Dipeptidyl peptidase IV (DPPIV) enzyme activity on immature T-cell line R1.1 is down-regulated by dynorphin-A(1-17) as a non-substrate inhibitor.

In this study we examined surface expression of CD26 and the corresponding enzyme activity of dipeptidyl peptidase IV (DPPIV) on the cells of immature murine T-cell line, R1.1. The data obtained have shown that R1.1 cells express high density of surface CD26 as compared to normal thymus cells. This was associated with strong enzyme activity, which, based on substrates and inhibitor specificity, corresponded to DPPIV. The DPPIV enzyme activity of R1.1 cells was 10 times stronger than that found on normal murine thymus cells (V(max) = 39 micromol/min/10(6) cells, vs 3.7 micromol/min/10(6) cells, respectively). Upon activation with anti-CD3, up-regulation of both membrane CD26, as well as of DPPIV enzyme activity on R1.1 cells were observed. The finding of strong DPPIV on R1.1 cells makes them suitable model for testing putative substrates/inhibitors of the enzyme in its natural microenvironment. Since in addition to strong DPPIV, R1.1 cells also express kappa opioid receptors (KOR) [European Journal of Pharmacology 227 (1992) 257], we tested the effect of dynorphin-A(1-17), an endogenous opioid peptide with KOR selectivity, on DPPIV of R1.1 cells. Dynorphin-A(1-17) down-regulated DPPIV in a dose-dependent manner, with the potency similar to that of substance P, a known natural DPPIV substrate [Journal of Pharmacology and Experimental Therapeutics 260 (1992) 1257]. DPPIV down-regulation was resistant to bestatin and thiorphan, the inhibitors of two cell surface peptidases (APN and NEP, respectively) with potential of dynorphin-A(1-17) degradation, suggesting that the mechanism underlying the observed effect does not involve degradative products of dynorphin-A(1-17). DPPIV down-regulation was also resistent to KOR antagonist, NBI, suggesting that the mechanism underlying the observed phenomenon involves neither cointernalization of KOR and DPPIV. Collectively, cells of immature T cell line, R1.1 exert strong DPPIV enzyme activity, which could be down-regulated in the presence of dynorphin-A(1-17) by mechanism that presumably includes non-substrate inhibition. By down-regulating DPPIV, dynorphin-A(1-17) may indirectly affect activity and/or specificity of natural substrates of DPPIV, such as substance P, RANTES, and endomorphins.

[Prognostic significance of newly formed blood vessels in the tumors of patients with breast cancer]. / Prognosticka vrijednost novostvorenih krvnih zila u tumorima bolesnica s rakom dojke.

In this study 96 specimens of ductal invasive breast carcinoma were analyzed. The following data of each patient were collected: age, tumor size, histological grade, axillary lymph node metastases, estrogen (ER) and progesterone (PR) receptor expression, percentage of cells in S-phase of cell cycle, cell ploidy status, and overall survival. From paraffin blocks 3-microns sections were stained using anti-factor VIII monoclonal antibody for detection of tumor neoangiogenesis. The number of new blood vessels/mm2 in the stained tumor tissue specimens was analyzed using a light microscope with Weibel graticule on the eyepiece. There was no statistically significant correlation of number of blood vessels/mm2 and age, axillary lymph node status, ploidy of tumor cells, size and tumor grade, ER and PR status, but there is a correlation with number of cells in the S-phase of cell cycle (p = 0.037). The cut-off value of tumor blood vessels/mm2 was 170. Univeriate analysis showed that overall survival correlated significantly with axillary lymph node involvement (p < 0.001), ER (p = 0.012) and PR (p = 0.001) status, and number of blood vessels/mm2 of tumor (p = 0.033). In multivariate analysis only axillary lymph node metastases (p = 0.015) and PR status (p = 0.026) were found to be independent and significant prognostic factors. When patients were stratified according to number of blood vessels/mm2 of tumor it was shown that those with the number of blood vessels/mm2 over 170, aged under 50 years (p = 0.011), number of cells in S-phase of cell cycle over 4% (p = 0.050), diploid tumor cells (p = 0.004), and negative PR (p = 0.059) had shorter survival than patients with tumors with less than 170 blood vessels/mm2 of tumor.

