IFN-gamma triggered STAT1-PKB/AKT signalling pathway influences the function of alloantigen reactive regulatory T cells.

CD4+CD25+Foxp3+ regulatory T cells (Tregs) play a key role in the induction and maintenance of peripheral tolerance. Rapid and transient production of IFN-gamma by Tregs from mice tolerized to alloantigen in vivo has been shown to be critical for their regulatory function. This IFN-gamma has the potential to affect the function of cells present in the same local microenvironment as the Tregs, including the Tregs themselves. Here we investigated the mechanism by which IFN-gamma produced by Tregs triggered signaling pathways in alloantigen reactive Tregs themselves thereby influencing their function in vivo. We show that IFN-gamma production and STAT1 activation was increased, while STAT1-dependent PKB/AKT activation was downregulated in alloantigen reactive Tregs. Further, the activation of STAT1 was blocked in IFN-gamma receptor deficient as well as IFN-gamma-deficient Tregs, suggesting that IFN-gamma produced by the alloantigen reactive Tregs might act in an autocrine manner to induce STAT1 activation. Importantly, STAT1-deficient Tregs failed to control allograft rejection in vivo. Overall, these findings suggest that the IFN-gamma-induced STAT1-PKB/AKT signaling pathway plays a key role in upregulating the ability of alloantigen reactive Tregs to control graft rejection in vivo.

Homeostatic repopulation by CD28-CD8+ T cells in alemtuzumab-depleted kidney transplant recipients treated with reduced immunosuppression.

Alemtuzumab (CAMPATH-1H) is a depleting agent introduced recently in transplantation and often used with reduced maintenance immunosuppression. In the current study we investigated the immune response of 13 kidney allograft recipients treated with alemtuzumab followed by weaned immunosuppression with reduced dose of mycophenolate mofetil (MMF) and tacrolimus. Tacrolimus was switched to sirolimus at 6 months and MMF withdrawn at 12 months after transplantation. We found that after alemtuzumab induction the recovery of CD8(+) T cells was much faster than that of CD4(+) T cells. It was complete 6 months posttransplant while CD4(+) T cells did not fully recover even 15 months posttransplant. Repopulating CD8(+) T cells were mainly of immunosenescent CD28(-)CD8(+) phenotype. In a series of in vitro experiments we showed that CD28(-)CD8(+) T cells might suppress proliferation of CD4(+) T cells. There were three successfully treated acute rejections during the study (first at +70 day, two others +12 months) that occurred in patients with the lowest level of CD28(-)CD8(+) T cells. We hypothesize that expanded CD28(-)CD8(+) T cells might compete for 'immune space' with CD4(+) T cells suppressing their proliferation and therefore delaying CD4(+) T-cells recovery. This delay might be associated with the clinical outcome as CD4(+) T cells, notably CD4(+) T effector memory cells, were shown to be associated with rejection.

Effects of oestrogen deprivation on interleukin-6 production by peripheral blood mononuclear cells of postmenopausal women.

