Search

KISS1 tumor suppressor protein regulates cancer cell invasion via MMP9 metalloproteinase. Downregulation of KISS1 gene expression promotes progression of breast cancer and melanoma, resulting in the development of distant metastases. In the current study, we investigated whether restoration of KISS1 expression in KISS1-deficient human metastatic breast cancer cells holds potential as an advanced anticancer strategy. To this end we engineered an infectivity-enhanced conditionally-replicative human adenovirus type 5 encoding KISS1 as an "arming" transgene in the Ad5 E3 region for an ectopic KISS1 expression in transduced cancer cells. The oncolytic potential of the vector was examined using brain-invading metastatic clones of CN34 and MDA-MB-231 breast cancer cells, which supported high levels of AdKISS1 replication, correlating with a robust CRAd-mediated cytotoxicity. Secretion of cellular factors responsible for tumor angiogenesis, cell-to-cell communication and anti-tumoral immune responses upon KISS1 expression in breast cancer cells was analyzed by a RayBiotech Kiloplex Quantibody array. Overall, our results indicate that KISS1 transgene expression provides an important benefit for CRAd-mediated cytotoxicity in breast cancer cells and holds potential as an anticancer treatment in conjunction with oncolytic virotherapy of breast and other metastatic cancers.

// Ilya V. Ulasov 1, 2, 3 , Natalya V. Kaverina 3, 4 , Dhimankrishna Ghosh 1 , Marya A. Baryshnikova 2, 3 , Zaira G. Kadagidze 3 , Apollon I. Karseladze 3 , Anatoly Y. Baryshnikov 2, 3 , Charles S. Cobbs 1 1 Swedish Neuroscience Institute, Center for Advanced Brain Tumor Treatment, Seattle, WA, 98122, USA 2 Institute of Experimental Diagnostics and Therapy of Tumors, N.N. Blokhin Russian Cancer Research Center, Moscow, 115478, Russia 3 NN. Blokhin Cancer Research Center, RAMN, Moscow, 115478, Russia 4 Current employment: Division of Nephrology, University of Washington, Seattle, 98109, WA, USA Correspondence to: Ilya V. Ulasov, email: Ilya.Ulasov@Swedish.org Charles S. Cobbs, email: Charles.Cobbs@Swedish.org Keywords: brain tumor, cytomegalovirus, glioma stem cells, miRNA Received: November 17, 2015 Accepted: July 26, 2016 Published: August 10, 2016 ABSTRACT Glioblastoma multiforme (GBM) is a rapidly progressive brain tumor with a median survival of 15–19 months. Therapeutic resistance and recurrence of the disease is attributed to cancer stem cells (CSC). Here, we report that CMV70-3P miRNA encoded by CMV increases GBM CSC stemness. Inhibition of CMV70-3P expression using oligo inhibitors significantly attenuated the ability of primary glioma cells to proliferate and form neurospheres. At the molecular level, we show that CM70-3P increases expression of cellular SOX 2. Collectively, these findings indicate that CMV70-3P is a potential regulator of CMV- mediated glioma progression and cancer stemness.

For the first time in Russia the immunophenotype of tumor infiltrated lymphocytes (TIL) was investigated with the use of a method of quantitative flow cytometry. Samples from 104 patients, suffering from solid tumors (such as breast cancer, ovarian cancer, melanoma and oral cavity squamous carcinoma), were analyzed in this investigation. In the difference of cytomorphological analysis, the method of flow cytometry identified TIL in 100% of cases. In the structure of CDS+Т- lymphocytes the ratio CD4/CD8 was equal to 1,1±0,1. Serial gating strategy allowed to assess minor subpopulations of regulatory T-lymphocytes CD45+CD4+CD127+low/neg and CD45+CD8+CD28-CD11b-. Regulatory mechanisms with involvement of CD8 T-cells played the main role in the generation of immune responses at the tissue level ofpatients, suffering from solid tumors, independent of nosological form of disease.

