Long-term (6 and 12 months) follow-up of two prospective, randomized, controlled phase III trials of photodynamic therapy with BF-200 ALA and methyl aminolaevulinate for the treatment of actinic keratosis.

BACKGROUND: Two phase III trials of photodynamic therapy (PDT) with BF-200 ALA, a recently approved nanoemulsion formulation of 5-aminolaevulinic acid (ALA) demonstrated high clearance rates in mild-to-moderate actinic keratosis (AK). The comparison to a registered methyl aminolaevulinate (MAL) cream demonstrated significantly superior total patient clearance rates. OBJECTIVES: To evaluate long-term efficacy and safety of PDT for AK 6 and 12 months after the last PDT with BF-200 ALA, MAL or placebo. METHODS: The follow-up phase (FUP) was performed with patients of two phase III studies. Both studies compared BF-200 ALA with placebo, one of the studies additionally with MAL. Overall recurrence rates and various subgroups (light source, lesion severity, lesion location, complete responders after first PDT) were assessed 6 and 12 months after the last PDT. RESULTS: Recurrence rates were similar for BF-200 ALA and MAL, with a tendency to lower recurrence rates for BF-200 ALA. The proportion of patients who were fully cleared during PDT and remained completely clear for at least 12 months after PDT were 47% for BF-200 ALA (both studies) and 36% for MAL treatment. The subgroup that was illuminated with narrow wavelength LED lamps reached 69% and 53% for BF-200 ALA (both studies, respectively) and 41% for MAL. No safety concerns were reported. CONCLUSIONS: The FUP data confirmed the high efficacy and safety of PDT with BF-200 ALA. The slightly lower recurrence rates after BF-200 ALA treatment compared with MAL treatment enhanced the better treatment outcome due to the significantly superior efficacy.

Photodynamic therapy with BF-200 ALA for the treatment of actinic keratosis: results of a multicentre, randomized, observer-blind phase III study in comparison with a registered methyl-5-aminolaevulinate cream and placebo.

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) or its methylester [methyl-5-aminolaevulinate (MAL) or 5-amino-4-oxopentanoate] was recently ranked as first-line therapy for the treatment of actinic keratosis (AK) and is an accepted therapeutic option for the treatment of neoplastic skin diseases. BF-200 ALA (Biofrontera Bioscience GmbH, Leverkusen, Germany) is a gel formulation of ALA with nanoemulsion for the treatment of AK which overcomes previous problems of ALA instability and improves skin penetration. OBJECTIVES: To evaluate the efficacy and safety of PDT of AKs with BF-200 ALA in comparison with a registered MAL cream and with placebo. METHODS: The study was performed as a randomized, multicentre, observer-blind, placebo-controlled, interindividual trial with BF-200 ALA, a registered MAL cream and placebo in a ratio of 3:3:1. Six hundred patients, each with four to eight mild to moderate AK lesions on the face and/or the bald scalp, were enrolled in 26 study centres in Germany, Austria and Switzerland. Patients received one PDT. If residual lesions remained at 3months after treatment, PDT was repeated. RESULTS: PDT with BF-200 ALA was superior to placebo PDT with respect to patient complete clearance rate (78·2% vs. 17·1%; P<0·0001) and lesion complete clearance rate (90·4% vs. 37·1%) at 3months after the last PDT. Moreover, superiority was demonstrated over the MAL cream regarding the primary endpoint patient complete clearance (78·2% vs. 64·2%; P<0·05). Significant differences in the patient and lesion complete clearance rates and severity of treatment-related adverse events were observed for the narrow- and broad-spectrum light sources. CONCLUSIONS: BF-200 ALA is a very effective, well-tolerated new formulation for AK treatment with PDT and is superior to a registered MAL medication. Efficacies and adverse events vary greatly with the different light sources used.

The highly attenuated vaccinia virus strain modified virus Ankara induces apoptosis in melanoma cells and allows bystander dendritic cells to generate a potent anti-tumoral immunity.

