Protumorogenic Potential of Pancreatic Adenocarcinoma-Derived Extracellular Vesicles.

Cancer development is a highly complicated process in which tumour growth depends on the development of its vascularization system. To support their own growth, tumour cells significantly modify their microenvironment. One of such modifications inflicted by tumours is stimulation of endothelial cell migration and proliferation. There is accumulating evidence that extracellular vesicles (EVs) secreted by tumour cells (tumour-derived EVs, TEVs) may be regarded as "messengers" with the potential for affecting the biological activities of target cells. Interaction of TEVs with different cell types occurs in an auto- and paracrine manner and may lead to changes in the function of the latter, e.g., promoting motility, proliferation, etc. This study analysed the proangiogenic activity of EVs derived from human pancreatic adenocarcinoma cell line (HPC-4, TEVHPC) in vitro and their effect in vivo on Matrigel matrix vascularization in severe combined immunodeficient (SCID) mice. TEVHPC enhanced proliferation of HPC-4 cells and induced their motility. Moreover, TEVHPC stimulated human umbilical vein endothelial cell (HUVEC) proliferation and migration in vitro. Additionally, TEVHPC influenced secretion of proangiogenic factors (IL-8, VEGF) by HUVEC cells and supported Matrigel matrix haemoglobinization in vivo. These data show that TEVs may support tumour propagation in an autocrine manner and may support vascularization of the tumour. The presented data are in line with the theory that tumour cells themselves are able to modulate the microenvironment via TEVs to maximize their growth potential.

Expression of the central obesity and Type 2 Diabetes mellitus genes is associated with insulin resistance in young obese children.

OBJECTIVES: The assessment of the health consequences associated with obesity in young children is challenging. The aims of this study were: (1) to compare insulin resistance indices derived from OGTT in obese patients and healthy control (2) to analyze central obesity and Type 2 Diabetes genes expression in obese children, with special attention to the youngest group (< 10 years old). PATIENTS AND METHODS: The study included 49 children with obesity (median age 13.5 years old), and 25 healthy peers. Biochemical blood tests and expression of 11 central obesity and 33 Type 2 Diabetes genes was assessed. RESULTS: A significant difference in insulin resistance between obese and non-obese adolescents was observed in all studied indices (mean values of the insulin levels: 24.9 vs. 9.71 mIU/L in T0, 128 vs. 54.7 mIU/L in T60 and 98.7 vs. 41.1 mIU/L in T120 respectively; AUC: 217 vs. 77.2 ng/ml*h, mean values of B% (state beta cell function), S% (insulin sensitivity), and IR were 255 (±97) vs. 135 (±37.8), 46.6 (±37.3) vs. 84.2 (±29.6) and 3 (±1.55) vs. 1.36 (±0,56); HIS, WBIS and ISIBel median 3.89, 44.7, 0.73 vs. 8.57, 110, 2.25. All comparisons differed significantly p<0.001). Moreover, insulin sensitivity was significantly better in the older obese group (>10 years old): median AUC 239 vs. 104 ng/ml*h, and HIS, WBIS and ISIBel 3.57, 38, 0.67 vs. 6.23, 75.6, 1.87 respectively in the obese older compared to the obese younger subgroup, p<0.05. The expression of 64% of the central obesity genes and 70% of Type 2 Diabetes genes was higher in the obese compared to control groups. The differences were more pronounced in the younger obese group. CONCLUSION: Insulin resistance may develop in early stage of childhood obesity and in very young children may be associated with higher expression of the central obesity and Type 2 Diabetes genes.

Human monocyte subsets exhibit divergent angiotensin I-converting activity.

