 in this disease. Here we describe four diverse keratin mutations that are all significantly associated with this disease. Two of these are in the helix 1A subdomain of the type II keratin 1, giving a serine-to-proline substitution in codon 185 and an asparagine-to-serine substitution in codon 187. In the analogous region of type I keratin 10, an arginine-to-proline and an arginine-to-serine transition in codon 156 have been identified. All four mutations create restriction fragment length polymorphisms that were used exclude the mutations from 120 normal chromosomes. Insertional polymorphism (in the V2 subdomains of the non-helical tails of K1 and K10) was excluded as the cause of the phenotypic heterogeneity observed within one family.
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Hiperqueratosis Epidermolítica/genética , Queratinas/genética , Mutación/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN/análisis , ADN/genética , Femenino , Humanos , Queratinas/análisis , Masculino , Microscopía Electrónica , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
We have identified mutations in keratins K5 (Arg331Cys) and K14 (Val270Met) in two kinships affected by the dominantly-inherited skin blistering disease, Weber-Cockayne epidermolysis bullosa simplex (EBS-WC). Linkage analysis, DNA sequencing and clinical and ultrastructural analysis are combined to provide the first detailed description of classical EBS-WC. Both phenotypes show similar blistering on trauma, indicating that both mutations compromise the structural resilience of the basal keratinocytes by affecting the keratin cytoskeleton. The location of these mutations in the L12 linker, which bisects the alpha-helical rod region of intermediate filament proteins, identifies another keratin mutation cluster leading to hereditary skin fragility syndromes.
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Citoesqueleto/fisiología , Epidermólisis Ampollosa Simple/genética , Queratinas/genética , Mutación , Edad de Inicio , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Niño , Preescolar , Citoesqueleto/química , Cartilla de ADN , Femenino , Humanos , Recién Nacido , Masculino , Microscopía Electrónica , Datos de Secuencia Molecular , Fenotipo , Piel/patología , Piel/ultraestructuraRESUMEN
The lymphocytes in the perivascular foci of tuberculin skin tests have a similar CD4:CD8 ratio to those in the peripheral blood, suggesting that these subsets do not show bias in their initial emigration. By contrast, the diffusely infiltrating lymphocytes show a relative preponderance of CD4 cells which is progressively greater in successive 250 micron layers into the dermis. A generally similar pattern is seen in healthy controls and in patients with untreated pulmonary tuberculosis, treated leprosy, haemophilia A and chronic obstructive lung disease (COLD) patients treated with prednisolone, but the gradient of increasing CD4:CD8 ratio with depth into the dermis is significantly less steep in patients with tuberculosis, haemophilia and prednisolone-treated COLD than in the healthy controls. Selective migration results in a relative preponderance of CD4 cells in the diffuse infiltrate and it is suggested that this is a mechanism likely to potentiate defensive reaction to Mycobacterium tuberculosis: any deficiency in selective migration may make immunological defences less effective and so contribute to the chronicity of the lesions of tuberculosis.
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Hipersensibilidad Tardía/inmunología , Piel/inmunología , Linfocitos T/inmunología , Prueba de Tuberculina , Movimiento Celular , Hemofilia A/inmunología , Humanos , Lepra/inmunología , Enfermedades Pulmonares Obstructivas/inmunología , Linfocitos T/fisiología , Tuberculosis Pulmonar/inmunologíaRESUMEN
Antigen preparations extracted from Mycobacterium tuberculosis by different procedures--PPD and 'New tuberculin'--were compared with respect to the cytology of the Type IV reaction they elicit in the dermis. The numbers and microanatomical localization of cells of the major lymphocyte and monocyte subsets were measured histometrically in immunocytochemically-stained sections of biopsies of the 48 h reactions. Despite the close similarity in size of the external appearance of the reactions provoked by the two preparations, 'New tuberculin' elicited a more extensive perivascular infiltrate and more numerous diffusely infiltrating M3-bearing cells: the number of diffusely infiltrating CD4 and CD8 lymphocytes was similar in reactions against the two antigen preparations. Thus the method used to prepare a skin test reagent may affect the nature of the cellular reaction it provokes: differences in content of various classes of antigenic macromolecules are probably important, but the relative content of other irritant constituents may also be important.