TNF-alpha production-enhancing activity of 2-(1-adamantylamino)-6-methylpyridine (AdAMP) in cultures of human normal and neoplastic cells.

The purpose of this study was to determine the TNF-alpha-stimulatory effect of a novel immunomodulator 2-(1-adamantylamino)-6-methylpyridine (AdAMP) on normal and neoplastic human cells. In a panel of several human ovarian cancer cell lines, almost half of them spontaneously secreted significant amounts of TNF-alpha. When incubated with AdAMP, a 3-fold enhancement of TNF-alpha production by cells was observed. Furthermore, the phorbol myristic acetate ester (PMA)-induced release of TNF-alpha in cultures of U937 cells was increased in the presence of AdAMP. Primary monocytes isolated from peripheral blood did not respond to AdAMP. Although cytokine release was not triggered in human peripheral blood monocytes, AdAMP co-stimulated these cells to produce TNF-alpha and IL-8 during incubation with lipopolysaccharide (LPS). No effect of AdAMP was found on IL-1beta and IL-6 production by monocytes. In cultures of peripheral blood T lymphocytes, AdAMP significantly decreased the adhesion of these cells to matrix proteins in an in vitro assay. The results suggest that AdAMP, as a stimulator of cytokine secretion, may have potential application in tumor therapy.

N-acetylphenylisoserinates of Lactarius sesquiterpenoid alcohols--cytotoxic, antiviral, antiproliferative and immunotropic activities in vitro.

In the present study a new group of derivatives of both paclitaxel and Lactarius sesquiterpenes, the N-acetylphenylisoserinates of Lactarius sesquiterpenoid alcohols, was tested for antiviral, cytotoxic, antiproliferative and immunotropic activities in vitro. Among the 13 compounds tested two, isolactarorufin 8-[ N-acetyl-(2' R,3' S)-3'-phenylisoserinate] and 3-O-ethylfurandiol 8-[ N-acetyl-(2' R,3' S)-3'-phenylisoserinate] inhibited Herpes simplex virus type 1 (HSV-1) replication at low cytotoxic concentrations. Selectivity indices were: > 12.2 and > 106.4, respectively. Both compounds decreased also the number of cell divisions. Mitotic indices of the cells submitted to these compounds were: 96/2000 and 48/1000, respectively, in comparison with control (169/2000). It seems that the antimitotic action of the compounds may be associated with their antiviral activity. Moreover, isolactarorufin 8-[ N-acetyl-(2' R,3' S)-3'-phenylisoserinate] inhibited PHA-induced T lymphocyte proliferation.

Immunomodulatory activity and influence on mitotic divisions of N-benzoylphenylisoserinates of Lactarius sesquiterpenoid alcohols in vitro.

Six N-benzoylphenylisoserinates of Lactarius sesquiterpenoid alcohols, which previously showed antiviral activities, were tested for their biological properties. Their influence on the mitotic division of the cells and on selected immunological parameters, e. g., T and B lymphocyte proliferation and synthesis of the cytokines: interleukin 2 (IL-2), tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) was assessed in vitro. All of the tested compounds significantly decreased the number of cell divisions. It appears that their influence on cellular divisions may be associated with anti-HSV activity. Moreover, one compound - isolactarorufin 8-epi-[N-benzoyl-(2' R,3' S)-3'-phenylisoserinate] significantly inhibited T lymphocyte proliferation and the synthesis of all tested cytokines.

Cytotoxic, antiviral (in-vitro and in-vivo), immunomodulatory activity and influence on mitotic divisions of three taxol derivatives: 10-deacetyl-baccatin III, methyl (N-benzoyl-(2'R,3'S)-3'-phenylisoserinate) and N-benzoyl-(2'R,3'S)-3'-phenylisoserine.

The aim of this study was to evaluate cytotoxic, antiviral (in-vitro and in-vivo) and immunomodulatory activity, as well as the influence on mitotic division, of three taxol derivatives representing modified parts of its molecule: 10-deacetyl-baccatin III, methyl (N-benzoyl-(2'R,3'S)-3'-phenylisoserinate) and N-benzoyl(2'R,3'S)-3'-phenylisoserine. The cytotoxicity of the compounds, assessed by the formazane method, was relatively low, with a 50% cytotoxic concentration (CC50)>500 microg mL-1. Moreover, all tested compounds inhibited Herpes simplex type 1 virus (HSV-1) replication in non-cytotoxic concentrations in-vitro. Selectivity indices were in the range 9.5-46.7. Anti-HSV-1 activity of the compounds may be associated with their influence on mitotic division. All of the compounds decreased the number of cell divisions. Mitotic indices ranged from 40/1000 (4.0%) to 62/1000 (6.2%). One compound, 10-deacetyl-baccatin III, influenced the growth of tumours induced in mice by infection with Moloney murine sarcoma virus. The effect of the tested compounds on T lymphocyte proliferation was evaluated by measurement of the activity of tritiated thymidine incorporated into DNA of dividing cells. One compound, methyl (N-benzoyl-(2'R,3'S)-3'-phenylisoserinate), inhibited T lymphocyte proliferation. This paper demonstrates that modified parts of the taxol molecule possess various types of biological activity in-vitro and in-vivo. Further experiments, focused on revealing their mechanisms of action, are necessary.