Vaccinia virus (VV) has been tested as oncolytic virus against malignant melanoma in clinical trials for more than 40 years. Until now, mainly strains comparable to viral strains used for smallpox vaccination have been probed for anti-tumoral therapy. We have shown recently that the wild-type strain Western Reserve (WR) can interfere with crucial functions of monocyte-derived dendritic cells (DCs). Our aim was to examine whether viral immune evasion mechanisms might be responsible for the ineffectiveness of WR-based vaccination strategies and whether the highly attenuated strain modified virus Ankara (MVA) differs from WR with respect to its possible immunostimulatory capacity after intratumoral injection. Using in vitro experiments, we compared the effect of both strains on melanoma cells and on local bystander DCs. We found that both VV-strains infected melanoma cells efficiently and caused disintegration of the actin cytoskeleton, as shown by fluorescence microscopy. In addition, both VV-strains caused apoptotic cell death in melanoma cells after infection. In contrast to MVA, WR underwent a complete viral replication cycle in melanoma cells. Bystander DCs were consecutively infected by newly generated WR virions and lost their capacity to induce allogeneic T cell proliferation. DCs in contact with MVA-infected melanoma cells retained their capacity to induce T cell proliferation. Immature DCs were capable of phagocytosing MVA-infected melanoma cells. Priming of autologous CD8(+) T cells by DCs that had phagocytosed MVA-infected, MelanA positive melanoma cells resulted in the induction of T cell clones specifically reactive against the model antigen MelanA as shown by enzyme-linked immunospot (ELISPOT) analysis. We conclude that the clinical trials with oncolytic wild-type VV failed probably because of suppression of bystander DCs and consecutive suppression of T cell-mediated anti-melanoma immunity. The attenuated VV-strain MVA facilitates the generation of tumour associated antigen (TAA)-specific T cell response as it is oncolytic for melanoma cells, but non-toxic for DC, and should be a promising candidate for intralesional metastatic melanoma therapy.

Interaction of cutaneous lymphoma cells with reactive T cells and dendritic cells: implications for dendritic cell-based immunotherapy.

BACKGROUND: Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of skin neoplasms that originate from T lymphocytes. An anti-CTCL T-cell immunity has been described but seems to be inefficient to clear CTCL cells. It is not known whether cutaneous dendritic cells (DCs) perpetuate the proliferation of the malignant CTCL cell clone or play a role in the control of this usually slowly progressing disease. OBJECTIVES: To characterize CTCL cell properties in the control of anti-CTCL T cells and to pave the way for a DC-based immunotherapy for CTCL. METHODS: We studied the interaction of a CTCL cell line with DCs and with allogeneic T cells. RESULTS: We found an antigen non-specific capacity of viable but not apoptotic CTCL cells to hamper CD4+ and CD8+ T-cell proliferation in a dose-dependent manner, indicating a suppressive potential of CTCL cells. Both viable and apoptotic CTCL cells were phagocytosed by immature DCs but only apoptotic CTCL cells induced an upregulation of DC maturation markers to a degree which enabled classification of these DCs as semimature. CTCL cells did not respond with proliferation when encountering allogeneic, mature DCs either loaded with CTCL cell material or unloaded, indicating a role for DCs in the induction of anti-CTCL T-cell immunity rather than in perturbation of clonal proliferation. For the loading of DCs with CTCL material lysate seems to be optimal as apoptotic cells were not phagocytosed extensively and necrotic CTCL material induced a partial cellular toxicity in DCs. DCs loaded with CTCL material were cryopreservable without significant loss of DC viability, surface marker expression or allostimulatory activity. CONCLUSIONS: Together, these data argue in favour for a DC-based immunotherapy for CTCL patients and provide an experimental protocol for preparing CTCL cell-loaded DCs.

Leptin concentrations in semen are correlated with serum leptin and elevated in hypergonadotrophic hypogonadism.

