The potential of gut microbiome as a non-invasive predictive biomarker for early detection of pancreatic cancer and hepatocellular carcinoma.

OBJECTIVE: The aim of this paper was to discuss the potency of gut microbiome as a non-invasive predictive biomarker for early detection of pancreatic cancer and hepatocellular carcinoma. MATERIALS AND METHODS: We analysed the available up-to-date literature (PubMed, Embase, Google Scholar databases) regarding the link between gut microbiome and early detection of pancreatic cancer, as well as hepatocellular carcinoma. The following search linked to gut microbiome and aforementioned cancers was used: 'gut microbiome', 'gut microbiota', 'pancreatic cancer', 'pancreatic ductal adenocarcinoma', hepatocellular carcinoma', 'microbial biomarkers', 'fungal microbiota', 'mycobiota'.  The search was conducted in English. RESULTS: The association between gut microbiota imbalance and development of pancreatic cancer and hepatocellular carcinoma has been recognized during last several years. The most common type of pancreatic cancer is pancreatic ductal adenocarcinoma, whose carcinogenesis is strongly related to oral microbial dysbiosis, H. pylori infection, bactibilia, hepatotropic viruses, and intrapancreatic microbiota. It is known that gut-liver axis exists and may affect hepatocarcinogenesis. Currently, the treatment strategies of these cancers are strongly limited and there are not well-recognized screening tools to early diagnose them. The growing attention towards the use of gut microbiome as a predictive non-invasive biomarker to detect pancreatic cancer and hepatocellular carcinoma in early stage has been observed. CONCLUSIONS: To conclude, the field regarding the link between gut microbiome as a non-invasive biomarkers and early detection of pancreatic cancer and hepatocellular carcinoma exists, however, it is not well-investigated. Additionally, many of the studies were conducted with small sample sizes, whereas biomarkers are ethnicity-dependent and should be validated in wide range of populations. Nevertheless, these aspects are promising and open up new diagnostic options.

Older age of rheumatoid arthritis onset is associated with higher activation status of peripheral blood CD4(+) T cells and disease activity.

Rheumatoid arthritis (RA) is a chronic inflammatory disease, with a clinical manifestation both systemic and in joints. It has been suggested that age at disease onset and/or patients' age have influence on disease activity and clinical outcome. The reasons for the different course of RA in older people are not known; however, the activation status of peripheral blood lymphocytes could be responsible. Our aim was to relate expression of activation markers in peripheral blood CD4(+) T cells of RA patients with patients' age and/or onset age and disease activity measured by DAS28. Seventy RA patients were included into the immunological study. Two separation criteria were performed: based on age of RA onset and on the biological age of patients. We examined different activation markers, CD69, CD25, CD95 and human leucocyte antigen D-related (HLA-DR), on the CD4(+) T cell surface. Division of RA patients in 10-year intervals at 40, 50 and 60 years revealed that RA patients with later disease onset were characterized by higher DAS28. This phenomenon was not limited to the division at 60 years of age but, surprisingly, the major differences were found for the 40-year onset division. Analysis of all four components of DAS28 revealed that disease activity in older disease onset was dependent on all components. Older-onset RA patients had a higher percentage of CD4(+) CD25(+) and CD4(+) CD95(+) T cells. Summarizing the major differences in DAS28 and activation status of CD4(+) T cells observed for onset of disease at 40 years seems to be the most informative about the immunological status of RA patients.

Mitochondrial transmembrane potential in spontaneous and camptothecin-induced apoptosis of melanotic and amelanotic melanoma cells.

In this work we tried to estimate the role of mitochondria in the ability of cells of two: melanotic and amelanotic transplantable melanoma lines to undergo spontaneous and camptothecin-induced apoptosis. We measured mitochondrial transmembrane potential (DeltaPsi) changes during culture without (spontaneous apoptosis) and with camptothecin (induced apoptosis) by using JC-1 staining and flow cytometry analysis. Cytochrome c release and PARP cleavage as the biological effects of DeltaPsim changes belonging to the phenomena observed during apoptosis were estimated by Western blotting. The results of our investigations showed in both transplantable melanoma cells the features indicating apoptosis: DeltaPsi changes, cytochrome c release and PARP cleavage, but the degree of observed changes depended on the phenotype of melanoma cells examined. After camptothecin treatment the changes were more pronounced in the amelanotic melanoma cells- the more aggressive line.

