Cytokine profiles during delivery affect cord blood hematopoietic stem and progenitors cells.

The study was aimed to determine the correlations between serum levels of cytokines (GM-CSF, IL-4, IL-10 and TNF) in maternal (MB) and cord blood (CB) and some features of cord blood hematopoietic stem and progenitor cells (CB HSPCs). Study material was MB and concomitant CB samples collected from 98 volunteers at the moment of delivery. The IL-4, IL-10, TNF and GM-CSF concentrations in serum and in supernatants from PMA-stimulated mononuclear cells isolated from both blood types were measured using BD Cytometric Bead Array Flex Set System. CB HSPCs (CD34(+)CD45(low)) proportion was also estimated by flow cytometry. The most relevant results concerned the tendency to down regulation of CB HSPCs number with an increase of IL-4, IL-10 and GM-CSF levels, only the TNF concentration seems to have no influence on HSPCs pole size. The strongest positive correlations were found between CD34(+)CD45(low) HSPCs number and IL-10 and GM-CSF in MB serum and GM-CSF and TNF from CB supernatants. The strongest negative correlations were found between CD34(+)CD45(low) HSPCs number and IL-4 and GM-CSF in CB serum and IL-10 in MB supernatants. Interestingly, we observed 'opposite correlation' between serum and supernatant from CB and MB. We concluded that elevated serum levels of IL-4, IL-10 and GM-CSF in CB are indicative of enhanced differentiation of HSPCs and characterize a normal perinatal development. Elevated levels of cytokines seem to stimulate differentiation of HSPCs what is advantageous for neonates during perinatal period.

Ex vivo expansion of CD4(+)CD25(+) T regulatory cells for immunosuppressive therapy.

Immunosuppressants are powerful drugs, capable of triggering severe adverse effects. Hence, there is tremendous interest in replacing them with less-toxic agents. Adoptive therapy with CD25(+)CD4(+) T regulatory cells (Tregs) holds promise as an alternative to immunosuppressants. Tregs have been described as the most potent immunosuppressive cells in the human body. In a number of experimental models, they have been found to quench autoimmune diseases, maintain allogeneic transplants, and prevent allergic diseases. A major stumbling block in their clinical application is related to Treg phenotype and the very limited number of these cells in the periphery, not exceeding 1-5% of total CD4(+) T cells. Recent progress in multicolor flow cytometry and cell sorting as well as cellular immunology has found ways of overcoming these obstacles, and has opened the doors to the clinical application of Tregs. In the review, we describe Treg sorting and expansion techniques that have been developed in recent years. In the experience of our laboratory, as well as some published reports, Treg adoptive therapy is a promising tool in immunosuppressive therapy, and should be considered for clinical trials.

From bench to bedside and back: the SENIEUR Protocol and the efficacy of influenza vaccination in the elderly.

Prophylaxis with vaccines is important in geriatrics as, apart from specific protection, it reduces incidence of potentially fatal infectious complications and exacerbations of existing medical conditions. Post-vaccination protection depends on immunity and therefore markers of immune status could be used to predict efficacy of vaccination. From the practical point of view, the determination of simple and robust methods for assessing immunity, which could enable practitioners to distinguish patients at risk of vaccination unresponsiveness, is desirable. Additional care, necessary to avoid possible complications, could be then targeted to such patients. Here, we discuss correlations between immune parameters and the clinical status of elderly people challenged with anti-influenza vaccination and ways to classify immune status, based on results obtained between 1999 and 2004 in the elderly immunized annually against influenza. These studies document a correlation between health status assessed according to the SENIEUR Protocol and response to the vaccination. Patients classified as SENIEUR-compatible responded to the vaccine, while non-SENIEURs did not. Factors most strongly associated with unresponsiveness were, perhaps unexpectedly, circulatory and psychiatric diseases, although both humoral and cellular immune responsiveness did correlate with levels of proinflammatory cytokines in the serum. Patients whose humoral responses improved during subsequent immunizations were characterized by well-preserved natural killer (NK)-mediated cytotoxicity. In contrast to the first-immunization responders, the second- immunization responders were characterized by elevated levels of proinflammatory cytokines and Cytomegalovirus seropositivity, which placed them in the non-SENIEUR group, although they were not yet frail and still lived independently. From this series, we conclude that detailed clinical data together with some simple markers such as IL 6 levels seem sufficient to provide clinicians with presumptive information on the condition of the immune system and to allow for initial prediction of vaccination efficiency. Those patients predicted to respond poorly could be considered for alternative treatment.

Analogues of muramyl dipeptide (MDP) and tuftsin limit infection and inflammation in murine model of sepsis.

