An efficient method for cloning human autoantigen-specific T cells

T-cell clones are valuable tools for investigating T-cell specificity in infectious, autoimmune and malignant diseases. T cells specific for clinically-relevant autoantigens are difficult to clone using traditional methods. Here we describe an efficient method for cloning human autoantigen-specific CD4+ T cells pre-labelled with CFSE. Proliferating, antigen-responsive CD4+ cells were identified flow cytometrically by their reduction in CFSE staining and single cells were sorted into separate wells. The conditions (cytokines, mitogens and tissue culture plates) for raising T-cell clones were optimised. Media supplemented with IL-2+IL-4 supported growth of the largest number of antigen-specific clones. Three mitogens, PHA, anti-CD3 and anti-CD3+anti-CD28, each stimulated the growth of similar numbers of antigen-specific clones. Cloning efficiency was similar in flat- and round-bottom plates. Based on these findings, IL-2+IL-4, anti-CD3 and round-bottom plates were used to clone FACS-sorted autoantigen-specific CFSE-labelled CD4+ T cells. Sixty proinsulin- and 47 glutamic acid decarboxylase-specific clones were obtained from six and two donors, respectively. In conclusion, the CFSE-based method is ideal for cloning rare, autoantigen-specific, human CD4+ T cells.     

Simultaneous turnover of normal and dysfunctional C1 inhibitor as a probe of in vivo activation of C1 and contact activatable proteases.

Simultaneous turnover of normal and dysfunctional C1-inhibitor (C1-INH) was carried out in 10 normal subjects and 13 patients with rheumatoid arthritis as a measure of the in vivo activation of C1 and the contact activatable enzymes. In the first series of experiments, dysfunctional protein We was used in simultaneous turnover studies in five normal subjects and nine patients. The fractional catabolic rate of the dysfunctional C1-INH, We, (FCR(d)) was unchanged in both groups but the fractional catabolic rate of the normal C1-INH (FCR(n)) was faster in the patients compared to the controls, in particular patients with vasculitis. The enzyme-dependent catabolism defined as FCR(n-d) X concentration of C1-INH X plasma volume, was raised in the patient group, and correlated with disease activity score (r = 0.83, P less than 0.05). Neither FCR(n) nor FCR(d) was dependent on C1-INH concentration. The latter was higher in the patients (206 mg/l compared with 155 mg/l) indicating a very high synthetic rate in the patients (280.81 micrograms/kg/h compared with 179.77 micrograms/kg). In the second series of turnovers in six patients and five normal subjects, another dysfunctional C1-INH, at, was used. The FCR of C1-INH was slower than C1-INH (We) (1.88%/h compared with 2.7%/h). Enzyme-dependent catabolism of C1-INH in these patients were raised and also correlated with disease activity score (r = 0.82, P less than 0.05).     

Pathogenesis of rabies in dogs inoculated with an Ethiopian rabies virus strain. Immunofluorescence,histologic and ultrastructural studies of the central nervous system

Summary Dogs were inoculated with either an Ethiopian or Mexican rabies virus strain. The distribution of viral antigen and lesions were studied by immunofluorescence, histologic and electron microscopic techniques. In all dogs inoculated with the Ethiopian rabies virus strain, tremendous whorls of filamentous fluorescing aggregates were observed throughout the brain; these were not observed in dogs inoculated with the Mexican virus.Lesions consisted of neuronal degeneration and neuronophagia, associated with large inclusion bodies and widespread inflammation in dogs inoculated with the Ethiopian isolate. All observed portions of the brain and spinal cord were affected. In general, lesions were much less severe with the Mexican isolate. Occasional astrocytes were observed to have inclusions in dogs inoculated both with the Ethiopian and Mexican strains.Most neurons examined electronmicroscopically showed signs of infection, varying from a small granular or finely fibrillar viral matrix to numerous matrices accompanied by prolific numbers of virus particles occupying much of the perikaryon. These were found in all dogs inoculated with the Ethiopian strain but were rare with the Mexican isolate. Viral budding occurred from membranes of the rough endoplasmic reticulum, outer lamella of the nuclear envelope, and rarely from the plasma membrane.With 15 FiguresThis work was done while the senior author (Visiting Scientist) was being supported by the Swedish Aid for Research Cooperation with Developing Countries Project 77/89 A 9–49 U-forsk, through the Department of Bacteriology and Epizootology, Biomedicum, P.O. Box 583, S-75123 Uppsala, Sweden.     