Down-regulation of CD28 expression by TNF-alpha.

Aging and chronic inflammatory syndromes, such as rheumatoid arthritis, are associated with high frequencies of CD4(+)CD28(null) T cells, which are rarely seen in healthy individuals younger than 40 years. Inasmuch as rheumatoid arthritis and aging are also associated with elevated levels of TNF-alpha, we examined whether this proinflammatory cytokine influences CD28 expression. Incubation of T cell lines and clones as well as Jurkat cells with TNF-alpha induced a reduction in the levels of cell surface expression of CD28. This effect of TNF-alpha was reversible; however, continuous culture of CD4(+)CD28(+) T cell clones in TNF-alpha resulted in the appearance of a CD28(null) subset. In reporter gene bioassays, TNF-alpha was found to inhibit the activity of the CD28 minimal promoter. Inactivation of the promoter was accompanied by a marked reduction in DNA-protein complex formation by two DNA sequence motifs corresponding to the transcriptional initiator of the CD28 gene. Indeed, in vitro transcription assays showed that nuclear extracts from TNF-alpha-treated cells failed to activate transcription of DNA templates under the control of a consensus TATA box and the CD28 initiator sequences. In contrast, similar extracts from unstimulated T cells supported transcription. These results demonstrate that TNF-alpha directly influences CD28 gene transcription. We propose that the emergence of CD4(+)CD28(null) T cells in vivo is facilitated by increased production of TNF-alpha.

Luteal phase of the menstrual cycle in young healthy women is associated with decline in interleukin 2 levels.

The aim of this study was to look at the possible changes in the blood levels of Interleukin 2 (IL2) during the sexual cycle in generally healthy, young, regularly menstruating women. The concentrations of progesterone and 17beta-estradiol were measured using radioimmunological assay. The bioactivity of interleukin 2 was measured using a biological test on the IL2-sensitive CTLL cell line. The percentage of lymphocytes with intracellular IL2 was determined by flow cytometry. Eighteen healthy volunteers (19-29 years old) were examined on days 5, 8, 14, 18 and 25 of the same cycle. All women were characterised by a regular menstrual cycle as per physiological levels of 17beta-es-tradiol and progesterone. The luteal phase of the cycle was characterised by both a decrease of IL2 blood levels and a decrease in the percentage of intracellular 1L2-containing lymphocytes stimulated in vitro. The IL2 level fluctuations observed during the menstrual cycle may be one factor causing pre-menstrual infections observed in young women. On the other hand, the decrease of IL2 may be seen as a start of the immune suppression necessary for an embryo's nidation.

Compensatory effect of TNFalpha on low natural killer activity in the elderly.

Regulatory effect of CD25, an activation antigen the alpha subunit of interleukin 2 receptor (IL2R) on the activity of natural killer (NK) cells was studied in fifty elderly (57-70 years old) and fifty young people (19-35 years old). Cytotoxic NK activity was assessed by 51Cr release assay, the levels of interleukin 2 (IL2) and tumour necrosis factors alpha (TNFalpha) were measured using bioassays and expression of CD16 and CD25 proteins by flow cytometry. Low NK activity in the elderly was associated with decline of full health, lowered serum concentration of IL2 and increased production of TNFalpha during NK reaction. Inhibition of TNFalpha activity by anti-TNF monoclonal antibody suppressed exclusively NK activity of low NK responders. Moreover, stimulation in vitro of blood mononuclear cells, with TNFalpha induced in the elderly low NK responders a significantly higher increase of the CD25 expression on the surface of NK cells as compared with that in the elderly high responders. Since the CD25 molecule constitutes a subunit of the high affinity receptor, binding IL2 to immunocompetent cells, its increased expression on NK cells of low NK responders would enable them to bind even low amounts of the endogenous IL2 available in this group of the elderly. Thus, an overproduction of TNFalpha seems to be a mechanism compensating, in the non-fully healthy elderly, for the decreased IL2 production, promoting efficient cytotoxic reaction.

Lower interleukin-2 and higher serum tumor necrosis factor-a levels are associated with perimenstrual, recurrent, facial Herpes simplex infection in young women.

