A simple and efficient method for attachment of a 40-base pair, GC-rich sequence to PCR-amplified DNA.
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Base Composition↗
Biomedical subjects
J S Beck
Publications and source records attributed to J S Beck.
Measurement of the tissue distribution of immunoperoxidase staining with polyclonal anti-BCG serum in lung granulomata of mice infected with Mycobacterium tuberculosis.
Mice inoculated with Mycobacterium tuberculosis, strain H37Rv were used as a model of human tuberculosis. The microanatomical location of immunoperoxidase staining with a polyclonal anti-BCG serum was within macrophages and appeared granular rather than delineating whole bacilli. Immunoperoxidase staining appears to demonstrate degraded mycobacterial antigens from disrupted organisms and so reflects prior turnover of bacilli. On Ziehl-Neelsen staining, intact or almost intact bacilli are seen and so the extent of this form of staining reflects the current bacillary load. Both methods have limited sensitivity, but with larger mycobacterial loads the area of immunoperoxidase stain measured on a semi-automated image analyser correlated with the numbers of bacilli observed. The immunoperoxidase method will be useful in the evaluation of residual antigen in studying the pathogenesis of experimental murine tuberculosis. In human mycobacterial granulomata, this immunohistochemical technique should provide an alternative method of estimating the extent of bacillary load: this approach may also provide evidence of mycobacterial infection from residual antigen deposits in the tissue when whole bacilli have been successfully cleared.
Animals↗
Skin changes in the tuberculin test.
This review describes the recent advances in knowledge of the nature and range of physiological changes that occur in the skin at the site of a positive tuberculin reaction. The infiltration of T-cells and monocyte/macrophages shows a marked compartmentalisation suggesting that the functions of particular cell types depend on their localisation. The extent of cutaneous oedema (detectable as induration) is not closely related to other features of the reaction or to systemic indicators of cell mediated immunity. The intensity of hyperaemia is maximal at the centre of the reaction and is correlated in most cases with the density of cellular infiltration in the dermis suggesting a functional coordination. Despite this correlation between cell numbers and velocity of blood flow, the reaction normally shows hypoxia, hypercapnia and local acidosis, but this metabolic modification may not be a wholly disadvantageous effect since these conditions appear to facilitate the growth and metabolism of activated lymphocytes and macrophages. In very strong reactions, there is central relative slowing of the circulation and this may lead to necrosis in extreme cases. There are however a minority of cases where cell infiltration occurs but induration is not palpable: this situation has been named pseudoanergy, and its pathogenesis has not yet been established. The occurrence of pseudoanergy must throw some doubt on the conventional criteria for positivity in the reading of tuberculin skin tests (induration greater than 5 mm) and this may have relevance to future strategies for assessment of new vaccines.(ABSTRACT TRUNCATED AT 250 WORDS)
Erythema↗
Inter- and intra-subject variation of the suppression of mitogen-induced proliferation of human lymphocytes by cyclosporin-A: reduction of response with delayed addition may be relevant to timing of therapy.
In vitro, CsA is capable of suppressing mitogen-stimulated growth of human peripheral blood lymphocytes. Dose--response studies on lymphocytes from normal volunteers have shown that the variation between subjects is greater than the variation between repeated studies on the same subject in the 72 h uptake of 3H-TdR. The tests are easy to perform and they could be used to help in deciding, prior to transplantation, the dose of CsA to be used and whether other immunosuppressive therapy is needed. Activation of lymphocyte proliferation in vivo is a continuous process and recruitment to growth in vitro takes place over 24 h after mitogen stimulation. CsA was most effective in suppressing the replicative growth of lymphocytes in vitro when added up to 4 h after the start of the culture, but it was much less effective when added at 8 h. The experiments suggest that 8 hourly administration of CsA will optimise suppression of activation of the lymphocytes in vivo. If the suppressive activity of the metabolite M17 and the individual variation in sensitivity of the patients' lymphocytes to CsA are also taken into account it may be possible to reduce the dose of CsA to achieve effective immunosuppression and thereby minimise toxicity.
