Leg ulcer in a patient associated with hydroxyurea therapy.

Hydroxyurea is a hydroxylated derivate of urea commonly used in the treatment of various hematologic disorders. Cutaneous side-effects such as alopecia, diffuse hyperpigmentation, scaling, poikiloderma, atrophy of the skin and subcutaneous tissues or nail changes can develop after long-term treatment with hydroxyurea. Painful leg ulcers in association with hydroxyurea have only rarely been reported. We present a report of a 52-year-old patient with essential thrombocythemia suffering from painful leg ulcers 3 years after starting therapy with hydroxyurea. We decided to treat the leg ulcers following a modern phase-adapted wound-healing strategy and continued hydroxyurea therapy until complete healing of the ulcers. In conclusion, cutaneous ulceration of the leg is one adverse effect in patients with essential thrombocythemia during hydroxyurea therapy. Healing does not necessarily require discontinuation of the drug. Therefore, therapists should first optimize a conservative and systematic wound-healing strategy. If these interventions fail, discontinuation of hydroxyurea therapy is advisable.

Micromanipulation of single cells from tissue imprints is an alternative to laser-assisted microdissection.

The characterization and analysis of single cells by molecular biological methods such as the polymerase chain reaction (PCR) is of increasing interest in biomedical research. Different techniques have been developed to obtain single cells from solid tissue. Currently, the most frequently used technique is laser-assisted microdissection (LAM). However, LAM of tissues cannot exclude contamination of the targeted cells by underlying cell fragments. Moreover, this technique can only be performed if a laser microscope is available. Thus, we developed a method to obtain single cells of fresh solid tissue by the simple technique of tissue imprints. After immunostaining of the imprints, single cells were transferred to a reaction tube using a 27-gauge needle guided by a mechanical micromanipulator. Consequently, we used these cells in a single cell PCR.

C-KIT expression in primary cutaneous T-cell lymphomas.

BACKGROUND: Mutations of the stem cell factor receptor C-KIT play a major pathogenetic role in the development of different malignant diseases like human mastocytosis, myeloproliferative disorders, gastrointestinal stromal tumors, acute myelogenous leukemia, and sinonasal lymphomas. Furthermore, the expression of C-KIT has been described in Hodgkin's disease and nodal CD30+ anaplastic large cell lymphomas (ALCLs). As it is possible to inhibit C-KIT by innovative kinase inhibitors like STI571, it may be an attractive target for new therapeutical approaches. Therefore, we screened more than 50 different types of cutaneous T-cell lymphomas (TCLs) for the presence of C-KIT. Immunohistochemical stainings were performed on paraffin-embedded tissue sections using a polyclonal rabbit anti-human C-KIT antibody. Naphtol-ASD-chloroacetate esterase (NASDCE)-control stainings were performed on every positive sample to distinguish C-KIT-positive lymphoma cells from C-KIT-positive mast cells. RESULTS: We found weak expression of C-KIT in seven of 18 patients with primary cutaneous CD30+ ALCL, two of eight patients with primary cutaneous pleomorphic TCL, six of 18 patients suffering from mycosis fungoides, and three of five patients with Sezary's syndrome. Generally, only a very small population of the lymphoma cells expressed C-KIT. This finding indicates a difference to the systemic variant of CD30+ ALCL. The potential use of C-KIT targeting new therapeutical approaches is therefore discussed critically, because C-KIT expression is very rare in all investigated types of primary cutaneous lymphoma.

CpG motifs are efficient adjuvants for DNA cancer vaccines.

