Long-lasting contact urticaria from castor bean.

Type I (immediate) and type IV (delayed) allergic reactions to castor bean developed in a stockroom worker in a coffee roasting plant. The exceptionally strong contact urticarial patch rest reaction persisted for more than 48 hours and was therefore called "long-lasting contact urticaria." Light and electron microscopic observations indicated that eosinophils and mast cells were activated and participated in patch test reactions, which include both type I and type IV allergic reactions. Although patch testing is an absolute prerequisite for an accurate diagnosis of delayed allergy, it should be stressed that skin tests should not be performed with castor bean because of its toxicity and potential danger.

Purification of MEA, a mast cell growth-enhancing activity, to apparent homogeneity and its partial amino acid sequencing.

A novel growth factor for bone marrow derived murine mucosal type mast cells has been isolated from the conditioned medium of the Mlsa-reactive mouse Th cell line MLS-4.2. In proliferation assays this growth factor synergizes, like IL-4, with IL-3 on established mast cell lines and was therefore termed MEA: mast cell growth enhancing activity. MEA was characterized as a glycoprotein with a Mr range between 37,000 and 43,000. Apparent homogeneity was obtained by using a four-step purification scheme including cation exchange chromatography, Procion red affinity chromatography, IEF, and gel filtration. Inasmuch as MEA was N-terminally blocked during automated Edman-degradation, peptide fragments after digestion with trypsin were used for partial amino acid sequence determination. All evaluable MEA peptide fragments showed complete sequence homology to a recently purified and cloned novel T cell growth factor (P40/TCGF III), the mouse homologue of human IL-9.

Ovine haemopoiesis: the development of bone marrow-derived colony-forming cells in vitro in the presence of factors derived from lymphoid cells and helper T-cells.

Techniques for the development of ovine bone marrow-derived haemopoietic progenitor cells and in situ identification of colony morphology are described. Both mitogen stimulated lymphoid cells and antigen stimulated helper T-cells generated potent colony-stimulating activity in conditioned medium. Monocyte/macrophage, neutrophil, eosinophil, basophil/mast cell, neutrophil/monocyte and mixed phenotype colonies developed in stimulated bone marrow cultures in a conditioned medium dose-dependent manner. Neutrophil, monocyte/macrophage and eosinophil colonies were detected in greater numbers than the other types, with mixed colonies representing only around 1% of the total. Eosinophil colonies were particularly abundant when compared to published reports of the numbers obtained with similar cultures of 'normal' mouse or human bone marrow cells. This culture technique will allow a detailed analysis of both ovine colony-stimulating factors and of the distribution of haemopoietic progenitor cells in vivo.

Characterization of peripheral blood and salivary gland lymphocytes in Sjögren's syndrome.

Primary Sjögren's syndrome (SS) is an autoimmune disease resulting from lymphocyte infiltration of lacrimal and salivary glands (SG). This study was designed to investigate the peripheral blood (PBL) and SG lymphocytes in 14 patients with primary SS and control subjects. With the use of monoclonal antibodies, cells were stained to identify T-cells and T-cell subsets (T-helper and T-suppressor) and cells positive for HLA-DR antigen, whereas B cells were determined by the Smlg (surface membrane immunoglobulin) method. Lymphocytes in SG biopsy specimens were characterized by means of monoclonal antibodies and the immunoperoxidase technique. In the peripheral blood lymphocytes, there was a significant reduction in T cells and suppressor T cells. T lymphocytes and mostly helper T cells were predominant around the ducts and within the lymphocytic infiltrates in the minor SG biopsy samples of patients with SS. Suppressor T cells and B cells were found in fewer numbers, HLA-DR(+) cell populations had increased, and IgG- and IgA-bearing plasma cells were also present within the infiltrates. These results may contribute to our understanding of the immunopathogenesis of primary SS.

Detection of individual mouse splenic T cells producing IFN-gamma and IL-5 using the enzyme-linked immunospot (ELISPOT) assay.