Immune cells may take part in the renin-angiotensin-aldosterone system (RAAS), which plays a pivotal role in the regulation of vascular tone and blood pressure. The aim of the study was to analyse the expression and activity of angiotensin-converting enzyme type 1 (ACE1) and ACE2 in human monocytes (MO) and their subsets. The highest relative level of ACE1-, as well as ACE2-mRNA expression, was observed in CD14(++)CD16(-) (classical) MO. Moreover, in these cells, mean level of ACE2-mRNA was almost two times higher than that of ACE1-mRNA (11.48 versus 7.073 relative units, respectively). In peripheral blood mononuclear cells (PBMC), MO and classical MO, ACE1 and ACE2 protein expression was stronger compared to other MO subpopulations. The highest level of Ang II generated from Ang I in vitro was observed in classical MO. In this setting, generation of Ang-(1-9) by PBMC and classical MO was higher when compared to the whole MO population (P < 0.05). The generation rate of vasoprotective Ang-(1-7) was comparable in all analysed cell populations. However, in CD14(+)CD16(++) (non-classical) MO, formation of Ang-(1-7) was significantly greater than Ang II (P < 0.001). We suggest that in physiological conditions MO (but also lymphocytes forming the rest of PBMC pool) may be involved in the regulation of vessel wall homeostasis via the RAAS-related mechanisms. Moreover, non-classical MO, which are associated preferentially with the vascular endothelium, express the vasoprotective phenotype.

Effect of tumour necrosis factor-α inhibitor on serum level of dickkopf-1 protein and bone morphogenetic protein-7 in ankylosing spondylitis patients with high disease activity.

OBJECTIVES: To examine changes in serum levels of the bone remodelling molecules dickkopf-1 (Dkk-1), sclerostin, wingless-type protein-3a (Wnt-3a), and bone morphogenetic protein-7 (BMP-7) during 6 months of anti-tumour necrosis factor (anti-TNF) treatment in ankylosing spondylitis (AS) patients with high disease activity. METHOD: We included 40 patients with axial AS: 20 patients with high disease activity were assigned to treatment with TNF inhibitor and 20 with low disease activity were assigned to non-steroidal anti-inflammatory drug (NSAID) treatment. Markers of bone remodelling and inflammation were assessed at baseline and after 6 months. RESULTS: In the TNF inhibitor-treated group Dkk-1 decreased significantly from 196.8 pg/mL [95% confidence interval (CI) 94.1-399.0] to 116.3 pg/mL (95% CI 38.0-301.6) and BMP-7 increased significantly from 1.4 pg/mL (95% CI 0-23.0) to 20.3 pg/mL (95% CI 4.9-41.3). There was a significant negative correlation between Dkk-1 and BMP-7 at 6 months (r = -0.64, p = 0.004) in this group. Non-parametric regression analysis adjusted for disease duration, age, sex, baseline modified Stoke's Ankylosing Spondylitis Spine Score (mSASSS), and baseline C-reactive protein (CRP) confirmed a statistically significant effect of treatment on time-related changes of Dkk-1 and BMP-7. Erythrocyte sedimentation rate (ESR), CRP, and also the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score decreased significantly in the anti-TNF-treated group. CONCLUSIONS: Among the potential biomarkers of bone remodelling in AS, Dkk-1 and BMP-7 displayed significant time alterations and correlative interactions during anti-TNF treatment.

Pentoxifylline does not affect nociception if administered postoperatively.

Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1) and interleukin-6 (IL-6), act as mediators of post-injury inflammation and increase pain sensitivity. Pentoxifylline (PTX) has the property of inhibiting TNF-alpha, IL-1, and IL-6 production. Previous studies revealed that the pre-injury or preoperative administration of PTX inhibited consequent hyperalgesia or postoperative pain. The aim of the study was to determine, if postoperative PTX administration affects postoperative pain. A group of 40 patients undergoing laparotomic cholecystectomy received postoperatively PTX at 10 mg/kg or placebo directly after the termination of general anesthesia. There were no differences in postoperative pain, analgesic drug requirement or TNF-alpha and IL-6 serum levels between the groups.

Cross-talk between human monocytes and cancer cells during reactive oxygen intermediates generation: the essential role of hyaluronan.