Cyclosporins↗
Antibodies to Mycobacterium tuberculosis-specific epitopes in lepromatous leprosy.
Sera from patients with leprosy or tuberculosis and healthy subjects have been analysed for the presence of antibodies to four species-specific mycobacterial epitopes, four different viruses and five autoantigens. Antibodies to the Mycobacterium leprae-specific 35-kD protein and phenolic glycolipid I epitopes were not present in patients with active pulmonary tuberculosis. In contrast, antibody levels to species-specific epitopes of the 38-kD and 14-kD antigens M. tuberculosis were significantly elevated in patients with lepromatous leprosy. Neither of the two antigens is cross-reactive with M. leprae at the B cell level. However, it was considered that cross-reactive helper T cells could recall the response of M. tuberculosis-specific memory B cells, which had been primed through prior self-healing tuberculous infection. As an alternative explanation, the possible role of polyclonal B cell stimulation was considered. This seemed unlikely, however, since: (i) antibody levels to autoantigens, except anti-smooth muscle, were not elevated, and (ii) antibody levels to four distinct viruses, unlike those to all mycobacterial epitopes, showed no correlation with titres, to M. tuberculosis-specific epitopes.
Antibodies↗
Early delayed hypersensitivity responses in tuberculin skin tests after heavy occupational exposure to tuberculosis.
The early (six hours) reaction to tuberculin skin testing was studied in 33 Indonesian hospital workers with frequent occupational exposure to M tuberculosis and compared with responses maximal at the usual time (48 hours) in factory workers, from the same locality but with only occasional occupational exposure, to determine the nature of the early reaction. The early reaction had the same general histopathological appearance as that seen in the conventional (48 hour) reaction, and both had an infiltrate consisting largely of T lymphocytes and macrophages. The cell densities were lower in the six hour reactions, but the relative concentration of macrophages was greater in the earlier response. These histometric measurements suggested that the six hour reaction was an accelerated delayed hypersensitivity reaction. Moreover, the absence of a specific IgE response or of particulate masses of Ig or complement, made it unlikely an anaphylactoid or Arthus-type reaction could have been responsible. It is concluded that those with frequent occupational exposure to M tuberculosis have larger numbers of circulating T cells reactive with mycobacterial antigens, so that the development of the skin test response to tuberculin is less dependent on "by-stander" cell infiltration to mediate the delayed hypersensitivity reaction than the reactions in those with less intense and less frequent natural exposure. The skin test response maximal at six hours is probably a hyperimmune reaction to an antigen recognised by T cells.
Adult↗
Impairment of vasomotor reflexes in the fingertips of leprosy patients.
A method is described for eliciting fingertip vasomotor reflexes by inspiratory gasp and contralateral hand cold challenge. The results of the two tests are reproducible on replicate testing and, when taken together, have proved reliable for detection of impairment of autonomic reflexes in 10 newly registered leprosy patients who did not have any obvious deformity. Similar, but less severe, impairment of vasomotor reflexes was noted in a group of 10 fully treated, apparently cured ex-leprosy patients, none of whom showed clinically obvious neuropathy. Both the new patients and the ex-patients were significantly different from healthy contacts and from healthy Europeans, who were indistinguishable by this test. Evidence is presented suggesting that impairment of these vasomotor reflexes is mainly due to damage to the efferent pathway in the peripheral nerves. The method might prove valuable for investigation of early nerve damage in new patients or during reversal reactions in leprosy at a stage before irreversible damage is done.
Adult↗
Role of chloride ions in liver cell volume regulation.
