Endogenous erythropoietin and erythropoietin receptors in colorectal cancer; can we answer the questions?

Erythropoietin (Epo) is glycoprotein hormone which binds on erythropoietin receptors (EpoR) promoting proliferation and differentiation. Studies have shown that EpoR, apart from erythrocyte precursors, is expressed on no hematopoietic tissue and various tumor cells. Despite the progress in modern medicine, colorectal carcinoma (CRC) is still the leading cause of increased morbidity and mortality between oncology patients worldwide. Its precursors are benign villous adenomas, which in certain percentage progress to cancer. Anemia of chronic disease is common finding in CRC patients. Some of them are treated with Epo. Epo/EpoR seems to correlate with tumor progression and metastasizing. Therefore, the identification of at-risk group remains a clinical challenge. Vascular endothelial growth factor (VEGF) is a signal protein that stimulates angiogenesis and concentration of VEGF is positive correlated with tumor growth in numerous tumors. The importance of Epo in tumor pathogenesis has led to a growing interest in the potential prognostic value. By our point of view there are many open questions about role of Epo/EpoR in CRC.

Indicators of Cellular and Developmental Disorders in Multiple Primary Cancers.

In human organism development is a very complex and highly regulated system that enables the functional balance of each organ in a whole body. Disorders and tumor micro-environment weaken host immune system that is not able to recognize the tumor as a unknown body and fight against its uncontrollable forces. Tumor avoids the immune system in a way that promotes immunosuppression and orientation cytokine production towards Th2 immune responses which are responsible for infection appearances. Some of infectious agents (viruses) can cause oncogene activation and inhibition of tumor suppressor genes. It is also known that oncology treatment can be detrimental to the host immune system. The drugs or radiation can activate different signaling pathways which lead to a vicious circle from which there is no return. Experimental models of tumor biology and molecular events in vivo are patients who have multiple primary cancers (MPC) diagnosed during life. Such patients confirm the complexity of disorders that occur in the cell and explain all the influences and contributions to developmental tumor cascade.

Role of tumor-associated glycoprotein-72 in the progression of endometrial adenocarcinoma: a proposed study.

Endometrial adenocarcinoma is on the basis of the molecular, immunohistological and clinicopathologic features broadly divided into two groups, referred as type I and type II. Type I appears more frequently and in principle patients have a good prognosis; however a significant number of patients develop local recurrences. We hypothesize that TAG-72, expressed on endometrial carcinoma binds and internalizes endocytic pattern recognition receptors on surrounding tissue antigen presenting cells (dendritic cells and macrophages), powers their anti-inflammatory maturation program and make them capable to elicit or modulated tolerogenic immune response mediated by local T and NK effectors. This could support uncontrolled local tumor growth, deeper tumor invasion into surrounding tissues, frequent local recurrences and/or lymph node metastasis. To test this hypothesis, we propose a semi-quantitative immunohistochemical analysis of TAG-72 expression in endometrial adenocarcinoma samples and to correlate the results with clinical and pathological parameters (age, type and histological grade of the tumor, estrogen and progesterone receptor expression, invasion into the myometrium and capillaries, presence of lymph node metastases, FIGO stage, and TNM classification). It would be worthwhile to investigate the local tissue immune response in the tumor environment using tissue samples removed during surgery. These studies could elucidate the underlying immunopathological mechanisms that govern the early recurrence and possibly distant metastases of TAG-72-expressing adenocarcinomas and might help in deciding the type of treatment to be applied in a selected group of cancer patients including application of biological therapy with anti-TAG-72 antibodies, according the principle of personalized oncology treatments.

Heat-Shock Proteins 70 Induce Pro-Inflammatory Maturation Program in Decidual CD1a(+) Dendritic Cells.

