Influence of an allogenic collagen scaffold on implant sites with thin supracrestal tissue height: a randomized clinical trial.

OBJECTIVES: This randomized clinical trial focused on patients with thin peri-implant soft-tissue height (STH) (≤ 2.5 mm) and investigated the impact of an allogenic collagen scaffold (aCS) on supracrestal tissue height and marginal bone loss (MBL). MATERIAL & METHODS: Forty patients received bone level implants and were randomly assigned to the test group with simultaneous tissue thickening with aCS or the control group. After three months, prosthetic restoration occurred. STH measurements were taken at baseline (T0) and reopening surgery (TR), with MBL assessed at 12 months (T1). Descriptive statistics were calculated for continuous variables, and counts for categorical variables (significance level, p = 0.05). RESULTS: At T1, 37 patients were available. At T0, control and test groups had mean STH values of 2.3 ± 0.3 mm and 2.1 ± 0.4 mm. TR revealed mean STH values of 2.3 ± 0.2 mm (control) and 2.6 ± 0.7 mm (test), with a significant tissue thickening of 0.5 ± 0.6 mm in the test group (p < 0.03). At T1, control and test groups showed MBL mean values of 1.1 ± 0.8 mm and 1.0 ± 0.6 mm, with a moderate but significant correlation with STH thickening (-0.34), implant position (0.43), history of periodontitis (0.39), and smoking status (0.27). CONCLUSION: The use of an aCS protocol resulted in soft tissue thickening but did not reach a threshold to reliably reduce MBL compared to the control group within the study's limitations. CLINICAL RELEVANCE: Peri-implant STH is crucial for maintaining peri-implant marginal bone stability. Marginal bone stability represents a crucial factor in prevention of peri-implantitis development. German register of clinical trial registration number DRKS00033290.

Rapid detection, enrichment and propagation of specific T cell subsets based on cytokine secretion.

T cell lines with defined cytokine profiles are an invaluable tool for assessing the control of immune responses both in vitro and in vivo. Production of such cell lines can be complex and time-consuming. Here we present a powerful technique to assay the cytokines produced by T cells activated polyclonally or with specific antigens. This paper presents a detailed methodology for the identification and isolation of cytokine-producing T cells activated with the artificial superantigen, CytoStim, or viral and fungal antigens. These cells can be analysed for different cytokines simultaneously, or cultured further to rapidly establish T cell lines making known cytokine types. We highlight the enumeration, isolation and phenotype of interleukin-17-producing T cells, and the rapid generation of virus-specific Th1 T cell lines.

Adoptive tumor therapy with T lymphocytes enriched through an IFN-gamma capture assay.

Successful adoptive T-cell therapy has been demonstrated in viral disease and selected forms of cancer. However, it is limited by the difficulty to efficiently isolate and amplify autologous tumor-reactive T-cell clones. Tetramers of major histocompatibility complex (MHC) class I and peptide have facilitated the characterization of CD8+ T cells specific for tumor-associated antigens. However, for adoptive T-cell therapy, MHC-tetramers have limitations: they require knowledge of tumor antigens, which is often not available; they select T cells with a single specificity, thereby posing risk for selection of tumor escape variants; they do not select for function, so that T cells may be anergic when isolated from cancer patients; and they do not allow the isolation of CD4+ T cells that can be essential for tumor rejection. Because interferon (IFN)-gamma is essential for tumor rejection, we isolated live T cells based on their IFN-gamma production. IFN-gamma secreted by previously activated T cells is retained on the cell surface, allowing their specific isolation and expansion. We show here that IFN-gamma+ but not IFN-gamma- T cells from tumor-immunized mice are cytolytic and mediate tumor rejection upon adoptive transfer. Importantly, tumor-specific T cells can be enriched from lymphocytes infiltrating human renal cell carcinoma by the IFN-gamma capture assay.

Induction of interleukin 4 (IL-4) expression in T helper (Th) cells is not dependent on IL-4 from non-Th cells.