Although several sensitive and specific assays have been developed to quantify murine cytokines, these assays do not allow individual cells to be correlated with the specific cytokines they produce. The purpose of this study was to develop a sensitive and reproducible method for the detection of individual T cells which secrete either interferon-gamma (IFN-gamma) or interleukin-5 (IL-5). We have used an adaptation of the enzyme-linked immunospot (ELISPOT) assay in which monoclonal antibodies to IFN-gamma (R4-6A2) and to IL-5 (TRFK-5) were used to coat 96-well plates with a nitrocellulose base. Mouse splenic T cells, either nonstimulated or activated with concanavalin A (ConA) or phytohemagglutinin (PHA), were cultured in individual wells. Following incubation, the cells were removed, and the bound cytokines probed with either biotinylated mAb anti-IFN-gamma (XMG 1.2) or anti-IL-5 (TRFK-4) followed by avidin-peroxidase. The spots which developed with 3-amino-9-ethylcarbazole were discrete and enumerated with a dissecting microscope. Although unstimulated splenic T cells contained low numbers of cytokine-specific spot-forming cells (SFC), 24-72 h activation with mitogen was required to induce significant numbers of cytokine producing cells. When mitogen-stimulated splenic CD4+ T cells were assessed, approximately equal numbers of IFN-gamma and IL-5 SFC were seen. Approximately 20-30% of all mitogen-activated splenic T cells produced at least one of these two cytokines. Pre-incubation of biotinylated anti-IFN-gamma with recombinant IFN-gamma (rIFN-gamma) or anti-IL-5 mAbs with rIL-5 completely inhibited cytokine-specific SFC. Further, use of nonrelevant antibodies did not result in spot formation, and treatment of mitogen-activated T cells with cycloheximide inhibited both IFN-gamma- and IL-5-specific SFC. A sensitive method has been developed which allows detection of individual T cells that produce either IFN-gamm or IL-5, and should be useful for detection of cytokine secretion at the single cell level.

Natural killer cells and Tac antigen in the hypertension of pregnancy.

Of the immunological alterations in pregnancy hypertension were studied peripheral blood lymphocyte subpopulation in normal and hypertensive pregnancies by means of monoclonal antibodies. In pregnant women with hypertension an increase was found in NK activity. It was also shown that an increase in circulating T cells expressing Tac antigen occurred in women with pregnancy hypertension. These preliminary data on Tac antigen suggest that there is an activation of Leu 3 cells; which may introduce the concept of Leu 3 activity like NK activity. Further studies on this subject could explain that the concept of Leu 3 activity is correct.

Lack of Leu-3a epitope on T-helper (CD4) lymphocytes.

Human T-helper cells express membrane-bound CD4 antigen whose many epitopes are recognized by different monoclonal antibodies. The epitope recognized by Leu-3a and similar clones has been shown to be the location for human immunodeficiency virus (HIV) receptor. We have found a unique blood donor whose CD4+ T-helper lymphocytes were lacking Leu-3a epitope. CD4+ T-helper cells lacking Leu-3a epitope might be resistant to HIV infection.

Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones.

A cytokine synthesis inhibitory factor (CSIF) is secreted by Th2 clones in response to Con A or antigen stimulation, but is absent in supernatants from Con A-induced Th1 clones. CSIF can inhibit the production of IL-2, IL-3, lymphotoxin (LT)/TNF, IFN-gamma, and granulocyte-macrophage CSF (GM-CSF) by Th1 cells responding to antigen and APC, but Th2 cytokine synthesis is not significantly affected. Transforming growth factor beta (TGF-beta) also inhibits IFN-gamma production, although less effectively than CSIF, whereas IL-2 and IL-4 partially antagonize the activity of CSIF. CSIF inhibition of cytokine synthesis is not complete, since early cytokine synthesis (before 8 h) is not significantly affected, whereas later synthesis is strongly inhibited. In the presence of CSIF, IFN-gamma mRNA levels are reduced slightly at 8, and strongly at 12 h after stimulation. Inhibition of cytokine expression by CSIF is not due to a general reduction in Th1 cell viability, since actin mRNA levels were not reduced, and proliferation of antigen-stimulated cells in response to IL-2, was unaffected. Biochemical characterization, mAbs, and recombinant or purified cytokines showed that CSIF is distinct from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IFN-gamma, GM-CSF, TGF-beta, TNF, LT, and P40. The potential role of CSIF in crossregulation of Th1 and Th2 responses is discussed.

T cell derived IL-6 is differentially required for antigen-specific antibody secretion by primary and secondary B cells.

