Psoriasis confined strictly to vitiligo areas--a Koebner-like phenomenon?

Vitiligo and psoriasis are both common skin disorders. However, psoriasis strictly confined to pre-existing vitiligo areas is rare and suggests a causal relationship. We report here on two patients with a strict anatomical colocalization of vitiligo and psoriasis. The histopathological examinations showed typical changes for both diseases together with a dense infiltrate of CD4+ and CD8+ T cells. By immunohistochemistry, intracytoplasmatic granzyme B and tumour necrosis factor alpha (TNF-alpha) were detected within the T-cell population, suggesting the functional activity of these cells and the creation of a local T helper 1 (Th1)-cytokine milieu. Additionally, in one patient we could identify anti-melanocytic T cells by tetramer staining and enzyme-linked immunospot (ELISPOT) analysis. These skin-infiltrating lymphocytes might trigger, by the local production of Th-1 cytokines such as TNF-alpha and interferon-gamma (IFN-gamma), the eruption of psoriatic plaques in patients with a genetic predisposition for psoriasis.

[Simultaneous onset of pemphigoid and factor VIII antibody hemophilia]. / Simultanes Auftreten eines Pemphigoids und einer Hemmkörperhämophilie.

A 47-year old patient who had been suffering from hypertension and chronic renal failure for many years developed progressive extensive haemorrhagic erosions of the mouth within 3 months and less severe erosions of the genital and nasal mucosa. Additionally, subcutaneous haematomas developed spontaneously. Laboratory investigations demonstrated circulating antibodies against factor VIII while direct and indirect immunofluorescent microscopy showed discrete tissue-bound and circulating IgG reactive with the epidermal basement membrane in a pemphigoid-like fashion. Immunoblot analysis of the patient's serum revealed an "atypical" IgG reactivity against a central portion of the extracellular domain of the BP180 antigen. These findings were unexpected, since the clinical aspect showed striking resemblance to (paraneoplastic) pemphigus. The patient developed life-threatening complications. Eventually, reduction of circulating autoantibodies by a combination of plasmapheresis and subsequent immunosuppressive therapy led to a stable remission of both autoimmune bullous skin disorder and acquired haemophilia.

A method for the production of cryopreserved aliquots of antigen-preloaded, mature dendritic cells ready for clinical use.

Dendritic cells (DC) are increasingly used as a vaccine. Unfortunately, a satisfactory cryopreservation of DC in the absence of FCS is not yet available, so that laborious repeated generation of DC from fresh blood or frozen peripheral blood mononuclear cells for each vaccination has been required to date. We now aimed at developing an effective cryopreservation method, and by testing several variables found that it was crucial to combine the most advantageous maturation stimulus with an improved freezing procedure. We generated monocyte-derived DC from leukapheresis products by using GM-CSF and IL-4 and showed that amongst several known maturation stimuli the cocktail consisting of TNF-alpha+IL-1 beta+IL-6+PGE(2) achieved the highest survival of mature DC. We then systematically explored cryopreservation conditions, and found that freezing matured DC at 1 degrees C/min in pure autologous serum+10% DMSO+5% glucose at a cell density of 10x10(6) DC/ml gave the best results. Using this approach 85-100% of the frozen DC could be recovered in a viable state after thawing (Table 1). The morphology, phenotype, survival as well as functional properties (allogeneic mixed leukocyte reaction, induction of influenza matrix or melan A peptide-specific cytotoxic T cells) of these thawed DC were equivalent to freshly prepared ones. The addition of CD40L or TRANCE/RANKL further improved DC survival. Importantly, we demonstrate that DC can effectively be loaded with antigens (such as Tetanus Toxoid, influenza matrix and melan A peptides) before cryopreservation so that it is now possible to generate antigen-preloaded, frozen DC aliquots that after thawing can be used right away. This is an important advance as both the generation of a standardized DC vaccine under GMP conditions and the carrying out of clinical trials are greatly facilitated.

Mage-3 and influenza-matrix peptide-specific cytotoxic T cells are inducible in terminal stage HLA-A2.1+ melanoma patients by mature monocyte-derived dendritic cells.

