Effect of immunosuppressive treatment on biomarkers in adult atopic dermatitis patients.

BACKGROUND: Biomarkers to objectively measure disease severity and predict therapeutic responses are needed in atopic dermatitis (AD). OBJECTIVE: Primary aim: To identify biomarkers reflecting therapeutic response in patients with AD treated systemically. Secondary aims: (i) To identify a biomarker pattern predicting responsiveness to systemic treatment. (ii) To identify differences in expression of biomarker in filaggrin gene (FLG) mutation carriers vs. non-FLG mutations carriers. METHODS: Thirty-eight severe AD patients treated with methotrexate or azathioprine participated. Serum levels of a proliferation-inducing ligand, B-cell activating factor of the TNF family, thymus and activation-regulated chemokine (chemokine (C-C motif) ligand 17) (TARC (CCl-17)), interleukin-1 receptor antagonist (IL-1RA), interleukin-1 bèta, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-18, IL-31, interferon gamma, tumour necrosis factor alpha, vascular endothelial growth factor (VEGF), monokine induced by interferon gamma (chemokine (C-X-C motif) ligand 9), interferon gamma-induced protein 10 (C-X-C motif chemokine Ligand 10), monocyte chemoattractant protein-1 (chemokine (C-C Motif) ligand 2), macrophage inflammatory protein-1 beta (chemokine (C-C motif) ligand 4), regulated on activation, normal T cell expressed and secreted (chemokine (C-C motif) ligand 5), Cutaneous T-cell-attracting chemokine (chemokine (C-C motif) ligand 27) (CTACK (CCL-27)), thymic stromal lymphopoietin, IL-5, interleukin-1 alpha and granulocyte-colony stimulating factor were analysed by ELISA and Luminex. The primary outcomes were differences in mean absolute change of SCORing Atopic Dermatitis (SCORAD) between groups after 12 weeks compared with baseline. Responders to treatment were defined by a SCORAD reduction in ≥50%. Buccal mucosa swabs were collected to determine FLG genotype status. RESULTS: Thymus and activation-regulated chemokine, CTACK, IL-13 and VEGF showed a significant decrease after treatment with methotrexate or azathioprine. However, no decrease in individual cytokine levels was significantly correlated with a change in any of the outcome parameters. In addition, baseline biomarker levels were not significantly different between responders and non-responders, and FLG and non-FLG mutants showed similar biomarker profiles. CONCLUSION: Thymus and activation-regulated chemokine and CTACK were confirmed as potential biomarkers. VEGF and IL-13 have a potential value as well. Biomarkers could not be used to discriminate at baseline between responders and non-responders, or FLG genotype status.

Cytokine profiles in interstitial fluid from chronic atopic dermatitis skin.

BACKGROUND: The in vivo levels of inflammatory mediators in chronic atopic dermatitis (AD) skin are not well-defined due to the lack of a non-invasive or minimally invasive sampling technique. OBJECTIVES: To investigate the cytokine milieu in interstitial fluid (ISF) collected from chronic lesional AD skin as compared to ISF from non-lesional AD skin and/or healthy donor skin. METHODS: ISF was obtained using a minimally invasive technique of creating micropores in the skin by a laser, and harvesting ISF through aspiration. We determined the levels of 33 cytokines by Luminex and ELISA in ISF and plasma from sixteen AD patients and twelve healthy individuals. In seven AD patients, we analysed the IL-13, IL-31, IL-17, IL-22 and IFN-γ production by T cells isolated from lesional skin. AD patients were genotyped for the filaggrin gene (FLG)-null mutations 2282del4, R501X, R2447X and S3247X. RESULTS: Twenty-five of 33 examined mediators were detected in the ISF. The levels of IL-1α, IL-1ß, IL-18, IL-1RA, IL-5, IL-13, IL-6, IL-8, TNF-α, RANTES(CCL-5), MIG(CXCL-9), IP-10(CXCL-10), TARC(CCL-17), VEGF and G-CSF showed significant differences between either lesional, non-lesional and/or healthy skin. IP-10 levels in ISF from lesional and non-lesional AD skin showed significant correlation with IP-10 blood levels. IP-10 also showed a significant correlation with clinical severity (SCORAD), as did IL-13. Levels of both IP-10 and IL-13 were more pronounced in patients with FLG-null mutations. Furthermore, FLG-null mutation carriers had more severe AD. CONCLUSION: The presented minimally invasive technique is a valuable tool to determine the in vivo cytokine profile of AD skin.

Downregulation of CD1 marks acquisition of functional maturation of human thymocytes and defines a control point in late stages of human T cell development.