e12581 Background: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Pathologic complete response (pCR) achievement during neoadjuvant chemotherapy (NACT) is very important for these pts as it correlates with long-term outcome. Predictive and prognostic factors for this group of pts are needed. Methods: Since 2014 we treated 98 pts with histologically confirmed stage II-III TNBC with following regimen of NACT: 6 cycles of Cisplatin 75mg/m2 day 1 and Paclitaxel 80mg/m2 days 1, 8, 15, every 4 weeks according to local protocol. After NACT pts proceeded to radical breast surgery with assessing of pathological response. Before the treatment we assessed TILs in biopsy samples of all patients. We also conducted subpopulation analysis of lymphocytes in peripheral blood before treatment. We analyzed CD8+, CD25+, NK and NKT cells as they were reported to be significant earlier. We compared results with median value in reference group (mRG) of healthy women. Results: Among all 98 pts 86 (87%) were operated, pCR was achieved in 60,5% of operated pts. pCR strongly correlated with TILs level: 38,5% for pts with TILs < 5% and 69,8% for pts with TILs > 5% (p = 0,05). We also found that NKT population was associated with pCR: 77,8% vs 45,8% among patients with NKT above mRG and up to mRG, respectively (p = 0,01). It correlated also with tpCR: 22,2% vs 70,6% (p = 0,01). When both CD25+ and NKT population were above mRG pCR rate achieved in 85,7% pts vs 50,0% pts if it was up to mRG (p = 0,049). NK, CD8+ and CD25+ did not show any correlation with pCR. Survival results are presented in table below. NK, NKT and TILs did not show any correlation with survival. Interestingly, CD8+ were also associated with lower incidence of visceral metastases: 42,1% in pts with normal CD8+ and 15,7% in pts with CD8+ above normal (p = 0,19). Conclusions: Not only TILs, but also NKT and CD25+/NKT population in the peripheral blood could be possible predictive factors for patients with stage II-III TNBC receiving NACT. CD8+, CD25+ and NKT populations could be further studied as prognostic markers for this group of patients. [Table: see text]

It is already established that verapamil inhibits Pgp expression in the brain tumor cancer cells such as medulloblastoma. The aim of this study is to investigate the sensitivity of glioblastoma cancer cells to verapamil and its combination with oncolytic adenoviral victor CRAd-S-pK7. In vitro using U87 and U251 human glioblastoma cell lines, we obtained experimental data suggesting a therapeutic effect of verapamil and CRAd-S-pK7. Moreover, we established that verapamil improves anti-glioma effect of oncolytic adenoviral vector in the presence of ionizing radiation, which results into more suppression of U251 cancer cells via inhibition of their proliferation.

Oncolytic gene therapy using viral vectors may provide an attractive therapeutic option for malignant gliomas. These viral vectors are designed in a way to selectively target tumor cells and spare healthy cells. To determine the translational impact, it is imperative to assess the factors that interfere with the anti-glioma effects of the oncolytic adenoviral vectors. In the current study, we evaluated the efficacy of survivin-driven oncolytic adenoviruses pseudotyping with adenoviral fiber knob belonging to the adenoviral serotype 3, 11 and 35 in their ability to kill glioblastoma (GBM) cells selectively without affecting normal cells. Our results indicate that all recombinant vectors used in the study can effectively target GBM in vitro with high specificity, especially the 3 knob-modified vector. Using intracranial U87 and U251 GBM xenograft models we have also demonstrated that treatment with Conditionally Replicative Adenovirus (CRAd-S-5/3) vectors can effectively regress tumor. However, in several patient-derived GBM cell lines, cells exhibited resistance to the CRAd infection as evident from the diminishing effects of autophagy. To improve therapeutic response, tumor cells were pretreated with tamoxifen. Our preliminary data suggest that tamoxifen sensitizes glioblastoma cells towards oncolytic treatment with CRAd-S-5/3, which may prove useful for GBM in future experimental therapy.

