Survivin is overexpressed in several malignancies and in tumor-associated endothelium making it an attractive target for therapeutic cytotoxic T-cell responses. Thus, it would be important to test this notion in preclinical models. Consequently, we screened the murine survivin sequence for potential binding K(b)-restricted octamer peptide epitopes. Two epitopes, which bind strongly to K(b), were selected to test their immunogenicity in vivo. Spleen cells from mice vaccinated by intradermal injection of mature DC pulsed with these peptides displayed reactivity to the respective epitopes. The natural processing and presentation of these epitopes by tumor cells was evident by the killing of murine melanoma cells by vaccination-induced T cells. Subcutaneous challenge with syngeneic melanoma demonstrated the protective immunity of this vaccination. Notably, analysis of the vessel density in subcutaneous tumors revealed that survivin-specific vaccination significantly reduced the number of intratumoral vessels. In summary, we demonstrated the immunogenicity of two K(b)-restricted peptide epitopes derived from the murine survivin protein; moreover, survivin-specific vaccination not only resulted in a reduction of tumor cells but also the tumor supplying blood vessels. The presented preclinical model for survivin-directed vaccination may serve as a valuable tool to improve already running clinical trials in a syngeneic tumor model.

We previously demonstrated that targeting lymphotoxin α (LTα) to the tumor evokes its immunological destruction in a syngeneic B16 melanoma model. Since treatment was associated with the induction of peritumoral tertiary lymphoid tissue, we speculated that the induced immune response was initiated at the tumor site.

BACKGROUND:Recent reports have demonstrated that the enzyme indoleamine 2,3-dioxygenase (IDO) is upregulated in human dendritic cells (DCs) upon in vitro maturation. IDO is supposed to convey immunosuppressive effects by degrading the essential amino acid tryptophan, thereby downregulating T-cell functions. Hence, we evaluated IDO expression in DC preparations used for therapeutic DC vaccination and its in vivo effects.PATIENTS, METHODS AND RESULTS:IDO expression was detected by real-time-PCR in a series of human clinical grade DCs (n = 28) prior to vaccination of advanced melanoma patients (n = 11). These analyses revealed an intra- and interpersonal variation in IDO mRNA levels. IDO was strongly upregulated in human DCs on RNA and on protein level upon in vitro maturation by Interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha), Interleukin-6 (IL-6) and Prostaglandin E2 (PGE2) over a time course of 24 h. The enzymatic activity of induced IDO was demonstrated by measuring tryptophan degradation. Moreover, in biopsies obtained 24 h after application of the DC vaccine a prominent infiltrate of IDO-positive cells was observed by immunohistochemistry. The inflammatory infiltrate of these sites stained positive for the transcription factor Forkhead box P3 (FoxP3), suggesting an IDO-mediated induction of regulatory T-cells. All analysed melanoma patients (n = 11) receiving DC based immunotherapy exhibited rapid disease progression with a short overall survival due to advanced tumour stage.CONCLUSION:The presented observations suggest a potential clinical relevance of IDO expression in DC-based therapeutic vaccines via the attraction or induction of FoxP3(+) T-cells.

An effective immunological eradication of tumors by the adaptive immune system depends on T cell priming, expansion of specific T cells and their effector function. It has been shown that either step may be impaired in the tumor-bearing host, and several strategies have been used to improve antitumor immune responses. In this regard, tumor-targeted IL2 therapy leads to the destruction of established melanoma metastases in fully immune competent mice as previously demonstrated. This effect has been attributed, but never directly confirmed, to the boost of antigen-experienced T cells. To this end, we demonstrate the absence of any antitumor effect of targeted IL2 in mice characterized by an impaired priming of T cell responses. Notably, in these animals tumor-targeted IL2 therapy induced tumor regression only after adoptive transfer of tumor-conditioned splenocytes. A detailed analysis revealed that T cells present within the transferred splenocytes were actively participating in the immune response as these were clonally expanded after targeted IL2 therapy. In summary, we demonstrate here that in LTα −/− mice lacking sufficient numbers of tumor-specific T cells only the passive transfer of such cells prior to therapy restores the efficacy of tumor-targeted IL2 therapy. Thus, the antitumor effect of tumor-targeted IL2 is indeed based on the boost of pre-existing T cell responses.

Prognosis of disseminated melanoma remains gloomy as neither chemotherapeutic nor unspecific immune modulatory approaches were able to improve the overall survival of these patients. Hence, specific immunotherapy has received increasing attention. Disappointing clinical results, however, indicate that the choice of suitable antigens is of special importance. To this end, the inhibitor of apoptosis (IAP) protein survivin, which is over-expressed in several tumours but is largely undetectable in adult tissues, appears to be a promising target for vaccination purposes, since down-regulation or loss of expression is associated with impaired tumour progression. Consequently, five heavily pretreated stage IV melanoma patients were vaccinated with the HLA-A2 restricted survivin96–104 epitope presented by autologous dendritic cells (DCs) in a compassionate use setting. Four of these patients mounted strong T cell responses to this epitope as measured by ELISPOT assay. Furthermore, in situ peptide/HLA-A2 multimer staining confirmed that these survivin reactive cells infiltrated both visceral and soft tissue metastases.