Various hormones can influence the expression of interleukin-6 (IL-6) and oestrogens are the most extensively studied. There is, however, controversy about the nature of the IL-6 secreted by human cells and its regulation by 17beta-oestradiol. The aim of this work was to clarify whether oestrogen deprivation after menopause may contribute to an enhanced IL-6 production by peripheral blood mononuclear cells (PBMC) in postmenopausal women. Twenty-two healthy postmenopausal women, age range 45-63 years, with clinical symptoms of oestrogen deficiency were enrolled in the study. The control group consisted of 16 healthy young women, age range 22-31 years, with regular menses and who were not taking oral contraceptives. Levels of IL-6 in the sera and PBMC culture supernatants were measured by the biological B9 cell-proliferation assay and expression of the IL-6 gene in non-stimulated PBMC was detected by RT-PCR. The effect of 17beta-oestradiol on spontaneous IL-6 production by the PBMC of postmenopausal women was also studied in vitro and in vivo. Seventeen out of the twenty-two postmenopausal women were given hormonal replacement therapy of 50 microg 17beta-oestradiol/day transdermally and the spontaneous production of IL-6 by the PBMC was analysed after 6 and 12 months of treatment. The postmenopausal women had significantly higher serum levels of IL-6 than the young controls. The spontaneous production of IL-6 by non-stimulated PBMC into the culture supernatants was also significantly higher in the postmenopausal women compared with the young. We also found that IL-6 gene expression was present in the non-stimulated PBMC isolated directly from the venous blood of the majority of the postmenopausal women. Women with IL-6 gene expression in the non-stimulated PBMC had significantly lower serum levels of 17beta-oestradiol compared with those where the IL-6 gene was not expressed in the PBMC. Our in vitro experiments showed that 17beta-oestradiol at concentrations of 10(-9) M and 10(-10) M decreased spontaneous IL-6 production by the PBMC of postmenopausal women. In vivo treatment with 17beta-oestradiol transdermally also significantly decreased spontaneous IL-6 production by the PBMC of postmenopausal women after 12 months of the therapy. Our results indicate that oestrogen deprivation after menopause may enhance IL-6 production by the PBMC of postmenopausal women. We suspect that the late complications of oestrogen deficiency, such as osteoporosis, coronary heart disease and Alzheimer's disease, may be mediated by an exaggerated production of IL-6 - a cytokine which seems to play a pivotal role in the pathogenesis of these age-related diseases.

Demonstration of iNOS-mRNA and iNOS in human monocytes stimulated with cancer cells in vitro.

Synthesis and localization of inducible nitric oxide synthase mRNA (iNOS-mRNA) and iNOS protein in the cultures of human monocytes (Mphi) and colon carcinoma cell line (DeTa) that resulted in nitric oxide (NO) synthesis has been studied. The iNOS-mRNA was observed around the sixth hour of culture and peaked at the twelfth hour. The iNOS-mRNA, as determined by the in situ hybridization and iNOS protein, as detected by staining with specific anti-iNOS monoclonal antibodies, were observed preferentially in the cytoplasm of some Mphi, but not in cancer cells. Mphi cultured alone did not show detectable iNOS-mRNA expression and iNOS protein. Mphi sorted out from tumor cells after 8 h of co-culture expressed iNOS protein and iNOS-mRNA, which were not detected in Mphi without previous contact with cancer cells. Prevention of NO synthesis by (L-N5-1-iminoethyl)-ornithine (L-NIO) partly inhibited Mphi cytotoxic activity against DeTa (NO-inducing cancer cell line) but not against the human pancreatic cancer (HPC-4) cell line that does not induce NO production in Mphi. This suggests that Mphi cytotoxic activity, at least in some cases, may be NO dependent. These observations provide further evidence that Mphi can be directly stimulated by cancer cells for de novo production of NO and suggest that iNOS occurring in the tumor-infiltrating macrophages may arise as a result of their interactions with tumor cells. However, because only some tumor cells are able to induce NO production in a small proportion of Mphi, its role in the anti-tumor response of the host is probably limited.

Preferential overexpression of CD44v5 in advanced gastric carcinoma Goseki grades I and III.

Tumor progression is associated with the clonal expansion of surviving cell variants. These results in cancer cell heterogeneity and selection of cells with a high malignant potential reflected also by the ability to metastasize. In seeding and implantation of cancer cells at the distant site cell adhesion molecules play a crucial role. Of particular interest is CD44 adhesion molecule, which possibly is involved in tumor metastasis development. Forty cases of an advanced gastric cancer were studied. Paraffin block were collected from the files. In addition to routine tumor typing, grading (Lauren and Goseki classifications) and staging, CD44 (standard, v5 and v6) was studied by immunohistochemistry and RT-PCR. CD44 immunoreactivity was found in 36 of the 40 studied cases. A significant overexpression of CD44v5 was noticed in gastric cancer. This was especially seen in Goseki's grades I and III (72.7% of cases) and was less common in Goseki's grades II and IV (44.4% of cases). CD44v6 was less commonly expressed. In some cases CD44 heterogeneity of neoplastic intravascular emboli was noticed and in some other cases stronger expression of CD44 was present in deeper parts of cancer infiltrate. Immunohistochemical expression was mostly in concert with CD44 gene expression as shown by RT-PCR results. Some discrepancies are discussed. These findings are interesting in view of better prognosis and different route of dissemination of Goseki's grades I + III compared with Goseki's grades II + IV of the gastric cancer. We have shown an overexpression of CD44v5 in an advanced gastric cancer, especially in Goseki's grades I and III. This could reflect a different malignant potential and a different route of dissemination of gastric well differentiated adenocarcinomas.