New insights into the possible role of bacteriophages in host defense and disease.

BACKGROUND: While the ability of bacteriophages to kill bacteria is well known and has been used in some centers to combat antibiotics - resistant infections, our knowledge about phage interactions with mammalian cells is very limited and phages have been believed to have no intrinsic tropism for those cells. PRESENTATION OF THE HYPOTHESIS: At least some phages (e.g., T4 coliphage) express Lys-Arg-Gly (KGD) sequence which binds beta3 integrins (primarily alphaIIbbeta3). Therefore, phages could bind beta3+ cells (platelets, monocytes, some lymphocytes and some neoplastic cells) and downregulate activities of those cells by inhibiting integrin functions. TESTING THE HYPOTHESIS: Binding of KGD+ phages to beta3 integrin+ cells may be detected using standard techniques involving phage - mediated bacterial lysis and plaque formation. Furthermore, the binding may be visualized by electron microscopy and fluorescence using labelled phages. Binding specificity can be confirmed with the aid of specific blocking peptides and monoclonal antibodies. In vivo effects of phage - cell interactions may be assessed by examining the possible biological effects of beta3 blockade (e.g., anti-metastatic activity). IMPLICATION OF THE HYPOTHESIS: If, indeed, phages can modify functions of beta3+ cells (platelets, monocytes, lymphocytes, cancer cells) they could be important biological response modifiers regulating migration and activities of those cells. Such novel understanding of their role could open novel perspectives in their potential use in treatment of cardiovascular and autoimmune disease, graft rejection and cancer.

Influence of clindamycin, metronidazole and polymyxin B on the expression of cell adhesion molecules stimulated by endotoxin and enterotoxin of Bacteroides fragilis.

The aim of this study was to compare the influence of antimicrobials (clindamycin, metronidazole and polymyxin B) on the expression of adhesion molecules (VCAM-1, ICAM-1 and E-selectin) on the HMEC-1 cell line stimulated by LPS and enterotoxin of B. fragilis. LPS was extracted from two reference: ATCC 43858 and NCTC 11295 and one isolated in our laboratory (W2) enterotoxigenic strains, and one nonenterotoxigenic reference strain--IPL E 323. Enterotoxin preparations (Tox 1 and Tox 2) were isolated from supematant of B. fragilis ATCC 43858 culture and purified. HMEC-1 cell line was stimulated with bacterial preparations at concentration of 10 mg/ml. For measuring the expression of adhesion molecules we used ELISA test. Clindamycin, metronidazole and polymyxin B supressed the ICAM-1 expression when endothelium was stimulated with B. fragilis LPS and augmented ICAM-1 expression by Tox 1 and Tox 2. The expression of VCAM-1 was augmented by antimicrobials when endothelium was stimulated with LPS or enterotoxin preparations. The expression of E-selectin was differentiated.

[Cytokines and growth factors serum level and renal allograft function (preliminary report)]. / Cytokiny i czynniki wzrostu w surowicy u biorców nerki allogenicznej a losy przeszczepu (doniesienie wstepne).

Long-term cyclosporine nephrotoxicity, subclinical rejections are risk factors of chronic allograft nephropathy. In a prospective, randomized study 44 pts. were randomized either to a reduced dose of CyA and daclizumab (group A, n = 22) or to a normal dose of CyA without daclizumab (group B, n = 22). Both groups were treated with MMF and prednisone. Number of rejection episodes was the primary endpoint. The secondary endpoints were renal function; histological parameters related to CyA; serum level of TGF-beta, PDGF-BB, blockade of CD25 molecule and surface expression of CD3, CD4, CD8, CD69, CD11a, CD49d, CD28, CD152 molecules in the subpopulations of T cells in the peripheral blood. A low incidence of clinically suspected rejection episodes were observed (19% in group A and 12.4% in group B; NS). The protocol biopsies at 3 month emerged 7 subclinical rejection episodes (4 in group A and 3 in group B). Serum creatinine level did not differ between examined groups. Chronic histopathologic changes related to CyA progressed significantly at the 3 month biopsies in both groups (with no differences between groups). Serum TGF-beta, PDGF did not differ between groups. Expression of CD25, CD152 molecule was significantly lower in group A than in group B. Immunosuppression regiment with low CyA dose with daclizumab, MMF, prednisone seems to be efficient and safe in low-risk rejection kidney allograft recipients.

Stimulation of TNF-alpha production by 2-(1-adamantylamino)-6-methylpyridine (AdAMP) - a novel immunomodulator with potential application in tumour immunotherapy.

