Immunosuppressive treatment for kidney transplantation.

Immunosuppressive treatment minimizes unwanted immune reactivity, but it also leads to complications such as metabolic disorders, cardiovascular diseases and malignant tumours. In this paper we summarise the recent developments in action mechanisms of available immunosuppressive drugs and their usage for renal transplantation. These drugs act at various levels of lymphocytic activation and proliferation, and they may have additive or synergic effects when combined. In the majority of patients, the immunosuppressive protocol includes a calcineurin inhibitor (tacrolimus or cyclosporin), an antimetabolite (mycophenolate mofetil or mycophenolic acid) and a corticosteroid. Most patients also receive induction with monoclonal or polyclonal antilymphocytic antibodies. These immunosuppressive drugs allow a one-year survival of renal allografts in over 90% of cases and an incidence of acute rejection episodes below 15%. In most cases, acute cell-mediated rejection can be reversed with pulse doses of methylprednisolone; less often antilymphocytic antibodies must be applied. Acute humoral rejection can be suppressed with high doses of intravenous immunoglobulines or low doses of cytomegalovirus hyperimmune globuline, in combination with plasmapheresis, to obtain a satisfactory reduction of anti-donor antibodies. This treatment also allows renal transplantation for sensitised recipients, or transplantation against a positive cross match or AB0 incompatibility. Less often, immunoadsorption, alemtuzumab, rituximab or splenectomy are applied. New immunosuppressive drugs and protocols are currently under investigation. Immunosuppressive agents and methods targeting the induction of immune tolerance to the donor organ are especially promising.

Clustering of colonic lamina propria CD4(+) T cells to subepithelial dendritic cell aggregates precedes the development of colitis in a murine adoptive transfer model.

Initial lesions in inflammatory bowel disease induced during the repopulation of immunodeficient RAG1(-/-) mice with immunocompetent CD4(+) T cells have not been previously described. In this transfer colitis model, we followed CD4(+) T cell repopulation in the host by injecting autofluorescent CD4(+) T cells from congenic, enhanced green fluorescent protein (eGFP)-transgenic mice. This allowed the direct, sensitive, and unambiguous histological detection of the repopulation of the intestinal tract, mesenteric lymph nodes, and spleen of the host with donor eGFP(+) CD4(+) T cells. We identified in RAG1(-/-) mice intestinal dendritic cell (DC) aggregates under the basal crypt epithelium at the mucosa/submucosa junction from which F4/80(+) macrophages were excluded. At Days 8 to 11 posttransfer (before colitis was manifest), CD4(+) T cells clustered and proliferated in CD11c(+) DC aggregates. T cell clustering was most pronounced in the cecum where histologically overt colitis became manifest 5 to 10 days later. Junctional DC aggregates were thus prevalent in the triggering phase of the disease. The data suggest that pathogenic T cell responses inducing inflammatory bowel disease are primed or restimulated in situ in junctional CD4(+) T cell/DC aggregates.

Modulatory effects of octreotide on anti-CD3 and dexamethasone-induced apoptosis of murine thymocytes.

In an attempt to elucidate the effects of somatostatin on two crucial processes that regulated T-cell differentiation and selection in thymus in this study, we investigated in vivo and in vitro the effects of octreotide (SMS 201-995) on dynamics of apoptosis, induced by dexamethasone (DEX) or by anti-CD3 monoclonal antibodies (mAb). The data were estimated by analysis of absolute cellularity, DNA fragmentation and maturational stage of thymocytes, detecting the CD4 and/or CD8 and T cell receptor (TCR) expression on thymocytes. The results, obtained by estimation of subdiploid peak of DNA and ladder DNA formation, have shown that SMS given in vivo, may potentiate the early phase of DEX-induced nuclear fragmentation (at 24 h), accelerating simultaneously the elimination of thymic cells with double positive (DP) CD4high CD8high phenotype (expressed both as percentage and absolute number). On the contrary, SMS, given both in vivo and in vitro, down-regulated the late process (at 72 h) of nuclear fragmentation, induced by anti-CD3 mAb, minimizing simultaneously the elimination of DP cells (expressed both as percentage and absolute number). In anti-CD3-treated cultures of thymocytes, SMS retarded also the elimination of immature thymocytes, expressing the TRC alpha/betalow or intermediate phenotype. The data emphasize that octreotide might have important regulatory effect on processes of thymic differentiation and maturation, which are crucial for T cell selection, induction of tolerance and prevention of autoimmune diseases.

Activating immunity in the liver. I. Liver dendritic cells (but not hepatocytes) are potent activators of IFN-gamma release by liver NKT cells.