Evaluation of circulating tumour cells expressing CD44 variants in the blood of gastric cancer patients by flow cytometry.

The occurrence of circulating tumour cells in the blood of 51 patients with gastric cancer (stages I-IV) was studied using flow cytometry, cell sorting, immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). The lysed whole blood samples were stained with monoclonal antibodies against common leukocyte antigen (CD45), epithelial membrane antigen (EMA), tumour associated glycoprotein 72kD (TAG72), CD44 variants (v5 and v6) and analysed by flow cytometry within ungated or CD45-gated populations. The frequency of detection of TAG72+, CD44v5+ and v6+ cells within CD45- gate was considerably increased in comparison to the ungated population. Furthermore, the presence of tumour cells was directly demonstrated by immunostaining for cytokeratin 18 of sorted CD45- population. The presence of CD44v5+, v6- cells and CD44v-mRNA in the blood was compared to their expression in the primary tumour. The occurrence of circulating CD44v+ cells was associated with their presence in the primary tumour and CD44v-mRNA in the blood. The method described may provide a sensitive tumour marker-independent tool for detection of circulating tumour cells in cancer patients.

Detection of cancer cells in the blood by FACS sorting of CD45- cells.

We describe a simple and sensitive method for detection of low number of cancer cells in the blood. The method is based on FACS sorting of leukocytes labelled with anti-CD45 monoclonal antibody and examining CD45- cells by conventional cytology and immunostaining for cytokeratin 18. In a model study, cancer cells seeded at the frequency of 1 per 106 and 1 per 107 leukocytes were detected in CD45- population. Sensitivity of this method was comparable to reverse transcription polymerase chain reaction (RT-PCR) used for detection of cancer cells expressing CD44 variants-mRNA. In a pilot study, cancer cells were also isolated from the blood of some patients with locally advanced gastric cancer. This method may be useful for detection of circulating tumour cells in cancer patients.

Detection of cytokine gene expression in human monocytes and lymphocytes by fluorescent in situ hybridization in cell suspension and flow cytometry.

The use of digoxigenin (DIG)- and biotin-labelled dsDNA probes to detect TNFalpha-mRNA accumulation in human peripheral blood mononuclear cells (PBMC) and isolated monocytes is described. The fragment of the glyceraldehyde-3-phosphate dehydrogenase GAPDH-cDNA was used as a control probe. The hybridization signals were detected by staining with fluorescein isothiocyanate (FITC)-labelled anti-DIG antibody and avidin-FITC, respectively. The cells were stimulated in vitro with lipopolysaccharide (LPS) for 0.5-6 h. The TNFalpha-mRNA was detected in monocytes 1 h after stimulation with LPS, and the highest accumulation was seen around 2 h. The TNFalpha-mRNA in stimulated PBMC was detected at the lower level peaking around 4 h. The TNFalpha-mRNA accumulation was lower in lymphocytes than in monocytes when PBMC were studied. There was no difference in the level of GAPDH-mRNA between unstimulated and stimulated cells. Finally, an enhanced accumulation of TNFalpha-mRNA was observed in PBMC from some patients with sepsis or cancer. Thus, this study shows that cytokine gene expression may be detected in cells ex vivo. This opens the possibility of studying the level of cytokine gene activation in PBMC of patients with diseases where the role of cytokines in their pathophysiology is implicated.

The IL-6 gene expression by leukemic cells from acute lymphoblastic leukemia common and T type and modulation of IL-6 production by TNF.