Interleukin 4 (IL-4) is essential for the induction of immunoglobulin E (IgE) responses in mice. Recent in vitro studies have suggested that IL-4 derived from non T helper (Th) cells, in particular from mast cells and basophils, may be essential for triggering of IL-4 expression in Th cells and may directly contribute to IgE isotype switch induction. Here, we have generated mice carrying a functional IL-4 gene only in Th cells or non-Th cells, respectively, by reconstitution of IL-4-deficient mice (IL-4T mice) with CD4+ or CD4- spleen cells from congenic wild-type animals. In mice in which only CD4+ cells are able to express IL-4, antigen-specific IgE is produced in a T cell-dependent immune response. Thus, induction of IL-4 expression in Th cells can occur in the absence of IL-4 from non-Th cells, which suggests that at least some Th cells can express IL-4 in response to another signal which has yet to be identified. No IgE is detectable, however, in mice in which only CD4- cells can express IL-4, suggesting that Th cells are the primary, if not the only source of IL-4 for initial induction of IgE synthesis.

An instructive component in T helper cell type 2 (Th2) development mediated by GATA-3.

Although interleukin (IL)-12 and IL-4 polarize naive CD4(+) T cells toward T helper cell type 1 (Th1) or Th2 phenotypes, it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate. To distinguish these instructive and selective models, we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral-based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels. We observe IL-4-dependent redirection of phenotype in cells that have already committed to a non-IL-4-producing fate, inconsistent with predictions of the selective model. Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate. These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.

Towards a commercial process for the manufacture of genetically modified T cells for therapy.

The recent successes of adoptive T-cell immunotherapy for the treatment of hematologic malignancies have highlighted the need for manufacturing processes that are robust and scalable for product commercialization. Here we review some of the more outstanding issues surrounding commercial scale manufacturing of personalized-adoptive T-cell medicinal products. These include closed system operations, improving process robustness and simplifying work flows, reducing labor intensity by implementing process automation, scalability and cost, as well as appropriate testing and tracking of products, all while maintaining strict adherence to Current Good Manufacturing Practices and regulatory guidelines. A decentralized manufacturing model is proposed, where in the future patients' cells could be processed at the point-of-care in the hospital.

Rapid enrichment and detection of melanoma cells from peripheral blood mononuclear cells by a new assay combining immunomagnetic cell sorting and immunocytochemical staining.

Commonly used methods for detection of melanoma cells in blood, including RT-PCR and immunocytochemistry, display only a limited sensitivity and specificity. Reliable detection of less than one melanoma cell per ml of blood is hardly possible using these methods. To obtain greater sensitivity so that a single melanoma cell in up to 25 ml of blood can be detected (5 x 10(7) peripheral blood mononuclear cells, or PBMC), we developed a new assay for combined enrichment and immunocytochemical detection of disseminated melanoma cells from PBMC of patients with malignant melanomas. Melanoma cells are directly magnetically labeled using colloidal superparamagnetic microparticles approximately 60 nm in diameter conjugated to the anti-melanoma monoclonal antibody 9.2.27, with no reactivity to normal cells in blood. Magnetically labeled melanoma cells are enriched from PBMC by magnetic cell separation and detected by a new approach for immunocytochemical staining with monoclonal mouse anti-melanoma antibodies (anti-MelanA and HMB-45). The efficiency of this assay was demonstrated in a model system in which 5-500 tumor cells from the melanoma cell line SK-MEL-28 were seeded into PBMC samples from healthy donors containing 5 x 10(7) leukocytes. Mean recovery of the seeded tumor cells was 47.4 +/- 13.99% (n = 15). Applying the assay to 20-50 ml blood samples of patients with stage III-IV malignant melanomas, we were able to detect melanoma cells in two of eight patients (25%).

Analysis and sorting of T cells according to cytokine expression.