Hypotonic swelling of liver cells is followed by regulatory volume decrease (RVD), which has been shown to involve facilitated release of K+. In this study, the role of C1- in RVD was examined by videoplanimetric analysis of cell volume and measurement of membrane potential (Vm) and resistance (Rm) in single isolated rat hepatocytes, and by measurement of 36Cl efflux in the isolated perfused liver preloaded with the isotope. Liver cells subjected to hypotonic stress by removal of 50 mM external NaCl (70% of control osmolality) swelled from an initial volume of 6.68 +/- 0.77 to 8.27 +/- 0.88 pl (24.3 +/- 3.4% increase) within 1 min and exhibited RVD at an initial rate of 0.26 +/- 0.01 pl/min. A step decrease in external Cl- accelerated the initial rate of RVD to 0.53 +/- 0.08 pl/min. RVD was abolished in cells that had been depleted of Cl-. Vm and Rm displayed biphasic responses to hypotonic stress. An initial (less than 15 s) hyperpolarization of Vm from -35.4 +/- 2.2 to -38.8 +/- 2.6 mV was followed by a gradual depolarization reaching -30.2 +/- 2.0 mV by 1 min. In parallel, Rm initially (less than 15 s) increased from 101 +/- 13 to 121 +/- 17 M omega (19 +/- 3% increase) and then declined to 55 +/- 4 M omega (59 +/- 4% of initial Rm) within 1 min. These changes were reversible upon return to isotonicity.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Volume regulation and intracellular calcium in the rabbit proximal convoluted tubule.
The hypothesis that an increase of calcium leads to activation of calcium-activated ionic conductances during cell swelling was examined in the isolated perfused proximal convoluted tubule of the rabbit. Reduction of bath and luminal osmolality by 90 mosmol/kgH2O caused the cells to swell by 23.6 +/- 1.5% (n = 5) and intracellular calcium to rise from 227 +/- 35 to 347 +/- 60 nM (n = 6). Both these increases were transient, with volume decreasing to 5.5 +/- 1.2% above control and intracellular calcium concentration decreasing to 272 +/- 46 nM after 5-9 min. The addition of glucose and alanine to the tubule lumen to increase transcellular sodium transport caused a sustained increase in cell volume of 15.6 +/- 3.4% (n = 4). In parallel experiments, no significant increase in intracellular calcium concentration was observed. Addition of 1 microM of the calcium ionophore, ionomycin, reversibly increased intracellular calcium by 224 +/- 60 nM from a control value of 301 +/- 29 nM (n = 7) and reversibly depolarized the basolateral membrane by 3.6 +/- 0.9 mV (n = 5). However, there was no initial increase in the apparent transference number for potassium or chloride and no significant change in cell volume. We conclude from these observations that the sustained increase in basolateral potassium conductance observed when cells are swollen by hypotonicity or increased sodium transport (J. S. Beck and D. J. Potts. J. Physiol. Lond. 425: 369-378, 1990) is not due to a calcium-activated potassium conductance.
Alanine↗
Relationship between sodium transport and intracellular ATP in isolated perfused rabbit proximal convoluted tubule.
The effect of alterations in sodium transport on cell ATP content and pH in the isolated perfused proximal convoluted tubule (PCT) of the rabbit was examined. Stimulating sodium transport by the addition of luminal glucose and alanine decreased cell ATP from 4.44 +/- 0.93 to 2.69 +/- 0.62 mM (n = 4), increased intracellular pH by 0.13 +/- 0.02 (n = 7), and increased cell volume by 0.10 +/- 0.02 nl/mm (n = 4). Blocking the sodium pump with 10(-4) M strophanthidin in tubules in which sodium transport had been stimulated increased cell ATP from 2.04 +/- 0.24 to 2.42 +/- 0.32 mM (n = 6). In parallel experiments the same dose of strophanthidin depolarized the basolateral membrane from -52.6 +/- 1.9 to -6.4 +/- 1.6 mV, depolarized the transepithelial potential from -3.2 +/- 0.3 to -0.1 +/- 0.1 mV, and reduced the basolateral membrane potassium transference number from 0.47 to 0.26 indicating a reduction in basolateral potassium conductance. Since strophanthidin caused a cell alkalinization of 0.15 +/- 0.03, this latter effect cannot be due to changes of intracellular pH. Strophanthidin caused no change in cell volume over the period studied, suggesting that stretch-activated potassium channels are not involved either. Instead, potassium conductance inhibition may be the result of the closure of ATP-sensitive potassium channels. These same channels might thus be partly responsible for the increase in potassium conductance commonly observed during stimulation of sodium transport.
