[Finding a developing intrauterine infection in preterm birth by using classic and modern infection indices].

A significant part of preterm births (PTB) are due to a developing intrauterine infection (IUI). This infection is often of subclinical nature, and its diagnosis is mainly based on laboratory measurements. The objective of our study was to compare the diagnostic potential of classic (clinical picture, leukocyte count, CRP) and modern (IL-6) indices of an infectious and inflammatory process. We found the established clinical symptoms of IUI in 4.2% of the women with PTB, and if symptoms such as subfebrile body temperature (37'-37.5 degrees C), and mother's pulse of 90-100 beats/minute are taken into consideration, then with them this infection were diagnosed 23% of the women with PTB. Based on reference values of blood markers, the following incidence rates of infection were established for the studied pregnant women: increased Leu--26.7% for PTB, and 3% for the full-term birth group; based on increased CRP--51.7% for PTB, and no available data on infection for the control group; based on IL-6 over 11 pg/ml--66.7% for the PTB group, and 16.7% for the full-term women. Laboratory markers have the following sensitivity, specificity and accuracy in finding an intrauterine infection (histologically confirmed): for Leu--93.3%, 25%, and 47.8%, respectively; for CRP--100%, 53.4%, and 69.3%; and IL-6--90%, 70%, and 76.7%, respectively. Obtained results demonstrate that clinical symptoms are rarely indicative of IUI. Of all laboratory measurements, IL-6 has the best diagnostic profile, followed by CRP and leukocyte count.

Dynamics of membrane translocation of phosphatidylserine during apoptosis detected by a monoclonal antibody.

Translocation of phosphatidylserine (PS) to the outer leaflet of the cellular membrane seems to be a key step in apoptosis and cell activation. In this paper, the production and characterization of a monoclonal antibody designated as Mab 1H6 is described which does not show cross reactivity with others anionic phospholipids. It is demonstrated that Mab1H6 can recognize externalized PS at early stages after the induction of apoptosis shown by both flow cytometry and immunofluorescence. Our results show that translocation of PS can be detected as early as 5 min by immunofluorescence and 10 min by flow cytometry after the treatment of cells and a specific dynamics is observed concerning the location and distribution of the staining. These data prove that antibody Mab 1H6 can be used as a specific probe for detection of PS translocation.

Presence of HLA-DR immunopositive cells in human fetal thymus.

Several kinds of thymic cells express MHC class II antigens, including human-leukocyte-associated antigen-DR (HLA-DR) during postnatal development. The present study was focused on the detection and analysis of HLA-DR immunoreactivity in human fetal thymuses (6-7th month of gestation). Using monoclonal antibodies, indirect immunoperoxidase staining (IIP), immunogold electron microscopy (IGEM), enzyme-linked immunosorbent assay (ELISA) and flow cytometry, HLA-DR immunopositive (IP) thymic cells were found in samples studied. IIP and IGEM demonstrated the presence of HLA-DR IP stromal cells (SCs): epithelial cells (ECs), dendritic-like cells (DCs) and macrophages (MCs) as well as HLA-DR IP lymphocytes (Lys) in all thymic regions. HLA-DR immunoreactivity was more prominent in the medullary ECs (mECs) than in the cortical ECs (cECs). Strong staining of Hassall's corpuscles and the adjacent mECs was seen. The differences in the intracellular distribution of HLA-DR molecules were detailed by IGEM as a first attempt to analyse HLA-DR IP cells at ultrastructural level. ELISA data and two-colour flow cytometric analysis revealed the presence of HLA-DR IP and HLA-DR/CD3 double IP Lys in accordance with the immunocytochemical assays. The results presented enrich the information about HLA-DR IP components of the thymic microenvironment in developing human thymus and raise the question of their role during prenatal T cell differentiation and selection processes.

In vivo transfection and/or cross-priming of dendritic cells following DNA and adenoviral immunizations for immunotherapy of cancer--changes in peripheral mononuclear subsets and intracellular IL-4 and IFN-gamma lymphokine profile.