Dendritic cell (DC) vaccination, albeit still in an early stage, is a promising strategy to induce immunity to cancer. We explored whether DC can expand Ag-specific CD8+ T cells even in far-advanced stage IV melanoma patients. We found that three to five biweekly vaccinations of mature, monocyte-derived DC (three vaccinations of 6 x 106 s.c. followed by two i.v. ones of 6 and 12 x 106, respectively) pulsed with Mage-3A2.1 tumor and influenza matrix A2. 1-positive control peptides as well as the recall Ag tetanus toxoid (in three of eight patients) generated in all eight patients Ag-specific effector CD8+ T cells that were detectable in blood directly ex vivo. This is the first time that active, melanoma peptide-specific, IFN-gamma-producing, effector CD8+ T cells have been reliably observed in patients vaccinated with melanoma Ags. Therefore, our DC vaccination strategy performs an adjuvant role and encourages further optimization of this new immunization approach.

Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma.

Dendritic cells (DCs) are considered to be promising adjuvants for inducing immunity to cancer. We used mature, monocyte-derived DCs to elicit resistance to malignant melanoma. The DCs were pulsed with Mage-3A1 tumor peptide and a recall antigen, tetanus toxoid or tuberculin. 11 far advanced stage IV melanoma patients, who were progressive despite standard chemotherapy, received five DC vaccinations at 14-d intervals. The first three vaccinations were administered into the skin, 3 x 10(6) DCs each subcutaneously and intradermally, followed by two intravenous injections of 6 x 10(6) and 12 x 10(6) DCs, respectively. Only minor (less than or equal to grade II) side effects were observed. Immunity to the recall antigen was boosted. Significant expansions of Mage-3A1-specific CD8(+) cytotoxic T lymphocyte (CTL) precursors were induced in 8/11 patients. Curiously, these immune responses often declined after the intravenous vaccinations. Regressions of individual metastases (skin, lymph node, lung, and liver) were evident in 6/11 patients. Resolution of skin metastases in two of the patients was accompanied by erythema and CD8(+) T cell infiltration, whereas nonregressing lesions lacked CD8(+) T cells as well as Mage-3 mRNA expression. This study proves the principle that DC "vaccines" can frequently expand tumor-specific CTLs and elicit regressions even in advanced cancer and, in addition, provides evidence for an active CD8(+) CTL-tumor cell interaction in situ as well as escape by lack of tumor antigen expression.

Environmental influence on age-related changes of human lymphocyte membrane viscosity using severe combined immunodeficiency mice as an in vivo model.

Peripheral blood lymphocytes (PBL) of healthy elderly people show increased plasma membrane viscosity compared to young subjects, that inversely correlates with lymphocyte proliferation after mitogen stimulation in vitro. Maintenance of a constant membrane viscosity, which is necessary for proper cell function, is crucially dependent on the membrane lipid composition. The cellular lipid metabolism, and thus lymphocyte function, may be subject to modulation by diet or drugs. To study the susceptibility of membrane viscosity to environmental conditions, we established an in vivo model using severe combined immunodeficiency (SCID) mice: human peripheral blood lymphocytes from healthy young and old subjects were engrafted for three days intraperitoneally into SCID mice to offer identical environmental conditions. First, we demonstrate that human lymphocytes can take up and utilize murine lipoproteins: engrafted human PBL can participate in the mouse lipid metabolism, and an exchange of membrane lipids in vivo is, therefore, possible. Second, plasma membrane viscosity was determined before and after engraftment: before engraftment, PBL from the elderly showed a significantly higher membrane viscosity than that from young controls, but this difference vanished during engraftment into SCID mice, wherein cells from both age groups exhibited nearly identical values. It was, therefore, concluded that lymphocyte membrane viscosity is influenced by environmental factors, and that the age-related increase is, in principle, reversible.

The production of the Alzheimer amyloid precursor protein (APP) in extraneuronal tissue does not increase in old age.