Human monocytes exhibit considerable cytocidal activity against tumor (but not normal cells) associated, at least partly, with the generation of reactive oxygen intermediates (ROIs). The present study examined the role of surface determinants and hyaluronan (HA) in the induction of ROI production by human monocytes stimulated with cancer cells, as measured by luminol-enhanced chemiluminescence (CL). The inhibitory effect of monoclonal antibodies (MAbs) indicated the engagement of CD18, CD29 and CD44 adhesion molecules. Preincubation of monocytes and tumor cells, expressing CD44 determinants, with either anti-CD44 MAb or HA inhibited CL generation. Addition of HA to monocytes decreased the expression of CD44 and induced CL response. Supernatants from the cultures of tumor cells stimulated CL response of monocytes, an effect that was abolished by treatment of the supernatants with hyaluronidase (HAase) or by preincubation of monocytes with an anti-CD44 MAb. These results indicate that several surface molecules of monocytes, including CD44, are required to trigger the generation of ROI after their contact with tumor cells, whereas HA overexpressed on some cancer cells may allow monocytes (via CD44) to distinguish between transformed and normal cells. However, blocking of CD44 on monocytes by free HA dampens their response to tumor cells. Taken together, these observations suggest that the presence of HA in the tumor stroma may modulate effector functions of infiltrating macrophages and their interactions with cancer cells in situ.

Role of Stat3 in lipopolysaccharide-induced IL-10 gene expression.

IL-10 is a unique cytokine because it is anti-inflammatory and immunosuppressive. IL-10 is regulated at the level of transcription, but the critical motifs and the relevant transcription factors controlling this gene have remained elusive to date. We now report that a sequence at -120 bp in the human IL-10 promoter binds Stat3 but no other Stat proteins. Mutation of this motif abrogates LPS-induced trans-activation. Overexpression of dominant negative Stat3 suppresses promoter activity, while wild-type Stat3 leads to an enhancement of this activity. Our results show that Stat3, by binding to a single motif in the IL-10 promoter, is controlling expression of the human IL-10 gene.

The M-DC8-positive leukocytes are a subpopulation of the CD14+ CD16+ monocytes.

Recently, a population of M-DC8-positive leukocytes has been described as a new subpopulation of human dendritic cells (DC). In view of the expression of the CD16 antigen on these cells, as well as the finding that DC can arise from blood monocytes, we hypothesized that the expression of M-DC8 is mainly associated with the CD14+ CD16+ phenotype of blood monocytes. Immunofluorescence analysis of whole blood showed that the percentage of M-DC8+ cells is about three times lower than the percentage of CD14+ CD16+ monocytes among all leukocytes (0.32% versus 1.10%). Further, in addition to the expression of CD16, these M-DC8+ cells were also positive for CD14 at low density. Multicolor flow cytometric analysis of whole blood revealed that more than one third of the CD14+ CD16+ monocyte population expressed the M-DC8 antigen (42.3%), and almost all M-DC8+ cells were CD14-CD16-double-positive (87.5%). Finally, the M-DC8 antigen was also expressed on alveolar macrophages from healthy individuals, i.e., cells that are phenotypically and functionally related to the CD14+ CD16+ monocytes. Taken together, the data presented here clearly demonstrate that the M-DC8+ leukocytes are a subpopulation of the CD14+ CD16+ monocytes and may represent DC

The effect of pentoxifiline on post-injury hyperalgesia in rats and postoperative pain in patients.

Recent studies demonstrate that activation of proinflammatory cytokines following injury intensifies the process of nociception. The present investigation assessed the influence of pre-injury pentoxifiline (PTFL, a non-specific cytokine inhibitor) on the development of post-injury nociception in animals and patients. It was established that intrathecal or intraperitoneal PTFL, elevated the nociceptive threshold for mechanical stimuli in the formalin test in rats. Pre-injury PTFL also inhibited pain-related behaviour. These findings correlate with a lower TNFalpha level in the serum of animals receiving pre-injury PTFL. In clinical investigations PTFL was administered intravenously before elective cholecystectomy. Patients who received preoperative PTFL had lower opioid requirements in the early postoperative period than control. At the same time, serum levels of TNFalpha and IL6 were lower in the PTFL group. Our results confirm the hypothesis as to the possibility of modulating of nociception through preemptive administration of a cytokine inhibitor.