The immunomodulatory effects of a recently synthesized adamantane derivative of aminopyridine - 2-(1-adamantylamino)-6-methylpyridine (AdAMP) - were tested on normal and neoplastic cells in vitro. When incubated with TNF-alpha gene-transduced mouse melanoma cells (B78/TNF), AdAMP significantly enhanced basal production of TNF-alpha by these cells, both by "high" and "moderate" TNF-alpha-producer cells. A similar TNF-alpha production-enhancing effect was observed in cultures of human ovarian carcinoma cells (CAOV1) which spontaneously produce TNF-alpha but not in cultures of tumour cells incapable of TNF-alpha secretion. RT-PCR analysis showed that the enhancement of TNF-alpha production by AdAMP was associated with an increase in TNF-alpha mRNA expression in the treated cells. The results of an electrophoretic mobility shift assay (EMSA) showed that AdAMP significantly activated nuclear factor kappaB (NF-kappaB) in both CAOV1 and B78/TNF cells. The role of NF-kappaB in enhancement of TNF-alpha production was confirmed in experiments in which MG132, an inhibitor of NF-kappaB activation, reversed the effect of AdAMP. Unexpectedly, dexamethasone, a potent antiinflammatory agent and a strong inhibitor of TNF-alpha production in vivo, increased both spontaneous and AdAMP-augmented production of TNF-alpha in in vitro cultures of ovarian carcinoma cells and B78/TNF cells. AdAMP also enhanced TNF-alpha secretion by LPS-induced monocytes. AdAMP-induced augmentation of TNF-alpha production by B78/TNF cells was accompanied by morphological changes in the treated cells and a decrease in their adherence to fibrinogen and collagen IV. In view of these properties, AdAMP seems to be a therapeutically promising compound with potential application as an adjuvant augmenting the efficacy of cancer vaccine-based therapies or in the local treatment of certain tumours.

[Interaction of T lymphocytes with elastin and their significance in immunopathology]. / Interakcje limfocytów T z elastyna i ich znaczenie w immunopatologii.

Recent data indicate that human T lymphocytes can adhere to elastin and respond to co-stimulatory signals of that protein. This reactivity is mediated by non-integrin receptor, elastin binding protein. In addition, another receptor belonging to integrin family may be also involved. T cell interactions with elastin (but not other extracellular matrix proteins) appear to be upregulated in healthy males and at least some patients with vasculitis. Interestingly, statins in pharmacological concentrations strongly and selectively block those interactions. Our data point to the potential role of T cell interactions with elastin in immunopathology of vasculitis and atherosclerosis.

[Stimulation of adhesion molecule expression in human vascular endothelium by Bacteroides fragilis toxins--influence of polymyxin B]. / Stymulacja ekspresji adhezyjnych czasteczek ludzkiego sródblonka naczyniowego przez toksyny Bacteroides fragilis--wplyw polimyksyny B.

The aim of this study was to examine the influence of polymyxin B on the level of expression of adhesion molecules E-selectin, ICAM-1, and VCAM-1 on human vascular endothelium activated with B. fragilis endotoxins or enterotoxin. Lipopolysaccharides were extracted by phenol-water method from one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF) B. fragilis strains. LPS preparations were purified with nucleolytic enzymes and ultracentrifugation. Enteotoxin (BFT) was prepared from the supernatant of reference B. fragilis ATCC 43858 culture by precipitation with ammonium sulphate. BFT preparations were purified with the application of ion-exchange chromatography and hydrophobic chromatography. Adhesion molecule expression on the surface of human vascular endothelial cells (HMEC-1 cell line) was determined after simultaneous stimulation with bacterial compounds at the concentration of 10 micrograms/ml and polymyxin B at the concentration of 20 micrograms/ml. Endothelial cells were activated for 4 hours (E-selectin expression) or for 24 hours (ICAM-1 and VCAM-1 expression). Adhesion molecules were detected in immunoenzymatic test (ELISA) with the use of mouse, monoclonal antibodies against human ICAM-1, VCAM-1, and E-selectin. The results of performed experiments suggest, that polymyxin B changes the level of adhesion molecule expression on human vascular endothelium. This antibiotic causes changes in the expression of endothelial ICAM-1, VCAM-1, and E-selectin during simultaneous stimulation of endothelium with B. fragilis endotoxins or enterotoxin. In the majority of cases the addition of polymyxin B leads to the up-regulation of examined adhesion molecules.

Expression of FasL gene in T cells of renal allograft recipients.

FasL molecule expressed on activated T cells induces apoptosis in Fas-expressing cells. It is possible that apoptosis induced by FasL is involved in the process of allograft destruction brought about by infiltrating T cells. The aim of our study was to evaluate expression of FasL gene in peripheral blood T cells of renal allograft recipients (RAR). We have studied 25 patients: 16 with uneventful stable course (RAR-S) and nine during biopsy proven chronic rejection (RAR-CH). The relative expression of FasL mRNA compared with that of beta-actin was established by semi-quantitative RT-PCR. We have found that FasL gene expression was significantly increased in T cells of RAR-CH compared to RAR-S (P<0.01). Our results suggest that T cell expression of FasL gene is increased during chronic rejection. Therefore, this phenomenon may pay a role in allograft injury associated with that process. Further studies are needed to unravel possible clinical consequences of observed differences in T cell expression of FasL.