PROBLEM: The aim of the study was to assess possible binding of a mixture of constitutive Hsc70 and inducible Hsp70 forms (HSP70) to Toll-like receptor (TLR) 4 and CD91 receptors on decidual CD1a(+) dendritic cells (DCs) and their influence on DCs maturation status. METHOD OF STUDY: Immunohistology and immunofluorescence of paraffin-embedded first trimetester and term pregnancy decidua were performed together with flow cytometry detection of antigens in DCs after stimulation of decidual mononuclear cells with HSP70. RESULTS: Hsc70 and Hsp70 labeling revealed intracellular and nuclear staining in trophoblast cells. The numbers of Hsc70(+) and Hsp70(+) cells of decidual tissue were higher in early pregnancy decidua than in decidua at term. HSP70 binds CD91 and TLR4 receptors on CD1a(+) DCs and increased the expression of CD83, HLA-DR, CD80, and CD86, but decreased CC receptor (CCR) 5. HSP70 increased CC ligand (CCL) 3 and CCL22. HSP70 in the concentration of 1 µg/mL increased the percentage of interferon-γ and interleukin (IL)-15-expressing cells over the cells expressing IL-4. CONCLUSION: HSP70 binds CD91 and TLR4 on decidual CD1a(+) DCs, causes their maturation, and increases IL-15 in the context of Th1 cytokine/chemokine domination, which could support immune response harmful for ongoing pregnancy.

Mystery Story about Erythropoietin (Epo) and Erythropoietin Receptor (EpoR) are Disguised?

In this review we would like to focus our attention upon very controversial reports on Erythropoietin (Epo) and Erythropoietin Receptor (EpoR) expression in cancer patients. The effects of Epo on cancerous tissues are poorly understood. Hypoxia results in an increase in the level of the production of both Epo and EpoR via activation of the hypoxia-inducible factor 1 (HIF-1) pathway. HIF-1α, promotes the expression of vascular endothelial growth factor (VEGF). The signaling through VEGF in both a paracrine and an autocrine manner is required for the homeostasis of adult vessels. Macrophages stimulate vessel sprouting via a soluble factor other than VEGF, rather than through direct contact with endothelial cells. The intriguing questions are set about many researches to link Epo/EpoR expression and function in order to establish one of the mechanisms of tumor growth, disease progression of cancer patient. However, it is uncertain role in tumour angiogenesis as promoter and stimulator of tumour growth which should need to be furtherly validated.

Nutrition in cancer patients.

Cachexia is defined as an unintended loss of stable weight exceeding 10%. Patients with advanced cachexia express anorexia, early satiety, severe weight loss, weakness, anemia, and edema. Anorexia represents the result of a failure of the usual appetite signals whereas cachexia is the debilitating state of involuntary weight loss. This syndrome, referred to as the cancer anorexia-cachexia syndrome, (CACS) and usually consists of a combination of anorexia, tissue wasting, malnutrition, weight loss and loss of compensatory increase in feeding. CACS represents the result of a complex interaction between cancer growth and host response and is associated with a poor response to chemotherapy and with an increase in drug-related toxicity. In advanced cachexia (mostly in metastatic cancer and terminally disease) any interventions with nutritional suplements are ineffective. Therefore, nutritional support in the reversion of tumor cachexia and in the importance of maintaining patient weight, muscle mass, quality of life, has the exceptional importance, becouse good nutritional status of patients leads to the possibility of more aggressive and longer treatment and thus to longer survival.

The approaches in the care for terminal cancer patients in radiotherapy and oncology clinic, Rijeka University Hospital Center.

We sought to determine the proportion of our admitted patients in terminal phase of ilness who recieved some kind of active oncological therapy. We conducted a pilot study on the records of patients who died in the University Hospital. We assessed the percentage of mortality, a therapeutic approach in terms of treating the underlying disease, and access to palliative treatment. Of 2097 patients hospitalized in the UHC Rijeka Department of Radiation Therapy and Oncology during 2010 and 2011, 44 pts died which accounts for 2.1%. The most common primary sites of cancer in patients who died in our Department were the lungs and then the breast. Ten (22.7%) patients were admitted exclusively to receive palliative care, while others (34-77.3%) were admitted for planned active chemo- and/or radiotherapy administration. Within three months before death, 18 (40.9%) patients underwent chemotherapy treatment. The number of patients hospitalized due to providing palliative care is extremely low, which could indicate a good supply of out-patient treatment of cancer patients in the terminal stage of the disease. However, concerned about the high percentage of patients who tried to provide oncology treatments in the three months before his death. The percentages referred to in their daily work is still guided by the principles of healing "to the end" and thus we plunge into the realm disthanasia.

Mucins help to avoid alloreactivity at the maternal fetal interface.