DNA vaccines can induce impressive specific cellular immune response (IR) when taking advantage of their recognition as pathogen-associated molecular patterns (PAMP) through Toll-like receptors (TLR) expressed on/in cells of the innate immune system. Among the many types of PAMP, immunostimulatory DNA, so-called CpG motifs, was shown to interact specifically with TLR9, which is expressed in plasmacytoid dendritic cells (pDC), a key regulatory cell for the activation of innate and adaptive IR. We now report that CpG motifs, when introduced into the backbone, are a useful adjuvant for plasmid-based DNA (pDNA) vaccines to induce melanoma antigen-specific protective T cell responses in the Cloudman M3/DBA/2 model. The CpG-enriched pDNA vaccine induced protection against subsequent challenge with melanoma cells at significantly higher levels than its parental unmodified vector. Preferential induction of an antigen-specific, protective T cell response could be demonstrated by (i) induction of antigen-dependent tumor cell protection, (ii) complete loss of protection by in vivo CD4+/CD8+T cell- but not NK cell-depletion, and (iii) the detection of antigen-specific T cell responses but not of relevant NK cell activity in vitro. These results demonstrate that employing PAMP in pDNA vaccines improves the induction of protective, antigen-specific, T cell-mediated IR.

Differential downregulation of endoplasmic reticulum-residing chaperones calnexin and calreticulin in human metastatic melanoma.

Characterization of the molecular basis of tumor recognition by T cells has shown that major histocompatibility complex (MHC) class I molecules play a crucial role in presenting antigenic peptide epitopes to cytotoxic T lymphocytes. MHC class Ia downregulation has been repeatedly described on melanoma cells and is thought to be involved in the failure of the immune system to control tumor progression. Proper assembly of MHC class I molecules is dependent on several cofactors, e.g. the chaperones calnexin and calreticulin residing in the endoplasmic reticulum. Alterations in the expression of these chaperones may have important implications for MHC class I assembly, peptide loading, and presentation on the tumor cell surface and thus may contribute to the immune escape phenotype of tumor cells. In the present study, we compared melanoma lesions representing different stages of tumor progression with regard to the expression of calnexin and calreticulin in tumor cells by means of immunohistochemistry. Metastatic melanoma lesions exhibited significant downregulation of calnexin as compared to primary melanoma lesions. In contrast, chaperone calreticulin was expressed in melanoma cells of primary as well as of metastatic lesions. Our data suggest that chaperone-downregulation, particularly calnexin-downregulation, may contribute to the metastatic phenotype of melanoma cells in vivo. Consistently, conserved chaperone expression in metastatic melanoma lesions may be a useful criterion for selection of patients for treatment with T cell-based immunotherapies.

[Options for debridement in the therapy of chronic wounds]. / Optionen des Debridements in der Therapie chronischer Wunden.

Debridement is defined as the removal of non-vital tissue from wounds. In chronic wounds, debridement means the elimination of necrosis as well as the clearing away of wound dressings, foreign bodies, and other non-vital substances. Sufficient debridement represents one basic prerequisite for a non-delayed wound-healing process. In addition to treating the causal factors for delayed wound healing, debridement should be the first step in an adequate phase-adapted wound-bed preparation for chronic wounds. This report aims to review the different options available in the execution of debridement in chronic wounds. The following therapeutic measures are available for the debridement of chronic wounds: surgery, maggot therapy, laser, ultrasound, hydrotherapy, wet-to-dry method, autolysis, proteolytic enzymes, osmotic or chemical debridement. There is no single correct debridement for all patients! Patients are individuals and therefore different methods of debridement may make sense. Which therapeutic option is chosen is determined by multiple factors. Moreover, depending on the wound milieu, the successive application of different methods can be useful.

[Imiquimod for the topical treatment of focal epithelial hyperplasia (Heck disease) in a child]. / Imiquimod-Creme für die topische Behandlung der fokalen epithelialen Hyperplasie (M. Heck) bei einem Kind.

Focal epithelial hyperplasia (FEH) or Heck disease is a rare skin disease caused by human papilloma viruses (HPV). The case of a 9-year old boy is presented to demonstrate the successful treatment of massive FEH with 5% imiquimod cream. Initially, the patient had noticed several separate papules, which spread and developed into multiple peri- and intraoral papillomatous nodules. The lesions were treated with carbon dioxide laser destruction. However, multiple, skin-coloured papillomatous nodules were found on the tongue, buccal mucosa and lips 1.5 years later. Treatment with imiquimod was initiated, because the patient suffered tremendously from the disease. 5% imiquimod cream was applied 3 times per week. Regression of lesions was obvious after 1 month of treatment. Complete clearance was achieved after 2 additional months of treatment and no recurrence was detected over a follow-up period of 5 months. Our case points out the clinical value of imiquimod for the non-traumatic and almost painless therapy of HPV-induced skin diseases in children.