Autophagy is an evolutionarily conserved process regulating cellular homeostasis via digestion of dysfunctional proteins and whole cellular organelles by mechanisms, involving their enclosure into double-membrane vacuoles that are subsequently fused to lysosomes. Glioma stem cells utilize autophagy as a main mechanism of cell survival and stress response. Most recently, we and others demonstrated induction of autophagy in gliomas in response to treatment with chemical drugs, such as temozolomide (TMZ) or oncolytic adenoviruses (Ads). As autophagy has been implicated in the mechanism of Ad-mediated cell killing, autophagy deficiency in some glioma tumors could be the reason for their resistance to oncolysis. Despite the observed connection, the exact relationship between autophagy-activating cell signaling and adenoviral infection remains unclear. Here, we report that inhibition of autophagy in target glioma cells induces their resistance to killing by oncolytic agent CRAd-S-5/3. Furthermore, we found that downregulation of autophagy inducer Beclin-1 inhibits replication-competent Ad-induced oncolysis of human glioma by suppressing cell proliferation and inducing premature senescence. To overcome the autophagy-deficient state of such glioma cells and restore their susceptibility to oncolytic Ad infection, we propose treating glioma tumors with an anticancer drug tamoxifen (TAM) as a means to induce apoptosis in Ad-targeted cancer cells via upregulation of BAX/PUMA genes. In agreement with the above hypothesis, our data suggest that TAM improves susceptibility of Beclin-1-deficient glioma cells to CRAd-S-5/3 oncolysis by means of activating autophagy and pro-apoptotic signaling pathways in the target cancer cells.

Formation of metastases, also known as cancer dissemination, is an important stage of breast cancer (BrCa) development. KISS1 expression is associated with inhibition of metastases development. Recently we have demonstrated that BrCa metastases to the brain exhibit low levels of KISS1 expression at both mRNA and protein levels. By using multicolor immunofluorescence and coculture techniques here we show that normal adult astrocytes in the brain are capable of promoting metastatic transformation of circulating breast cancer cells localized to the brain through secretion of chemokine CXCL12. The latter was found in this study to downregulate KISS1 expression at the post-transcriptional level via induction of microRNA-345 (MIR345). Furthermore, we demonstrated that ectopic expression of KISS1 downregulates ATG5 and ATG7, 2 key modulators of autophagy, and works concurrently with autophagy inhibitors, thereby implicating autophagy in the mechanism of KISS1-mediated BrCa metastatic transformation. We also found that expression of KISS1 in human breast tumor specimens inversely correlates with that of MMP9 and IL8, implicated in the mechanism of metastatic invasion, thereby supporting the role of KISS1 as a potential regulator of BrCa metastatic invasion in the brain. This conclusion is further supported by the ability of KISS1, ectopically overexpressed from an adenoviral vector in MDA-MB-231Br cells with silenced expression of the endogenous gene, to revert invasive phenotype of those cells. Taken together, our results strongly suggest that human adult astrocytes can promote brain invasion of the brain-localized circulating breast cancer cells by upregulating autophagy signaling pathways via the CXCL12-MIR345- KISS1 axis.

BACKGROUND: Metastases to the brain represent a feared complication and contribute to the morbidity and mortality of breast cancer. Despite improvements in therapy, prognostic factors for development of metastases are lacking. KISS1 is a metastasis suppressor that demonstrates inhibition of metastases formation in several types of cancer. The purpose of this study was to determine the importance of KISS1 expression in breast cancer progression and the development of intracerebral lesions. METHODS: In this study, we performed a comparative analysis of 47 brain metastases and 165 primary breast cancer specimens by using the antihuman KISS1 antibody. To compare KISS1 expression between different groups, we used a 3-tier score and the automated score computer software (ACIS) evaluation. To reveal association between mRNA and protein expression, we used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Significance of immunohistochemistry stainings was correlated with clinicopathological data. RESULTS: We identified that KISS1 expression is significantly higher in primary breast cancer compared with brain metastases (P < .05). The mRNA analysis performed on 33 selected ductal carcinoma brain metastatic lesions and 36 primary ductal carcinomas revealed a statistically significant down-regulation of KISS1 protein in metastatic cases (P = .04). Finally, we observed a significant correlation between expression of KISS1 and metastasis-free survival (P = .04) along with progression of breast cancer and expression of KISS1 in primary breast cancer specimens (P = .044). CONCLUSIONS: In conclusion, our study shows that breast cancer expresses KISS1. Cytoplasmic expression of KISS1 may be used as a prognostic marker for increased risk of breast cancer progression. Cancer 2012;. © 2011 American Cancer Society.