The fine structure of chordoma with particular reference to the physaliphorous cell

A chordoma was examined by electron microscopy. It was composed of stellate and physaliphorous cells. The fine structure of these cells is described. Transitional type cells are also described and it is suggested that the physaliphorous cells develop from the stellate cells through a process of vacuolation. Two types of vacuoles are described, `smooth-walled' and `villous' endowed with numerous structureless microvilli. Some observations on stained sections are discussed.     

Vitamin B12 levels in erythrocytes in hypochromic anaemia

Vitamin B(12) levels in erythrocytes were low in untreated hypochromic anaemia, rose to abnormally high levels during therapy with iron alone, and finally slowly fell to normal. These changes were similar to those previously found in pernicious anaemia in response to vitamin B(12) therapy and in folate-deficiency anaemia in response to folic acid, thus changes in erythrocyte B(12) levels are not always due directly to changes in B(12) metabolism but may be secondary to changes in the levels of other haematinic factors.     

Histochemical and immunohistochemical study of diffuse large-cell lymphomas.

Ninety diffuse large-cell lymphomas (diffuse histiocytic lymphoma) were subclassified into B-cell, T-cell, and histiocytic types according to their enzyme histochemical and immunohistochemical characteristics. The B-cell type was characterized by presence of intracellular monoclonal immunoglobulin; negative or weakly positive diffuse acid phosphatase activity; and an occasional focal nodular pattern or preceding nodular lymphoma. The T-cell type was characterized by moderate, focal acid phosphatase activity; convoluted nuclear structure; and frequent preceding cutaneous manifestations. The histiocytic type was characterized by strong nonspecific esterases and diffuse acid phosphatase activity and presence of lysozyme and phagocytic activity. Most of the lesions (74 cases) were of the B-cell type. This group was further subdivided into follicular center cell type and B-cell immunoblastic sarcoma, according to the stage of cellular transformation. Preliminary clinical correlation suggests that the histiocytic type is most resistant to treatment. B-cell immunoblastic sarcomas were much more aggressive than neoplasms of the follicular center cell type.     

Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm

The ability to maintain cellular volume is an important general physiological function. Swelling induced by hypotonic stress results in the opening of channels, through which ions exit with accompanying water loss (regulatory volume decrease, RVD). RVD has been shown to occur in mammalian sperm, primarily through the opening of quinine-sensitive potassium channels. However, as yet, direct evidence for the participation of anion channels in sperm RVD has been lacking. The chloride channel type ClC-3 is believed to be involved in RVD in other cell types. Using electronic cell sizing for cell volume measurement, the following results were obtained. (i) The anion channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), tamoxifen and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) increased hypotonic swelling in concentration-dependent fashion, whereas verapamil (P-glycoprotein inhibitor) had little effect. The most potent, NPPB and DIDS, blocked RVD without affecting cell membrane integrity at effective concentrations. (ii) When gramicidin was included to dissipate Na+/K+ gradients, major secondary swelling was observed under hypotonic conditions. This secondary swelling could be reduced by NPPB, and suppressed completely by replacing chloride in the medium with sulphate, an ion which does not pass through chloride channels. It was deduced that the initial hypotonic swelling activated an anion channel through which chloride ions could then enter freely down a concentration gradient, owing to the lack of a counter-gradient of potassium. (iii) Taurine, an osmolyte often involved in RVD, does not appear to play a role in sperm RVD because lengthy preincubation with taurine did not alter sperm RVD response. Our observations provide direct evidence that a chloride channel (possibly ClC-3) is involved in the process of volume regulation in mammalian sperm.