Course of HIV disease does not depend on risk group: 7.5-year follow-up in 296 patients.

Several reports on the course and progression of HIV infection with regard to the mode of transmission have yielded different results. Since comparative studies of homosexuals and intravenous drug abusers are rare, we wanted to determine the course of HIV infection and survival in an HIV-seropositive patient cohort in a retrospective study at a single tertiary care referral centre. Two hundred and ninety-six HIV patients infected by one of the three predominant modes of HIV transmission (male homosexual contact, n = 193; injecting drug use, n = 64; or heterosexual contact, n = 39) were analysed. Using multivariate Cox regression analysis the overall survival did not depend on the patient's risk group. However, with regard to antiretroviral therapy, monotherapy was shown to shorten patients' lives, whereas only triple-therapy led to prolonged survival, when treatment usage was incorporated as a time-updated covariate. The course of HIV disease in a cohort study outside of clinical trials seems not to depend on the mode of transmission.

Immunoproteasome subunits LMP2 and LMP7 downregulation in primary malignant melanoma lesions: association with lack of spontaneous regression.

Recently, expression of the immunoproteasome subunits low molecular protein (LMP) 2 or LMP7 was shown to reduce the presentation of certain major histocompatibility complex (MHC) class I-restricted tumour peptide epitopes in renal cell carcinoma and melanoma cells. This may provide the tumour cells with an immune escape mechanism. To test the relevance of this hypothesis, we have taken advantage of the fact that spontaneous regression of human primary melanoma is thought to be the result of a successful peptide-specific cellular immune response in vivo. Immunohistochemical staining with anti-LMP2 and anti-LMP7 xenoantibodies showed a significantly higher expression of these immunoproteasome subunits in primary melanoma lesions exhibiting histological signs of tumour regression than in primary melanoma lesions without regression phenomena. In spontaneously regressing melanoma lesions, LMP2 and LMP7 expression was significantly associated with the presence of tumour-infiltrating lymphocytes. Our results are compatible with the possibility that the expression of the immunoproteasome subunits LMP2 and LMP7 rather than their downregulation in melanoma cells is associated with the presence of a successful anti-melanoma immune response.

Association of TAP1 downregulation in human primary melanoma lesions with lack of spontaneous regression.

Spontaneous regression of primary melanoma lesions is regarded as the result of the recognition of melanoma-associated antigen (MAA)-derived peptides by cytotoxic T-lymphocytes and destruction of melanoma cells. The transporter associated with antigen processing (TAP1/2) is likely to play a crucial role in this process since it loads antigen peptides onto MHC class I molecules. To determine the impact of TAP defects on the spontaneous regression of melanoma lesions, we have compared the expression of TAP1 and TAP2 in 39 primary melanoma lesions exhibiting clinical and histological signs of tumour regression and in 35 primary melanoma lesions without regression phenomena. TAP1 expression was significantly associated with regression of melanoma lesions, since the staining pattern with anti-TAP1 antibody was positive in 38 of the 39 lesions exhibiting regression phenomena and in only 24 of the 35 lesions without histopathological signs of tumour regression. In the latter group, six lesions were stained with a heterogeneous pattern and five with a negative pattern. Furthermore, in lesions with a heterogeneous staining pattern, a clear association was found between TAP1 expression in melanoma cells and the presence of tumour-infiltrating lymphocytes. These results suggest that TAP1 plays an important role in the MAA-specific cytotoxic T-lymphocyte response, which has been suggested to underlie the spontaneous regression of primary melanoma.

Optimizing the patch-test concentration of para-tertiary-butylcatechol: results of a prospective study with a dilution series.