Leptin seems to play a role in both food intake and energy balance as well as in the regulation of reproductive features. In order to investigate the regulation of testicular functions by leptin we analysed leptin concentrations in the semen of men with different andrological diseases. It was demonstrated that semen leptin concentrations were inversely correlated with serum testosterone levels and directly with serum leptin concentrations. Furthermore, semen leptin concentrations display only a fraction of serum leptin levels. Semen leptin levels of patients with azoospermia due to hypergonadotrophic hypogonadism, associated with increased follicle-stimulating hormone levels and of high-grade oligozoospermia, were significantly elevated (1.19 +/- 0.46 and 1.09 +/- 0.54 microg l(-1), respectively), while semen levels of leptin in patients with obstructive azoospermia (0.54 +/- 0.41 microg l(-1)) and low-grade oligozoospermia (0.54 +/- 0.34 microg ml(-1)) were comparable with those of normozoospermic men (0.21 +/- 0.21 microg l(-1)). Our data suggest that dysfunction of testicular epithelia as found in hypergonadotrophic hypogonadism and high-grade oligozoospermia with decreased testosterone levels causes elevated spermal leptin concentrations. However, the correlation of semen with serum leptin concentrations indicates that leptin is not actively transported but rather leaks through the blood-testis barrier.

Viral vectors for dendritic cell-based immunotherapy.

Dendritic cells (DCs) constitute a specialised system of antigen-presenting cells with a high capacity to induce and to modulate the immune response against microbial, tumour and self-antigens. New techniques to generate large amounts of DCs together with the molecular identification of human tumour-associated antigens (TAA) have opened new ways for antigen-specific cancer immunotherapies. DCs loaded either with TAA-derived MHC class I-specific synthetic peptides or with whole tumour cell preparations have been used in numerous clinical trials evaluating the efficacy of DCs in patients with cancer. However, the disadvantages of DCs pulsed with synthetic peptides from TAA include the uncertainty regarding the longevity of antigen presentation, the restriction by the patient's haplotype and the relatively low number of known MHC class I and in particular of MHC class II helper cell-related epitopes. Whole tumour cell preparations are difficult to standardise, and they depend on the availability of tumour cells. Thus the utilisation of viral vectors genetically modified to express TAA for the ex vivo transduction of DCs is an attractive alternative to achieve a MHC I- and MHC II-restricted presentation of tumoural antigens. To induce protective anti-tumoural immune response an increasing number of modified viral vectors have been used to transduce DCs. Although high transduction efficacies were reported for several viruses, analysis of the interaction of viral vectors with DCs has revealed several viral mechanisms that interfere with main functions of DCs, dampening somewhat the initial optimism in the field of DC transduction. However, promising results with different vectors have been achieved. In this review we summarise available data and discuss advantages and drawbacks of currently available vectors.

Dendritic cells pulsed with unfractionated helminthic proteins to generate antiparasitic cytotoxic T lymphocyte.

Dendritic cells (DC) are sentinels of immunity. We determined their role in the induction of immunity against alveolar echinococcosis, caused by the larval stage of the cestode Echinococcus multilocularis. Furthermore, we evaluated if unfractionated protein from E. multilocularis (Em-Ag) can be used as loading agent for DC (comparable to unfractionated tumour proteins) in order to generate antiparasitic cytotoxic T lymphocyte (CTL). Interestingly, immature DC did not mature in the presence of 1 microg/ml Em-Ag as analysed by FACS and mixed leucocyte reactions. Yet, their capacity to take up dextran was markedly reduced. Further maturation of immature Em-Ag pulsed DC could be induced by proinflammatory cytokines. These mature DC were slightly better inducers of T cell proliferation when compared with unpulsed mature DC. Importantly, by repetetive stimulation of autologous CD8+ lymphocytes with the Em-Ag pulsed mature DC, we were able to generate specifically proliferating CTL lines. Thus, immunotherapy with ex vivo generated Em-Ag pulsed DC might be of benefit for patients inheriting this incurable disease.

Viral vectors for dendritic cell-based immunotherapy.

Transduction of dendritic cells (DCs) by viral vectors genetically engineered to express tumor-associated antigens (TAAs) or cytokines can produce a high level of transgene expression and is an attractive approach for DC-based immunotherapy. Ex vivo transduction allows the control of DC quality, antigen quantity and site of DC reinjection. This review evaluates the viral vectors currently being developed for use in DC-based immunotherapy.

