Maria Nowaczyk

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

BackgroundWhile 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 hypothesisAt least some phages (e.g., T4 coliphage) express Lys-Arg-Gly (KGD) sequence which binds β3 integrins (primarily αIIbβ3). Therefore, phages could bind β3+ cells (platelets, monocytes, some lymphocytes and some neoplastic cells) and downregulate activities of those cells by inhibiting integrin functions.Testing the hypothesisBinding of KGD+ phages to β3 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 β3 blockade (e.g., anti-metastatic activity).Implication of the hypothesisIf, indeed, phages can modify functions of β3+ 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.

Immunomodulatory action of human recombinant erythropoietin in man

Recent findings suggest that recombinant human erythropoietin (rhEpo) may have an immunomodulating action. We have studied the in vitro and in vivo effects of rhEpo on immune functions in man. Low pharmacological concentrations of the hormone inhibit T-cell activation and proliferation, while higher ones are without that effect. The same Epo concentrations inhibit mitogen- and alloantigen-driven B-cell differentiation and immunoglobulin synthesis and, to a lesser extent, B-cell proliferation. In vivo treatment with rhEpo causes an initial inhibition of T- and B-cell proliferation, but with prolonged administration improved responsiveness is observed. Our data support the notion that rhEpo can regulate immune functions, a fact of potential clinical application.

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.

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 μg 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.

Immunoregulatory factors controlling terminal differentiation of malignant B-lymphocytes

B-lymphocytes of patients with chronic lymphocytic leukemia differentiate in vitro towards plasma cells upon activation with Pokeweed mitogen (PWM) and Cowan. The level of response is much lower than that of normal cells and so is the helper T-cell function. However, concanavalin A-activated suppressor T-cell function of Ig synthesis is normal. Malignant B-cells have a normal capacity to stimulate Ig synthesis in the mixed lymphocyte cultures, but they respond poorly to an allogeneic stimulus. Thymosin (TFX) causes a marked enhancement of PWM-driven differentiation of malignant B-cells.

Polysaccharide and lipid components of Bacteroides thetaiotaomicron lipopolysaccharide as stimulators of endothelial adhesion molecule expression

Bacteroides thetaiotaomicron , a Gram-negative anaerobic rod and a member of the Bacteroides fragilis group (BFG) causes many systemic and local infections in humans, most of which are endogenous and suppurative. The microorganism produces two high-molecular weight, carbohydrate-containing cell-surface antigens, both of which have been implicated as virulence factors for this organism; these consist of lipopolysaccharide (LPS) and capsular polysaccharide (CPS). Adhesion molecules ICAM-1, VCAM-1 and E-selectin can be stimulated to be expressed on the surface of endothelial cells (ECs) by a variety of mediators of inflammation. The aim of this study was to assess the ability of polysaccharide (PS) and lipid (lipid A) components of three B. thetaiotaomicron LPS preparations to induce adhesion molecule expression on the surface of human vascular endothelial cells. The HMEC-1 cell line has been employed along with ELISA assays to examine the relative activity of B. thetaiotaomicron LPS. ELISA was performed using monoclonal mouse anti-human ICAM-1, VCAM-1 and E-selectin antibodies. The lipid A moieties of the three LPS revealed the ability to stimulate ICAM-1, VCAM-1 and E-selectin on endothelial cells. Their relative activities were similar and stronger than the biological activity of the lipid A-depleted polysaccharide (PS) components of LPS that were, nevertheless, significantly above background levels. In contrast, the PS moiety of LPS extracted from a reference strain B. thetaiotaomicron NCTC 10582 was totally unable to induce the expression of any adhesion molecule under investigation. The lipid A of B. thetaiotaomicron LPS is, therefore, and probably not unexpectedly, involved in the stimulation of adhesion molecules that are expressed on HMEC-1 to a greater extent than the PS moiety. Importantly, however, the PS components of the two LPS preparations tested manifest a weak but, nevertheless, significant activity in this process. It is possible that the PS moiety may modify the immunobiological effects of the complete LPS molecule.