Para-tertiary-butylcatechol (PTBC), which has long been patch tested internationally at 1% and 0.5% concentrations, was recently shown to induce patch-test sensitization at the 1% patch-test concentration. In order to determine a safe patch-test concentration, we performed a prospective study with lowered patch-test concentrations. A dilution series of PTBC 0.25%, 0.1%, 0.01% and 0.001% (petrolatum, pet.) was tested on the upper back. Additionally, 0.25% PTBC was tested on the left upper arm to allow patients to carry out self-examination daily. Patch tests were read on D1-3, D7, D14 and D21 after patch-test application. Patients who were not able to return for all scheduled readings were telephoned and asked to report any reaction at the patch-test sites. 65 out of the 101 patients included completed the study. A positive patch-test reaction was observed in 4 patients during D1-D3, indicating previous sensitization. 1 patient had a doubtful reaction at D3 reading. Negative patch-test results were noted in 60 patients. None of the patients developed a positive patch-test reaction during the late readings (D7-D21). Thus, patch-test sensitization was not observed in any case. Para-tertiary-butylcatechol 0.25% pet. is recommended for patch testing internationally.

HIV-positive men differ in immunologic but not catecholamine response to an acute psychological stressor.

Acute psychological stress in humans induces sudden alterations in catecholamine plasma levels and in the distribution of peripheral blood lymphocytes. The purpose of this study was to investigate whether infection with the human immunodeficiency virus (HIV) had an impact on the psychoneuroimmunologic axis. Twelve asymptomatic HIV-positive homo- or bisexual men (CD4 cell counts>400/mm3) and 13 healthy HIV-negative control subjects were exposed to an acute psychological stressor consisting of a 30 min semi-structured stress interview using emotion- and client-centered conversation techniques surrounded by two relaxation periods. Changes in neuroendocrine and immunological, as well as cardiovascular parameters, were intermittently monitored. Under the influence of the stressor plasma norepinephrine (NE) levels increased significantly in HIV-positive patients (+30.6%; p<0.05) and in HIV-negative individuals (+17.5%; n.s.). These changes were paralleled by significant increases in blood pressure and heart rate. Plasma cortisol decreased continuously from initially high levels during the entire experimental setting in both groups, compatible with an adaption reaction. Concomitantly, WBC and neutrophilic granulocytes increased significantly in HIV-negative subjects, while they were blunted in HIV-positive patients. Interestingly, NK cell increases were significantly higher during the stress experiment in HIV-positive patients than in HIV-negative controls. CD4+ and B cell counts remained unaffected by the stressor. These results suggest that catecholamine secretion induced by an acute psychological stressor is preserved in HIV-infected patients with the responsiveness of WBC and neutrophilic granulocytes being diminished, while NK cells showed an increased response.

Double-blind, randomized, placebo-controlled phase III trial of oxymetholone for the treatment of HIV wasting.

BACKGROUND: Despite highly active antiretroviral therapy (HAART), chronic involuntary weight loss still remains a serious problem in the care of HIV patients. Various alterations in energy metabolism and endocrine regulation have been found to cause loss of lean body mass (LBM) and body cell mass (BCM). Previous studies in HIV-positive men undergoing androgen replacement therapy or treatment with recombinant growth hormone (rGH) have shown partial restoration of LBM, but these treatments have largely been ineffective in eugonadal individuals. STUDY DESIGN: Double-blind, randomized, placebo-controlled trial of 89 HIV-positive women and men with wasting assigned to the anabolic steroid oxymetholone [50 mg twice (BID) or three times daily (TID)] or placebo for 16 weeks followed by open-label treatment. STUDY ENDPOINTS: Body weight, bioimpedance measurements, quality of life parameters and appetite. RESULTS: Oxymetholone led to a significant weight gain of 3.0 +/- 0.5 and 3.5 +/- 0.7 kg in the TID and BID groups, respectively (P < 0.05 for each treatment versus placebo), whereas individuals in the placebo group gained an average of 1.0 +/- 0.7 kg. Body cell mass increased in the oxymetholone BID group (3.8 +/- 0.4 kg; P < 0.0001) and in the oxymetholone TID group (2.1 +/- 0.6 kg; P < 0.005), corresponding to 12.4 and 7.4% of baseline BCM, respectively. Significant improvements were noted in appetite and food intake, increased well-being and reduced weakness by self-examination. The most important adverse event was liver-associated toxicity. Overall, 35% of patients in the TID, 27% of patients in the BID oxymetholone group and no patients in the placebo group had a greater than five times baseline increase for alanine aminotransferase during the double-blind phase of the study. CONCLUSIONS: Oxymetholone can be considered an effective anabolic steroid in eugonadal male and female patients with AIDS-associated wasting. The BID (100 mg/day) regimen appeared to be equally effective as the TID (150 mg/day) regimen in terms of weight gain, LBM and BCM and was associated with less, but still significant liver toxicity.