Interaction of large DNA viruses with dendritic cells.

Dendritic cells (DC) with their unique capacity to prime naïve T cells are crucial in the induction of immunological responses, including anti-tumoral and anti-viral immunity. DC based immunotherapies are thus currently considered a particularly promising approach for cellular immunotherapy. The cloning of tumor associated antigens (TAAs) together with the possibility of manipulating viral genomes by biotechnological techniques has sparked the interest of using genetically modified viruses to transduce DC in order to achieve antigenic expression of TAA with the aim of inducing a protective immune response. An increasing number of modified viral vectors has been designed for gene therapy purposes and consecutively has been used for the ex vivo transduction of DC. It has been shown that viral vectors genetically engineered to express TAA or immune modifiers like cytokines or costimulatory molecules can lead to a high level of transgene expression. Furthermore, these studies have also revealed that viruses have developed several immune evasion mechanisms specifically targeting DC. Therefore, analysing the interactions of viruses with DC is crucial for the development of new viral vectors suitable for the transduction of DC. In this report we describe the interaction of two large DNA viruses, herpes simplex virus type 1 (HSV-1) and vaccinia virus (VV), with DC generated from peripheral blood mononuclear cells.

Poxvirus as a vector to transduce human dendritic cells for immunotherapy: abortive infection but reduced APC function.

Dendritic cells (DC) are potent antigen-presenting cells (APC). Ongoing preclinical and clinical studies exploit this capacity for the immunotherapy of tumors. We tested vaccinia virus (VV) as a vector to transduce human DC. Immature and mature DC were prepared from blood monocytes and infected with (1) recombinant VV expressing GFP to analyze infection rates, virus replication in DC and the effect of infection on DC phenotype and (2) recombinant VV expressing beta-galactosidase (betaGAL) under the control of viral early, intermediate and late promoters to analyze the poxvirusdriven gene expression. While the infection rate in DC was comparable to a permissive fibroblast cell line, viral betaGAL gene expression was limited to early promoters. Genes under the control of virus late promoters were not expressed by VV in DC, indicating an abortive infection. VV infection selectively reduced the surface expression of the costimulatory molecule CD80 and the DC maturation marker CD83 on mature DC while other surface molecules including CD86 and MHC remained unchanged. In line with this finding, there was a pronounced reduction in the capacity of VV-infected DC to stimulate allogeneic or autologous T cells in mixed lymphocyte reactions. Furthermore, VV infection inhibited the maturation of immature DC after exposure to proinflammatory cytokines. These results indicate that VV-derived vectors may have complex effects on their target cells. In the case of DC used for immunotherapy, this may be detrimental to their function as potent APC and particularly their capacity to activate T helper cells.

Dendritic cells containing apoptotic melanoma cells prime human CD8+ T cells for efficient tumor cell lysis.

Dendritic cells (DCs) phagocytose apoptotic influenza-infected monocytes and cross-present influenza antigen to CD8+ T cells, generating a specific CTL response. We investigated whether apoptotic melanoma cells, presented by this mechanism, can lead to CTL responses to tumor-associated antigens and melanoma cells. Apoptotic HLA-A2- MEL-397 melanoma cells were internalized by HLA-A2+ immature monocyte-derived DCs but failed to induce maturation of DCs. When exposed to interleukin 6, interleukin 1beta, tumor necrosis factor alpha, and prostaglandin E2, DCs containing apoptotic MEL-397 cell material matured normally [cross-presenting DCs (cp-DCs)]. Autologous CD8+ CTL lines generated with cp-DCs produced tumor necrosis factor when stimulated with HLA-A2-binding immunodominant peptides from MelanA/MART1 and MAGE-3 (expressed by MEL-397 cells) but not tyrosinase (absent in MEL-397). T2 target cells loaded with the respective peptides were lysed by these cell lines, although to a lesser extent than by CTL lines generated in the presence of mature DCs and peptides from melanoma-associated h antigens. In contrast, lines generated with cp-DCs lysed HLA-A2+ MEL-526 melanoma cells or allogenic HLA-A2+ cp-DCs efficiently, whereas the CTL generated with DCs and peptides had little lytic activity. Mature DCs containing apoptotic tumor cells may thus represent an alternative approach for the therapy of malignant tumors.