A prominent subset of the hepatic innate immune system is alpha-galactosylceramide (alphaGalCer)-reactive, (CD4(+) and CD4(-)CD8(-)) CD1d-restricted NKT cells. We investigated in C57BL/6 (B6) mice which hepatic cell type stimulates hepatic NKT cell activation. Surface expression of CD1d but not CD40, CD80, or CD86 costimulator molecules was detected in hepatocytes. Pulsed in vitro or in vivo with alphaGalCer, hepatocytes triggered IL-4 release by liver NKT cells but required exogenous IL-12 to trigger IFN-gamma release by NKT cells. Liver dendritic cells (DC) isolated from nontreated mice showed low surface expression of MHC, CD1d, and CD40, CD80, or CD86 costimulator molecules that were strikingly up-regulated after alphaGalCer injection. Although liver CD11c(+) DC displayed lower CD1d surface expression than hepatocytes, they were potent stimulators of IFN-gamma and IL-4 release by liver NKT when pulsed with alphaGalCer in vitro or in vivo. Liver DC are thus potent stimulators of proinflammatory cytokine release by NKT cells, are activated themselves in the process of NKT cell activation, and express an activated phenotype after the NKT cell population is eliminated following alphaGalCer stimulation.

In situ characterization of genetically targeted (green fluorescent) single cells and their microenvironment in an adoptive host.

Stable expression of transgene-encoded enhanced green fluorescence protein (eGFP) was used as a sensitive and specific marker to detect in situ donor cells engrafted into different tissues of adoptive hosts. eGFP(+) lymphoid or myeloid cells (eg, CD4(+) T cells or bone marrow-derived dendritic cells) from eGFP-transgenic C57BL/6 donor mice were injected into congenic, immunodeficient RAG1(-/-) C57/BL6 hosts. eGFP(+) cells were detected in the adoptive host from 2 days to 4 weeks after transfer using an optimized method of fixed cryopreservation to process the tissue. This allowed the simple, sensitive, and specific detection of eGFP(+) donor cells in histological sections of transplanted hosts. We further demonstrate that this technique can be combined with other established labeling methods such as 1) immunofluorescent labeling to characterize the host cells interacting with engrafted cells and to determine the phenotype of the engrafted cells in situ; 2) terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining to detect apoptotic death of engrafted and autochthonous cell populations; and 3) fluorescent antibody labeling of incorporated bromodeoxyuridine to measure the fraction of proliferating cells in the graft.

MHC-II-independent CD4+ T cells induce colitis in immunodeficient RAG-/- hosts.

CD4(+) alpha beta T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice engrafted into congenic immunodeficient RAG1(-/-) B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4(+) T cells from CD1d(-/-) knockout (KO) B6 donor mice but not those from MHC-I(-/-) (homozygous transgenic mice deficient for beta(2)-microglobulin) KO B6 mice induced a colitis in RAG(-/-) hosts. Abundant numbers of in vivo activated (CD69(high)CD44(high)CD28(high)) NK1(+) and NK1(-) CD4(+) T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-alpha and IFN-gamma but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4(+) T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice. cLP CD4(+) T cell populations from homozygous transgenic mice deficient for beta(2)-microglobulin KO B6 donor mice engrafted into RAG(-/-) hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4(+) T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.

Prognostic significance of DNA ploidy pattern and nucleolar organizer regions (AgNOR) in colorectal carcinoma.

AIM: To investigate the prognostic significance of DNA ploidy and silver stained nucleolar organizer regions (AgNOR), as well as their relation to the histological grade and Dukes' stages of colorectal carcinomas, and the relation of tumor cells proportion in the S-phase and Dukes' stage, histologic grade, DNA ploidy, or AgNOR count. METHODS: DNA flow cytometric analysis and AgNOR were performed on 94 surgically removed colorectal carcinomas. The mean AgNOR count was calculated in 200 tumor cells for each case. Survival rates and tests for significance were evaluated using the log-rank test and Cox regression model. RESULTS: There were no significant correlations between the ploidy pattern, histological grade, and Dukes' stage. Diploid tumors had a significantly lower AgNOR count (median 2.5, range 2.1-7.7) than aneuploid (median 6.2, range 2.0-7.9). Dukes' C stage tumors exhibited higher AgNOR count than Dukes' A or B stages. The proportion of tumor cells in S-phase did not correlate with any other parameter. Each of these parameters failed to show any correlation with survival. After dividing the tumors into those with high (>5) and low AgNOR count (<5), no correlation was found in the latter group between AgNOR and any other studied parameters, whereas in the group with high AgNOR count correlations to Dukes' stage, DNA ploidy, and histological grade were established.Conclusions. The difference in survival between well, moderately, and poorly differentiated tumors were significant in the group with high AgNOR counts. Dukes' C stage and aneuploid tumors had the worst prognosis.