Matrix metalloproteinases (MMPs) are essential for tumor progression, invasion and metastases formation. Expression of these proteinases is not only restricted to the tumor cells themselves, but also is found in normal stromal cells. Moreover, immunohistochemistry suggests stromal cells as the major source. To scrutinize this hypothesis we established a slowly growing, syngeneic tumor model using the B16-melanoma cell line B78D14. In vitro analysis demonstrated that B78D14 cells secreted MMP-2, MT1-MMP, and to a lesser degree MMP-9; in addition they expressed both MT1-MMP and EMMPRIN on their surface. In subcutaneous (s.c.) tumors of these cells MMP-2 expression was predominantly present at the tumor-stroma border indicating stromal cells as primary source for this protease in vivo. Indeed, double staining experiments and in situ zymography confirmed that tumor adjacent stromal cells at the invasive front expressed MMP-2 and only at this site activated MMP-2 was detectable. Notably, in an experimental pulmonary metastases model neither tumor nor stromal cells expressed MMP-2, suggesting that the capacity of stromal cells is largely dependent on the surrounding microenvironment.

Background: Imaging of the anorectal region has drastically changed over the past decade by the introduction of anal endosonography. This method has been used to localize injuries to the anal sphincters, fistula tracts and abscesses as well as to assess local invasion of cancerous lesions. It allows a quick and easy visualisation of the anorectal mucosa and the surrounding muscular structures. Limitations of endosonography include strictures and acute painful conditions. These caveats are largely caused by the size of regular rectal probes with an average diameter of 17 to 20 mm. Patients and Method: We compared a new rectal probe with an external diameter of only 7 mm to a regular probe with 20 mm. From April 1999 to March 2000 80 patients with acute and chronic anal pain were examined. Results: Even patients with acute anal pain could be examined without anaesthesia or sedation. The minimal compression and the absence of stretching artefacts of the 7 mm probe enabled us to depict the anal canal in an almost physiologic state. Marginal fistulas and haemorrhoidal knots in their anatomical localisation in the anal canal were easily identified. Conclusion: The introduction of endoanal ultrasound probes with a diameter below 1 cm represent a major improvement particularly for imaging all layers off the anal canal even under acute pain.

Die Prognose von Patienten mit fernmetastasiertem Melanom ist außerordentlich schlecht. Weder Chemotherapien, noch unspezifische Immunstimulatoren haben bislang zu einer signifikanten Verlängerung der Gesamtüberlebenszeit der Betroffenen geführt, daher finden aktuell spezifische Immuntherapien besonderes Interesse. Trotz erfolgversprechender theoretischer Konzepte sind die klinischen Ergebnisse bislang jedoch zumeist enttäuschend. Die Wahl der antigenen Zielstrukturen erwies sich in Tiermodellen von herausragender Wichtigkeit. Survivin, ein Mitglied der Inhibitoren der Apoptoseproteine (IAP), das von nahezu allen Tumorentitäten überexprimiert wird, erscheint als ein erfolgversprechendes Zielantigen, da eine Herabregulierung oder der Verlust seiner Expression mit einer verminderten Tumorprogression assoziiert sind. Fünf bereits mehrfach chemo- und/oder immuntherapeutisch vorbehandelte Stadium-IV-Melanompatienten wurden im Rahmen eines Heilversuches mit einem HLA-A2 restringierten Survivinepitop (Sur95–104), welches von autologen dendritischen Zellen präsentiert wurde, vakziniert. Mittels ELISPOT konnte gezeigt werden, dass vier dieser Patienten starke T-Zellantworten gegenüber dem Survivinepitop entwickelten. Darüberhinaus gelang mittels in-situ Peptid/HLA-A2 Multimerfärbung der Nachweis Survivin-reaktiver T-Zellen im Infiltrat von Metastasen. Bemerkenswerterweise traten unter Vakzinierungstherapie keinerlei therapieassoziierte Toxizitäten auf. Zusammenfassend legen unsere Daten nahe, dass sogar bei Patienten mit fortgeschrittener Melanomerkrankung T-Zellantworten gegenüber Survivin mittels DC-basierter Vakzinierung induziert werden können.

Imaging of the anorectal region has drastically changedover the past decade by the introduction of anal endosonography.This method has been used to localize injuries to the analsphincters, fistula tracts and abscesses as well as to assesslocal invasion of cancerous lesions. It allows a quick and easyvisualisation of the anorectal mucosa and the surroundingmuscular structures. Limitations of endosonography includestrictures and acute painful conditions. These caveats arelargely caused by the size of regular rectal probes with anaverage diameter of 17 to 20 mm.