Pharmacological manipulation of the balance between pro- and anti-inflammatory mediators emerges as a key aspect of a successful treatment of sepsis. A murine model of septic shock was developed and chosen conjugates (1a, 1b, 8a, 8c) and analogs (T2) of muramyl dipeptide and tuftsin were tested in this model as prospective anti-bacterial drugs or adjuvants. The phagocytic activity of monocytes/macrophages was determined (flow cytometry, bacterial clearance from vital organs). To evaluate cytokines levels (TNFalpha, IFNgamma, IL6, IL10) we used real-time PCR. The most promising immunomodulatory properties were displayed by the analogue T2 and two conjugates: 8a, 8c.

[Tuftsin--new analogues and properties]. / Tuftsyna--nowe analogi i wlasciwosci.

Tuftsin, a natural tetrapeptide of sequence TKPR, occuring in the blood of humans and other mammals, capable of stimulating certain white blood cells (monocytes, macrophages, and neutrophils), was isolated at Tufts University in 1970 by Najjar and Nishioka. Tuftsin is a compound with a wide spectrum of biological activities, notable enhances phagocytosis, immune response, bactericidal, tumoricidal and antifungal activities. This article concerns new analogues and properties of tuftsin.

Anti-tumor action of tumor necrosis factor against Bomirski Ab melanoma in hamsters.

INTRODUCTION: Tumor necrosis factor (TNF) is a cytokine able to exert anti-tumor activity in various models and modes of applications. However, the exact mechanism mediating the in vivo anti-tumor effect of TNF has not yet been clarified. MATERIALS AND METHODS: The effects of intratumoral injection of rat TNF into hamsters bearing Bomirski Ab amelanotic melanoma, a fast growing tumor of high metastatic potential, were tested. Subcutaneous injections of the anti-angiogenic compound TNP-470 allowed analysis of its influence on the effects of TNF administration. RESULTS: TNF application resulted in a significant inhibition of tumor growth and changes in metastasis pattern. Accelerated hemorrhagic necrosis was also observed, indicating the effect of the cytokine on tumor vessels. Moreover, the synergistic anti-tumor effect of TNF and anti-angiogenic agent TNP-470 suggested a cooperative activity of both substances on tumor vasculature. Microscopically, the effect of TNF injections was expressed by an increase in the amount of tumor cells with nuclear pyknosis and karryorrhexis. In vitro assays indicated a direct cytotoxic effect of TNF against Ab melanoma cells, most probably as an outcome of apoptosis. Intratumoral application of TNF also caused some modulation of cytokine response in melanoma-bearing hamsters as evidenced by increased levels of IL-6 in blood serum. CONCLUSIONS: This study established Bomirski Ab melanoma as a useful model for complex analysis of the anti-tumor activity of TNF.

Treatment with recombinant human erythropoietin is associated with rejuvenation of CD8+ T cell compartment in chronic renal failure patients.

BACKGROUND: A growing body of evidence suggests an impact of rHuEpo on the immune system. METHODS: We assessed the impact of recombinant human erythropoietin (rHuEpo) on the immunity of 11 chronic renal failure patients who did not require haemodialysis. Naïve (Tn), central (Tcm) and effectory memory (Tcm, Tem, TemRA) subsets of CD8+ T cells, memory-CMV-specific CD8+ T cells, titres of anti-CMV antibodies and activity of NK cells were evaluated during the first year of rHuEpo administration. RESULTS: While the number of CD8 T cells did not change, significant change was found in their proportions. Percentage of Tn cells increased at the expense of Tcm cells. Appearing Tn cells were CD28+ increasing the total pool of CD28+ T cells. Together with decreasing number, Tcm cells changed to mainly CD28- Tcm cells. A 'move' towards the naïve compartment was also confirmed as the level of memory-CMV-specific CD8+ T cells decreased. Humoral immunity analysed as titres of anti-CMV antibodies as well as innate immunity measured as cytotoxicity of NK cells did not change during the follow-up. CONCLUSIONS: We found that the administration of rHuEpo caused rejuvenation of cellular CD8+ T-dependent immunity in our patients.

Mycobacterial heat shock protein-induced blood T lymphocytes subsets and cytokine pattern: comparison of sarcoidosis with tuberculosis and healthy controls.