During gestation, many different mechanisms act to render the maternal immune system tolerant to semi-allogeneic trophoblast cells of foetal origin, including those mediated via mucins that are expressed during the peri-implantation period in the uterus. Tumour- associated glycoprotein-72 (TAG-72) enhances the already established tolerogenic features of decidual dendritic cells with the inability to progress towards Th1 immune orientation due to lowered interferon (IFN)- γ and interleukin (IL)-15 expression. Mucine 1 (Muc 1) supports alternative activation of decidual macrophages, restricts the proliferation of decidual regulatory CD56(+) bright natural killer (NK) cells, and downregulates their cytotoxic potential, including cytotoxic mediator protein expression. Removing TAG-72 and Muc 1 from the eutopic implantation site likely contributes to better control of trophoblast invasion by T cells and NK cells and appears to have important immunologic advantages for successful implantation, in addition to mechanical advantages. However, these processes may lead to uncontrolled trophoblast growth after implantation, inefficient defence against infection or tumours, and elimination of unwanted immunocompetent cells at the maternal-foetal interface. The use of mucins by tumour cells to affect the local microenvironment in order to avoid the host immune response and to promote local tumour growth, invasion, and metastasis confirms this postulation.

The significance of heat-shock protein gp96 and its receptors' CD91 and Toll-like receptor 4 expression at the maternal foetal interface.

PROBLEM: Differences in the expression of gp96 and its receptors were analysed in normal and pathological human pregnancy. MATERIAL AND METHODS: Immunohistology and immunofluorescence of sections from decidual part of term placenta, first trimester normal decidua, missed abortion and blighted ovum decidua were performed together with reverse transcriptase-quantitative polymerase chain reaction and flow cytometry. RESULTS: In missed abortion, gp96 was intensively stained, when compared to normal early pregnancy. The intensity of CD91 and TLR4 was higher in the first trimester pregnancy and blighted ovum, when compared to missed abortion. Decidual part of the term placenta is invaded with gp96⁺ , CD91⁺ and TLR4+ trophoblast. Progesterone-induced blocking factor (PIBF) decreased the frequency of TLR4⁺ T lymphocytes, CD91⁺ T, natural killer (NK) and mature dendritic cells after an 18-h culture. Decidual mononuclear cells (DMCs) treated with PIBF down-regulated CD91, TLR4 and gp96 gene expression. CONCLUSION: The presence of gp96, CD91 and TLR4 at the maternal-foetal interface provides a molecular basis for their interaction, particularly in the absence of PIBF.

Specific decidual CD14(+) cells hamper cognate NK cell proliferation and cytolytic mediator expression after mucin 1 treatment in vitro.

Mucin 1 (MUC1) forms a glycocalyx on the surface of decidual epithelial cells that needs to be removed for successful embryo attachment. We investigated whether MUC1 affects human early pregnancy decidual CD14(+) cells and their interactions with cognate decidual natural killer (NK) cells. FITC-dextran internalisation, surface and intracellular antigen levels, and proliferation of CD14(+) and/or CD56(+) cells were analysed by flow cytometry. Magnetic separation was used to purify CD56(+) and CD14(+) cells. Uncultured CD14(+) cells expressed a negligible percentage of CD1a and CD83 molecules. They expressed lower levels of CD16, and higher levels of endocytic mannose receptors (MR), dendritic cell-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN), proinflammatory chemokine CC receptor 5 (CCR5), and CD163 receptor, than their peripheral blood counterparts. Lipopolysaccharide stimulation did not affect FITC-dextran internalisation in CD14(+) cells. MUC1 bound and internalised, in a dose-dependent manner, the carbohydrate recognition domain of MR, increasing the decoy IL-1 receptor type II and decreasing IL-15 expression in CD14(+) cells. In the presence of MUC1-treated macrophages, the expression levels of the proliferation and cytotoxic mediators (perforin, Fas ligand and TNF-related activation-induced ligand or TRAIL) was attenuated, while that of the anti-inflammatory chemokine CCL17 was increased, in NK cells compared with untreated macrophages. In conclusion, MUC1 supports the alternative activation of tissue-specific CD14(+) cells, and may restrict proliferation of NK cells and regulate their content of cytotoxic mediators. Based on the experiments with first-trimester decidual cells in vitro, we conclude that removing MUC1 from decidual tissue might help control trophoblast invasion by NK cells.

Tumor-associated glycoprotein (TAG-72) is a natural ligand for the C-type lectin-like domain that induces anti-inflammatory orientation of early pregnancy decidual CD1a+ dendritic cells.