We have investigated whether in the human thymus transition of CD4+CD8+ double positive (DP) to CD4+ or CD8+ single positive (SP) cells is sufficient for generation of functional immunocompetent T cells. Using the capacity of thymocytes to expand in vitro in response to PHA and IL-2 as a criterion for functional maturity, we found that functional maturity of both SP and DP thymocytes correlates with downregulation of CD1a. CD1a- cells with a persistent DP phenotype were also found in neonatal cord blood, suggesting that at least a proportion of mature DP cells can emigrate from the thymus. The requirements for generating functional T cells were investigated in a hybrid human/mouse fetal thymic organ culture. MHC class II-positive, but not MHC class II-negative, mouse thymic microenvironments support differentiation of human progenitors into TCR alpha beta+CD4+ SP cells, indicating that mouse MHC class II can positively select TCR alpha beta +CD4+ SP human cells. Strikingly, these SP are arrested in the CD1a+ stage and could not be expanded in vitro with PHA and IL-2. CD1a+CD4+ SP thymocytes do not represent an end stage population because purified CD1a+CD4+ SP thymocytes differentiate to expandable CD1a- cells upon cocultivation with human thymic stromal cells. Taken together these data indicate that when CD1a+ DP TCR alpha beta low cells mature, these cells interact with MHC, but that an additional, apparently species-specific, signal is required for downregulation of CD1a to generate functional mature TCR alpha beta + cells.

Identification of a committed T cell precursor population in adult human peripheral blood.

Here, we report data concerning the discovery in adult human peripheral blood of a precursor cell population able to differentiate into CD4+CD3+ alpha beta + mature T cells. These cells, which represent 0.1-0.5% of total peripheral blood mononuclear cells (PBMC), express substantial levels of CD4, but lack CD3 surface expression. At a molecular level, they express the pre-T cell receptor alpha (pT alpha) gene, CD3-gamma, CD-delta and CD-epsilon, and RAG-1 recombination enzyme and have initiated rearrangements in the T cell receptor (TCR)-beta locus (D-J). Moreover, low levels of CD3 epsilon protein, but not of TCR-beta chain, can be detected in their cytoplasm. Our results suggest that CD4+CD3- cells identified in peripheral blood are different from CD3-CD4+CD8- thymocytes and may contain precursors of an extrathymic T cell differentiation pathway.

Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3.

Bipotential T/natural killer (NK) progenitor cells are present in the human thymus. Despite their bipotential capacity, these progenitors develop predominantly to T cells in the thymus. The mechanisms controlling this developmental choice are unknown. Here we present evidence that a member(s) of the family of basic helix loop helix (bHLH) transcription factors determines lineage specification of NK/T cell progenitors. The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer. Constitutive expression of Id3 completely blocks development of CD34+ cells into T cells in a fetal thymic organ culture (FTOC). In contrast, development into NK cells in an FTOC is enhanced. Thus, the activity of a bHLH transcription factor is necessary for T lineage differentiation of bipotential precursors, in the absence of which a default pathway leading to NK cell development is chosen. Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.

Selection of a human T helper type 1-like T cell subset by mycobacteria.

Mycobacteria elicit a cellular immune response in their hosts. This response usually leads to protective immunity, but may sometimes be accompanied by immunopathology due to delayed type hypersensitivity (DTH). A striking example in man is tuberculoid leprosy, which is characterized by high cellular immunity to Mycobacterium leprae and immunopathology due to DTH. Skin lesions of patients suffering from this disease have the characteristics of DTH reactions in which macrophages and CD4+ T lymphocytes predominate. In animal models, it has been shown that DTH responses are associated with the presence of a particular subset of CD4+ T cells (T helper type 1 [Th1]) that secrete only certain cytokines, such as interleukin 2 (IL-2), interferon gamma (IFN-gamma), and lymphotoxin, but no IL-4 or IL-5. We studied the cytokine release of activated M. leprae-reactive CD4+ T cell clones derived from tuberculoid leprosy patients. These T cell clones, which were reactive with mycobacterial heat shock proteins, exhibited a Th1-like cytokine secretion pattern with very high levels of IFN-gamma. Half of these clones secreted low levels of IL-4 and IL-5, but the ratio of IFN-gamma to IL-4 and IL-5 was much higher than that of T cell clones reactive with nonmycobacterial antigens. A Th1-like cytokine secretion pattern was also observed for T cell clones and polyclonal T cell lines from control individuals that recognized both heat shock and other mycobacterial antigens. The levels of IFN-gamma secreted by these clones were, however, significantly less than those of patient-derived T cell clones. This Th1-like pattern was not found with T cell clones from the same patients and healthy individuals generated in the same manner, but reactive with nonmycobacterial antigens. Our data thus indicate that mycobacteria selectively induce human T cells with a Th1-like cytokine secretion profile.