Functional distinct populations of T helper cells can be defined according to the expression of cytokines. A remarkable diversity of cytokine expression has been demonstrated in single T cells even from clonal populations and up to now no stable surface markers have been described which on the single-cell level directly correlate with the secretion of a certain cytokine. Since cytokines are the major parameters of T cell effector function we have developed strategies which now allow to separate cells according to the specific cytokines they secrete. The "affinity matrix technology"--secreted molecules are relocated to the cell surface by an artificially created antibody matrix--allows to isolate cells according to a distinct secreted product. In addition to this universal, but laborious technology, we could demonstrate by high-sensitivity immunofluorescence that IFN-gamma and IL-10 but not IL-2 and IL-4 are specifically expressed in low copy number on the surface of cells secreting these cytokines. Surface IFN-gamma and IL-10 are the first unambiguous surface markers for pro-inflammatory IFN-gamma-secreting Th 0/1 cells and IL-10-producing anti-inflammatory Th2/3 cells. We have used purified cytokine-secreting T cells to study in individual T cells the sequential production of IL-2, IFN-gamma, and IL-10, the stability of IFN-gamma expression and the selective homing of IFN-gamma-producing cells into inflamed tissues.

Surgery in patients with coronary artery disease--silent ischaemia during transurethral resection of tumors of prostate or bladder.

BACKGROUND: Asymptomatic episodes of myocardial ischemia in clinically stable patients seem to occur frequently and may hint at a worse prognosis. HYPOTHESIS: This study was undertaken to determine whether surgical patients with coronary artery disease (CAD) have a higher risk of cardiac ischemia during the perioperative period compared with the late postoperative period and compared with patients without CAD. METHODS: In all, 14 patients with and 14 patients without CAD were examined by Holter monitoring during the perioperative and three days later during the postoperative periods for the presence of ST-segment depression as a marker of silent myocardial ischemia. RESULTS: While patients without CAD did not show ST-segment depression, patients with CAD were found to have had 143 episodes of ST-segment depression, 49% in the perioperative and 51% in postoperative recordings. CONCLUSION: Though patients were asymptomatic with antianginal therapy, there were episodes of ST-segment depression indicating silent myocardial ischemia in patients with CAD. Surgical interventions such as transurethral resection of tumors of prostate or bladder did not produce an increase of ischemic burden registered by Holter monitoring.

Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi.

Foxp3(+) regulatory T cells (Treg) have a central role for keeping the balance between pro- and anti-inflammatory immune responses against chronically encountered antigens at mucosal sites. However, their antigen specificity especially in humans is largely unknown. Here we used a sensitive enrichment technology for antigen-reactive T cells to directly compare the conventional vs. regulatory CD4(+) T-cell response directed against two ubiquitous mucosal fungi, Aspergillus fumigatus and Candida albicans. In healthy humans, fungus-specific CD4(+)CD25(+)CD127(-)Foxp3(+) Treg are strongly expanded in peripheral blood and possess phenotypic, epigenetic and functional features of thymus-derived Treg. Intriguingly, for A. fumigatus, the strong Treg response contrasts with minimal conventional T-cell memory, indicating selective Treg expansion as an effective mechanism to prevent inappropriate immune activation in healthy individuals. By contrast, in subjects with A. fumigatus allergies, specific Th2 cells were strongly expanded despite the presence of specific Treg. Taken together, we demonstrate a largely expanded Treg population specific for mucosal fungi as part of the physiological human T-cell repertoire and identify a unique capacity of A. fumigatus to selectively generate Treg responses as a potentially important mechanism for the prevention of allergic reactions.

Flow cytometric determination of cytokines in activated murine T helper lymphocytes: expression of interleukin-10 in interferon-gamma and in interleukin-4-expressing cells.

In an immune response, effector functions are controlled by T helper (Th) 1 cytokines [interferon-gamma (IFN-gamma), interleukin (IL)-2 and tumor necrosis factor-beta] and Th2 cytokines (IL-4, IL-5 and IL-10). Here we analyze by multiparameter immunofluorescence to what extent IL-2, IL-4, IL-5, IL-10 and IFN-gamma are co-expressed in individual normal murine Th cells upon activation in vitro with the bacterial superantigen Staphylococcus aureus enterotoxin B, presented in the context of major histocompatibility complex class II. IL-2 and IFN-gamma are co-expressed by some, but not by other Th cells. Expression of IL-4 and IFN-gamma is exclusive. IL-10 is co-expressed in individual cells either with IL-4 or with IFN-gamma. No IL-5-expressing cells are detected. While IL-10- and IL-4-co-expressing Th cells correspond to classical Th 2 cells, cells co-expressing IL-10 and IFN-gamma could be involved in negative-feedback regulation of a Th1 response. Apart from such functional implications, our results show that IL-2, IL-4, IL-5, IL-10 and IFN-gamma are expressed independently of each other in individual murine Th cells.