The tumour necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) mRNA accumulation and the release of the cytokines TNF-alpha and IL-6 was determined in leukemic cells isolated from bone marrow biopsy from patients with acute lymphoblastic leukemia: ALL-common type (cALL), 11 patients; ALL-T type, nine patients. The non-leukemic bone marrow cells (BMMC) and peripheral blood mononuclear cells (PBMC) from healthy donors were used as a control. The mRNA was assessed by fluorescent in situ hybridization in cell suspension and analyzed with flow cytometry. The accumulation of cytokine mRNA was higher in cALL cells as compared to ALL-T and PBMC (control) and was comparable to cytokines mRNA accumulation in BMMC. The production of IL-6 by leukemic cells from both types of leukemia was significantly lower than in BMMC. The bioactive TNF was not detected in either of the leukemia groups studied. TNF-alpha protein was produced by ALL-T cells and BMMC but not by cALL type of leukemic cells. The synthesis of IL-6 was significantly enhanced by TNF-alpha in BMMC and ALL-T while the presence of TNF-alpha had no effect on IL-6 synthesis in the culture of cALL leukemic cells. It was concluded that despite IL-6 and TNF-alpha mRNA contents, leukemic cells representing early stage of B-cell development (CD10+) showed disregulation of production of these cytokines.

Vancomycin down-regulates lipopolysaccharide-induced tumour necrosis factor alpha (TNF alpha) production and TNF alpha-mRNA accumulation in human blood monocytes.

The cytokines play an important role in the cascade of the pathological events leading to septic shock. The TNF alpha produced by monocytes/macrophages upon stimulation with bacterial fragments may contribute to induction of this cytokine cascade. Moreover, the antibiotics used for antimicrobial therapy may cause the increase of TNF alpha production due to massive bacterial killing and exposure of monocytes/macrophages to bacterial cell constituents. To investigate the effect of Vancomycin on TNF alpha production, an in vitro model of LPS-stimulated monocytes was used. The level of TNF alpha protein or TNF biological activity were tested in the culture supernatants of monocytes with LPS. Vancomycin down-regulated, in dose-dependent manner, the TNF alpha production. Vancomycin also inhibited TNF alpha-mRNA accumulation in LPS-stimulated monocytes, as assessed by fluorescence in situ hybridization (FISH) in cell suspension. The down-regulation of TNF alpha production in LPS-stimulated monocytes may indicate that inhibition of this cytokine release is one of the important therapeutic effects of Vancomycin in sepsis.

Isotype-specific regulation of MHC class II gene expression in human monocytes by exogenous and endogenous tumor necrosis factor.

The control of expression of MHC class II molecules on antigen-presenting cells is important for the induction of immunity, while aberrant expression of these molecules plays a role in the immunopathology of autoimmune diseases. This study explored the role of tumor necrosis factor alpha (TNF) in controlling the level of HLA class II mRNA in human monocytes. Exposure of monocytes to exogenous recombinant TNF (rTNF) selectively up-regulated DR alpha-mRNA but not DP or DQ alpha-mRNA. Inhibitors of TNF synthesis, pentoxifylline (PTX) and thalidomide, inhibited TNF mRNA accumulation in LPS-activated monocytes and down-regulated DR mRNA but not DP or DQ mRNA. The inhibitory effect of anti-TNF monoclonal antibody (MAb) indicated that endogenously generated TNF acted extracellularly. Anti-p75 TNF-R2 receptor and to a lesser extent anti-p55 TNF-R1 MAbs inhibited TNF-mediated up-regulation of DR mRNA and TNF mRNA. Taken together, this implies that endogenously generated TNF plays a role in controlling isotype-specific MHC class II gene expression in human monocytes/macrophages. These results may have some implications for anti-tumor response and autoimmunity.

Molecular analysis of alleles segregation at RFLPs within RB-1 gene in four families with hereditary retinoblastoma.

Four families with suspected hereditary retinoblastoma in proband and one other family member were examined by genetic segregation analysis of the RB-1 gene loci with specific RFLPs of chromosome 13. Two families, TA-6 and partially CK-46, were informative using this method. In TA-6 family healthy sister (TA-6/10) of the proband was shown not to be a carrier of the mutant RB-1 gene. These results show that the segregation analysis of RB-1 gene loci with specific RFLPs, used as the DNA markers, could be helpful in the genetic diagnosis and counselling in families with hereditary retinoblastoma.