The aim of this study was to look at a possible relationship between the recurrent perimenstrual dermatosis - facial Herpes simplex infection and the serum concentrations of interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-alpha). Twenty-one volunteers (19-26 year olds) were examined at five points of the menstrual cycle. Ten volunteers were characterised by recurrent Herpes simplex infection lasting either from the 18th or the 25th day of the menstrual cycle until a few days after menstruation. Eleven young women without symptoms formed the control group. Both groups were similar as regards blood levels of 17beta-estradiol and progesterone. The group with the frequent infectious symptoms was characterised, however, by lower concentrations of IL-2 throughout the whole menstrual cycle, as compared to those without the symptoms. Levels of IL-2 in this group additionally dropped significantly on the 18th and on 25th day of the cycle. Moreover, the group with symptoms was characterised by higher level of TNF-alpha on the 18th day. These changes were found during the menstrual cycle of the women with recurrent herpes infection who however, at the time of the examination were free of the clinical symptoms. There was a similar tendency in both groups towards an increase in the levels of TNF-a around menstruation. Measurement of the other serum pro-inflammatory marker - IL-6 showed higher levels of this cytokine during the menstrual cycle in the group with the clinical symptoms. The results indicate that a decrease of IL-2 together with an increase of TNF-alpha and IL-6 in the serum seem to be related to recurrent perimenstrual Herpes simplex infection.

Nitric oxide, heparin and procaine treatment in experimental ceruleine-induced acute pancreatitis in rats.

The aim of the study was to investigate the impact of L-arginine (nitric oxide donor), L-NNA (NO synthase inhibitor), heparin and procaine on the pancreas' microcirculation, serum interleukin 6 (IL-6) level, and microscopic alterations of the pancreatic gland in acute pancreatitis (AP) in rats. AP was induced by 4 i.p. injections of cerulein (15 micrograms/kg/h). Microcirculatory values of the pancreas were measured by means of laser Doppler flowmetry 5 h after the first cerulein injection. Remarkable morphologic changes in the pancreas, including parenchymal necrosis, an elevation of serum IL-6 activity, and significant drop of pancreatic capillary perfusion was observed in rats with NO synthase inhibition. L-arginine improved the pancreatic microcirculation but worsened the microscopic alterations within the pancreas. Heparin had a beneficial effect on the microcirculatory values, serum IL-6 activity, and morphologic changes. Procaine had no effect on the course of AP. Authors conclude that heparin, improving the pancreatic capillary blood perfusion, may be considered as a promising therapeutic agent in acute pancreatitis.

The upregulation of TNF alpha production is not a generalised phenomenon in the elderly between their sixth and seventh decades of life.

The aim of this paper was to analyse the link between the intensity of tumor necrosis factor alpha (TNF alpha) production and the health status of the elderly and to find out whether the age between sixty and seventy is a 'turning point' for the changes in the production of this cytokine. Fifty elderly volunteers (age range: 60-70), twenty-five middle-aged (age range: 36-59) and fifty young (age range: 20-35) were enrolled into the study. Their health status was graded as 'healthy' and 'almost-healthy'. The level of TNF alpha was determined by bioassay, the activity of the TNF alpha gene was analysed in non-stimulated PBMC by the RT-PCR method. The results showed that the production of TNF alpha in the 'healthy' elderly people is not upregulated until the age of sixty-seventy. The 'almost-healthy' elderly are characterised by a higher release of TNF alpha from the cultures of non- and stimulated PBMC and an activation of the TNF alpha gene in the non-stimulated PBMC. Summarising, our results indicate that the deterioration of health status is a prerequisite for an exaggerated TNF alpha production by peripheral blood mononuclear cells of people between the sixth and seventh decades of life.

Heparin and nitric oxide treatment in experimental acute pancreatitis in rats.