We developed and described a new approach to vital analysis of functional activity of multidrug resistance markers (ABC transporters) in intact biopsy specimens from human solid tumors by the method of flow cytofluorometry. The algorithm of the study underwent revision, and the cell suspension was obtained in the final stage. Intensification of intracellular doxorubicin accumulation (fluorescence) and increase in the number of fluorescent cells and total fluorescence of doxorubicin-accumulating cells produced by ABC transporter inhibitor sodium azide served as the criteria of expression of these transporters in tumor tissue. Informative value of changes in various parameters of doxorubicin fluorescence is discussed. The increase in the count of fluorescent cells in the suspension of tumors cells after treatment with the inhibitor indicates the presence of tumor cells absolutely resistant to this preparation. The proposed method is technically simple, suitable for structurally different tumors, requires small amounts of the biopsy material and, therefore, can be used for routine analysis. The results of our analysis and spectrofluorometric assay of ABC transporters agree very closely, which suggest that this method is adequate for the purpose.

The main goal of cancer immunotherapy is to induce or boost tumor-specific effector cells able to eliminate or reduce tumor progression. In this study, we characterized lymphocyte phenotypes in melanoma patients receiving dendritic cell (DC)-based vaccinotherapy. We found that several biological markers served as unfavorable prognostic factors for patients' response to therapy. This included decrease of CD4+ and CD8+ lymphocyte levels, 10% and higher increase of CD16+CD3+CD8+ lymphocyte population, and increase of CD16+CD8+perforin+ T lymphocytes, especially in combination with decreased levels of CDI6+CD8(-)perforin+ and CD8+CD16(-)perforin+ cells. Increase in CD8+CD16(-)perforin+ T lymphocytes with normal levels of CD16+CD8(-)perforin+ cells and the absence of CD16+CD8+perforin+ and regulatory lymphocytes were shown to be the positive prognostic markers for patients' response to DC vaccines.

Tumor markers CA-19-9 (a carcinoembryonic antigen) and α-fetoprotein are measured in sera of 437 patients hospitalized for diseases other than cancer. The CA-19-9 level is increased in patients with peptic ulcer and cholelithiasis. The study demonstrates the advantages of immunological methods of examination in the diagnosis of latent tumors of the kidneys, stomach, pancreas, liver, gallbladder, and lungs. Tumor markers can be used as prognostic criteria in cirrhosis of the liver, chronic hepatitis, and circulatory diseases.

s / The Breast 20 (2011) S12–S55 S25 Results: Similarly, association between p53 protein and disease response was evaluated. With 90% confidence it was established, that p53 protein overexpression influences disease progression (Fisher‘s p 1⁄4 0.096). Wealso estimated the impactof proteinexpressionondisease outcomes in 24 patients who underwent only first-line treatment against metastatic breast cancer. If p53 protein overexpression was found, mean survival was 28.5 months (SEM1⁄4 5.4, 95% CI 17.9–39.0) andmedian 25months (SEM1⁄4 6.9, 95% CI 11.4–38.6), if itwasabsent– survivalwas slightly longer:mean33.6months (SEM1⁄4 4.2, 95%CI 25.3–41.8) andmedian 30months (SEM1⁄4 2.6, 95%CI 24.9– 35.0). Kaplan-Meiermethod did not detect statistically significant differences between the groups (log-rank p > 0.05, Breslow p > 0.05). For small samples, associations among molecular markers were evaluated using Fisher‘s exact test. It was established, that p53 protein has a trend to be associated with underexpression of ER (OR1⁄4 5.83, 95% CI 0.9–37.81, p1⁄4 0.064). Associations among other factors were non-significant. Analysis of combinations of ER and PR (ER-PR-, ER-PR+, ER+PR-, ER+PR+) with p53 protein and HER2 was also conducted. However, there were no significant association between them. Additionally, combination of negative expression in 3 markers (ER, PR and HER2) with p53 protein expressionwas checked. Thus, significant associationwas found between positive p53 protein expression and combination of molecular markers ER-PRHER2(Fisher‘s p 1⁄4 0.041). Conclusion: It was established, that combination of molecular markers ER-PR-HER2is 7.7 times (95% CI 1.16–51.17; p 1⁄4 0.035) more likely in case of positive expression of p53 protein and it is associated with the worse prognosis of the patients.