Alzheimer's disease (AD) is characterized by the cerebral deposition of beta-amyloid (A beta). A beta plaques also occur in the brains of healthy aged individuals, and A beta concentrations are increased in the cerebrospinal fluid (CSF) in old age. Based on results from an in vitro senescence model on human fibroblasts, it was proposed that the production of the beta-amyloid precursor protein (APP) was increased during aging. No information was available as to whether APP production was also augmented in aged humans. It was therefore the aim of the present study to analyze APP in connective tissue, skeletal muscle, peripheral blood mononuclear cells, and serum samples from young and aged healthy individuals. APP production was assessed by Northern and Western blotting. The expression of the different APP isoforms was studied by reverse transcription-polymerase chain reaction (RT-PCR) technique. The results demonstrate that APP messenger ribonucleic acid (mRNA) and protein concentrations were identical in blood and tissue samples from young and aged individuals and that there were no age-dependent changes in the APP isoform production pattern. Thus, our data strongly argue against the possibility of an altered production of APP during healthy aging and underline the point that in vitro aging models may not accurately reflect the in vivo situation.

Co-expression of ICAM-1, VCAM-1, ELAM-1 and Hsp60 in human arterial and venous endothelial cells in response to cytokines and oxidized low-density lipoproteins.

T-cells and monocytes are the first cells infiltrating the arterial intima during the early stages of atherogenesis. Recently our laboratory has provided evidence that T-cells isolated from atherosclerotic intima reacts against heat shock protein 60 (Hsp60). Transmigration of activated T-cells into the intima is mediated by adhesion molecules (ICAM-1; VCAM-1; ELAM-1) expressed on activated endothelial cells. Here we studied the potential of cytokines (TNF-alpha, IFN-gamma, IL-1). Escherichia coli lipopolysaccharide (LPS), native and oxidized low-density lipoprotein (LDL; oxLDL) and high temperature to induce adhesion molecules as well as Hsp60 and Hsp70 expression in human endothelial cells (EC). On Northern blots, a strong signal for ICAM-1, VCAM-1 and ELAM-1 was detected after 4 h, which thereafter declined, but did not reach the basal level of untreated control cells. Heat shock induced the expression of Hsp60 and Hsp70 but not of adhesion molecules. EC were cultivated in serum-free medium, which led to the expression of adhesion molecule transcripts. Addition of LDL or oxLDL to these ECs did not alter the expression of these transcripts. The production of adhesion molecule proteins was analysed by flow cytometry. In human venous endothelial cells (HVEC) and human arterial endothelial cells (HAEC) ICAM-1 and VCAM-1 production was permanently highly induced, whereas the high level of ELAM-1 production at 4 h disappeared after 24 h. Furthermore, only HAEC, but not HVEC, produced ICAM-1, VCAM-1 and ELAM-1 after stress by moderately and highly oxLDL. LDL and oxLDL did not induce the production of Hsp60 and Hsp70. The present study demonstrates the co-expression of Hsp60 and adhesion molecules in arterial and venous EC in response to cytokine and LPS exposure, and that oxLDL is an efficient inducer of adhesion molecules in arterial EC and not in venous EC. These features provide the prerequisites for a cellular immune reaction against Hsp60 expressed by stressed EC in the initial stages of atherosclerosis.

Cytokine production in response to stimulation with tetanus toxoid, Mycobacterium tuberculosis and influenza antigens in peripheral blood mononuclear cells and T cell lines from healthy elderlies.

Little is known about the type 1/type 2 T cell dichotomy in old age. Peripheral blood mononuclear cells (PBMC) and T cell lines from old and young healthy individuals were therefore analyzed for their production of interferon gamma (IFN-gamma) and interleukin 4 (IL-4). Tetanus toxoid (TT), purified protein derivative of Mycobacterium tuberculosis, inactivated influenza virus and OKT-3 were used as stimuli. RT-PCR and ELISA determinations were performed. When stimulated with TT, PBMC from young and old individuals expressed IL-4, but produced little IFN-gamma. All other stimuli induced a pronounced IFN-gamma production, while little or no IL-4 was expressed. T cell lines, regardless of their specificity or the donor age, produced IFN-gamma and IL-4. The quantities of cytokines produced did not significantly differ between the age groups. The capacity of the immune system to trigger type 1 and type 2 T cell responses is thus well preserved in old age.