A 25-year-old female patient from Kosovo presented with a slowly progressive cicatricial alopecia which had started when she was 6 years old. Her brother in Kosovo had similar lesions. At the erythematous border of the hairless area, crusts, erosions and pustules were apparent. Mycological examination identified Trichophyton schönleinii, the causative pathogen of favus. Histological examination revealed hyphae and showed no features suggestive of other causes of cicatricial alopecia. Systemic terbinafine combined with topical ciclopiroxolamine resulted in rapid improvement of this disease which is seldom seen in Central Europe.

BACKGROUND:Narrow-band ultraviolet (UV) B (311 nm) phototherapy is an effective treatment for psoriasis. In order to reduce cumulative UV doses and to enhance clearance of psoriasis plaques, combination therapies with topical agents such as dithranol and calcipotriol have been established.OBJECTIVES:To compare the clinical efficacy, in a half-side manner, of UVB (311 nm) in combination with either calcitriol or dithranol.METHODS:Ten patients with symmetrical stable plaque psoriasis were treated with narrow-band UVB (311 nm) five times a week. In addition, topical calcitriol was applied twice daily to one arm, whereas the other arm and the rest of the body were treated once daily with dithranol. The follow-up period was at least 4 weeks. Efficacy was assessed separately for both arms prior to treatment and once weekly thereafter by a modified Psoriasis Area and Severity Index (PASI) score. The cumulative irradiation dose and the number of treatment sessions required for clearance of psoriasis lesions were determined for each patient. Additionally, all patients completed a quality of life questionnaire.RESULTS:Both treatment modalities notably reduced the PASI score. A clinical comparison of UVB (311 nm) in combination with either calcitriol or dithranol revealed no significant therapeutic differences between the regimens.CONCLUSIONS:Combination of narrow-band UVB (311 nm) therapy with calcitriol is equally effective as the combination with dithranol for the treatment of psoriasis. However, patients preferred calcitriol rather than dithranol when both quality of life and treatment acceptability were assessed.

Fabry disease is an X-linked lysosomal storage disorder that results from a deficiency of the enzyme alpha-galactosidase A (alpha-Gal A). The lack of alpha-Gal A causes an intracellular accumulation of glycosphingolipids, mainly globotriaosyceramide (GL3). Affected organs include, among others, the vascular endothelium, heart, brain, and kidneys, leading to end-stage renal disease (ESRD). Since Fabry disease cannot be cured at present, clinical management is symptomatic. Enzyme replacement therapy (ERT) with recombinant alpha-Gal A has been introduced as a new therapeutic option for the treatment of Fabry patients. Short-term (one year) clinical studies have positively correlated ERT with improvement of clinical symptoms and microvascular endothelial cell clearance. Treatment outcome concerning severe organ manifestations such as proteinuria and renal function impairment, left ventricular hypertrophy, and heart failure in the long run has yet to be shown. In our studies we used sensitive and noninvasive techniques such as ultrasound-based strain rate imaging and magnetic resonance imaging (MRI), combined with MR-spectroscopy (MR-S), for the quantification of functional abnormalities at an early stage of the disease and during long-term follow-up. Future issues should determine the appropriate timing to start therapy and how children and heterozygous females should be managed. Given the diagnostic and therapeutic potential today, it is of importance to identify patients at an early stage and to start therapeutic intervention before progression of organ damage is inevitable.

Intact human pregnancy can be regarded as an immunological paradox in that the maternal immune system accepts the allogeneic embryo without general immunosuppression. Because dendritic cell (DC) subsets could be involved in peripheral tolerance, the uterine mucosa (decidua) was investigated for DC populations. Here we describe the detailed immunohistochemical and functional characterization of HLA-DR-positive antigen-presenting cells (APCs) in early pregnancy decidua. In contrast to classical macrophages and CD83(+) DCs, which were found in comparable numbers in decidua and nonpregnant endometrium, only decidua harbored a significant population of HLA-DR(+)/DC-SIGN(+) APCs further phenotyped as CD14(+)/CD4(+)/CD68(+/-)/CD83(-)/CD25(-). These cells exhibited a remarkable proliferation rate (9.2 to 9.8% of all CD209(+) cells) by double staining with Ki67 and proliferating cell nuclear antigen. Unique within the DC-family, the majority of DC-SIGN(+) decidual APCs were observed in situ to have intimate contact with CD56(+)/CD16(-)/ICAM-3(+) decidual natural killer cells, another pregnancy-restricted cell population. In vitro, freshly isolated CD14(+)/DC-SIGN(+) decidual cells efficiently took up antigen, but could not stimulate naive allogeneic T cells at all. Treatment with an inflammatory cytokine cocktail resulted in down-regulation of antigen uptake capacity and evolving capacity to effectively stimulate resting T cells. Fluorescence-activated cell sorting analysis confirmed the maturation of CD14(+)/DC-SIGN(+) decidual cells into CD25(+)/CD83(+) mature DCs. In summary, this is the first identification of a uterine immature DC population expressing DC-SIGN, that appears only in pregnancy-associated tissue, has a high proliferation rate, and a conspicuous association with a natural killer subset.