In order to provoke an immune response, a tumor vaccine should not only maximize antigen-specific signals, but should also provide the necessary "co-stimulatory" environment. One approach is to genetically manipulate tumor cells to either secrete lymphokines (GM-CSF, IL-12, IL-15) or express membrane bound molecules (CD80, CD86). Furthermore, patient dendritic cells can be loaded with tumor-associated antigens or peptides derived from them and used for immunotherapy. Genetic modification of dendritic cells can also lead to presentation of tumor-associated antigens. Transfection of dendritic cells with DNA encoding for such antigens can be done in vitro, but transfection efficiency has been uniformly low. Alternatively, dendritic cells can also be modulated directly in vivo either by "naked" DNA immunization or by injecting replication-deficient viral vectors that carry the tumor specific DNA. Naked DNA immunization offers several potential advantages over viral mediated transduction. Among these are the inexpensive production and the inherent safety of plasmid vectors, as well as the lack of immune responses against the carrier. The use of viral vectors enhances the immunogenicity of the vaccine due to the adjuvant properties of some of the viral products. Recent studies have suggested that the best strategy for achieving an intense immune response may be priming with naked DNA followed by boosting with a viral vector. We have successfully completed a phase I and phase II clinical trials on immunotherapy of prostate cancer using naked DNA and adenoviral immunizations against the prostate-specific membrane antigen (PSMA) and phase I clinical trial on colorectal cancer using naked DNA immunization against the carcinoembryonic antigen (CEA). The vaccination was tolerated well and no side effects have been observed so far. The therapy has proven to be effective in a number of patients treated solely by immunizations. The success of the treatment clearly depends on the stage of the disease proving to be most efficient in patients with minimal disease or no metastases. A panel of changes in the phenotype of peripheral blood lymphocytes and the expression of intra-T-cell lymphokines seems to correlate with clinical improvement.

Naked DNA and adenoviral immunizations for immunotherapy of prostate cancer: a phase I/II clinical trial.

INTRODUCTION AND OBJECTIVES: Animal studies have indicated that the use of syngeneic dendritic cells that have been transfected ex vivo with DNA for tumor-specific antigen results in tumor regression and decreased number of metastases. Additional studies have also suggested the possibility to modulate the dendritic cells in vivo either by 'naked' DNA immunization or by injecting replication-deficient viral vectors that carry the tumor-specific DNA. Using the prostate- specific membrane antigen (PSMA) as a target molecule, we have initiated a clinical trial for immunotherapy of prostate cancer. The primary objective of the study was to determine the safety of the PSMA vaccine after repeated intradermal injections. METHODS: We have included the extracellular human PSMA DNA as well as the human CD86 DNA into separate expression vectors (PSMA and CD86 plasmids), and into a combined PSMA/CD86 plasmid. In addition, the expression cassette from the PSMA plasmid was inserted into a replication deficient adenoviral expression vector. Twenty-six patients with prostate cancer were entered into a phase I/II toxicity-dose escalation study, which was initiated in spring 1998. Immunizations were performed intradermally at weekly intervals. Doses of DNA between 100 and 800 microg and of recombinant virus at 5x10(8) PFUs per application were used. RESULTS AND CONCLUSION: No immediate or long-term side effects following immunizations have been recorded. All patients who received initial inoculation with the viral vector followed by PSMA plasmid boosts showed signs of immunization as evidenced by the development of a delayed-type hypersensitivity reaction after the PSMA plasmid injection. In contrast, of the patients who received a PSMA plasmid and CD86 plasmid, only 50% showed signs of successful immunization. Of the patients who received PSMA plasmid and soluble GM-CSF, 67% were immunized. However, all patients who received the PSMA/CD86 plasmid and sGM-CSF became immunized. The patients who did not immunize during the first round were later successfully immunized after a boost with the viral vector. The heterogeneity of the medical status and the presence in many patients of concomitant hormone therapy does not permit unequivocal interpretation of the data with respect to the effectiveness of the therapy. However, several responders, as evidenced by a change in the local disease, distant metastases, and PSA levels, can be identified. A phase II clinical study to evaluate the effectiveness of the therapy is currently underway.

Immunologic studies in patients with glomerulonephritis.

Blood levels of E-rosette forming cells, HbsAg, C3 and immunoglobulins G, A and M were determined in a series of 101 patients with different forms of glomerulonephritis. Cell-mediated immunity to glomerular basement membrane (GBM) antigens and renal tubular epithelial (RTE) antigen was studied in 39 patients with glomerulonephritis and in 53 patients with Balkan nephropathy (BEN). The patients with glomerulonephritis had statistically higher serum levels of IgG, IgA and IgM than normal individuals. Decreased serum C3 values were recorded in 40.6 per cent of the patients and circulating immune complexes (CIC) were detected in 47.5 per cent; 6.9 per cent of the patients were HBsAg positive. The patients' levels of E-rosette forming cells were significantly lower than those of healthy controls. Cell-mediated immunity to GBM was recorded in 53.8 per cent of the patients with chronic glomerulonephritis and to RTE-antigen in 58.5 per cent of the patients with BEN. These changes are interpreted as being a manifestation of an abnormal immune response, characterized by disorders of T lymphocyte function and activation of B lymphocytes. Evidence for this includes the presence of CICs, the low C3 levels the high number of positive tests for HBsAg.