T lymphocyte cytokine profile at a single cell level in alveolar Echinococcosis.

The metacestode Echinococcus multilocularis causes a life-threatening disease in humans, named alveolar echinococcosis (AE). A comparative analysis of the early activation marker CD69 on peripheral blood mononuclear cells (PBMC) of patients with AE and healthy controls after in vitro culture with crude E. multilocularis antigen revealed that specific expression of CD69 was induced in CD4(+)T lymphocytes as well as in CD8(+)T lymphocytes. Using a protocol for intracellular staining of cytokines followed by fluorescence activating cell sorting (FACS) analysis, production of interleukin (IL)-2, IL-5 and IL-10 was detected in CD4(+)as well as in CD8(+)lymphocytes. Most notably, there was a definite increase in the expression of IL-10 in CD8(+) lymphocytes from patients with alveolar echinococcosis. The data support an important role of CD8(+) lymphocytes in the long persistence of the metacestode.

Clinical efficacy of and immunologic alterations caused by interferon gamma therapy for alveolar echinococcosis.

Alveolar echinococcosis (AE) is a rare and often fatal disease characterized by a tumorlike expansion of the metacestode Echinococcus multilocularis in the liver. Because of the severe side effects of therapy with benzimidazoles, we treated a patient with recombinant interferon gamma at a dose of 250 microg over a 3-day period once a month. Disease progression was not detected during the observed period of 18 months. Following stimulation with crude Echinococcus antigen, mRNA from interleukin 5 was still detected in peripheral blood mononuclear cells by means of reverse transcriptase polymerase chain reaction analysis, and expression of interleukin 10 in T lymphocytes (as measured by fluorescence-associated cell sorting of intracellular cytokines) was elevated. These results indicate that bolus therapy with interferon gamma has some clinical effect but does not result in a change in the T helper 2 lymphocyte-dominated immune response to this parasite.

IL-5 expressed by CD4+ lymphocytes from Echinococcus multilocularis-infected patients.

IL-5 is a major factor inducing differentiation of B lymphocytes into immunoglobulin-producing cells as well as a main regulator of eosinophils. Recently, we have shown that peripheral blood mononuclear cells (PBMC) from patients with alveolar echinococcosis (AE) express IL-5 mRNA after stimulation with crude Echinococcus multilocularis (E.m.) antigen. To characterize the observed response in lymphocyte subpopulations, we cultured patients' PBMC in the presence of E.m. crude antigen for 18 h. PBMC were separated from seven patients by fluorescence-activated cell sorting (EPICSorter) into CD4+ and CD8+ subpopulations and from an additional seven patients by magnetic cell sorting (MACS) into CD4+, CD8+ and the CD4+/CD8+ depleted fractions. mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) for the cytokines IFN-gamma, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, as well as for beta-actin as control. IL-4 and IFN-gamma expression was positive in all of the patients in the stimulated CD4+ subgroup. IL-5 mRNA expression was detected in eight out of 14 CD4+ samples (58%) and not observed in the other subpopulations, or the unstimulated and healthy controls. Co-expression of other Th2 cytokines in the eight patients expressing IL-5 mRNA was found in five patients for IL-3 and in seven for IL-10. Expression of IL-5 and both Th2 cytokines (IL-3 and IL-10) was only observed in patients judged as critically ill. Out of the six patients who were regarded as cured after radical operation or as stabilized with or without chemotherapy, only two expressed IL-5. Out of those eight patients considered as critically ill, six expressed IL-5 mRNA and five of these co-expressed IL-3 and IL-10. Thus, we conclude that specific antigenic challenge of PBMC from patients with active or previous AE induces an IL-5 response of CD4+ lymphocytes. The expression of Th2-type interleukin mRNA is significantly more frequent in patients clinically judged as progressive. Furthermore, IgE was elevated only in patients regarded as critically ill (six out of eight). In none of the patients were eosinophils elevated. These data support a Th2-type immune response in patients with chronic E. multilocularis infection.

