Akacid medical formulation induces apoptosis in myeloid and lymphatic leukemic cell lines in vitro and in vivo.

Akacid medical formulation (AMF) is an oligoguanidine that exerts biocidal activity against airborne and surface microorganisms including bacteria, viruses, fungi, and molds, while showing relatively low toxicity to humans. We have previously shown that AMF exerts antiproliferative effects on a variety of solid tumor cell lines. In this study we raised the question whether AMF could also substantially inhibit cell growth or induce apoptosis in cell lines derived from hematologic malignancies such as leukemia or lymphoma. We found that AMF has antiproliferative effects on various hematologic cell lines derived from human leukemia and lymphoma. Additionally, we show that AMF induces apoptosis in leukemia cell lines not only via the extrinsic and intrinsic pathway, but also in a caspase-independent manner. This effect was found also in G0-arrested cells. Finally, in our animal experiments utilizing male nu/nu Balb/c mice we found a significant growth retardation, which was immunohistochemically associated with a significantly lower number of KI67-positive cells and caspase-3 induction in AMF-treated mice.

Interferon-gamma-mediated pathways are induced in human CD34(+) haematopoietic stem cells.

Pro-inflammatory cytokines like interferon-gamma (IFN-gamma) are considered to be important in the development of anaemia of chronic disease (ACD). Both, inhibitory and stimulatory activities of IFN-gamma on erythropoiesis have been observed in vitro earlier. IFN-gamma induces several biochemical pathways in human monocytes, among them neopterin formation by GTP-cyclohydrolase I (GTP-CH I) and tryptophan degradation by the enzyme indoleamine 2,3-dioxygenase (IDO). IDO-mediated tryptophan deprivation efficiently inhibits the growth of proliferating cells and microbes, thus we wanted to examine whether enhanced tryptophan degradation by monocytic precursor cells also suppresses erythropoiesis. Therefore, IFN-gamma-mediated pathways were investigated in human CD34(+) progenitor cells, and effects of IFN-gamma on the proliferative activity of different progenitor subpopulations were studied. Cells were either cultivated in agar-conditioned medium (ACM) or in medium containing erythroid growth factors interleukin-3 (IL-3) and stem cell factor (SCF; EGFCM). Stimulation of CD34(+) cells with IFN-gamma in different doses (either 5000U/ml once or 200 and 400U/ml every other day) induced tryptophan degradation and in parallel also neopterin formation. Unstimulated cells cultured with ACM produced higher amounts of neopterin and kynurenine (all p<0.05). IFN-gamma stimulated higher kynurenine and neopterin formation in cells cultivated in EGFCM, stimulation with 400U IFN-gamma every other day was most effective. IFN-gamma stimulated the growth and proliferation of CFU-E and BFU-E (3-8) in both media. In conclusion, stimulation of haematopoietic stem cells with IFN-gamma activates IDO and neopterin formation, and it also exerts an influence on the proliferation of various stem cell populations.

Sphingosine-1-phosphate receptor type-1 agonism impairs blood dendritic cell chemotaxis and skin dendritic cell migration to lymph nodes under inflammatory conditions.

SEW2871 is a potent sphingosine-1-phosphate receptor type-1 (S1P(1))-selective agonist that induces peripheral lymphopenia through sequestration of lymphocytes into secondary lymphoid organs, similar to the non-selective sphingosine-1-phosphate (S1P) receptor agonist FTY720. FTY720 has been reported to interfere with human dendritic cell (DC) effector functions and both FTY720 and SEW2871 have been shown to modulate murine DC trafficking in vivo. Little is known about the possible effects of SEW2871 on human and murine DC functions. Here, we demonstrate that in contrast to FTY720, SEW2871 does not induce down-regulation of S1P(1) in human DCs and thus does not exert a functional antagonism at S1P(1). Notably, the compound was found to impair chemotaxis of immature and mature human DCs in vitro, possibly by interfering with the activation of p44/p42 and p38 mitogen-activated protein kinase signaling pathways. Comparative FACS analyses show that SEW2871 mediates CD18 down-regulation on mature human DCs. The influence on DC migration could be confirmed with in vivo assays using BALB/c mice in which SEW2871 impairs the migration of CD11c+ DC and CD207+ Langerhans cells (LC) to the draining lymph nodes (LNs) under inflammatory conditions. These results suggest that the S1P-S1P(1) axis might not only control lymphocyte trafficking but also play a pivotal role in DC migration from the skin to LN.

Akacid-medical-formulation, a novel biocidal oligoguanidine with antitumor activity reduces S-phase in prostate cancer cell lines through the Erk 1/2 mitogen-activated protein kinase pathway.