Regulation of T helper cell cytokine expression: functional dichotomy of antigen-presenting cells.

A bias to either cell-mediated or antibody-mediated effector mechanisms is induced in an immune response against a pathogen, if activated T helper cells (Th) predominantly express Th1 [interleukin (IL)-2, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-beta] or Th2 (IL-4, IL-5, IL-6 and IL-10) cytokines. Here we provide evidence that, due to the capability to secrete IL-1, macrophages, but not B cells, as antigen-presenting cells (APC) induce production of IFN-gamma in resting Th cells. Normal murine splenic Th cells were activated in vitro with the superantigen Staphylococcus aureus enterotoxin B (SEB) presented by macrophages as compared to other APC from murine spleen. As determined by immunofluorescence, Th cells producing IL-2 but almost none producing IL-4 and IL-5 are generated, irrespective of the type of APC. Generation of IFN-gamma-producing Th cells is largely dependent on presentation of SEB by macrophages. The requirement for macrophages, however, is overcome if IL-1 is provided. Expression of IFN-gamma by Th cells is not induced, if production of IL-1 by macrophages is inhibited by IL-10. Our results suggest a functional dichotomy of APC: normal resting Th cells differentiate into IL-2 and IFN-gamma secreting cells (Th1 cells) if antigen is presented by macrophages, whereas presentation by B cells generates Th cells secreting IL-2, which might differentiate into Th2 cells upon re-stimulation.

Analysis and sorting of live cells according to secreted molecules, relocated to a cell-surface affinity matrix.

We have developed a technology for analysis and sorting of live cells according to secreted molecules. An artificial affinity matrix, specific for the secreted product of interest, is created on the cell surface, and the cells are allowed to secrete for a defined time period. The secreted molecules bind to the affinity matrix on the secreting cell and are subsequently labeled with specific fluorescent or magnetic staining reagents for cytometric analysis and cell sorting. Crossfeeding of the secreted products to other cells is prevented by decreasing the permeability of the incubation medium. This approach will have a wide range of applications in biotechnology and biomedical research. Here, we describe analysis and sorting of hybridoma cells, according to secreted antibodies, and of activated T lymphocytes, according to secreted cytokines.

Specific expression of surface interferon-gamma on interferon-gamma producing T cells from mouse and man.

Interferon (IFN)-gamma is a potent immunoregulatory protein secreted by CD4+ and CD8+ T cells and by natural killer cells. Here, we show that IFN-gamma is specifically displayed at a low concentration on the cell surface of those activated T cells from mouse and man which express IFN-gamma. It is transiently expressed on the cell surface with kinetics similar to those of intracellular IFN-gamma expression. Detectable surface IFN-gamma is not expressed by activated T helper (Th) cells producing other cytokines but which do not express IFN-gamma. Thus, surface IFN-gamma is the first available marker for live T lymphocytes expressing IFN-gamma, e.g. Th1 cells.

Commitment of individual Th1-like lymphocytes to expression of IFN-gamma versus IL-4 and IL-10: selective induction of IL-10 by sequential stimulation of naive Th cells with IL-12 and IL-4.

Commitment of Th lymphocytes to the Th1 phenotype, as characterized by the expression of the major proinflammatory cytokine IFN-gamma, may be critically involved in the establishment of chronic inflammation and inflammatory autoimmune disease. To date, it has been shown that in IL-12-stimulated murine Th cell lines containing a major fraction of Th1 cells, Th2 cells can be induced by IL-4 until about 2 wk after initial activation, but not later. Here we analyze, based on the magnetic isolation of viable Th1 cells according to their specific expression of IFN-gamma, the cytokine commitment of individual Th1 cells. After activation of naive Th cells with Ag and IL-12 for up to 5 wk, isolated IFN-gamma-producing cells were restimulated with Ag and IL-4. Within the first 3 to 4 wk of IL-12 stimulation, some IFN-gamma+ cells stopped expression of IFN-gamma when restimulated with IL-4. However, within only 1 to 2 wk of IL-12 stimulation, few IFN-gamma+ cells could be converted to produce IL-4. Others continued to express IFN-gamma and thus were already committed to a proinflammatory, Th1-like phenotype. Surprisingly, within 3 wk of IL-12 stimulation, many of the IFN-gamma-producing cells responded to IL-4 restimulation by expression of IL-10, but neither IFN-gamma nor IL-4, i.e., by conversion to a suppressive, anti-inflammatory phenotype.