The MHC class-II and CD44 molecules are involved in the induction of tumour necrosis factor (TNF) gene expression by human monocytes stimulated with tumour cells.

Tumour necrosis factor alpha (TNF) mRNA is detected in the macrophage infiltrate surrounding the tumour, but the cellular/molecular interactions leading to TNF gene expression in macrophages are unknown. The in vitro system in which human blood monocytes are stimulated with human cancer cells for TNF release was used to study such interactions. Monoclonal antibodies (MAbs) against various adhesion molecules (LFA-1, LFA-3, ICAM-1, VNR, VLA beta I chain) were unable to block TNF production in co-culture of monocytes with a human pancreatic carcinoma (HPC) cell line. However, anti-CD44 and anti-HLA-DR MAbs effectively blocked TNF release and TNF-mRNA induction in monocytes. Pre-incubation of monocytes with anti-HLA-DR and tumour cells with anti-CD44 MAbs had a similar effect. It was concluded that CD44 molecules are involved in tumour-monocyte interactions and that HLA-DR determinants of monocytes are engaged in signal transduction for TNF gene activation. These findings may suggest that certain surface determinants of tumour cells act as ligands for MHC class-II molecules and induce TNF production in monocytes.

Tumour-cell-induced production of tumour necrosis factor by monocytes of gastric cancer patients receiving BCG immunotherapy.

Human peripheral blood monocytes cocultured with tumour cells were used as an in vitro model of in situ interactions between tumour-infiltrating macrophages and the tumour. Tumour cells stimulated de novo expression of the human tumour necrosis factor alpha (TNF) gene in monocytes and caused the release of TNF into the culture supernatant. A group of 14 patients with stage IVA gastric cancer receiving adjuvant chemotherapy (5-FU, Adriamycin, mitomycin C: FAM) or immunochemotherapy (BCG+FAM) was investigated for the ability of monocytes to produce TNF in vitro upon stimulation with tumour cells or purified protein derivative of tuberculin (PPD). Patients were followed at biweekly intervals, i.e. before each instillation of BCG epicutaneously over a period of 10 weeks. It was found that monocytes of some patients receiving BCG at the end of the observation period had an enhanced ability to produce TNF following stimulation with tumour cells. In contrast, such production was not substantially altered during the study period in patients on chemotherapy. PPD-induced TNF production was much weaker and was not significantly changed during this observation time. We infer that BCG immunotherapy may induce the subtle changes in some cancer patients that lead to an increased interaction between monocytes and tumour cells and result in enhanced production of cytokine(s) with antitumour properties.

Detection, by in situ hybridization using sulphonated cDNA probe, the specific mRNA for HLA-DR alpha induced in monocyte cell lines by recombinant interferon gamma.

We adopted the nonradioactive method used for blot hybridization for the detection of inducible mRNA for HLA-DR alpha by the in situ hybridization. Unstimulated and interferon gamma stimulated MonoMac6 and U937 human monocytic cell lines were used as target cells. Sulphonation of plasmid pBR322 with HLA-DR alpha cDNA insert (2 x 700 bp, in Pstl restriction site) was performed according to the manufacturer's procedure (SulfoProbe Kit, Sigma). The hybridization signals were detected with mouse monoclonal, anti-sulphonated DNA antibody, followed by immunovisualization with anti-mouse IgG-alkaline phosphatase conjugates. Unstimulated MonoMac6 and U937 cells showed few granular reaction products only in small percentage of cells (1-5%), while in IFN gamma stimulated cells the fine granular immunoenzymatic reaction was observed in the cytoplasm of majority of cells (> 80%). This method seems to be easy and rapid to perform, making it applicable for routine diagnostic purposes in tissue sections and biopsies.

Instability of hybrid plasmids in Bacillus subtilis.