Aim: The present study was aimed to investigate whether KISS1 suppressor protein could be used as a prognostic marker in development of breast cancer metastases. Methods: To test whether breast cancer brain metastases (BMHB) have altered expression of KISS1, we created a TMA (tissue microarray) recipient block containing 136 specimens: 103 specimens of primary breast cancer cells (invasive ductal carcinoma, IDC), 7 specimens from draining lymph nodes (LN), 39 brain metastases and 14 samples of normal breast tissue (NBreast). Each core was duplicated. Sections from this block were stained with antibodies that recognize KISS1. Immunoassays were graded based on: A) intensity of immunoreactive cells staining (Negative, 0% of the cells have staining; 1+, 5-20% of total cells have staining (Mild); 2+, 20-50% of total cells have staining (Moderate); 3+, 50% or more of total cells have staining(High)) and B) score calculated by ACIS system (Chromavision/Clarient, Aliso Viejo, CA). Positive staining in the present study was defined as visibility at low magnification (10x) and was confirmed by two pathologists. To validate the IHC findings, relative expression of KISS1 was evaluated by real time PCR technique. Results: Based on TMA expression results, we found that breast cancer metastases exhibited statistically significant less KISS1 expression vs. primary specimens (Kruskal-Wallis p value = 0.011). In validation set, 84.2% of IDC samples had either moderate (38/76) or high (26/76) expression of KISS1 protein whereas weak (negative or mild) expression was observed in 15.8% cases (12/76). In contrast, metastatic cases demonstrated weak expression in 100% cases (19/19). The real time PCR data confirmed IHC findings. We analyzed the correlation between the level of KISS1 expression and breast cancer prognosis based on the IHC data. The group of patients with high expression of KISS1 protein (1x and more) was found to have a trend toward slower disease progression. Those patients developed brain metastases 51.0+15.0 (CI: 17.7-84.3) months after tumor resection whereas the group of patients with no KISS1 expression had metastases after only 27.0+4.93 (CI: 15.39-38.69) months. Conclusions: These results suggest that high level of KISS1 protein expression is advantageous for breast cancer patients and that low levels correlate with metastases towards secondary organs such as brain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1439. doi:10.1158/1538-7445.AM2011-1439

Solid tumour cells were shown to express VLA-beta and Thy-1 antigens. For identification of these molecules two monoclonal antibodies, K-20 and ICO-10, characterised in detail previously, were used. Four groups of solid tumours have been identified according to their immunophenotype: VLA-beta+ and Thy-1-; VLA-beta+ and Thy-1+; VLA-beta- and Thy-1+; VLA-beta- and Thy-1-. To a certain extent these groups have been shown to reflect tumour histogenesis: tumours of epithelial origin never expressed an ICO-10+, K20-phenotype while soft tissue sarcomas and neuroblastoma cells never expressed the beta-chain of VLA molecular complexes.

3077 Background: Vaccination with dendritic cells (DC) expressing tumor antigens is considered to be a possible strategy to induce anti-tumour immune responses in treatment of melanoma. Methods: 42 stage III and IV melanoma patients were enrolled into the study receiving autologous monocyte-derived DCs primed with autologous tumor lysate. DCs displayed mature phenotype. 75% of recruited pts had disease progression after conventional treatments modalities. Pts demographic characteristics wear: age 26–82 years (mean 54), male/female ratio = 24/18. Arm A included 17 pts treated with DC vaccine in therapeutic regimen (III stage - 2 pts, IV stage - 15 pts). Arm B consisted of 25 pts receiving DC vaccine after radical metastasectomy (III stage - 10 pts, IV stage - 15 pts). DC were administered s.c. q 2–4 weeks until disease progression in a dose of 1,0–5,0x10(6) cells. Pts who received = 4 vaccinations were evaluated, totally - 33 pts (79%) were considered evaluable. The vaccine therapy was evaluated for safety, tolerability and clinical effectiveness. Results. DCs vaccine was safe and well tolerated. Toxicity included flu-like syndrome (1 grade CTC), transitory hyperemia and itching in the sites of injections (1 grade CTC). 13 of 17 pts were evaluable in arm A, they received 210 vaccinations (from 4 to 21 per pts, mean 10,5). We observed 15,4% overall clinical response (1 complete and 1 partial response) and 38% stabilization of disease (lasting = 2 mos). 1- year survival, median survival were 42,5% and 9,5 mos, respectively. Follow-up 4–39+ mos. Among 20 evaluable pts in arm B who received 205 vaccinations (from 4 to 38 per pts, mean 15,8) median time to progression was 10 mos, 1-year survival - 84%, at a median follow- up of 12 mos (3–20+) disease free survival was 45%. Conclusion: DCs vaccine therapy is safe and lacks serious adverse effects. Melanoma pts with regional and distant metastasis demonstrated moderate clinical effectiveness in response to specific vaccine therapy. No significant financial relationships to disclose.