Peripheral blood dendritic cells reinduce proliferation in in vitro aged T cell populations.

Dendritic cells (DC) are professional antigen presenting cells which are essential for the initiation of an immune response. Recently we demonstrated that DC, which had been propagated from the peripheral blood of healthy elderly people, were morphologically and functionally intact. It was the aim of the present study to analyze how DC from young and old healthy individuals could affect T cell responsiveness to antigen in an in vitro senescence model. Tetanus toxoid (TT)-specific T cell lines were derived from 3 young (< 30 years) and 3 old (> 65 years) individuals and were kept in long term culture. T cell proliferation in response to stimulation with antigen presented by either autologous peripheral blood mononuclear cells (PBMC) or DC was assessed at three different time points, once soon after the initiation of the cultures and twice after 20 to 30 population doublings at a stage when growth, was slow and programmed cell death imminent. Antigen presentation by DC enhanced T cell proliferation at each time point and reinduced proliferation in in vitro aged T cell populations which had stopped dividing. Terminal apoptosis was thus prevented. DC from old individuals were as effective as cells from young donors. Our results demonstrate that DC stimulate the clonal expansion and postpone the clonal elimination of antigen-specific T cell populations. As a consequence they may increase immunoreactivity, prolong immunological memory and be of particular importance for the maintenance of the T cell repertoire in old age.

[Immune reaction to tetanus in the elderly: what is the duration of vaccine protection?]. / Die Immunreaktion gegenüber Tetanus im Alter: Wie lange hält der Impfschutz?

Infectious diseases represent one of the most frequent causes of morbidity and mortality in the elderly. Little information is yet available on the state of immunization against well-known antigens such as tetanus toxoid (TT) in old age. It was, therefore, the aim of this study to analyze antibody titres and peripheral blood mononuclear cell (PBMC) reactivity to TT in healthy SENIEUR compatible young (< 30 years, n = 25) and old (> 65 years, n = 32) blood donors. TT-specific antibodies were measured by the ELISA technique; PBMC proliferation was assessed by 3H-thymidine incorporation analysis. In the young group TT antibody titres were detectable in all but two individuals, whereas 60% of the old persons had no detectable TT antibodies. This seemed partly to be due to a shortened immunological memory in old age, since 32% of the aged persons without TT antibodies had been vaccinated within the past 10 years, 21% even 3 to 6 years prior to investigation. TT antibody concentrations were normal in aged individuals vaccinated within the past two years. Peripheral blood lymphocytes from all but two persons without antibody titres did not proliferate when stimulated with TT in vitro, indicating that no memory T cells were available to reinduce an efficient immune response. Our results suggest that the tetanus vaccination strategy practised in Austria does not guarantee full protection in the elderly.

Morphologically and functionally intact dendritic cells can be derived from the peripheral blood of aged individuals.

Dendritic cells are antigen-presenting cells (APC), which are crucial for the initiation of an immune response. In spite of the well known decline of immune function in old age, no information is yet available on whether dendritic cells are also affected by the ageing process. It was therefore the aim of this study to compare peripheral blood dendritic cells (DC) from old and young healthy individuals. Using granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4, DC were propagated from peripheral blood mononuclear cells (PBMC). The obtained cell populations had a typical dendritic morphology and expressed HLA class I and class II, CD23, CD32, CD40, CD44 and CD54, but not CD3 and CD19. Larger numbers of DC were obtained from old individuals than from young ones in spite of a similar expression pattern of surface molecules. DC from aged persons also survived better under in vitro culture conditions. When tested for their antigen-presenting capacity, DC from young and old individuals were equally effective in inducing the proliferation of tetanus toxoid-specific T cell clones after antigenic stimulation. Peripheral blood DC from aged individuals may thus still function as powerful APC. They may represent useful tools for immunotherapy in the aged.

Regulation of CD95 (APO-1) expression and the induction of apoptosis in human T cells: changes in old age.

