[Frontiers in Live Bone Imaging Researches. In vivo imaging of immune tissues].

In vivo imaging analysis of the immune tissues, especially secondary lymphoid tissues such as lymph nodes, has greatly increased our understanding of how immune responses are promoted and regulated by immune cell trafficking. Recently, in vivo tracking of follicular helper T (Tfh) cells, a vital T cell subset for B cell responses to produce antibodies, by imaging analysis and light-induced cell labeling not only revealed their migration dynamics, but also provided new insights into how Tfh cells may be involved in the generation of immunological memory.

Nanoscale organization of CD4 molecules of human T helper cell mapped by NSOM and quantum dots.

CD4 molecule, the surface marker of T helper cell, has been confirmed to be the main cellular receptor for the human immunodeficiency viruses HIV-1, HIV-2 and SIV. Recent research demonstrated the importance of the spatial arrangement of CD4 on the cell membrane to its binding efficiency to virus. In this article, the combined near-field scanning optical microscopy (NSOM) and quantum dots (QDs) fluorescent labeling technology were performed to investigate the nanoscale organization of CD4 molecules with a spatial resolution about 100 nm. Simultaneous topographic image of the T helper cell and fluorescent image of QDs have been directly gained by NSOM/QDs-based system. Intensity- and size-distribution histograms of the QDs fluorescent spots verify that approximately 80% of the CD4 molecules are organized in nanosized domains randomly distributed on the cell surface. Intensity-size correlation analysis revealed heterogeneity in the molecular packing density of the domains. Our results also illustrated the combination of NSOM imaging and QDs labeling is an ultrasensitive, high-resolution technique to probe nanoscale organization of molecules on the cell surface.

[The drug "Selena" in the complex treatment of patients with chronic inflammatory diseases of the biliary system]. / Preparat "Selena" v kompleksnomu likuvanni khvorykh na khronichni zapal'ni zakhvoriuvannia biliarnoï systemy.

The drug selena of the Novamed firm has been shown to be of high clinical, biochemical, and immunological effectiveness in a combined treatment of patients with chronic inflammatory diseases of the biliary system presenting with immunological disorders, that was evidenced by a sooner than in controls disappearance or alleviation of clinical manifestations of the disease, normalization of clinical indices for blood, ultrastructure of the immunocompetent cells, and by less time for the functional state of the liver to return to normal. It is suggested that the drug effect on the immune system might be mediated through normalization of lipid peroxidation and of the system for antioxidant defence.

Dynamics of antigen-specific helper T cells at the initiation of airway eosinophilic inflammation.

Bronchial asthma is characterized by chronic eosinophilic inflammation of the bronchial mucosa in which Th2 cells play crucial roles. Ovalbumin-reactive Th2 clones were labeled with a fluorescent-probe then infused into unprimed mice to elucidate the dynamics of antigen-specific T cells involved in allergic inflammation. Infiltration of not only labeled antigen-specific T cells, but also unlabeled nonspecific CD4(+) T cells into the bronchial mucosa following inhaled antigen challenge was detectable under confocal microscopy and flow cytometry. Accordingly, labeled T cells in the spleen were decreased, whereas those in hilar lymph nodes were increased upon antigen challenge. Approximately 45% of antigen-specific T cells that migrated into the lungs bore CD25, while another early activation marker, CD69, was expressed on 80% of the migrated T cells. Accordingly, antigen challenge to the mice induced in situ proliferation of antigen-specific T cells as well as bronchial epithelial cells in the lungs. Expression of vascular cell adhesion molecule (VCAM)-1, but not intercellular adhesion molecule (ICAM)-1, on the vascular endothelium in the lungs was enhanced following antigen challenge. Nevertheless, treatment with anti-VCAM-1 antibody, and also anti-ICAM-1 antibody strongly suppressed the accumulation of T cells, suggesting that both VCAM-1 and ICAM-1 are essential for antigen-stimulated T cell mobilization into peripheral tissues. Our current study visualized the kinetics and the mechanism of antigen-specific T cell migration in response to local challenge with a protein antigen.

Molecular analysis of highly enriched populations of T-cell-depleted monocytes.