Oligomeric guanidines are highly efficient biocides against a broad spectrum of microorganisms. However, their antitumor effects have not been studied so far. We investigated an antiproliferative effect of Akacid-medical-formulation (AMF), a member of the oligoguanidine family of biocides, against solid cancer cell lines and primary cells by measuring [3H]-thymidine incorporation. Additionally, we examined cell cycle distribution in two AMF-sensitive prostate cancer cell lines (DU-145, LNCaP) using flow cytometry. Finally, the influence of AMF on cell cycle regulatory molecules and intracellular kinase cascade-related signaling molecules was assessed. We found that AMF has variable antiproliferative effects on all tested cells. In DU-145 and LNCaP cells, flow cytometric studies showed a reduction of S-phase with a maximum extent of 24 and 58%, respectively. This was associated with a decrease in expression of cyclin D1, cyclin-dependent kinases 2 and 4, while having varying effects on expression of cyclin E and p27. Additionally, reduced phosphorylation of Erk1 and Erk2 was found, whereas expression of phospho-Akt1 remained unchanged. Herein we report for the first time that AMF exerts potent antiproliferative activity against various malignant cell lines, including those of prostate. We therefore recommend further investigation of the anticancer activity of this biocidal oliguanidine.

Therapeutic effects of gemcitabine on cutaneous manifestations in an Adamantiades-Behçet's disease-like mouse model.

The aim of this work was to study the effects and side effects of gemcitabine (2',2'-difluorodeoxycytidine, dFdC), a pyrimidine synthesis inhibitor, on skin lesions of a herpes simplex virus (HSV)-induced Adamantiades-Behçet's disease (ABD)-like mouse model. For the dose-escalation study, ICR mice were treated intraperitoneally with dFdC over 5 days. For the efficacy study, ICR mice were inoculated with HSV and classified as having ABD according to a revised Japanese classification, and then 18 ABD mice were randomly assigned to placebo, 0.06 or 0.12 microg of dFdC/day over 5 days. Serum levels of interleukin-4 (IL-4), IL-6, IL-10, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) were determined using enzyme-linked immunosorbent assay. After application of 3 microg of dFdC over 5 days, alanine aminotransferase increased (P = 0.032), but all other kidney and liver parameters were unchanged. In ABD mice, 5 days of dFdC treatment with 0.06 or 0.12 microg of dFdC/day resulted in a dose-dependent improvement of cutaneous manifestations by more than 60% (P = 0.017). There was no significant change in cytokine levels, and none of the cytokine levels correlated with response to treatment. Moreover, dFdC shows promising effects to improve cutaneous lesions in the HSV-induced ABD-like mouse model. In this animal model, effects of dFdC on the cytokine profile remained inconclusive.

Possible role of cytokine-induced tryptophan degradation in anaemia of inflammation.

Anaemia of inflammation (AI) is a frequent complication in patients suffering from chronic inflammatory disorders including infections, autoimmune and malignant disease. Cytokine imbalance with a shift towards T-helper (Th)1-type immune response seems to be important in the pathogenesis of this type of anaemia. Interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha may affect the growth and differentiation of erythroid progenitor cells. In macrophages, IFN-gamma strongly induces indoleamine (2,3)-dioxygenase, an enzyme which degrades tryptophan (trp) to kynurenine (kyn). Trp availability is rate limiting for protein biosynthesis and thus cell growth, including erythropoiesis. In this study, trp and kyn concentrations and their relationship to haemoglobin concentrations and to immune activation was examined in 22 patients with AI. Patients with AI presented with lower trp concentrations than healthy controls of similar age, and a significantly higher kyn to trp ratio, suggesting enhanced trp degradation and, because of a positive correlation with neopterin, immune activation. The kyn to trp ratio was inversely correlated to haemoglobin levels. Thus, the limitation of trp availability to erythroid progenitors may be a key mechanism in cytokine-mediated inhibition of erythropoiesis, and the therapeutic modulation of indoleamine (2,3)-dioxygenase and trp levels may be promising targets for AI therapy.

Measurement of intracellular versus extracellular prostate-specific antigen levels in peripheral macrophages: a new approach to noninvasive diagnosis of prostate cancer.

Although prostate-specific antigen (PSA) is a useful screening marker in prostate cancer, it has limited specificity. Previously it was shown that the amount of surface-bound PSA present on circulating macrophages was different between patients with localized prostate cancer and those with metastatic prostate cancer. It was recently demonstrated that intracellular PSA in macrophages can be measured by flow cytometry. In the context of searching for a noninvasive, highly reliable method for prostate cancer diagnosis, we assessed the extent to which extracellular (ie, surface-bound) and intracellular PSA-positive macrophages might differentiate patients with benign versus malignant prostatic disease. In a pilot study, the levels of complexed, surface-bound, and intracellular PSA were measured in 25 patients with elevated serum PSA values and histologically confirmed disease. In this group, no significant differences for serum PSA and complexed PSA levels, respectively, could be detected among patients with benign prostatic hyperplasia, prostatitis, and prostate cancer. Significant differences, however, were detected in intracellular PSA, although not in surface-bound PSA, among the 3 groups of patients. Intracellular PSA was measured prospectively in a second cohort of 189 patients who had a transrectal biopsy because of a serum PSA constellation suspicious for prostate cancer. In the expanded cohort, highly significant differences in intracellular PSA were observed between benign and malignant disease of the prostate, even in patients with serum PSA level<4 ng/mL. Screening of serum PSA alone or in combination with complexed PSA does not clearly distinguish patients with prostate cancer from those with prostatitis or benign prostatic hyperplasia. Macrophage intracellular PSA might represent a more.