The aim of this study was to investigate the impact of L-arginine (nitric oxide synthase substrate), L-NG-nitro-L-arginine (nitric oxide synthase inhibitor), and heparin on the pancreas microcirculation, serum IL-6 level and microscopic alterations of the pancreas in acute pancreatitis in rats. Acute pancreatitis was induced by 4 i.p. injections of cerulein (15mg/kg). Microcirculatory values were measured by means of laser Doppler flowmetry 5 h after the first cerulein injection. Remarkable histopathological changes in the pancreas, including parenchymal necrosis, an elevation of serum IL-6 level, and a significant drop of pancreatic capillary perfusion was observed in rats with nitric oxide synthase inhibition. L-arginine improved the pancreatic microcirculation but worsened the microscopic alterations within the pancreas. Heparin had a beneficial effect on the microcirculatory values, serum IL-6 concentration, and morphologic changes. Authors conclude that inhibition of nitric oxide synthase aggravates acute pancreatitis. L-arginine treatment improves pancreatic perfusion but potentiates morphological alterations. Heparin, improving the microcirculation and inflammatory changes within the pancreatic gland, may be considered as a promising therapeutic agent in acute pancreatitis.

Increase of interleukin 6 and decrease of interleukin 2 production during the ageing process are influenced by the health status.

The ageing process is accompanied by the disregulation of interleukin 2 (IL2) and interleukin 6 (IL6) production. In our paper, we asked whether the age between 60 and 70 years is a turning point for the disregulation of both IL2 and IL6 production. Fifty volunteers 60-70 years old, 25 aged 36-59, and 50 of 20-35 years old were enrolled into the study. Their health status was graded according to the criteria of the Senieur Protocol (SP) as 'healthy' and 'almost-healthy'. The cytokines level was determined in the sera of the volunteers. Moreover, the spontaneous release of IL6 by peripheral blood mononuclear cells (PBMC) and the activity of the IL6 gene in non-stimulated PBMC were also analysed. Cytokine levels were measured by biological assays, mRNA for IL6 was detected by RT-PCR method. The results showed that the production of IL2 is not disregulated in the 'healthy' people until the age of 60-70. People not fulfilling all SP criteria are characterised by a lower level of IL2 in the sera. The overproduction of IL6 into the sera and supernatants from non-stimulated PBMC and PBL as well as the activation of IL6 gene start between the ages 36 and 59 and is more pronounced in the 'almost-healthy'.

The influence of recombinant human erythropoietin on tumor necrosis factor alpha and interleukin-10 production by whole blood cell cultures in hemodialysis patients.

Impaired immunological response in hemodialysis (HD) patients, which leads to inappropriate cytokine production, is partially caused by the hyperstimulation of both T lymphocytes and monocytes/macrophages. Recent data suggest that human recombinant erythropoietin (rhEPO) may have an immunological action. The goal of our study was to estimate the influence of rhEPO treatment on the production of the inflammatory cytokine tumor necrosis factor alpha (TNFalpha) and antiinflammatory cytokin interleukin-10 (IL-10) in 10 HD patients receiving rhEPO for 6 months. The levels of cytokines were measured in the in vitro cultures of whole blood. The level of IL-10 increased in all treated patients during the therapy, and it was accompanied by a transitory decrease of TNFalpha. The results of our studies suggest that rhEPO may reduce the inflammatory process by decreasing production of TNFalpha and increasing production of IL-10.

Overactivity of tumor necrosis factor-alpha but not interleukin 6 is associated with low natural killer cytotoxic activity in the elderly.

In this paper we asked whether the increased production of two proinflammatory cytokines-tumor necrosis factor-alpha (TNF alpha) and interleukin-6 (IL6) in the absence of the inflammatory stimulus, a phenomenon frequently occurring in the elderly, may contribute to the decrease of natural killer (NK) cytotoxic activity. NK cell activity is a part of a nonspecific immunity directed against infected and transformed cells. We measured the levels of bioactive TNF alpha and IL6 during cytotoxic NK reactions and analyzed the effect of neutralizing monoclonal antibodies anti-TNF alpha and anti-IL6 on the outcome of cytotoxic reaction. Bioactive TNF alpha was produced in higher amounts during cytotoxic reactions of the elderly with low NK activity in comparison with its production by the elderly with high NK activity. Anti-TNF alpha decreased already low cytotoxic responses of the elderly while not having an effect on the cytotoxic potential of high responders. More of the bioactive IL6 was released during cytotoxic reactions of the elderly as compared with young people. There was, however, no relation between the height of NK cytotoxic activity and the amount of IL6. Our results suggest that the increased level of TNF alpha seen in the elderly low NK responders may pose a compensatory mechanism, necessary to keep the highest possible level of NK responses. The increased bioactivity of IL6 during NK reaction of the elderly does not seem to participate in the regulation of the level of NK cytotoxic activity.