CD4+ T lymphocytes and monocytes/macrophages are important components of the immune system. Blood monocytes are usually targeted for studies of the human macrophage lineage cells because of their accessibility through blood sampling. Most separation techniques currently available to obtain human monocytes either require large volumes of blood or do not yield a monocyte fraction sufficiently depleted of other cell types. We have developed a simple strategy to isolate a highly enriched population of monocytes from small volumes (< 6 ml) of peripheral blood by using an anti-CD14 monoclonal antibody and magnetic microspheres. Yields of monocytes ranged from 75 to 80% of CD14+ cells in peripheral blood. CD4+ T cells were subsequently selected from the monocyte-depleted peripheral blood by using an anti-CD4 monoclonal antibody and immunomagnetic beads. The effectiveness of immunomagnetic selection to yield a monocyte population highly depleted of T cells was analyzed by using a sensitive molecular strategy based on PCR amplification and detection of T-cell receptor (TCR) gene rearrangements. The relative frequency of rearranged TCRs within the monocyte population was compared with the frequency of rearranged TCRs within the CD4+ T-cell fraction from the same individual. Molecular analysis indicated that a viable monocyte population which contains fewer than 2% residual T lymphocytes can be consistently selected from small aliquots of blood.

Thyrotropin receptor T cell epitopes in autoimmune thyroid disease.

The human TSH receptor represents the primary target of thyroid-stimulating immunoglobulins responsible for the hyperthyroidism of Graves' disease. In the present series of investigations, the distribution of T cell epitopes has been mapped using synthetic peptides spanning the entire extracellular region of the human TSH receptor. In vitro proliferative responses of the mononuclear cells were measured using flow cytometric analysis of bromodeoxyuridine incorporation into nuclei. In 8 of 11 samples from patients with Graves' disease, at least one (and up to 9) regions of the human TSH receptor induced proliferation, with the mean stimulation index being 39.8 +/- 47.3. No single universal stimulatory peptide was identified. In contrast, stimulation was not observed in three control subjects, while one control subject showed minimal stimulation (index of 5.7) to peptides encompassing a limited area (amino acids 31-65). The immunodominant epitope of patients with recent-onset Graves' disease was localized between amino acids 271 and 365, whereas the immunodominant epitope of patients with disease duration greater than 1 year localized between amino acids 91 and 215. We conclude that the bromodeoxyuridine incorporation method is a useful and important tool for detecting antigen-induced lymphocyte proliferation. The TSH receptor-specific T cells from different Graves' disease patients recognize variable distinct sites within the extracellular region of the TSH receptor, and the immunodominant epitope apparently shifts toward the N-terminus of the receptor protein during the course of treated Graves' disease.

Antigen recognition by helper T cells elicits a sequence of distinct changes of their shape and intracellular calcium.

BACKGROUND: Helper T-cell activation is initiated in vivo when the T-cell receptor complex recognizes an antigen fragment associated with MHC class II molecules on the surface of an antigen-presenting cell. In most previous studies of this phenomenon, T cells were stimulated not with antigen-presenting cells, but with CD3-specific antibodies. This approach provided considerable understanding of the cascade of molecular events triggered by T-cell receptor stimulation. However, the specific consequences of cell-cell interactions are still poorly understood. We therefore used a dual imaging system that provides simultaneous transmission and fluorescence images to study the morphological changes and variations of intracellular calcium concentration ([Ca2+]i) triggered in a human CD4+ antigen-specific T-cell clone in response to antigen presented by a class II-transfected murine fibroblast. RESULTS: T cells loaded with the Ca(2+)-sensitive fluorescent dye Fura-2 were individually monitored for half an hour following their contact with a monolayer of antigen-pulsed antigen-presenting cells. The response was found to have three distinct phases. During the first few minutes after contact, the T cell moves over the antigen-presenting cells, as if 'scanning' them. After several minutes, an oscillating [Ca2+]i response begins, accompanied by the immobilization of the cell and the retraction of pseudopodia. This rounding-up was probably Ca(2+)-dependent, as it could also be triggered by ionomycin or thapsigargin. Later during the [Ca2+]i response, the T cell becomes flattened and further elongated, suggesting increased adhesion to antigen-presenting cells. CONCLUSIONS: The physiological signal for T-cell activation, antigen recognition, is a three-step process reminiscent of the three steps previously observed in the interaction between neutrophils and endothelial cells. During these successive steps, a mobile, weakly interacting T cell is transformed into an immobile cell fully engaged in the activation pathway. Thus, antigenic recognition is not instantaneous, but evolves slowly by progressive amplification of the signal given by a few antigen molecules, eventually resulting in T-cell activation.