Adenosine Triphosphate↗
Impairment of fingertip vasomotor reflexes in leprosy patients and apparently healthy contacts.
Fingertip blood-flow velocity and its control by vasomotor reflexes were studied in leprosy patients and in healthy controls with a laser Doppler flowmeter. In newly registered patients, the flow was significantly lower than in the healthy controls, and even lower values were recorded in the long-standing patients with lower limb ulcers and/or deformity. The newly registered patients showed substantially impaired vasomotor reflex responses in the fingertips to cold challenge of the opposite hand or deep inspiratory gasp. Low blood flow and impairment of vasomotor reflexes were more prominent in those leprosy patients who showed clinical evidence of neuropathy and/or histological evidence of reaction in a punch biopsy of leprosy skin lesions. This aspect of dysautonomia to cold challenge was particularly prominent in apparently healthy, fully treated ex-patients. There was an unexpectedly high prevalence of impairment of vasomotor reflexes in newly registered and apparently healthy, adequately treated leprosy patients. The method is very sensitive, and it remains to be established whether the lesions it detects are nonprogressive residues, or previous nerve damage, or an indication of on-going nerve damage. A minority of leprosy contacts showed impairment of vasomotor reflexes. Those with two or more affected fingers were more likely to have had a higher level of exposure to Mycobacterium leprae than those with one or no affected fingers. The cause of this unexpected impairment of fingertip vasomotor reflexes in a minority of leprosy control workers has not yet been determined.
Adolescent↗
Identification of novel rhodopsin mutations associated with retinitis pigmentosa by GC-clamped denaturing gradient gel electrophoresis.
Retinitis pigmentosa (RP) is a group of disorders characterized by progressive degeneration of the outer retina, resulting in night blindness, visual field loss, an abnormal electroretinogram, and characteristic retinal pigmentary changes. An important step in the understanding of RP has been the recognition that some cases of autosomal dominant RP (ADRP) are caused by mutations in the rhodopsin gene. Multiple different point mutations within the coding sequence of the rhodopsin gene have been associated with ADRP. We have developed a GC-clamped denaturing-gradient-gel electrophoresis (DGGE) assay for the coding region of the rhodopsin gene and have used this assay to screen ADRP patients for mutations. The assay consists of amplifying with PCR the five exons of the rhodopsin gene and then analyzing each PCR product by DGGE. We have used this assay to detect three previously unreported rhodopsin base substitutions associated with ADRP. The use of this assay to identify ADRP patients who have various rhodopsin mutations has allowed us to begin studies seeking to correlate molecular genotype with clinical phenotype. Furthermore, GC-clamped DGGE has allowed us to identify families with ADRP not caused by a rhodopsin mutation. Such families will be important in the search for other genes involved in ADRP.
Amino Acid Sequence↗
The influence of histamine and PGE2-induced hyperaemia and oedema on respiratory metabolism in normal human forearm skin.
Transcutaneous measurements of pO2 and pCO2 were made on the forearm skin after intradermal injection of histamine, PGE2, and saline. The mediators, used at concentrations which induce intense hyperaemia, did not modify the steady state tcpO2/pCO2 levels measured with a sensor head temperature of 44 degrees C when breathing air or hyperbaric (2ATA) oxygen. It was deduced that gas transport is unaffected by mediator-induced conditions in the skin. The rates of fall of tcpO2 and of rise of tcpCO2 after arresting the forearm circulation by cuff occlusion of the arm were significantly less at the histamine site than at the PGE2 and saline sites. The values over the PGE2 and saline injection sites were less than those over undisturbed skin. The dynamic tests of respiratory gas exchange indicate that the skin metabolic rate is reduced at all injection sites and the greatest effect was seen with histamine. Measurement of dermal thickness after saline injection has shown that the excess interstitial fluid persists at the time of maximal hyperaemia: this is further accentuated at the histamine site through active oedema formation. Accumulation of excess interstitial fluid (persistence of aqueous injection or oedema generated by the action of mediator) separates the tissue cells. The reduction in the number of cells per unit volume is sufficient to explain the observed reduction in oxygen consumption per unit volume of skin. It is concluded that the increased diffusional distances in mediator-induced oedema are unimportant for the respiration of otherwise normal tissues, but that oedema by reducing oxygen flux may contribute appreciably to the hypoxia of inflamed tissue infiltrated with metabolically active cells.