Cellular and humoral immunity to purified protein derivative (PPD) in PPD skin reactive and nonreactive patients with pulmonary tuberculosis: comparative analysis of antigen-specific lymphocyte proliferation and IgG antibodies.

AIM: To evaluate in vitro reactivity against tuberculin purified protein derivative (PPD) in patients with active pulmonary tuberculosis scoring either positive or negative upon intradermal PPD application (PPD-DTH). METHOD: Two groups of patients with pulmonary tuberculosis, 22 PPD+ and 22 PPD-, were studied. Peripheral blood mononuclear cells (PBMC) were assayed for in vitro proliferation to PPD antigen, phytohaemagglutin, concanavalin A, and pokeweed mitogens. In the proliferation assay PBMC were incubated in a medium supplemented with serum (20% concentration) from healthy donors, autologous serum, or allogenic serum. Anti-PPD IgG concentration in patients sera were analyzed by ELISA. CD3+ lymphocytes from 10 patients in each group were tested for the expression of surface activation markers (HLA-DR and CD25/IL-2 receptors) by flow cytometry. RESULTS: PPD- patients showed clinically and radiologically more advanced forms of pulmonary tuberculosis as compared with PPD+ patients. PBMC from both groups of patients proliferated in response to PPD effectively, but significantly higher de novo DNA synthesis was observed in PPD+ patients (p<0.001). Proliferative activity was not affected by the type of the serum supplement (autologous or allogenic) in the culture medium. Mitogen stimulation elicited similar proliferative responses in both groups. Similar percentages of T-lymphocytes and T-lymphocytes expressing CD25 activation markers were observed in both groups of patients. There was a borderline difference in the percentage of CD3+HLA-DR+ lymphocytes between these two groups of patients (p=0.05). At 1:1000 serum dilution a significant difference (p=0.002) in anti-PPD IgG concentrations was found between PPD- and PPD+ patients. CONCLUSION: Patients with active pulmonary tuberculosis with a more favorable clinical course have a more potent specific cell-mediated immunity to PPD (positive skin reactivity in vivo and significantly greater lymphocyte proliferative response in vitro) than patients with a clinically more severe form of the disease. The concentration of PPD specific IgG in the serum appears to be higher in patients with relatively more severe forms of the disease.

Acute promyelocytic leukemia M3: cytomorphologic, immunophenotypic, cytogenetic, and molecular variants.

Acute promyelocytic leukemia (APL) M3 is an acute myeloid leukemia (AML) subtype characterized by proliferation of malignant promyelocytes with mature myeloid immunophenotype and the translocation t(15;17)(q22;q11), which results in the fusion of retinoic acid receptor-alpha (RARalpha) gene on chromosome 17 and the gene PML on chromosome 15. There are three M3 morphologic variants: the typical hypergranular form and the microgranular and basophilic variants. Although most leukemic cells in M3 patients express t(15;17), other cytogenetic abnormalities have also been reported. Also, there are three molecular variants of the PML/RARalpha transcript (bcr1, bcr2, bcr3). Blasts had typical hypergranular appearance (13 patients) with a mature myeloid immunophenotype (HLA-DR(-),CD13(+), and/or CD33(+)) (10 patients) in the majority of patients with M3 followed in this study. The typical translocation [t(15;17)(q22;q11)] was detected by cytogenetic analysis in 5 M3 patients, but PML/RARalpha was positive in 13 out of 15 patients, as assessed by RT-PCR (8 patients with bcr1 and 5 with bcr3 subtype). Cytogenetic diversity was found in three patients (1 with t(17;17), 1 with +8, and 1 with add (7)(q22); -7; +8). According to many studies, leukemic cell heterogeneity in APL influences the clinical outcome of disease. The analysis of certain leukemic cell characteristics on the clinical outcome in our study revealed that patients with bcr3 had shorter medians of first remission and survival in comparison to patients with the bcr1 isoform of PML/RARalpha. Also, the clinical relapse of disease in 4 APL patients with reverted PML/RAR alpha positivity is consistent with the view that detection of PML/RARalpha by RT-RCR in patients in remission implies a poor prognosis. On the contrary, lack of detection of PML/RARalpha by RT-PCR at least three times is a sign of long remission and survival.