Chlorinated dibenzo-p-dioxins and dibenzofurans and the human immune system. 1. Blood cell receptors in volunteers with moderately increased body burdens.

Using monoclonal antibodies (mAbs) and flow cytometry, we studied a variety of surface receptors on lymphocyte subpopulations of workers with moderately increased body burdens of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and of other polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDF), expressed here as International-Toxicity Equivalencies (I-TE). The hypothesis to be tested was whether or not humans exhibit a similar susceptibility to PCDDs/PCDFs with respect to the surface receptors found previously to respond to small doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Callithrix jacchus. These are: helper-inducer (memory) T cells (CD4+CD45R0+CD45RA-CD29highCD11a+), CD20+ B cells, and cytotoxic T cells (CD8+CD56+/CD57+). Furthermore, 68 triple-labellings with mAbs were performed on the cells of each volunteer to possibly generate further hypotheses. It was evaluated whether any of the variables might be used as a biomarker of effects for this class of compounds. There were two main goals: (1) to evaluate whether workers with a moderately increased PCDD/PCDF-body burden [25-140 ppt TCDD or 104-522 ppt I-TE in blood fat] exhibit changes in the surface receptors of white blood cells, as observed in previous studies in non-human primates, and (2) to clarify whether persons at the upper range [10-23 ppt TCDD or 30-90 ppt I-TE in blood fat] of the body burden reference values of a not particularly exposed population show detectable deviations in these immunological variables, when compared with persons at the lower and medium range [1-3 ppt TCDD or 9-29 ppt I-TE] of these body burden reference values. Regression analysis of our data revealed slight trends for some of the biomarkers (e.g. CD45R0+). With one exception, these were all increases. None of the alterations observed are of medical relevance. The slight increase in the percentage of CD4+CD45R0+ cells remained significant even after covariant analysis taking age-related changes into account. Altogether, the data do not provide any evidence to support an assumption that moderately increased body burdens of PCDDs/PCDFs in adults induce decreases in the cellular components of the human immune system. Adult humans certainly are less susceptible to this action of PCDDs/PCDFs than adolescent Callithrix jacchus.

Cognate interaction between T helper cells and B cells. VII. Role of contact and lymphokines in the expression of germ-line and mature gamma 1 transcripts.

Complete reconstitution of Th cell function for B cell growth and differentiation was provided by plasma membranes (PM) derived from activated Th (PMAct) in combination with IL-4 and IL-5 (IL-4/IL-5). IL-5 has been shown to be essential for the secretion of all Ig isotypes by resting, conventional B cells activated by PMAct and IL-4. It was shown that in the presence of PMAct/IL-4/IL-5, a high frequency of resting B cells differentiated to express IgG1. IL-4 alone transiently induced the expression of germ-line gamma 1 transcripts. PMAct alone were ineffective at inducing germ-line gamma 1 transcript expression by resting B cells suggesting that Th-B cell contact was an insufficient signal to cause a detectable increase in the steady-state levels of gamma 1 germ-line transcripts. PMAct alone or PMAct/IL-4 did not induce the appearance of transcripts for secreted mu or mature gamma 1. IL-5, in combination with PMAct/IL-4, provided the necessary signal(s) required for the expression of secreted mu and mature gamma 1 transcripts. Therefore, IL-5 appeared to be an important if not essential differentiation factor for conventional B cells that have been activated by cognate help. It appeared that IL-5 promoted the secretion of Ig by inducing the synthesis of mature Ig transcripts.

T helper cell membranes promote IL-4-independent expression of germ-line C gamma 1 transcripts in B cells.