On the role of T lymphocytes in stimulation of humoral immunity induced by peptidoglycan-monomer linked with zinc.

Effects of peptidoglycan linked with zinc (PGM-Zn) were investigated on plaque-forming cell (PFC) generation to sheep red blood cell (SRBC) and SRBC-unrelated antibody production in primary and secondary immune response in mice depleted in vivo of CD4+ and/or CD8+ T lymphocytes. PGM-Zn in nondepleted mice stimulated the PFC generation and IgM or IgG and IgG1 production in primary and secondary reaction. Single depletion of CD4 or CD8+ T cells did not change this ability. The effects of PGM-Zn after CD8+ depletion were even greater than those in nondepleted mice. Depletion of both T cell subsets, however, completely abrogated immunostimulatory effects of PGM on PFC generation (primary and secondary response), as well as on primary SRBC-unrelated antibody production, leaving only the increase of IgG in secondary response unchanged. Immunostimulatory effects and isotype switching to IgG1 and IgG2a correlated with the changes in splenic CD4+, CD8+, CD5+ cells, pointing to the regulatory role of these cells and/or their cytokines in PGM-Zn-induced immunostimulation. Altogether the data suggest that PGM-Zn may potentiate the costimulatory signals coming from activated T cells and act on B cells without the T cell help.

Treatment of medial and lateral epicondylitis--tennis and golfer's elbow--with low level laser therapy: a multicenter double blind, placebo-controlled clinical study on 324 patients.

BACKGROUND AND OBJECTIVE: Among the other treatment modalities of medial and lateral epicondylitis, low level laser therapy (LLLT) has been promoted as a highly successful method. The aim of this clinical study was to assess the efficacy of LLLT using trigger points (TPs) and scanner application techniques under placebo-controlled conditions. STUDY DESIGN/MATERIAL AND METHODS: The current clinical study was completed at two Laser Centers (Locarno, Switzerland and Opatija, Croatia) as a double-blind, placebo controlled, crossover clinical study. The patient population (n = 324), with either medial epicondylitis (Golfer's elbow; n = 50) or lateral epicondylitis (Tennis elbow; n = 274), was recruited. Unilateral cases of either type of epicondylitis (n = 283) were randomly allocated to one of three treatment groups according to the LLLT technique applied: (1) Trigger points; (2) Scanner; (3) Combination Treatment (i.e., TPs and scanner technique). Bilateral cases of either type of epicondylitis (n = 41) were subject to crossover, placebo-controlled conditions. Laser devices used to perform these treatments were infrared (IR) diode laser (GaAlAs) 830 nm continuous wave for treatment of TPs and HeNe 632.8 nm combined with IR diode laser 904 nm, pulsed wave for scanner technique. Energy doses were equally controlled and measured in Joules/cm2 either during TPs or scanner technique sessions in all groups of patients. The treatment outcome (pain relief and functional ability) was observed and measured according to the following methods: (1) short form of McGill's Pain Questionnaire (SF-MPQ); (2) visual analogue scales (VAS); (3) verbal rating scales (VRS); (4) patient's pain diary; and (5) hand dynamometer. RESULTS: Total relief of the pain with consequently improved functional ability was achieved in 82% of acute and 66% of chronic cases, all of which were treated by combination of TPs and scanner technique. CONCLUSIONS: This clinical study has demonstrated that the best results are obtained using combination treatment (i.e., TPs and scanner technique). Good results are obtained from adequate treatment technique correctly applied, individual energy doses, adequate medical education, clinical experience, and correct approach of laser therapists. We observed that under- and overirradiation dosage can result in the absence of positive therapy effects or even opposite, negative (e.g., inhibitory) effects. The current clinical study provides further evidence of the efficacy of LLLT in the management of lateral and medial epicondylitis.

Tissue zinc dynamics during the immune reaction in mice.