Prediction of accelerated cure in Plasmodium falciparum malaria by the elevated capacity of tumor necrosis factor production.

Cytokine regulation was compared in three groups of Gabonese patients with Plasmodium falciparum malaria before and after therapy; adults with uncomplicated malaria, children with uncomplicated malaria, and children with severe malaria. Plasma levels of tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-8, TNF receptors (TNF R), and the TNF/TNF R ratios were significantly higher in severe malaria compared with uncomplicated malaria. High plasma levels of all immunoregulatory molecules were associated with slow cure after therapy. In all patients, phytohemagglutinin-induced cytokine production was depressed on admission compared with convalescence. A significant difference was the higher TNF production capacity in patients with severe malaria on day 2 and day 5 compared with that in patients with uncomplicated malaria. In contrast to IL-6 and IL-8, a high TNF production capacity during the acute phase of malaria predicted a rapid clinical and parasitologic cure in the patients. These findings illustrate the dual role of TNF in the protection and pathology of malaria.

Quinine plus clindamycin improves chemotherapy of severe malaria in children.

In a randomized trial, a 4-day quinine-clindamycin regimen was compared with the standard 7-day quinine regimen for 100 Gabonese children (50 children in each group) with severe Plasmodium falciparum malaria. In each group, only one patient died. Parasite clearance and fever clearance times were significantly shorter in the quinine-clindamycin group (P = 0.03 and P = 0.01, respectively) than in the quinine group, and significantly more recurring fever episodes occurred in the quinine group than in the quinine-clindamycin group shortly after initial fever clearance and parasite clearance (P < 0.001).

Malaria epidemiology in the province of Moyen Ogoov, Gabon.

In the course of epidemiological and immunological baseline studies parasitological surveys were conducted, in 1992, in three localities situated in our near rain forest in the area of Lambaréné, Gabon, western Central Africa. Anopheles gambiae s.s. and A. funestus are considered to be the main vectors of malaria. The three localities represent strata with obvious differences in the intensity of malaria transmission. The lowest parasite rates were recorded in the village around the Albert-Schweitzer-Hospital where environmental sanitation and easy access to diagnostic and therapeutic facilities afford a fair measure of malaria control. The villages of Bellevue and Tchad show a much higher prevalence of Plasmodium falciparum, followed by P. malariae and P. ovale. In all three villages parasite rates and geometric mean parasite densities of P. falciparum showed the age pattern typical for areas with stable, hyperendemic malaria. Analysis by season showed the period of the long rains to be the epidemiologically calmest while the dry season and even more the short rainy season produced an increase of parasite rates and densities. In Tchad, the most affected of the three villages, the parasite rates in female adults were significantly lower than in male adults. This was accompanied by lower parasite densities in female adults.

Comparison of micronized halofantrine with chloroquine-antibiotic combinations for treating Plasmodium falciparum malaria in adults from Gabon.

Multidrug resistance of Plasmodium falciparum is spreading throughout Africa. In Lambarene, Gabon where chloroquine-resistant malaria is prevalent, a randomized comparative trial with three regimens for treating P. falciparum malaria in adults was performed. One hundred two patients evaluated received either a new micronized formulation of halofantrine (8 mg/kg every 6 hr in three doses) (group H) or chloroquine (25 mg/kg for a 48-hr period) plus clindamycin (5 mg/kg every 12 hr in six doses) (group CC1), or chloroquine (as above) plus doxycycline (2 mg/kg every 12 hr in six doses) (group CD). All treatment regimens were well-tolerated. In group H, 100% of the patients were cured, and in group CC1, 97% of the patients were cured by day 28 of follow-up. In group CD, a significantly lower cure rate of 75% (P < 0.01) and a slower parasite clearance was observed, but only low grade (RI) resistance occurred.