IL-6 (formerly PCTGF, HP-1, BSF-2, HGF, IFN-beta 2, 26 kDa) is a recently defined lymphokine demonstrating activity on multiple cell types, including hepatocytes, thymocytes, T cells, plasmacytomas, and B cells. The biologic effects of IL-6 on lymphocytes, particularly B cells, suggest this factor may be involved in the regulation of normal immune responses. Accordingly, we have investigated the role of IL-6 in Ag-specific responses of B cells from both naive and Ag-primed mice. When Ag-primed splenic T cells were used as a source of help, naive (primary) B cell responses specific for the hemagglutinin molecule of the influenza A virus (PR8) were fully inhibited by the addition of an anti-IL-6 antiserum, and are thus IL-6 dependent. In contrast, secondary B cell responses were essentially IL-6 independent, being unaffected by this antiserum even at concentrations 10-fold higher than required to completely inhibit primary responses. This differential IL-6 requirement was further investigated by using a panel of hemagglutinin molecule-specific Th clones. Consistent with the above findings, a Th1 clone secreting biologically active IL-6 enables antibody secretion by both primary and secondary B cells, whereas Th1 clones that do not produce IL-6 support secondary responses, but fail to help primary B cell responses unless exogenous IL-6 is added. These results provide the first instance of differential lymphokine requirements among primary vs secondary B cell responses, and suggest T cell-derived IL-6 plays a critical role during the regulation of humoral immune responses. Moreover, functionally distinct Th1 clones were identified that differed in IL-6 secretion and their corresponding ability to induce Ig secretion by primary and secondary B cells.

Flow cytometric monitoring of human immunodeficiency virus-infected patients. Simultaneous enumeration of five lymphocyte subsets.

The utility of CD4 lymphocytes in monitoring disease progression and prognosis of human immunodeficiency virus (HIV)-infected patients is well established. We have modified a previously described antibody cocktail to provide complete lymphocyte subset analysis on 100-200-microL samples of whole blood. This method optimizes accuracy of CD4 lymphocyte assessments and provides simultaneous assessment of four other lymphocyte subtypes of interest in specimens with absolute lymphocyte counts as low as 300 X 10(6)/L. Lymphocytes are classified as Thelper (CD3+CD4+); Tsuppressor (CD3+CD8+); Tnull (CD3+CD4-CD8-, putative gamma delta T-cell receptor); B (CD19+CD20+); or natural killer (CD3-CD16+CD56+). The method positively discriminates against contamination of lymphocyte scatter gates by monocytes and unlysed erythrocytes and is compatible with a variety of cell preparation procedures. Increased accuracy of CD4 lymphocyte determinations and simultaneous identification of other lymphocyte subsets whose relationship to disease progression is under study make this an efficient and informative method for disease monitoring and evaluation of therapy in HIV-infected patients.

[Immunohistochemical studies in fungal diseases of skin].

The aim of the present work was to characterize the infiltrating cells in fungal diseases of the skin by using immunohistochemical methods. The lesions of superficial mycosis were characterized by a increase in the number of Langerhans cells (OKT6, HLADR+). In the upper dermis more OKT4 cells stained than OKT8, and in the deeper dermis the was a more decreased ratio of OKT4+/OKT8+ cells. On the other hand, the granulomatous reactions of deep mycosis mainly consisted of cells that were labeled positively for lysozyme and alpha 1-antichymotrypsin. In cases with secondary or local immunodeficiency, however, the infiltrating cells wer negative for these enzymes.

Predominance of T cells that express CD45R in the CD4+ helper/inducer lymphocyte subset of neonates.

Neonates have an increased risk of severe infections. For several in vitro and in vivo immune responses, neonates have been shown to have significant differences when compared to normal adults. To indirectly study immune cellular defects, we compared cell surface markers on cord blood lymphocytes (CBL) from 58 term infants to peripheral blood lymphocytes (PBL) from 17 healthy adults using flow cytometry with standard as well as newly defined monoclonal antibodies (Mab) that distinguish regulatory T cells. CBL had significantly smaller percentages of lymphocytes that express the CD2 and CD8 markers (total T cells, and suppressor/cytotoxic T cells, respectively), although absolute numbers of CD2+ and CD8+ cells were comparable in neonates and adults. CBL and PBL were similar in terms of the percentage of CD4+ cells (helper/inducer T cells), although the absolute numbers of CD4+ cells were higher in CBL than in PBL. The CD4+ population was subdivided into cells bearing the virgin and memory T cell phenotypes using anti-2H4 and anti-4B4 Mab and dual parameter analysis with anti-CD4. Neonates were deficient in the percentage of CD4+, 4B4+ (3.8 +/- 2.8 vs 13.4 +/- 7.5, P less than 0.001), but equivalent to adults in the percentage of CD4+, 2H4+ T cells (21.4 +/- 9.8 vs 18.8 +/- 12.8). In absolute numbers, neonates had fewer CD4+, 4B4+ cells (178 +/- 173 vs 344 +/- 152 cells/microliters, P less than 0.001), but more CD4+,2H4+ cells (978 +/- 572 vs 542 +/- 518 cells/microliters, P less than 0.01) than adults. The predominance of 2H4+ virgin T cells in the CD4 population whose function is associated with that of the induction of suppression rather than the up-regulation of immune responses may contribute to the observed susceptibility of neonates to infection.