Studies using plasma membranes from activated Th cell clones (Th membranes) to stimulate B cells have shown that both a contact-mediated activation signal plus Th-derived cytokines are required for antibody production. In order to clearly separate and define the role of these two signals in isotype switching, B cells were stimulated with Th membranes in the presence or absence of cytokines, and the transcriptional activity of the unrearranged H chain loci was determined. In the presence of Th membranes, two known switch factors were shown to specifically induce germ-line transcription of the same H chain loci as in LPS-stimulated B cells (IL-4 induced C gamma 1 and C epsilon transcription, transforming growth factor-beta induced C alpha transcription). The contact-mediated activation signal provided by the Th membranes, in the absence of any added cytokines, resulted in the specific induction of C gamma 1 germ-line transcription, and thus functioned as a switch signal for IgG1. These findings provide a mechanism for previously observed IL-4-independent isotype switching to IgG1.

Heart allografts in murine systems. The differential activation of Th2-like effector cells in peripheral tolerance.

Activated CD4+ Th2 cells release cytokines (IL-4,-10) that block activation & cytokine (IL-2/IFN-gamma) release by proinflammatory T (CD4+,CD8+) effector cells. To test the hypothesis that peripheral tolerance to alloantigen is linked to differential activation of CD4+ Th2 cells we measured cytokine transcripts in heart grafts (C57BL/10----C3H/HeJ) and spleens of mice rendered "tolerant" by donor-specific blood transfusion, anti-CD4 mAb pretreatment, and cyclosporine administration. The expression of IL-2/IFN-gamma transcripts was reduced greater than 90% in grafts from tolerant recipients. IL-4/IL-10 transcripts were generally enhanced and persisted in graft and recipient spleen. Accordingly adoptive transfer studies were performed to determine whether Th2-like effectors, which express Fc receptors (FcR), mediate suppression in this model. Unfractionated mononuclear cells (MC) (5 x 10(6), isolated from spleens of heart graft recipients made tolerant by DST, prolonged the survival of test grafts greater than 90 days in irradiated (680 rads) recipients reconstituted with a sufficient number of MC from spleens of naive C3H to precipitate rejection of the test graft in 18.2 days (MST, n = 5). Conversely adoptive transfer of inocula depleted of FcR+ cells on immune complex columns or with anti-FcR mAb 24G2 caused test grafts to be rejected in 8-11 days. These results suggest that peripheral tolerance to alloantigen may be linked to differential activation of Th2 cells that induce anergy by suppression. The possibility that Th2-like effectors mediate peripheral tolerance to self is discussed.

Pathogenic autoantibody-inducing gamma/delta T helper cells from patients with lupus nephritis express unusual T cell receptors.

In previous work, we found that only 59 (15%) of 396 "autoreactive" T cell clones derived from five patients with lupus nephritis had the ability to selectively augment the production of pathogenic anti-DNA autoantibodies and the majority (49/59) of those autoimmune T helper (Th) clones were CD4+. Surprisingly, 7 of those Th clones were CD4-/CD8- and gamma/delta TCR+, capable of augmenting the production of pathogenic anti-DNA autoantibodies up to 125-fold. The gamma/delta Th clones responded in a MHC-nonrestricted manner to some endogenous autoantigen associated with heat shock proteins (HSP60) on the lupus B cells. The gamma/delta TCR genes expressed by 4 of these Th clones were amplified and sequenced here. Three of the 4 Th clones, each from a different lupus patient, expressed a gene from the V gamma 1 subgroup. Moreover, 2 of the Th clones expressed V delta 5, and the others V delta 1 or V delta 3. These TCRs are rarely expressed by peripheral blood gamma/delta T cells of normal adult humans. The predominant gamma/delta T cells in human peripheral blood express V gamma 2 (V gamma 9) and V delta 2 TCR genes, including HSP-responsive T cells. None of the lupus Th clones expressed this combination of TCR genes. In addition, some of these pathogenic autoantibody-inducing Th clones from the lupus patients had limited diversity and few N-nucleotide additions in their gamma/delta TCR junctional regions (CDR3), thus resembling fetal gamma/delta thymocytes early in ontogeny.

Soluble interleukin 2 receptor release, interleukin 2 production, and interleukin 2 receptor expression in activated T-lymphocytes in vitro.