Expression of matrix metalloproteinases (MMPs) and their activation in tumor cells, as well as tumor surrounding stromal cells have been implicated in tumor cell invasion and metastasis. By means of a syngeneic tumor model for either experimental or spontaneous metastases, the differential expression of MMPs and tissue inhibitors of MMPs (TIMPs) in relation to the microenvironment and the way of metastasis induction was characterized. In vitro characterization revealed that increased levels of secreted MMP-2, MMP-9, and TIMP-1 were only detectable in the most aggressive cell line, B16G3.12BM2. Remarkably, active MMP-2 was restricted to this cell line, whereas TIMP-2 and membrane type (MT) 1-MMP expression was comparable in all three of the spontaneously metastasizing melanoma cell lines investigated. In vivo analysis demonstrated that MMP-2, MMP-9, and MT1-MMP were predominantly expressed at the tumor-stroma border of s.c. tumors. Furthermore, functional active MMP-2 was restricted to this invasive front. In spontaneous lymph node or lung metastases, however, MMP-9 was expressed both in the center and the periphery of tumors; these areas were largely negative for MMP-2 and MT1-MMP. Notably, tumor cells of experimental lung metastases did not express MMP-9 at all. These results indicate that expression of MMPs in melanoma metastases is not only influenced by their localization but also the nature of tumor induction, suggesting that individual MMPs serve specific roles during the different stages of metastasis formation.

During the past few years, sentinel lymph-node (SLN) excision has become a standard staging procedure for solid tumours such as breast cancer or melanoma. 1 Gershenwald JE Thompson W Mansfield PF et al. Multi-institutional melanoma lymphatic mapping experience: the prognostic value of sentinel lymph node status in 612 stage I or II melanoma patients. J Clin Oncol. 1999; 17: 976-983 Crossref PubMed Google Scholar Although some scientists believe SLN might be both a diagnostic and a therapeutic measure, concerns have been raised about the safety of removing the lymphoid tissue responsible for initial induction of specific immune responses. Indeed, Romero and colleagues have shown that tumour-specific T cells are present in excised SLNs. 2 Romero P Rod DP Valmori D et al. Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes. J Exp Med. 1998; 188: 1641-1650 Crossref PubMed Scopus (469) Google Scholar Thus, it seems possible that immune responses to the tumour may be impaired by this procedure. To investigate this, we did a thorough immunological workup of a patient who developed two consecutive melanomas. This analysis showed that the SLN actually harboured T cells that recognise the tumour, but the same clones present in the excised tissue were nevertheless maintained for almost 2 years in other compartments. This was established by analysis of the inflammatory infiltrate of a second melanoma which arose 2 years later; the same T-cell clones were present.

Melanoma, despite its aggressive growth characteristics, is an antigenic tumor expressing several characterized neo- and differentiation antigens. Dendritic cells (DC) when pulsed with defined peptides have been shown to effectively induce melanoma-specific T cell responses in humans and mice. These protect animals from challenge with melanoma, but so far have failed to induce significant tumor regressions. To study the efficacy of DC-based anti-tumor vaccinations, we set up a therapeutic model using C57BL/6J mice with established pulmonary and subcutaneous metastases induced by the B16-melanoma cell line B78-D14. Mice were vaccinated twice with 20,000 antigen-presenting cells, either bone marrow-derived DC or epidermal Langerhans cells (LC), which were loaded with the tyrosinase-related protein 2 (TRP2) peptide. Generally, DC cultured with fetal calf serum (FCS) induced a dominant unspecific response. This was not seen using LC cultured without serum; however, vaccination with TRP2-loaded FCS-free LC alone failed to influence the growth of established B16 tumors. A reproducible reduction of tumor size and weight was only obtained if LC vaccinations with TRP2 were followed by a 5-day treatment of mice with 200,000 IU IL-2 intraperitoneally twice/daily. Omitting the TRP2 peptide abolished the efficacy of this combined treatment, demonstrating the crucial role of priming a melanoma specific T cell response. Microcytotoxic assays performed with spleen-derived T cells and melanoma as well as congenic fibroblast lines as targets confirmed the TRP2-dependent specificity of LC-induced immune responses. Thus, despite the fact that tumor-specific T cells were primed, an additional IL-2-dependent stimulus was needed to translate this immune response into a therapeutic effect against established tumors.