BACKGROUND AND OBJECTIVE: Sarcoidosis (SA) is a disorder of unknown aetiology. Mycobacterium tuberculosis heat shock proteins (Mtb-hsp) have been considered as causative agents of SA. The role of Mtb-hsp in the immune response in SA has not been investigated. METHODS: Mtb-hsp-stimulated T-cell subsets and Th1/Th2 cytokine patterns in the supernatant from peripheral blood mononuclear cell cultures from 22 SA patients, 20 tuberculosis (TB) patients and 20 healthy volunteers were compared using flow cytometry. RESULTS: In unstimulated cultures, a significantly higher percentage of CD8(+)alphabeta(+)T-cells were present in SA versus controls. Similarly there was a significantly increased IL-6 and decreased IL-4 level in SA and significantly lower INF-gamma, IL-2, IL-4, IL-10 production in TB versus controls. After Mtb-hsp stimulation, there was a significantly increased TNF-alpha, IL-6, IL-10 and decreased INF-gamma, IL-2, IL-4 production in SA and significantly increased TNF-alpha, IL-6 concentrations in TB versus controls. CD8(+)gammadelta(+)IL-4(+)T-cells were detected significantly less often in Mtb-hsp-induced cultures in SA versus controls. Comparing SA versus TB, CD4(+)gammadelta(+)TCR-cells were significantly increased in Mtb-hsp-induced cultures in TB versus controls and SA. Before stimulation, significantly increased IL-6, IL-10 and decreased IL-4 level in SA versus TB was revealed, whereas Mtb-hsp stimulation caused significantly increased IL-10 and decreased IL-4 concentrations in SA. CONCLUSIONS: After Mtb-hsp stimulation, increased levels of pro-inflammatory cytokines, TNF-alpha and IL-6 were found in sera from SA and TB patients in comparison with healthy controls; SA patients demonstrated the lowest levels of IL-4 and the highest levels of IL-10.

NK cell compartment in patients with coronary heart disease.

BACKGROUND: Viral and bacterial infections have been considered as a risk factor for Coronary Heart Disease (CHD). NK cells, as a first line of defense against those infections, may play a role in CHD development. Thus, the main aim of our study was to determine NK cell compartment in patients with CHD undergoing coronary artery by-pass grafting. RESULTS: Ninety three patients with CHD were included into the study; the control group consisted of 49 healthy volunteers. As compared to controls, CHD patients had lower NK cytotoxic activity. CHD group had also a decreased absolute number and percentage of total NK cells and CD3-CD56dim cytotoxic NK subset. In addition, we observed tendency toward lower percentage of the CD3-CD56bright regulatory NK subset and CD3-CD56+IFN-gamma+ cells in CHD patients. CONCLUSION: These data indicate that CHD is associated with an impairment of NK cells compartment.

Quantifying division of aortal smooth muscle cells in culture stimulated by 1,25(OH)2D3.

Inhibitory effect of 1alpha,25dihydroxycholecalciferol (1,25D(3)=calcitriol) in different cell type is well recognized but its promoting effect on vascular smooth muscle cells (SMCs) is poor established. Therefore, the aim of this study was to determine stimulatory effect of calcitriol on aortal SMCs proliferation in culture. We used the cell division analysis procedure based on the quantitative sequential halving of the stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE). This technique allowed the visualization of cycles of SMCs division by flow cytometry. Rat aortal SMCs were labeled with CFSE and cultured for up to 10 days with defined concentration of calcitriol in medium. Proliferative activity as the percentage of SMCs in different phases of the cell cycle using propidium iodide was determined. Apoptosis was assessed using Annexin-V/CFDA method. The results suggest that low concentrations of an active form of vitamin D-1alpha,25dihydroxycholecalciferol applied in supraphysiological concentration of 10 nmol/l is a mitogenic factor for aortal SMCs. None of the applied concentrations of calcitriol caused apoptosis. The findings well support our morphological (LM) and ultrastructural (TEM and SEM) observations.

Long-term prognosis after coronary bypass surgery depends on interleukin 6 polymorphism and past acute infections.

We were seeking for a mutual link between the -174G>C IL6 promoter polymorphism, history of the past acute respiratory infections and the long-term post-coronary artery bypass grafting (CABG) major adverse cardiovascular events (MACE) incidence. Two hundred thirty seven post-CABG patients have been followed up for a median period of 36 months. We found that past acute infections, influenza-like illness and lack of vaccination against influenza confer a significant risk of the post-CABG MACE incidence in the -174G allele carrying patients.

Interleukin 6 polymorphism corresponds to the number of severely stenosed coronary arteries.