Heat shock proteins in immunopathology.

In recent years, studies have suggested that autoimmunity and/or immunopathology may sometimes result from the immune response to heat shock proteins of autologous cells and microorganisms. Focusing on the T-cell mediated responses, we review the latest literature on this issue with regard to three hypothetical concepts of immunopathology in which heat shock proteins might play a role.

T cell precursors in man and mice.

The thymus is seeded at week 7-8 of gestation with hematopoietic progenitor cells derived from the liver. By week 15-16 of gestation a fully differentiated thymus with a cortical/medullary junction and Hassal's corpuscles has been formed. The thymus is continuously populated by progenitor cells first from the liver and then from bone marrow. This process continues in childhood after which the thymus starts to involute. Recent information indicates that the cells that populate the thymus are not committed to the T cell lineage. When developing to T cells these progenitor cells traverse a series of cellular stages that can be discriminated on the basis of cell surface and cytoplasmic markers, status of TCR gene rearrangements and precursor cell activities. The early stages of T cell development in the mouse thymus have been described in detail. The recent development of assays to measure the T cell precursor activity of human thymic and extrathymic progenitor cell subsets has led to a rapid accumulation of data on early events in human thymic development as well. The information available now permits a comparison of early cellular stages of T cell development in mice and man. Some of the extrinsic and intrinsic factors that govern T cell differentiation will be discussed. Data on the role of the cytokine, interleukin-7, in human and mouse T cell development will be summarized. Furthermore, recent data on the involvement of transcription factors in early T cell development are reviewed.

Characterization of CD34+ thymic stromal cells located in the subcapsular cortex of the human thymus.

In this paper we report that suspensions of human fetal thymocytes contain cells that express high levels of CD34 and Thy-1. These cells were characterized with regard to location within the thymus, phenotype, and function. Confocal laser scan analysis of frozen sections of fetal thymus with anti-CD34 and Thy-1 antibodies revealed that the double-labeled cells were located in the pericortical area. In addition, it was found that the CD34+Thy-1+ cells lacked CD45 and CD50, indicating that these cells are not of hematopoietic origin; this was confirmed by the finding that these cells could be cultured as adherent cells in a medium with cholera toxin and dexamethasone, but failed to grow in mixtures of hematopoietic growth factors. Further analysis indicated that most cultured CD34+Thy-1+ cells expressed cytokeratin (CK) 14 but lacked CK 13, suggesting that these cells are immature epithelial cells. Cultured CD34+Thy-1+ cells were able to induce differentiation of CD1-CD34+CD3-CD4-CD8- thymic precursors into CD4+CD8+ cells in a reaggregate culture in the absence of exogenous cytokines. The CD4+CD8+ cells that developed in these cultures did not express CD3, indicating that CD34+Thy-1+ thymic stromal cells are not capable of completing full T cell differentiation of thymic hematopoietic progenitor cells.

The crucial role of human dendritic antigen-presenting cell subsets in nickel-specific T-cell proliferation.

In the majority of patients, allergic nickel contact dermatitis is associated with a proliferative response of peripheral blood T lymphocytes to nickel sulfate. Optimal proliferation was found in a concentration range of 1-2 X 10(-4) M nickel sulfate. Nickel-specific response of purified peripheral blood T cells requires the presence of antigen-presenting cells (APC). Both peripheral blood monocytes and skin-derived epidermal cells could function as APC, but epidermal cells were shown to be more potent than monocytes. By testing FcR+ monocytes and FcR- circulating dendritic cells for their antigen-presenting capacities, it was found that the critical APC within the fraction of monocytes is the circulating dendritic cell. Testing highly purified T6+ (CD 1) skin-specific dendritic cells (Langerhans cells, LC) and T6- epidermal cells as APC, the critical APC within the fraction of epidermal cells appeared to be the LC. The crucial role of LC was stressed in experiments using T cells from patients exhibiting a positive patch test to nickel but a low or absent proliferative response to nickel by unpurified peripheral blood cells. Whereas addition of peripheral blood APC was ineffective, addition of LC to purified peripheral T cells was shown to overcome this low responsiveness to nickel. These results indicate the crucial role of dendritic APC subsets in nickel-specific T-cell proliferation.

Inflamed joints of patients with rheumatoid arthritis contain T cells that display in vitro proliferation to antigens present in autologous synovial fluid. Functional analysis on the basis of synovial-fluid-reactive T-cell clones and lines.