Following activation in vitro, peripheral blood mononuclear cells (PBMC) express cell-associated interleukin-2 receptors (IL2R). The present study was undertaken to define the proportion of T lymphocyte subsets that express the IL2R (CD25 antigen) upon different mitogenic stimulation. Double immunofluorescence staining with different fluorochromes, fluorescein isothiocyanate and phycoethyrin, was applied for identification of IL2R positive cells and individual lymphocyte subset. The exact percentage of individual activated lymphocyte subset bearing IL2R was enumerated by photographic counting. There was paucity of IL2R in freshly isolated, unstimulated peripheral blood, PBMC cultured without mitogen, and cultured B lymphocytes. Following pokeweed mitogen stimulation in vitro, 19% of CD4 (T-helper/inducer) lymphocytes and 14% of CD8 (T-suppressor/cytotoxic) lymphocytes expressed IL2R. Similarly, 25% of CD4 lymphocytes and 19% of CD8 lymphocytes expressed IL2R following phytohemagglutinin stimulation in vitro. Contrary to the reported data of Tac-positive cells in human lymphoid tissues, our study revealed that, upon lectin mitogen stimulation, approximately 55% of IL2R positive PBMC were CD4 lymphocytes, and 45% of them were CD8 lymphocytes. These observations imply the plausible notion that interleukin-2 mediated immune activation of T lymphocytes in PBMC is different from that in local lymphoid organs. It was also demonstrated that the release of soluble IL2R (sIL2R) and IL2 production in supernatant from cultured PBMC varied with different lectin stimulation. A significant correlation was demonstrated between the cellular and soluble IL2R but the production of IL2 from activated mononuclear cells bore no good correlation with either the cellular IL2R expression or the release of sIL2R.

Study of the separate and combined effects of the non-planar 2,5,2',5'- and the planar 3,4,3',4'-tetrachlorobiphenyl in liver and lymphocytes in vivo.

Polychlorinated biphenyls (PCBs) are a group of industrial chemicals that are widely distributed in the environment. Because these compounds occur as mixtures, studies of their possible interactive effects are essential for an understanding of the mechanism of the toxicity of these mixtures. For the determination of a possible interaction of the effects in vivo of 2,5,2',5'-tetrachlorobiphenyl (TCB) and 3,4,3',4'-TCB, rats were exposed to a single dose of diethylnitrosamine (DEN) and subsequently to 0.1 p.p.m. 3,4,3',4'-TCB and/or 10 p.p.m. 2,5,2',5'-TCB in the feed for 1 year. The two major targets of PCB toxicity, the liver and the peripheral blood, were examined after these treatments. TCB treatment after DEN exposure caused a predominance of increased placental glutathione S-transferase (PGST) and deficiencies of ATPase as preneoplastic markers in focal hepatic lesions. When 0.05% phenobarbital (PB) was administered after DEN exposure, the distribution of markers in altered hepatic foci (AHF) was essentially equal for increased PGST and gamma-glutamyltranspeptidase (GGT) and for ATPase deficiency. Many of these AHF also exhibited increased P450 b/e expression. Our results demonstrated that the two PCB congeners interacted in vivo to produce an increase in AHF that were PGST positive and ATPase negative. PGST-positive and ATPase-negative AHF correlated best with focal areas of P450 b/e expression. The combination of the two PCBs caused a greater than additive decrease in the total number of lymphocytes and antibody-producing B-cells. Also the thymocyte-dependent T-helper cells isolated from the animals receiving the combination of TCBs demonstrated a morphologically abnormal subpopulation. The results indicate that the interaction of 2,5,2',5'-TCB and 3,4,3',4'-TCB in vivo induced much greater toxicity and mutagenicity in peripheral lymphocytes and hepatocytes than treatment with either congener alone.

Omental dendritic cells: Ia expression and relation to macrophages.

Rat omental dendritic cells (ODC) occur in two forms, mainly spindle-shaped, Ia-, on the surface, and stellar, Ia+ within the omentum. Like macrophages, most ODC label with W3/25 mAb and have a demonstrable activity of non-specific esterase, acid phosphatase and ATPase. Up to 30% of ODC are actively phagocytic. The proportion of Ia+ ODC rises 7 days after ip antigenic stimulation and there seems to be a reciprocal development of Ia positivity and phagocytic capacity between ODC and macrophages 3 and 5 days after ip immunization. ODC contribute to the overall fluctuation of Ia expression in the entire omentum after immunization. Ia+ ODC display a linear arrangement along preilymphatic spaces and may be related to the formation of new lymph vessels. They also constitute the stroma of pseudofollicles containing clusters of what are presumably T-helper lymphocytes closely attached to ODC, scattered suppressor T lymphocytes and B cells accumulating in later stages at the immune response.