IL6 gene promoter polymorphisms may influence the outcome of cardiovascular diseases. The aim of our study was to find out whether the -174G>C polymorphism, as well as the IL6 secretory profile, may be linked to the number of severely (> or = 75%) occluded coronary arteries in patients with advanced coronary heart disease (CHD). Three hundred and twenty patients awaiting elective coronary artery bypass grafting were enrolled into the study. Blood was taken the day before surgery. The PCR-RFLP method was used for IL6 gene polymorphism analysis. Spontaneous IL6 release was measured by bioassay in supernatants of whole blood cell cultures (WBCC) incubated for 24 h and 48 h. We found that significantly more patients with triple vessel disease were found within the -174GG group as compared to the -174GC and CC genotype carriers. The highest IL6 serum levels were found in the -174GG and the lowest in the -174CC genotype patients. Spontaneous in vitro IL6 secretion appeared to be significantly higher at all time points in the -174GG as compared to the CC and GC genotype carriers. The serum concentration of IL6 and the spontaneous IL6 secretion were directly related to the number of obstructed coronary vessels. Our results emphasize the role of IL6 as an important, non-classical risk factor predicting the number of severely affected coronary vessels.

The atypical pattern of cell death in B16F10 melanoma cells treated with TNP-470.

TNP-470 is an acknowledged anti-angiogenic factor, and was studied clinically as an anti-cancer drug. We previously reported on an additional property of this molecule: the intracellular generation of reactive oxygen species in B16F10 melanoma cells. We showed that a massive generation of ROS occurred in the first few hours after treatment with TNP-470 and that this event was critical to subsequent cell death. In this study, we analyzed the process of cell death and noticed an atypical pattern of death markers. Some of these, such as DNA fragmentation or condensation of chromatin, were characteristic for programmed cell death, while others (the lack of phosphatidylserine flip-flop but permeability to propidium iodide, the maintenance of adhesion to the substratum, no change in mitochondrial transmembrane potential, no effect of the panspecific caspase inhibitor) rather suggested a necrotic outcome. We concluded that TNP-470 induced at least some pathways of programmed cell death. However, increasing damage to critical cell functions appears to cause a rapid switch into the necrotic mode. Our data is similar to that in other reports describing the action of ROS-generating agents. We hypothesize that this rapid programmed cell death/necrosis switch is a common scenario following free radical stress.

Comparative analysis of mycobacterial heat shock proteins-induced apoptosis of peripheral blood mononuclear cells in sarcoidosis and tuberculosis.

Sarcoidosis (SA) is a granulomatous disorder of an unknown etiology. Mycobacterium tuberculosis heat shock proteins (Mtb-hsp), considered as causative agents, play an important role in apoptosis. A role for apoptosis has been proposed in pathogenesis of SA and tuberculosis (TB) granuloma formation but results remain controversial. Differences in Mtb-hsp-induced apoptosis between SA, TB, and healthy subjects found in this study might put some light on the etiology of SA. Early apoptotic peripheral blood mononuclear cells (PBMC) were determined in 22 SA patients, 20 TB patients, and 20 healthy volunteers by flow cytometry (Annexin-V-FITC). Our results revealed that spontaneous apoptosis of monocytes and CD8+ T-cells was comparable between tested groups. Apoptosis of unstimulated CD4+ T-cells was significantly lower in TB versus controls and insignificantly lower versus SA. Mtb-hsp- and PHA (Phytohemagglutinin)-induced monocytes apoptosis was significantly lower in TB versus controls and SA. Mtb-hsp-induced CD4+ T-cell apoptosis was significantly lower in TB versus controls and SA. There were no differences of PHA-induced CD4+ T-cell and CD8+ T-cell apoptosis between tested groups. Apoptosis of Mtb-hsp-induced CD8+ T-cells was significantly lower in TB and SA versus controls. Analysis of PBMC apoptosis before and after stimulation in each tested group revealed that, in contrast to TB, sarcoid monocytes were resistant to Mtb-hsp- and PHA-induced apoptosis and CD4+ T-cells were resistant to PHA- but not Mtb-hsp-induced apoptosis. CD8+ T-cell apoptosis, before and after Mtb-hsp or PHA stimulation, was significantly increased in all tested groups. It seems likely that dysregulated apoptosis of CD4+ T-cells and resistant apoptosis monocytes may be involved in pathogenesis of SA.

CD4+CD25+ T regulatory cells inhibit cytotoxic activity of CTL and NK cells in humans-impact of immunosenescence.