We studied the mechanisms responsible for instability of hybrid derivatives of staphylococcal R plasmids in B. subtilis cells. Four pBCH plasmids were constructed from staphylococcal chloramphenicol-resistance plasmid pC756 ligated with pBR322 or pUC8 vectors, and two pAEC plasmids contained pC756 plasmid, erm gene from S. aureus pE3692 and pBR322 plasmid vector. The level of instability of these recombinant plasmids was clearly related to their sizes, the larger derivatives were segregationally highly unstable. The four hybrid pBE plasmids constructed from erythromycin-resistant pE3692 plasmid or its deletion derivative and pBR322 vector were poorly maintained in B. subtilis rec+ strain in comparison with the B. subtilis (recE4) strain. Moreover, the absence in pBE-hybrids of a part of palA sequence led to considerable reduction of plasmid stability in B. subtilis cells. However, the clones of B. subtilis harbouring pBE plasmids integrated into the bacterial chromosome presented much higher stability of the plasmid erm gene than the clones maintaining autonomously replicating plasmids.

[Integration of hybrid plasmids-derivatives of staphylococcal plasmid pE3692 and pBR322 vector with Bacillus subtilis chromosome]. / Integracja do chromosomu Bacillus subtilis hybrydowych plazmidów pochodnych gronkowcowego plazmidu pE3692 i wektora pBR322.

Bacillus subtilis is a nonpathogenic microorganism which certainly for also that feature is widely used in several biotechnological processes. Certainly, because of that an elaboration of gene cloning methods in Bacillus subtilis cells is of great significance both from scientific and practical point of view. It was found hybrid plasmids constructed by ligation of pE3692 and pE3692-9 staphylococcal plasmids with pBR322 vector can be integrated with B. subtilis chromosome. Selection of cells in the presence of erythromycin at 50 degrees C allows to isolate clones containing exclusively integrated plasmids. Growth rate of cells containing these plasmids in the selective medium is the same or slightly higher at 50 degrees C than at 37 degrees C. It seems that hybrid plasmid tested pBE4 and pBE91 after appropriate reconstruction in vitro could serve as vectors for B. subtilis gene cloning.

[Cloning and identification of erm gene and genes responsible for replication and stability of staphylococcal plasmid pE3692 determining the resistance to antibiotics of the MLS group]. / Klonowanie i identyfikacja genu ERM oraz genów odpowiedzialnych za replikacje i stabilnosc gronkowcowego plazmidu pE3692 determinujacego opornosc na antybiotyki z grupy MLS.

Genetic elements coding for resistance to MLS group of antibiotics occur also in plasmids of staphylococcal cells. Wide distribution of such plasmids and their high segregational stability justify undertaking of detailed studies on their molecular structure and stability. It was shown that erm gene from pE3692 plasmid is located inside ClaI-HindIII segment of 1.3 kb dimension. Constructed pBE-9/1 plasmid, derivative of pE3692-9, contains inserted pBR322 vector in position ClaI. This plasmid replicates in E. coli and B. subtilis cells. It seems that Hind III-ClaI segment (0.8 hb) from pE3692 plasmid contains all indispensable genes for pE3692 replication. Excicion of Sta t (0.3 kb) fragment from pE3692 plasmid reduced considerably its stability. This segment may constitute a longer sequence fragment responsible for the stability of pE3692 plasmid.

Stability and expression of the staphylococcal constitutive macrolide-lincosamide-streptogramin B resistance plasmid pE3692 in Staphylococcus aureus and Bacillus subtilis.

The constitutive MLS resistance plasmid pE3692 (2.4 kb) from S. aureus and pE3692-9 plasmid derived from pE3692 by in vitro deletion of the small HindIII-HindIII fragment Sta (0.3 kb) were introduced into S. aureus RN450 and B. subtilis UOT0277 strains. The pE3692 plasmid was completly stable in S. aureus RN450, however, in B. subtilis UOT0277 this plasmid was segregationally unstable. Deletion of the Sta t fragment from the pE3692 reduced plasmid stability considerably both in S. aureus and B. subtilis host strains. The lower expression of tylosin-resistance of the erm gene of the pE3692-9 plasmid in B. subtilis UOT0277 strain was also observed.