The immunopathology of inflamed joints in patients with RA is thought to result from an antigen-driven T-cell response. The antigen(s) responsible for the activation of synovial T cells, however, are as yet unidentified. In this study, we tested SF as a potential source of (auto)antigen(s). Five of 15 IL-2-expanded T-cell lines generated from SF cells of RA patients displayed a proliferative response to autologous SF. Five CD4+CD8-alpha beta TCR+SF-reactive T-cell clones obtained from responder T-cell lines were studied in more detail. Three T-cell clones from one RA patient were found to recognize epitopes in autologous SF in the context of DR4(Dw4), and two T-cell clones of another RA patient responded to autologous SF in the context of the HLA-DPB1*0401 gene product. The two DP-restricted clones and one of the DR-restricted clones did not proliferate to 50 SF samples of other RA patients, whereas the remaining DR-restricted clones responded to one allogeneic sample. Sequence analysis demonstrated that the latter clones expressed identical V beta 6.9 + TCR beta chains. This was also found for the (V beta 19+) DP-restricted clones. Proliferation of SF-reactive T cells was not only obtained with SF of the joint that had contained the T cells, but also with autologous SF of other affected joints. Together, these findings indicate that epitopes able to stimulate synovial T cells differ among RA patients, but may be similar within multiple joints of an individual patient. The presence of T cells able to respond to SF antigens in inflamed joints suggests that these T cells play an active role in the pathogenesis of RA.

Real-time on-line flow cytometry for bioprocess monitoring.

As the understanding of variation is the key to a good process and product quality one should pay attention to dynamics on the single-cell level. The basic idea of this approach was to qualify and quantify variations on the single-cell level during bioreactor cultivations by monitoring the expression of an eGFP tagged target protein (human membrane protein) using fully automated real-time, flow injection flow cytometry (FI-FCM). The FI-FCM system consists of a sampling- and defoaming- as well as of a dilution-section. It allows a very short monitoring interval (5 min) and is able to dilute the reactor sample by a factor ranging up to more than 10,000. In bioreactor cultivations of recombinant Pichia pastoris expressing the eGFP tagged target protein, high correlations (R(2)≥ 0.97) between the FI-FCM fluorescent signal and other, however, population-averaged fluorescence signals (off-line fluorescence, in situ fluorescence probe) were obtained. FI-FCM is the only method able to distinguish between few cells with high fluorescence and many cells with low fluorescence intensity and proved that cells differ significantly from each other within the population during bioreactor cultivations. Single-cell fluorescence was distributed over a broad range within the cell population. These distributions strongly suggest that (a) the AOX-I promoter is leaky and (b) a fraction of the population is able to express more protein of interest within shorter time and (c) a fraction of the population does not express the fusion protein at all. These findings can help in the selection of high producing, stable strains. To show the platform-independency of the system, it has successfully been tested during bioreactor cultivations of three different strains (P. pastoris, Saccharomyces cerevisiae, Escherichia coli). Along with its applications in PAT, the FI-FCM could be used as a platform-independent (prokaryotes and eukaryotes) method in various other applications; for example in the closed-loop-control of bioprocesses using different kinds of fluorescent reporters, (waste- and drinking-) water analysis, clone selection in combination with FACS or even for surgery applications.

[Sterilization of the male]. / Sterilizacija muskaraca.

The authors describe conditions in the field of sterilization in Slovenia from July 1977 to December 1979 in view of the new law on medical measures for the realization of the right to free decision on the birth of children. During this time 163 females and 62 males were sterilized. A general approach to sterilization is described, as well as the results of vasectomy.

Developmental stages in the human thymus.

The thymus is populated by hematopoietic cells that have the capacity to develop into at least three different hematopoietic lineages, T, NK and dendritic cells. While developing into T cells these cells pass a series of developmental stages that can be discriminated on the basis of expression of a number of antigens. The availability of a myriad of monoclonal anti- bodies against human differentiation antigens has permitted a detailed analysis of the various cellular stages in the human thymus. This analysis not only comprised investigation of molecular but also of functional features of purified thymocyte subsets, since more recently assays were set up that allowed investigation of the hematopoietic precursor activities of human thymic progenitor cells. Here we review the current status of knowledge with regard to early developmental stages in the human thymus. In addition, we discuss recent data on later developmental stages, in particular concerning positive selection and maturation of T cells.

Colonization of human genital tract by Streptococcus agalactiae.