Sequential production of IL-2, IFN-gamma and IL-10 by individual staphylococcal enterotoxin B-activated T helper lymphocytes.

Upon primary activation, T helper (Th) cell populations express different cytokines transiently and with different kinetics. Stimulation of naive murine splenic Th cells with the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) in vitro results in expression of IL-2, IFN-gamma and IL-10 with fast, intermediate and slow kinetics, respectively. This first report of a functional analysis of cells separated alive according to cytokine expression shows that these cytokines are not produced by different Th cell subpopulations, but can be expressed sequentially by individual Th cells. Th cells, activated with SEB for 1 day and isolated according to expression of IL-2, using the cellular affinity matrix technology, upon continued stimulation with SEB later secrete most of the IFN-gamma and IL-10. Likewise, after 2 days of SEB culture, cells expressing IFN-gamma, separated according to specific surface-associated IFN-gamma as detected by magnetofluorescent liposomes, 1 day later secrete IL-10. Thus, individual Th1 cells can contribute to the control of their own IFN-gamma expression by sequential expression of first IL-2, supporting their proliferation, and later IL-10, down-regulating the production of IFN-gamma-inducing monokines and limiting the pro-inflammatory effects of IFN-gamma.

Systemic T-cell unresponsiveness during rush bee-venom immunotherapy.

By rush bee-venom immunotherapy, subjects reacting allergically to the venom can be effectively anergized, although the mechanism of action is not known. Here we analyzed the systemic effects of rush desensitization on the T cells of allergic patients. In most patients, we found reduced frequencies of T cells recalled to express CD69 and the cytokines interleukin (IL)-4 and interferon-gamma (IFN-gamma) after stimulation of peripheral blood mononuclear cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin, as compared with normal donors. These frequencies are progressively reduced during immunotherapy. The frequency of cells expressing IL-2 does not change. A few patients show a different response to immunotherapy: frequencies of cells expressing CD69, IL-4, or IFN-gamma do not change, and remain similar to those of normal donors. However, the frequency of cells able to express IL-2 is increased. The analysis of cytokine expression in CD45RO+ vs CD45RO- T-cell populations revealed differences between normal and allergic donors. In allergic patients, higher frequencies of IL-4- and IFN-gamma-expressing cells among the CD45RO- subpopulation were found than in normal donors. This situation is not modified by immunotherapy. The results reveal a certain degree of heterogeneity in the response of allergic patients to bee-venom rush immunotherapy; however, all are clearly differentiated from normal controls as judged by cytokine expression of CD45RO- T cells. In most allergic patients, a considerable percentage of Th cells become unresponsive to mitogenic stimulation, and may be responsible for the desensitization itself.

Stat6-independent GATA-3 autoactivation directs IL-4-independent Th2 development and commitment.

The initial source of IL-4-inducing Th2 development and the mechanism of stable Th2 commitment remain obscure. We found the reduced level of IL-4 production in Stat6-deficient T cells to be significantly higher than in Th1 controls. Using a novel cell surface affinity matrix technique, we found that IL-4-secreting Stat6-deficient T cells stably expressed GATA-3 and Th2 phenotype. Introducing GATA-3 into Stat6-deficient T cells completely restored Th2 development, inducing c-Maf, Th2-specific DNase I hypersensitive sites in the IL-4 locus, and Th2 cytokine expression. The fact that GATA-3 fully reconstitutes Th2 development in Stat6-deficient T cells indicates it is a master switch in Th2 development. Finally, GATA-3 exerts Stat6-independent autoactivation, creating a feedback pathway stabilizing Th2 commitment.