Owing to the importance of zinc for the functioning of the immune system, the role of endogenous Zn, located both in lymphoid and nonlymphoid organs, was investigated during the standard humoral and cellular types of immune response. For this purpose, the dynamics of hepatic, thymic, splenic, and renal Zn content was determined in mice sensitized with (a) sheep red blood cells and (b) semiallogeneic lymphocytes during the local host vs graft reaction (HVGR). The data obtained by ion-coupled plasma spectrometry revealed that the humoral type of immunity is characterized by a significant increase of Zn concentration in the liver and in the thymus. Simultaneously, linear regression analysis showed that the generation of plaque-forming cells in the individual mouse was highly positively correlated with Zn concentration in the liver (r=0.897), and spleen (r=0.833), and negatively with Zn concentration in the thymus (r=-0.624). Similar relationships between the intensity of local immune reaction and tissue Zn levels were found in local HVGR at the fifth day in the liver and spleen (r=0.861 and r=0.695, respectively), at the seventh day in the thymus (r=-0.797), and at the tenth day in the liver (r=-0.859). The data emphasize the necessity of Zn for the development of normal immune response and point to the existence of a Zn-dependent hepato-thymic axis during the humoral and cellular types of immune reactivity.

IL-13 supports differentiation of dendritic cells from circulating precursors in concert with GM-CSF.

Dendritic cells (DC) can be obtained from peripheral blood mononuclear cells (PBMC) by in vitro culture with IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF). IL-13 shares properties with IL-4 but its receptor does not involve the common gamma chain present in the receptor complex of IL-4 and other cytokines. The present study was aimed to elucidate whether IL-13 can substitute for IL-4 in DC cultures and to compare the phenotypic and functional characteristics of cells obtained using these two cytokines. Monocyte-enriched PBMC were cultured with GM-CSF and IL-4 or GM-CSF and IL-13. Cell yields and DClike morphology were similar. The cells showed a membrane phenotype typical of DC (MHC II+; CD1a+; CD14-; CD3-; CD20-). IL-13-derived and IL-4-derived DC were similar in terms of macropinocytosis, stimulatory capacity of cord blood lymphocytes in mixed leukocyte reaction (MLR), and responsiveness to chemotactic signals. It is concluded that IL-13 is as effective as IL-4, combined with GM-CSF in sustaining DC differentiation from PBMC and that activation of the common gamma chain-driven transduction pathways is dispensable for DC differentiation and function.

Immunosuppressive and antiproliferative effects of somatostatin analog SMS 201-995.

The effects of long acting somatostatin analog SMS 201-995 were examined in vivo on: 1) lymphoid morphostasis and functional reactivity of cells obtained from SMS treated donors, 2) on humoral, and 3) cellular type of immunity; and in vitro on: 1) blastic transformation of lymphocytes stimulated by activators of different transmembrane pathways (CD2 by PHA and CD3/TCR by anti-CD3 monoclonal antibody and by allogeneic cells) and 2) on growth and secretory activity of several hybridoma cell lines. The data have shown that SMS in vivo decreases the proportion of CD4+, CD5+ and Ig+ cells in spleen. The reactivity of these cells to Con A was suppressed, but their spontaneous blastic transformation was increased. SMS suppressed also the plaque forming cells generation and proliferation of cells in popliteal lymph nodes during the local host versus graft reaction. The former immunosuppression was abrogated with the use of growth hormone, while in the latter, the time dependent changes in spleen composition were also noticed. The data obtained in vitro revealed that SMS may inhibit only the CD2-induced blastogenesis (in early and late interval after the use of PHA). SMS inhibited also the spontaneous growth and/or secretion of antibodies in some hybridoma cell lines.

Immunoregulating effects of peptidoglycan monomer linked with zinc in adult mice.

Since it is well known that both zinc ions and bacterial immunostimulants influence the function of the immune system, in the present study we investigated the immunomodulating activity of a new analog of peptidoglycan monomer (PGM), in which the basic molecule was linked to zinc (PGM-Zn). Its effects in BALB/c mice, aged 10-12 months, were compared with the effects of equimolar doses of PGM and ZnCl2. The treatment lasted 26 days (one i.p. injection every fifth day). The results showed that PGM-Zn may markedly enhance antibody production to sheep red blood cells, as well as spontaneous and concanavalin A (ConA)-induced blastogenesis. The generation of plaque-forming cells in individual mouse was positively correlated with the expression of class II antigens in the liver and negatively correlated with the total quantity of hepatic proteins. PGM-Zn also induced the appearance of peritoneal macrophages, which in cocultures with syngeneic splenocytes were less able to enhance the spontaneous, and particularly the ConA-induced blastic transformation. The enhancing activities of PGM-Zn were in some respects more closely correlated with the action of PGM, whereas the induction of suppressive macrophages resembled more the activity of ZnCl2. The data emphasize that PGM-Zn may both stimulate and inhibit immunoregulative pathways by mechanisms which are not identical to those of PGM or ZnCl2.