The development of a transplantable tumor (Zajdela ascitic hepatoma) is studied in pregnant rats. Pregnancy is shown to retard tumor growth and influence its course. It prolongs the survival of tumor-bearing rats and induces total regression of the tumor in 15–20% of cases; pregnancy causes the ascitic growth to be transformed into a solid form, and prevents the development of hemorrhagic ascites.

A modifying effect of a catecholamine precursor (L-DOPA, 20 mg/kg, intraperitoneally) on various stages (initiation and promotion) of hepatocarcinogenesis, induced by N-nitrosodiethylamine (NDEA, 85 mg per 1 litre of tap water) was studied in chronic experiments on 150 male rats. L-DOPA administration prior to NDEA (influence on initiation) stimulated hepatocarcinogenesis considerably, while its administration after NDEA (influence on promotion) inhibited the realization of a carcinogenic effect. A statistically significant decrease in noradrenaline hypothalamus content was identified during early stages of chemically induced neoplastic transformation of hepatic cellular elements (stages of diffuse and focal proliferation). The results are discussed in terms of the regulatory role of the tone of the adrenergic autonomous nervous system component at the level of presynaptic inhibitory receptors in realization of the chemical carcinogenic effect.

The action of dexamethasone on lymphoid tissue in a culture of partially purified peripheral blood lymphocytes was biphasic in character. After incubation of lymphocytes in vitro with the hormone for 6 h stimulation of RNA synthesis was found. Sedimentation analysis of labeled RNA fractionated on a column containing poly-V-sepharose indicated an increase in the mRNA content and enrichment of cytoplasmic RNA with polyA sequences. Meanwhile Mn++-dependent RNA-polymerase, sensitive to α-amanitine, was activated. After cultivation of the lymphocytes with the hormone for 24 h, RNA synthesis was inhibited. The biphasic character of the action of the steroid also was observed in the rosette-formation test.

Subpopulations of T- and B- lymphocytes may be distinguished by their sensitivity to Dexamethasone (9L-fluoro-16L-methyl-11β, 17, 21-trihydroxypregna-1,4-diene-3,20-dione). The RNA-synthesizing system of lymphoid cells at different stages of differentation displays distinct sensitivity to Dexamethasone. The rate of RNA synthesis in thymocytes of normal organisms is eventually lowered under the action of the hormone. The glucocorticoid effect on RNA biosynthesis in T1-lymphocytes, however, has been found to be of a biphasic character (stimulation followed by inhibition), whereas the RNA synthesizing system of B- and T2- lymphocytes proved to be nonresponsive to the hormone under similar conditions. It is concluded that a complete loss of sensitivity of the RNA-synthesizing system to the hormone or a drastic diminution of the capacity to respond to it observed in peripheral blood lymphocytes taken from cancer patients may be due to a shift in the relative proportion of various types of lymphocyte populations in favour of the nonreactive lymphocytes.

Biosynthesis of RNA catalyzed by DNA-dependent RNA polymerase was demonstrated in a reconstructed system containing isolated lymphocyte nuclei, Mg2+ or Mn2+ salts, and ammonium sulfate, in the presence of four nucleoside triphosphates. Both Mg2+- and Mn2+-dependent forms of this enzyme were found in the nuclei of normal lymphocytes and of lymphocytes from patients with melanoma, lung carcinoma, and sarcoma. The activity of both forms of RNA polymerase in the nuclei of the patients' lymphocytes was higher than in normal analogs. The sensitivity of DNA-dependent RNA polymerase to dexamethasone and phytohemagglutinin was less marked in the nuclei from patients with lung carcinoma, melanoma, and sarcoma than in normal lymphocytes.