Mizolastine provides effective symptom relief in patients suffering from perennial allergic rhinitis: a double-blind, placebo-controlled study versus loratadine.

BACKGROUND: Mizolastine is a nonsedating H1 histamine receptor antagonist with additional antiallergic properties currently marketed in Europe for the treatment of seasonal and perennial allergic rhinitis (PAR) and urticaria. OBJECTIVE: This multicenter, randomized, double-blind, parallel-group study was conducted to evaluate the efficacy and safety of mizolastine in PAR compared with loratadine and placebo. METHODS: After a 1-week placebo run-in period, 428 adult PAR patients received placebo (146 of 428), mizolastine 10 mg (141 of 428), or loratadine 10 mg (141 of 428) once daily for 28 days. Symptoms were evaluated by patients and physicians using a total nasal score, evaluating itching, rhinorrhea, nasal blockade, and sneezing severity. RESULTS: Mizolastine treatment resulted in a significantly greater decrease in patient-rated total nasal score than placebo after 2 weeks (D14; -42%, P < 0.001) and at the end of the treatment period (-46%, P = 0.01), and significantly greater than that observed with loratadine at D14 (P = 0.031). No significant difference in change in total nasal score was observed between loratadine and placebo at 2- and 4-week visits. The global safety was satisfactory and the incidence of adverse events was similar in the three treatment groups. CONCLUSIONS: Mizolastine provides effective symptom relief in PAR together with a satisfactory safety profile. Improvement with mizolastine was significantly greater than placebo throughout the study despite a large placebo effect. Also mizolastine's effects were greater those observed with loratadine after 2 weeks of treatment.

Lymphocytes proliferate in blood and lymph nodes following interleukin-2 therapy in addition to highly active antiretroviral therapy.

BACKGROUND: Substantial redistribution of lymphocytes occurs upon the initiation of highly active antiretroviral therapy (HAART) and immune-based HIV therapies. OBJECTIVE: To evaluate the relative contribution of apoptosis and proliferation to changes in lymphocyte populations in peripheral blood and lymph node resulting from interleukin-2 (IL-2) therapy in patients receiving stable HAART. METHODS: Lymphocyte apoptosis was analyzed on various subtypes using fluorescence activated cell sorting with an annexin-V antibody in peripheral blood and by the TUNEL (terminal uridine nucleotide end labelling) method in corresponding lymph node sections. Lymphocyte proliferation was evaluated using an antibody against the cell cycle-associated marker Ki-67 (MIB-1) in peripheral blood and lymph nodes. RESULTS: A transient increase in apoptosis was seen in peripheral blood and lymph nodes during a cycle of subcutaneous IL-2. A pronounced proliferative effect of IL-2 (from 6.4% of total lymphocytes in patients only treated with HAART to 23.4% in those treated with HAART + IL-2) was detected in peripheral blood, affecting the CD4, CD8 and CD16/56 subsets to a similar extent. Remarkably, the proliferative effect also occurred in lymphoid tissues. While the lymph node structure gradually disintegrated over 24 months in some individuals, the amount of proliferating lymphocytes, including CD4 cells, B cells and follicular dendritic cells, greatly increased upon IL-2, while HIV RNA load in lymph nodes remained unaffected. CONCLUSION: These results show that IL-2 leads to lymphocyte proliferation in peripheral blood and lymph nodes without an impact on viral load in lymphoid tissue. These results have important implications for attempts to reconstitute the immune system in HIV disease.