This bacteriological study involved 92 young married couples who required medical help because of infertility. Colonization of male urethra or the presence of Streptococcus agalactiae in the ejaculate was detected in 9.78% of men. Vagina or vagina and cervix were colonized in 4.35% of women. Oral application of phenoxymethylpenicillin temporarily eliminated S. agalactiae from the genital tract of all colonized partners. Repeated colonization occurred 3-7 months later, in three cases by different serotypes compared with pretreatment period, in one case by the same serotype.

Heat-shock proteins and autoimmunity in humans.

T cells and antibodies against self and non-self hsp are present in both patients and healthy controls. T cells responding to hsp65 can be involved in autoimmune diseases, this was demonstrated for two site-specific animal autoimmune diseases: AA in Lewis rats and diabetes (IDDM) in NOD mice. In human ReA there is evidence for a direct stimulation of joint T cells by antigens of the organisms causing the infection which precedes the joint inflammation. The individual antigens of the triggering bacteria still have to be defined, but hsp65 may be of importance since this is one of the molecules recognized by synovial T cells in ReA patients. In RA there are no clear data implicating an infection in the initiation of joint inflammation, but mycobacteria have been suggested to be involved. We have discussed experimental findings which are in favor of, or in contradiction with, a role of mycobacterial antigens--particularly hsp65--in the etiology of RA. T cells recognizing hsp65 and other mycobacterial antigens are present in the joint, but there is no indication for a specific involvement of one or a limited set of (myco)bacterial antigens in the pathogenesis of RA.

Prethymic CD34+ progenitors capable of developing into T cells are not committed to the T cell lineage.

Progenitor cells that seed the fetal thymus are derived from the fetal liver and the bone marrow. These cells migrate through the fetal blood to the thymus. In this work, we address which peripheral progenitor cells have the potential to become T cells and whether these progenitor cells are already committed to the T cell lineage. All CD34+CD38- precursor cells, regardless of their origin, are able to develop into T cells in a hybrid human/mouse fetal thymic organ culture. Previously, we found that the more differentiated CD34+CD38+ progenitor cells from fetal liver cannot develop into T cells. In this work, we show that CD34+CD38+ cells from fetal bone marrow and cord blood are capable of T cell development. In spite of the T cell-developing potential, we did not detect rearrangements of TCR-delta or TCR-beta loci in any of the CD34+ peripheral precursors. CD34+ fetal bone marrow cell subpopulations express pre-TCR-alpha. However, we could not detect expression of pT alpha or of recombination-activating gene 1 in CD34+ cord blood cells. Since cord blood CD34+ cells should contain the direct progenitors of the CD34+ thymocytes, our data do not support the notion that in humans commitment to the T cell lineage occurs before the cells migrate into the thymus.

Birth after treatment of a male with seminoma and azoospermia with cryopreserved-thawed testicular tissue.

The case of an infertile couple in which a testicular seminoma and azoospermia were discovered in the husband during infertility treatment is described. A small piece of testicular tissue, obtained by biopsy from the healthy testis [testicular sperm extraction (TESE)], was deep-frozen before oncology therapy was initiated. The patient's lymphocyte karyotype was normal and no Y microdeletions were found. After conclusion of oncology treatment, the tissue was thawed and successfully used in the intracytoplasmic sperm injection (ICSI) procedure. A healthy girl was born. Testicular tumours are known to impair fertility in the majority of patients, and fertility deteriorates further after cytotoxic and surgical oncology treatment. Until recently in Slovenia, for young oncology patients cryopreservation was applied only to high quality ejaculate fulfilling the criteria for intrauterine insemination or in-vitro fertilization after thawing. Failing that, the only remaining options were fertilization by donor spermatozoa or child adoption. New assisted reproductive technologies, of which the ICSI procedure is the most successful, are suitable for the treatment of only the most severe cases of male infertility. It is reasonable to cryopreserve even poor quality ejaculate prior to the oncology therapy, as well as testicular tissue in cases of azoospermia.

Expression of pTalpha mRNA in a committed dendritic cell precursor in the human thymus.

We have characterized dendritic cell precursors (pre-DC) in the human thymus. These CD1a(-)CD3(-)CD4(+)CD8(-) cells express high levels of interleukin-3Ralpha (IL-3Ralpha) on the membrane and are able to develop into mature DC upon culture with IL-3 and CD40 ligation. The DC precursors are predominantly located in the thymic medulla. Interestingly, the pre-DC express pTalpha mRNA, which is also present in CD1a(+)CD3(-)CD4(+)CD8(-) pre-T cells. Yet, the pre-DC lack expression of recombination activating gene-1 mRNA and fail to develop into T cells in appropriate assays. The thymic pre-DC are very similar to the recently characterized pre-DC found in the T cell areas of the tonsil, and it is suggested that these pre-DC populations are of lymphoid origin.