Phytohemagglutinin (PHA) stimulates RNA synthesis 6 h after the beginning of incubation with blood lymphocytesin vitro. Dexamethasone, in a concentration of 60 μg/ml, inhibits RNA synthesis in PHA-treated lymphocytes from donors' blood 6 h after the beginning of incubation of the cells with the hormone and PHAin vitro. Changes in the mechanism of action of the hormone which may lead to changes in the sensitivity of the same cells, at different stages of the cell cycle, to glucocorticoids are analyzed.

Employing the immunohistochemical method with the use of specific antisera to melatonin and to N-acetylserotonine the authors showed the presence of these substances in the enterochromaffine cells of the gastrointestinal. It is emphasized that in the organism there exists a group of melatonin-producing cells whose principal link is served by the enterochromatoffine cells playing an important role in the maintenance of homeostasis due to their function--that of melatonin production.

Investigation of acid phosphatase activity during interaction between lymphocytes and target cells showed that its greatest increase is observed in lymphocytes in 1-h and sometimes 3-h cultures, whereas in the cells it was observed after 3–6 h. Initial acid phosphatase activity was higher in immune lymphocytes, but later, on their addition to a culture of L-cells, no significant difference was found between the change in enzyme activity in immune and normal lymphocytes. Acid phosphatase activity in L-cells was lower on the addition of normal than of immune lymphocytes. Activity of the dehydrogenases in the lymphocytes increased until 3 h of incubation and the increase was greater in immune than in normal lymphocytes. Activity of succinate and dehydroorotate dehydrogenases in the L-cells changed at virtually the same times as in the lymphocytes. Increased activity of oxidoreductases and acid phosphatase in the first few hours of contact is evidence of the rapid activation of effector lymphocytes.

Six hybridoma clones were obtained secreting monoclonal antibodies (Mab) against differentiating antigens of human hemopoietic cells. ICO-1 Mab detect Ia-like (Dr) antigens. Mab react with B-lymphocytes and monocytes without detecting antigens on granulocytes and T-cells. Antigen expression was enhanced following cell activation in a blast cell transformation test and mixed lymphocyte culture (MLC). ICO-1 Mab blocked MLC. The molecular weights of the antigen were 29 and 34 kilodaltons. Comparative studies of ICO-1 Mab with other Mab against Ia-like antigens revealed their identical reactivity. ICO-11 Mab detect the antigen on 4% of blood lymphocytes, 75% thymocytes, monoblasts, and CFU-GM. These Mab block NK-cell activity of blood mononuclear cells. ICO-GM-1 Mab detect the antigen on myelomonocytic cells and their precursors, but not CFU-GM. These Mab block binding of the C3Bi complement component to CR3 receptor and NK-cell activity. ICO-G2 Mab detect the antigen expressed at final stages of granulocyte differentiation. ICO-10 Mab detect the antigen on early thymocytes and ICO-02 on undifferentiated blast cells. Mab were shown to be applicable for human leukemia and lymphoma immune diagnosis.

IL-6, or cytokines of the IL-6 family using gp130 as transducer chain receptor, have been suggested to play a role in certain B lymphoid neoplasia. The presence of cell membrane gp80 and gp130 IL-6 receptors was studied in 98 patients with various leukaemia and non-Hodgkin's malignant lymphoma using flow cytofluorometry and immunohistology. Except neoplasia of immature B cells which expressed neither of the receptors, the majority of B cell tumours expressed one or both of them, mantle cell lymphoma being found to express the highest density of receptors. Using IL-6-dependent XG myeloma cell lines and mAb recognizing various gp80 and gp130 functional epitopes, it has been shown that IL-6 activation leads to a modified expression of some epitopes. In particular, the decrease or the disappearance of a gp130 epitope called A1 signed gp130 dimerization which is the first step of the gp130 activation pathway. Gp80 and gp130 epitope analysis was achieved in 17 of the patients. In four, an epitope phenotype compatible with a cytokine-induced activation was found. The cells of five B-CLL patients which expressed both gp80 and gp130 receptors were incubated with IL-6 to induce activation. In three of the cases they were found to rearrange their receptors in activated forms but not in the two others, showing that cells able to be activated or not can be found. These results confirm that gp130 signalling might play an important role in the pathogenesis of certain B cell neoplasia.