We hypothesized that advanced age and medical conditions had an impact on the accumulation of CD4+CD25+ T regulatory cells (Treg), which in turn could deteriorate cytotoxic activity of CD8+ T and NK cells. Volunteers were divided according to the Senieur Protocol into healthy young and elderly and non-healthy young and elderly subjects. The numbers of Treg cells in peripheral blood, their influence on CD8+ T and NK cells and production of IL2 as well as apoptosis intensity of Treg cells were measured. The number of Treg cells was higher in both elderly groups than in respective young ones. Compared to healthy subjects, those with medical conditions were revealed to have higher numbers of Treg cells. In addition, the highest accumulation of Treg cells in non-healthy elderly could be a result of their resistance to undergo apoptosis. The frequency of Treg cells correlated inversely with the activity of autologous cytotoxic cells in PBMC and production of IL2 by autologous CD4+CD25- Th cells. Thus, these parameters were the most highly decreased in non-healthy subjects, notably in the elderly. However, these parameters improved in the cultures of pure sorted cells. The only subset capable of decreasing them to the levels noted in PBMC when added back was Treg cells, which proved the link between the number of Treg cells, cytotoxic activity and production of IL2. Concluding, we found that Treg accumulated as a result of ageing and/or medical conditions were capable of decreasing cytotoxic activity of CD8+ T and NK cells and production of IL2.

Serum and urinary cytokine homeostasis and renal tubular function in children with type 1 diabetes mellitus.

AIM: To explore the relationships between tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6) and urinary N-acetyl-beta-D-glucosaminidase (NAG) and the function of renal proximal tubules in children with type 1 diabetes mellitus (DM1). METHODS: Fifty-six children with DM1 and 35 healthy controls were analyzed. We measured NAG (A and B isoforms) in urine as well as serum TNFalpha and urinary IL-6. RESULTS: The children with DM1 with microalbuminuria (group A) had significantly higher urinary IL-6 and serum TNFa than the children without microalbuminuria (group B). The diabetic patients with no sign of nephropathy showed significantly higher TNFalpha and NAG and its A and B isoforms in urine compared to the healthy group. Additionally, groups A and B both showed a positive significant correlation between serum TNFalpha and urinary isoform B. CONCLUSIONS: From our pilot results it appears that TNFalpha might be a sensitive marker of damage to the renal proximal tubules occurring prior to microalbuminuria. Conversely, the increase in NAG and its isoform B activity in patients with no clinical sign of diabetic nephropathy may indicate the onset of microalbuminuria.

The biological activity of new tuftsin derivatives--induction of phagocytosis.

Phagocytosis plays a crucial role in a host defense against invading microorganisms. This process can be induced by many phagocytosis stimulating factors. One of them is an endogenous tetrapeptide - tuftsin that occurs in the blood of mammals including human beings. Tuftsin is capable of potentiating granulocyte and macrophage functions such as: phagocytosis, motility, and chemotaxis as well as bactericidal and tumoricidal activity. The other particle able to induce phagocytosis is muramyl dipeptide (MDP), the smallest synthetic glycopeptide of bacterial origin that possesses an immunogenic activity. MDP is known to affect most functions of macrophages. Phagocytosis stimulating properties of a new group of tuftsin and MDP analogues (one tuftsin analogue and four conjugates of tuftsin/retro tuftsin and muramyl dipeptide or nor-muramyl dipeptide) were tested. The results of the study show that all of the examined conjugates are able to generate oxidative burst. The most promising analogues proved to be kd6 and kd7.

Effects of fasting and refeeding on serum leptin, adiponectin and free fatty acid concentrations in young and old male rats.

BACKGROUND: Regulatory mechanisms of metabolic homeostasis undergo important alterations during ageing. The age-related changes become often evident only during stimulation of basic functions that occurs, e.g. during fasting and refeeding which represent natural challenge to energy metabolism. OBJECTIVE: To determine the effect of short-term fasting and subsequent refeeding on serum levels of key hormones and metabolites in young adult 5- and 24-month-old male Wistar rats. METHODS: Control rats were fed ad libitum. Animals were fasted for 48 h or fasted and refed for 24 h. Metabolite serum concentrations were measured by standard methods. Leptin and insulin were determined by rat-specific RIA, and adiponectin serum levels by mouse/rat-specific ELISA. RESULTS: (1) Control serum levels of key metabolites and hormones were similar in both age groups except for increased triglycerides (TG) in old fed rats. (2) Fasting caused a significant decrease of leptin, insulin, glucose, and TG serum levels in both age groups, and an increase of free fatty acids (FFA) concentration, however, only in young animals. (3) Upon refeeding serum glucose, TG and insulin reversed to control levels in both age groups, however, FFA concentration decreased to control values only in young rats. (4) In contrast to young animals, refeeding of old rats did not increase serum leptin concentration to control level. (5) Neither fasting nor refeeding changed adiponectin serum levels in both age groups. CONCLUSION: Aging suppresses leptin secretion and metabolism of FFA during refeeding that follows short-term starvation. In old rats serum levels of FFA are refractive to the alterations induced by fasting/refeeding in young ones.