Adoptive transfer of diabetes in BB rats induced by CD4 T lymphocytes.

Unseparated splenocytes (SPCs) or purified SPC subsets from diabetes-prone BB (BBdp) or diabetic BB (BBd) rats were activated in vitro with either phorbol myristate acetate (PMA) and ionomycin (I) or concanavalin A (ConA). Such activated SPCs were then injected intravenously into 30-day-old BBdp rats, and their capacity to induce adoptive transfer (AT) of diabetes was studied. The proliferative response in vitro of BBd unseparated SPCs or purified W3/13+ SPCs (i.e., T lymphocytes + large granular lymphocytes) to PMA + I far exceeded that of ConA, resulting in mean stimulation indices of 68 and 112 (PMA + I) and 1.9 and 30 (ConA). The incidence of AT was similar when equal numbers of unseparated SPCs from the same BBd donor were injected after activation by either PMA + I + interleukin 2 (PII) or ConA (57 vs. 50%, respectively); however, injection of PII-activated and macrophage-depleted W3/13+ SPCs from BBd animals resulted in a significantly higher incidence of AT (90%, P less than 0.05). As few as 0.5 x 10(6) PII-activated W3/13+ SPCs were sufficient to induce AT. Sixteen percent of recipients developed diabetes after injection of activated W3/13+ cells from 40-day-old BBdp donors. To determine which W3/13+ cells might mediate such transfer, purified and PII-preactivated CD4 T lymphocytes from BBd rats were injected, and they succeeded in AT in 44% of the recipients. Preactivated BBd B lymphocytes were unable to induce AT. Although a possible role for large granular lymphocytes cannot be excluded, the results demonstrate that in the BB rat, the beta-cell destruction can be induced by CD4 T lymphocytes.

[Mycosis fungoides--report of a case].

A 61-year-old female suffered from scaly, violaceous, reddish patches and plaques on the face, trunk and upper arms for two years. Histopathology showed that atypical lymphocytes had infiltrated the epidermis and dermis. Epidermotropism was noted but Pautrier microabscesses was not identified. The electron microscopy revealed the atypical lymphocytes had convoluted contour and peripheral hyperchromatic nuclei. By immunohistochemical study, the labelling of OKT4, OKT8 and OKT11 was positive for the cells, which appeared in the majority of helper T-cells, infiltrating the dermis. The diagnosis was mycosis fungoides, plaque stage. The patient received UVA therapy for 3 months but discontinued due to a worsening of her physical condition.

Mechanism in 1,25(OH)2D3-induced suppression of helper/suppressor function of CD4/CD8 cells to immunoglobulin production in B cells.

On the basis of previous data that 1,25(OH)2D3 suppressed both helper and suppressor activities of CD4 and CD8 cells in the pokeweek mitogen-stimulated culture, we examined the further effect of 1,25(OH)2D3 on both cells to define how 1,25(OH)2D3 is involved in the deterioration of their functions. 1,25(OH)2D3 suppressed the pokeweed mitogen and phytohemagglutinin-induced DNA synthesis of CD4 and CD8 cells. The suppression by 1,25(OH)2D3 of DNA synthesis was caused by a time lag in reaching maximal response. 1,25(OH)2D3 also suppressed interleukin-2 production of CD4 and CD8 cells. 1,25(OH)2D3 did not, however, affect their interleukin-2 receptor expression detected within 24 hr after phytohemagglutinin stimulation. In addition, 1,25(OH)2D3 failed to suppress DNA synthesis of CD4 and CD8 cells when cultured with a large amount of interleukin-2. Suppression by 1,25(OH)2D3 of proliferation and interleukin-2 production in CD4 and CD8 cells would bring about the decrease of their helper or suppressor functions by inhibiting their expansion or maturation.