Selective replication of simian immunodeficiency virus in a subset of CD4+ lymphocytes.

Although all CD4+ cells theoretically are at risk for infection by human immunodeficiency viruses or the related simian immunodeficiency viruses found in Old World monkeys, only a small proportion of CD4+ lymphocytes from infected individuals have detectable virus. This suggests that immunodeficiency viruses may replicate predominantly in a minor subset or activated form of CD4+ T cells, a possibility we examined in macaques infected with a simian immunodeficiency virus isolate, SIV/Mne. Macaque CD4+ lymphocytes could be divided into two subtypes that differed in their level [high (hi) or low (lo)] of expression of a class of heterotypic adhesion receptors (HARs). In blood from animals infected with SIV/Mne, HARhi CD4+ T cells were lost selectively compared to HARlo CD4+ cells and, when cultured, exhibited 50-fold more recoverable reverse transcriptase activity. The HARhi CD4+ subset was also markedly more susceptible to productive infection following exposure to SIV/Mne in vitro. Both subsets are composed primarily of small resting lymphocytes. However, HARhi cells respond differentially to mitogenic stimulation and may thus be more likely to provide the cellular factors necessary to initiate or enhance virus replication. Thus, HAR expression may prove useful both as a prognostic indicator in immunodeficiency virus infection and as a tool to analyze pathogenesis of immunodeficiency viruses.

T cell receptor gene segment usage in a panel of hen-egg white lysozyme specific, I-Ak-restricted T helper hybridomas.

We have studied the relationship between MHC-restricted, Ag-specific recognition and TCR structure in a panel of seven Th-hybridomas specific for the foreign protein Ag, hen egg-white lysozyme, and the I-Ak class II MHC molecule. The fine specificity of these Th cells had been determined previously by their reactivity to a panel of APC lines bearing mutant I-Ak molecules and to proteolytic fragments of HEL. TCR gene segment composition was determined by cDNA cloning and DNA sequencing. A heterogeneous, yet repetitive usage of gene segments was observed within the panel. The same V alpha C10-J alpha MD13 rearrangement was used in three of the hybridomas, two with identical Ag and MHC-restriction fine specificities. The prevalent usage of the V beta 14 gene segment and members of J beta 2 cluster was noted. Inasmuch as gene segment usage did not correlate with MHC-restriction or Ag fine specificity alone, these results favor an interactive Ag model of T-cell recognition, in which Ag and MHC are recognized as a bimolecular complex.

Demonstration of surface antigens on bronchoalveolar lavage cells using the immunoalkaline phosphatase method.

The immunoalkaline phosphatase procedure is described as a method for labelling bronchoalveolar lavage cellular specimens with monoclonal antibodies. This method has several advantages over conventional immunofluorescent techniques: it can be performed on cytocentrifuge preparations stored for long periods before staining; cell morphology can be observed in detail in positive and negative cells; the staining is permanent and stable, and, the reaction can be evaluated with a light microscope. Normal values for lymphocyte subpopulations in smokers and nonsmokers are also reported.

Cyclosporin A: preliminary results in the treatment of psoriasis.

Cyclosporin A (CYA) has recently attracted the attention of the dermatologists with regard to its use in the treatment of severe psoriasis. We report four cases of psoriasis resistant to conventional therapy. Three patients were affected by chronic plaque psoriasis and one by erythrodermic psoriasis. The initial dosage of CYA was 5 mg/kg/day per os, and was gradually reduced according to the course of the disease. The drug was administered for a period of 6 weeks. All patients showed a significant improvement of the clinical picture. No remarkable clinical side effects were observed. During the trial we took skin biopsies on which we conducted histological and immunohistochemical studies. The efficacy of CYA in the treatment of psoriasis demonstrates the important role of the cell mediated immunity in the pathogenesis of psoriasis.