CD95 (APO-1) is a member of the TNF/nerve growth factor receptor superfamily, which is expressed on the surface of different types of cells. Cross-linking of CD95 leads to the induction of apoptosis. This may be of importance in many physiological systems, but seems to play a special role for the maintenance of immunological homeostasis. In view of the known decline of immune function in old age it seemed of interest to study the expression and inducibility of CD95 in peripheral blood T lymphocytes from young and old healthy subjects selected according to the guidelines laid down in the Senieur protocol of the European Community's Concerted Action Programme on Aging. Resting T cells did not express CD95. T cell activation by anti-CD3 monoclonal antibody (OKT3) did, however, lead to a rapid increase in the number of CD95 expressing cells. This increase was slower and less pronounced in old healthy subjects than in young ones. The activation-induced increase in CD95 expression was followed by a decrease, which was observed in both age groups, but was less pronounced in old subjects. Under long-term culture conditions T cell lines derived from both young and old individuals progressively lost the capacity to decrease the expression of CD95 at the end of their activation cycle and an increasing susceptibility to activation-driven programmed cell death was noted. The latter change was more pronounced in T cell lines derived from aged donors. The results suggest that a lowered sensitivity in the regulation of CD95 as well as an increased susceptibility to apoptosis-inducing mechanisms during clonal expansion are features of T cell senescence.

Comparison of low density lipoprotein uptake by different human lymphocyte subsets: a new method using double-fluorescence staining.

A method to determine low density lipoprotein (LDL) uptake of distinct lymphocyte subpopulations was developed using fluorescent LDL and subsequent staining of lymphocyte subsets with biotinylated monoclonal antibodies plus streptavidin-CyChrome. LDL uptake was detected on a single cell level and semiquantified by FACS analysis. This method allows comparison of defined lymphocyte subsets from different individuals and excludes the falsifying influence of individual differences in subset distribution, which may occur in studies on total peripheral blood lymphocytes (PBL). Investigation of total PBL and lymphocyte subsets of 20 healthy volunteers (8 male, 12 female) showed the following. i) Different lymphocyte subsets exhibited highly significant differences in LDL uptake, with NK cells (CD16) showing a higher uptake than T (CD3) and B cells (CD19); CD8-positive cells exhibited higher values than CD4-positive cells. ii) These differences are due to specific, LDL-receptor (LDL-R)-mediated LDL uptake. iii) Inter-individual differences in LDL uptake are reflected on all lymphocyte subsets.

Reference intervals for human peripheral blood lymphocyte subpopulations from 'healthy' young and aged subjects.

Some lymphocyte subpopulations change during aging but age-related reference limits from a healthy reference population are still lacking. In this study, we compiled 90% reference intervals for commonly determined lymphocyte subpopulations in 'healthy' (Senieur-compatible) young (20-32 years) and elderly (65-74 years) subjects. The most striking age-related changes included increases in HLA-DR+ T lymphocytes, and the shift in the expression of CD45 isoforms from the CD45RA+CD45RO-to the CD45RA-CD45RO+ subset. Both age-related alterations occurred in the CD4+ as well as in the CD8+ subpopulations and most of them were present in the relative and absolute number of lymphocyte subsets. We compare our data with those from previous investigations on lymphocyte subpopulations from the elderly and comment on useful presentation of reference values.

Target organ susceptibility and autoantibody production in an animal model of spontaneous autoimmune thyroiditis.

F1-hybrids of Obese strain (OS) chickens, afflicted with spontaneous autoimmune thyroiditis (SAT), and normal, inbred CB chickens, do not develop severe thyroiditis. About 50% of these crosses show circulating autoantibodies to thyroglobulin (TgAAb), but the thyroid glands are only slightly infiltrated, suggesting that the target organ is not susceptible to autoimmune attack. In the present study we show that despite this mild infiltration TgAAb are only synthesized by lymphoid cells within the thyroid gland. Furthermore, we demonstrate that immunization with chicken thyroglobulin (Tg) in complete Freund's adjuvant causes severe experimental autoimmune thyroiditis (EAT) in F1(OSxCB) hybrids.