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.

Involvement of protein kinases in signalling for nitric oxide (NO) and tumour necrosis factor alpha (TNF) production by monocytes stimulated with colorectal DeTa cancer cells: the lack of evidence for the role of TNF in the regulation of NO production.

Kinases that are involved in NO and TNF production by human monocytes (MO) stimulated by colorectal cancer (DeTa) cells and effects of exogenous and endogenously synthesized TNF on NO induction were studied. The results based on the use of various inhibitors of protein kinases suggest that different signalling pathways operate in MO during induction of TNF and NO release after stimulation by DeTa cells. Stimulation of NO production required at least PTK, PKC and PKA, but only PTK and PKC were engaged in signal transduction for TNF production. Exogenous TNF and TNF produced by MO upon contact with DeTa cells was not sufficient for the induction or enhancement of NO synthesis in MO. The TNF synthesis was not influenced by neither exogenous nor endogenous NO produced by MO in the co-culture. Therefore, signal transduction pathways operating in MO during NO induction seem to be different from these engaged in TNF production, and both regulatory pathways probably operate in MO independently.

Induction of reactive oxygen intermediates in human monocytes by tumour cells and their role in spontaneous monocyte cytotoxicity.

The present study examined the ability of human monocytes to produce reactive oxygen intermediates after a contact with tumour cells. Monocytes generated oxygen radicals, as measured by luminol-enhanced chemiluminescence and superoxide anion production, after stimulation with the tumour, but not with untransformed, cells. The use of specific oxygen radical scavengers and inhibitors, superoxide dismutase, catalase, dimethyl sulphoxide and deferoxamine as well as the myeloperoxidase inhibitor 4-aminobenzoic acid hydrazide, indicated that chemiluminescence was dependent on the production of superoxide anion and hydroxyl radical and the presence of myeloperoxidase. The tumour cell-induced chemiluminescent response of monocytes showed different kinetics from that seen after activation of monocytes with phorbol ester. These results indicate that human monocytes can be directly stimulated by tumour cells for reactive oxygen intermediate production. Spontaneous monocyte-mediated cytotoxicity towards cancer cells was inhibited by superoxide dismutase, catalase, deferoxamine and hydrazide, implicating the role of superoxide anion, hydrogen peroxide, hydroxyl radical and hypohalite. We wish to suggest that so-called 'spontaneous' tumoricidal capacity of freshly isolated human monocytes may in fact be an inducible event associated with generation of reactive oxygen intermediates and perhaps other toxic mediators, resulting from a contact of monocytes with tumour cells.

Intravesical interleukin-2 in T1 papillary bladder carcinoma: regression of marker lesion in 8 of 10 patients.

PURPOSE: We evaluate the therapeutic effect of intravesical interleukin-2 (IL-2) on T1 papillary bladder carcinoma after incomplete transurethral resection. MATERIALS AND METHODS: After incomplete transurethral resection we treated 10 patients in whom the marker lesion was left in place with 3 x 10(6) Chiron units IL-2 in 50 ml. saline plus 0.1% human serum albumin. The solution remained in the bladder for 2 hours and it was instilled on 5 consecutive days. The effect of IL-2 treatment on the marker lesion was evaluated by cystoscopy and repeat biopsy of the marker site 2 months after treatment. In addition, the effect on the recurrence of bladder tumors was studied. RESULTS: At 2 months 8 of the 10 marker lesions (80%) had completely regressed and there were no tumor cells on repeat biopsy. Four patients remained tumor-free after 30 to 54 months. We noted no toxic effects. In 1 patient with a 7-year history of bladder cancer the marker was only partially regressed after 2 months. After removal of the marker this patient remained tumor-free at a followup of 54 months. CONCLUSIONS: To our knowledge this report represents the first study of the effect of IL-2 on marker lesions left in place after transurethral resection. The results indicate that IL-2 instillations are feasible, and the combination of transurethral resection and IL-2 instillation may have a powerful antitumor effect. The therapeutic effects may not simply be due to intravesical IL-2, because previous transurethral resection probably caused some influx of infiltrating cells and the marker may have had tumor associated antigens. Consequently these effects may be due to the interaction of tumor associated antigens, infiltrating cells and IL-2.