Tumor-associated glycoprotein-72 (TAG-72) is physiologically present in secretory phase endometrium, but its presence and possible immunological role in early normal human pregnancy decidua has not received attention. The double labeling of paraffin-embedded early pregnancy decidua sections using B-72.4 anti-TAG-72 mAb and MNF 116 anti-cytokeratin mAb revealed the absence of TAG-72 in uterine decidua of normal and pathological pregnancies (non-embryonic pregnancy and missed abortion) at the implantation sites, although it was present in epithelial cells at and away from the tubal implantation site of an ectopic pregnancy. TAG-72 binds and internalizes by reacting with the mannose receptor (MR-CD206) or with DC-specific ICAM reacting non-integrin (DC-SIGN-CD209) on decidual CD1a+ cells. Decidual CD1a+ cells stimulated with TAG-72 decreased CD83 expression and diminished IL-15 and IFN-γ intracellular production. TAG-72-treated CD1a+ cells decreased IFN-γ production in syngenic decidual and allogenic cord blood T cells even in the presence of lipopolysaccharide. TAG-72- and lipopolysaccharide-pre-treated CD1a+ cells significantly increased IL-4 expression in allogenic cord blood T cells. TAG-72 increased allogenic cord blood T cell proliferation, mediated by decidual CD1a+ cells, compared with its effect on the proliferation of syngenic decidual T cells. All these data emphasize the anti-inflammatory properties of TAG-72-treated decidual CD1a+ cells in terms of their interaction with T cells. Thus, the absence of TAG-72 at the maternal-fetal interface during early pregnancy could lead to a mild pro-inflammatory response that may be beneficial for pregnancy success and trophoblast growth control.

Phenotype of NK cells and cytotoxic/apoptotic mediators expression in ectopic pregnancy.

PROBLEM: The expression of cytotoxic/apoptotic mediators and the phenotype characteristics of uterine NK cells (uNK) in tubal ectopic pregnancy (EP) were investigated. METHOD OF STUDY: Samples of uterine decidua and tubal mucosa as well as peripheral blood (PB) of the same women with EP were used for phenotype characterization of NK cells and detection of cytotoxic/apoptotic mediators and IL-15. RESULTS: In tubal mucosa, perforin, FasL, granulysin and IL-15 were almost completely absent, but they were present in normal and EP uterine deciduas. TRAIL was present on trophoblast and tubal mucosa, contrary to its lack in normal and EP uterine decidua. CD16⁻ CD56(dim) NK cells, mostly CD94⁻ and NKG2A⁻, predominate in tubal mucosa, whereas CD16⁻ CD56(bright) NK cells, predominantly CD94(+) and NKG2A(+) prevail in EP uterine decidua. NK cells at the EP implantation site express lower percentages of perforin and granulysin, but they express a higher percentage of TRAIL than do EP uterine decidual and PB NK cells. Lower percentage of TNF-α-expressing and IL-4-expressing NK cells were found at the implantation site compared to EP uterine decidua. CONCLUSIONS: Authentic uNK cell population seems to be insufficient to restrict trophoblast invasion because of low expression of cytotoxic/apoptotic mediators.

Decidual natural killer cell tuning by autologous dendritic cells.

PROBLEM: Dendritic cells (DC)/natural killer (NK) cells interactions in the deciduas of early human pregnancies were analyzed in vitro. METHOD OF STUDY: Phenotype, cytokine expression and/or cytolytic mediators' expression were measured by flow cytometry in NK and DC from the freshly isolated decidual mononuclear cells or after their purification and co-culture in vitro. Proliferation of 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labeled CD56(+) cells was analyzed by flow cytometry after the co-culture with CD1a(+) or CD83(+) DC. RESULTS: Decidual CD1a(+) cells show less mature phenotype with no expression of CD197, lower expression of CD80 and CD86 and higher expression of CD206 and CD195 in comparison to CD83(+) cells. Interleukin (IL)-15, interferon-gamma and tumor necrosis factor-alpha productions were higher in immature than mature DC, whereas IL-10 and IL-18 were equally produced in both subpopulations. Immature DC increase perforin, FasL and TRAIL protein expression and proliferation of NK cells, but decrease their intracellular IL-15 production. Mature DC caused less efficient proliferation of NK cells, and did not affect cytokine and cytolytic mediator expression. CONCLUSION: These results suggest that decidual CD1a(+) cells regulate and shape NK cell function more profoundly than CD83(+) cells in decidua.