Adult↗
Cell swelling, co-transport activation and potassium conductance in isolated perfused rabbit kidney proximal tubules.
1. Isolated, perfused rabbit proximal tubules were used to study the effects of activation of the apical membrane sodium co-transporters, and of the effects of osmotically induced cell swelling, upon cell volume, basolateral membrane potential and apparent partial conductance of potassium. 2. Activation of electrogenic apical sodium co-transport caused a depolarization of the basolateral membrane and a reduction of the basolateral apparent potassium transference number. This was followed by a spontaneous partial recovery of potential and increase in apparent potassium transference number. 3. Stimulation of apical sodium co-transport led to a sustained increase in cell volume. 4. A sustained increase in cell volume (of similar magnitude to that seen after activation of apical membrane sodium co-transporters) was also caused by reduction of bath and perfusate osmolality by removal of 89 mmol l-1 mannitol from both lumen and bath solutions. 5. This reduction in bath and perfusate osmolality also led to a basolateral membrane hyperpolarization and an increase in basolateral apparent potassium transference number. 6. These observations support the possibility that some of the partial recovery of basolateral membrane potential (Vb1) during apical sodium co-transport stimulation is due to a cell volume sensitive change in basolateral potassium conductance.
Animals↗
Observer variability in histopathological reporting of transitional cell carcinoma and epithelial dysplasia in bladders.
Sections from 90 urinary bladder biopsy specimens were examined by 11 consultant histopathologists with varying experience to determine the appropriateness of existing pathology terminology. Analysis with kappa statistics showed fair to good agreement in the grading and staging of transitional cell carcinoma. There was also reasonable agreement in the diagnosis of high grade dysplasia in random biopsy specimens from the urothelium adjacent to the neoplasm, but very poor agreement for lesser degrees of dysplasia. It is concluded that the present classification of bladder carcinomata is reliable and that pathologists can determine stage with a high degree of reproducibility and grade with a fair degree of reproducibility.
Carcinoma, Transitional Cell↗
Cooperative studies on the immunology of tuberculosis at Airlangga University, Surabaya, Indonesia.
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Acute-Phase Proteins↗
A quantitative study of the relationship between systemic and histological parameters of immunity in individual leprosy patients.
A group of 52 untreated leprosy patients were examined to determine the relationship between local and systemic immunological parameters across the clinico-pathological spectrum. The Ridley-Jopling classification, bacterial index (BI), and granuloma fraction (GF) were assessed in biopsies from 40 cases. The densities of apoptoses, mitoses, and plasma cells were also measured. Systemic immunity to mycobacteria was assessed by skin tests with leprosin A and PPD, and by measurement of the serum antibody responses to Mycobacterium leprae, M. tuberculosis, and M. scrofulaceum. The serum responses to phenolic glycolipid-I (PGL-I) of M. leprae was assessed using a glycoconjugate which mimics an immunodominant epitope. The serum antibody levels and skin test results showed the expected inverse relationship. The BI within lesions showed an inverse correlation with the skin test results, but none of the other histological parameters studied showed a significant relationship with the other measurements of systemic immunity. Our findings suggest that the inverse relationship between delayed-type hypersensitivity and humoral immunity in leprosy patients, which is strong in groups of patients across the leprosy spectrum, is less strong in individual patients than is often thought. The lack of correlation of many histological and systemic parameters suggests that local factors modulate systemic immunity in the pathogenesis of leprosy lesions.
Adolescent↗