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.

MHC class I and class II determinants and some adhesion molecules are engaged in the regulation of nitric oxide production in vitro by human monocytes stimulated with colon carcinoma cells.

Surface molecules that are involved in tumor-monocyte interactions were studied. The in vitro system in which human blood monocytes are stimulated with human colon carcinoma cells for nitric oxide (NO) production was used. Monoclonal antibodies (mAbs) against various adhesion molecules (LFA-1, ICAM-1, VNR) were unable to block NO production in coculture of monocytes with carcinoma cells. However, anti-CD44, -LFA-3, and -VLA beta 1 chain mAbs effectively blocked NO production. Also mAbs against MHC class I and HLA-DR molecules inhibited, in a dose-dependent manner, No release. It was concluded that some adhesion molecules and MHC class I and/or class II determinants of monocytes may be involved in tumor-monocyte interactions leading to signal transduction for NO production.

Characterization of human pancreatic adenocarcinoma cell line with high metastatic potential in SCID mice.

The CD44 molecule and CD44 isoforms are expressed on some malignant tumours and it has been suggested that their expression may correlate with tumour spread. Human pancreatic carcinoma cell line (HPC-4) expressing CD44 was established from a patient with adenocarcinoma of pancreas. This line showed a rapid growth in vitro, several chromosome abnormalities and surface expression of some adhesion molecules (ICAM-1, LFA-3, beta 1-chain of VLA integrins, VNR). Xenotransplanted HPC-4 cells were able to grow rapidly in SCID mice as subcutaneous tumour, leading to 100% mortality within 3-5 weeks when 1 x 10(5)-1 x 10(7) cells were inoculated. Spontaneous metastases in the liver, lung, spleen and kidney of SCID mice were observed. Interestingly enough, HPC-4 cells in vivo and ex vivo also expressed HLA-DR molecules, but these were rapidly lost upon culture in vitro. It is suggested that the appearance of HLA-DR may be the result of interaction of the tumour with a local environment of the host, while CD44 expression may explain the rapid growth and occurrence of distant metastases in SCID mice. The ability of HPC-4 cells to form spontaneous metastases in SCID mice may prove to be a potentially interesting model of human carcinoma for testing new treatment modalities.

Human monocytes are stimulated for nitric oxide release in vitro by some tumor cells but not by cytokines and lipopolysaccharide.

Nitric oxide (NO) has been recently identified as a potent mediator of tumoricidal activity of activated macrophages. Macrophages can be activated for tumor cell killing by microbial products, including lipopolysaccharide (LPS) and various cytokines. Here we report that in contrast to mouse macrophages, human peripheral blood monocytes stimulated with cytokines or LPS failed to release NO. Also priming of monocytes with interferon-gamma followed by activation with cytokines or LPS did not cause NO secretion. However, monocytes responded with NO production to stimulation with some human cancer cells but not with untransformed cells. NO production by monocytes was inhibited by NG-monomethyl-L-arginine, specific inhibitor of NO synthase and emetine, an irreversible blocker of protein synthesis. This may imply that human monocytes are unique in their restricted capacity to produce NO following interaction with some tumor cells, but not with other stimulators, and in this respect they may be able to distinguish between malignant and normal cells.

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.

[Clinical probes using tumor necrosis factor in treatment of neoplastic diseases]. / Próby kliniczne zastosowania czynnika martwicy nowotworów (tumor necrosis factor alfa-TNF) w leczeniu chorób nowotworowych.

Tumor necrosis factor-TNF is a cytokine that is predominantly produced by the activated macrophages. It was shown that TNF has the antineoplastic activity in vitro or in tumor xenografts on the animals in vivo. Clinical trials phase I and II carried out in many centers assessed toxic effects of TNF administration, as well as its pharmacokinetic and therapeutic effect. The following paper is a survey of the results of clinical trials which could be useful for the assessment of TNF role in oncological immunotherapy.