Christian Demanet

Proinflammatory cytokines and their role in the development of major transplant-related complications in the early phase after allogeneic bone marrow transplantation.

Serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor (TNF)-alpha were frequently measured during the first 30 days after allogeneic bone marrow transplantation (BMT) in 84 consecutive adult patients. Major transplant-related complications (MTCs) occurred in 33% of cases and included veno-occlusive liver disease, idiopathic pneumonia syndrome, severe endothelial leakage syndrome and >grade II acute graft-versus-host disease. Compared with patients having minor complications, those with MTCs developed higher levels at times of maximal clinical signs (all cytokines, P<0.001), between days 0–5 post-BMT (IL-6 and IL-8, P<0.05) and days 6–10 (L-6, P<0.001; IL-8 and TNF, P<0.01) post-BMT. We could not discriminate patterns of cytokine release that were specific for any subtype of MTC. Higher levels of IL-8 during days 0–5 were associated (P=0.044) with early (<40 days) death. Multivariate analysis including patient and transplant characteristics as well as post-BMT levels of C-reactive protein showed that high average levels of one or more of the cytokines within the first 10 days post-BMT were independently associated with MTC (Odds ratio: 2.3 [1.2–4.5], P=0.011). This study shows that systemic release of proinflammatory cytokines contributes to the development of MTC and provides a rationale for pre-emptive anti-inflammatory treatment in selected patients.

Elevated serum heat-shock protein 70 levels in patients with acute infection: use of an optimized enzyme-linked immunosorbent assay.

Heat‐shock proteins (Hsps) are highly conserved throughout evolution and evoke great interest both in basic biology and in medicine. They are expressed in small quantities under normal conditions, and their expression can be strongly induced by several stressors. Although their action is basically intracellular, it is now obvious that these proteins can be released into the extracellular environment from viable cells. In this study, the human Hsp 70 serum concentrations were determined using an optimized, cost‐effective enzyme‐linked immunosorbent assay (ELISA). The average intra‐assay variation was 6%, whereas the average interassay variation was 9%. The sensitivity of the assay was 10 ng/ml, and spiking experiments showed recoveries between 101 and 109%. As an application of the technique, we have investigated the serum levels of human Hsp 70 in patients with infection and in healthy subjects. Our data show significantly higher levels of Hsp 70 (P  = 0.003) in patients compared to control subjects. Positive correlations were noticed between the serum levels of Hsp 70 and various markers of inflammation (IL‐6; r = 0.579, P = 0.009, TNF‐α; r  = 0.552, P  = 0.012, IL‐10; r  = 0.361, P = 0.002). We conclude that Hsp 70 is involved in inflammation of infectious origin. The interindividual variation in the serum concentration of Hsp 70 precludes the use of serum Hsp 70 levels to distinguish patients from healthy subjects.

Biochemical Changes in Response to Intensive Resistance Exercise Training in the Elderly

Background: It is assumed that low-grade inflammation, characterized by increased circulating IL-6 and TNF-α, is related to the development of sarcopenia. Physical exercise, especially high intensity resistance training, has been shown to be effective in restoring the strength deficit in the elderly. Intensive exercise is accompanied by significant release of IL-6 and TNF-α into the blood circulation, but does not result in muscle wasting. Exercise-induced changes in heat-shock protein (Hsp), responsible for cellular protection during stressful situations, might interfere with the acute phase reaction and muscle adaptation. Objective: To investigate if intensive strength training in elderly persons induces changes in Hsp70 expression, and if these changes are related to changes in the acute phase reaction or muscle adaptation. Methods: 31 elderly persons (aged 68.4 ± 5.4 years) performed 6 weeks’ intensive strength training. At baseline and after 6 weeks, muscle strength, functional performance (physical activity profile, 6-min walk, 30- second chair stand, grip strength, chair sit & reach and back scratch), linear isokinetic leg extension, circulating IL-6, TNF-α, IL-10 and TGF-β, and Hsp70 in monocytes (M) and lymphocytes (L) immediately after sampling (IAS), after incubation at 37 and 42°C were determined. In 12 participants, cytokines were determined in untrained and trained conditions before and after a single training session. Results: After 6 weeks’ training, muscle strength and functional performance improved significantly, together with decreased Hsp70 IAS and Hsp70 37°C and increased Hsp70 42°C (all p < 0.05). Strength gains correlated positively with baseline Hsp70 37°C and training-induced changes of Hsp70 42°C in M and L. In an untrained condition, training induced an increase of IL-6 (p < 0.05) and a tendency of IL-10 to decrease (p = 0.06). In a trained condition the decrease of IL-10 disappeared. Baseline physical activity and 6-min walk distance correlated negatively with circulating IL-6 (p < 0.05); except for a negative correlation between TGF-β and Hsp70 37°C L (p < 0.05), no significant relationships were found between cytokines and Hsp70. After the training program, Hsp70 37°C was negatively related to circulating TNF-α, IL-10 and TGF-β. Conclusion: Strength training in the elderly induces changes in Hsp70 expression, associated to strength gains and circulating cytokines.

Age-Related Decrease in the Inducibility of Heat-Shock Protein 70 in Human Peripheral Blood Mononuclear Cells

We have investigated the effect of age and of the presence of proinflammatory cytokines on Hsp 70 production in human peripheral blood mononuclear cells, using flow cytometry. Twenty-seven women and 23 men, all apparently healthy, participated in the study. At 37°C, the percentage of Hsp 70-producing monocytes and lymphocytes, as well as the level of Hsp 70 in monocytes, were negatively influenced by age. After exposure of the cells to 42°C, the increase of Hsp 70 production was more pronounced in monocytes than in lymphocytes; both the intensity of Hsp 70 production and the percentage of Hsp 70-producing cells were negatively influenced by the age of the subjects, as well for monocytes as for lymphocytes. There was a negative correlation between the intensity of Hsp 70 production by monocytes exposed to 42°C and the serum levels of tumor necrosis factor-α and interleukin-6. In conclusion, in human monocytes and lymphocytes, heat-induced Hsp 70 production is reduced with increasing age and is negatively influenced in monocytes by proinflammatory cytokines.

Inflammatory status as an important determinant of heat shock protein 70 serum concentrations during aging.

Heat shock proteins (Hsp) form a large family of proteins that are ubiquitously present in all organisms. In the absence of destabilising stimuli, Hsp are expressed at low levels, but their expression can be highly induced by various noxious conditions such as heat, oxygen stress and infection. Hsp have been reported to interfere with inflammatory processes and their induction is well known to decrease with aging. In the present study we have investigated Hsp70 serum concentrations using an optimised ELISA in elderly patients, recruited from a geriatric University Hospital ward. Our resultsportray positive correlations between the serum levels of Hsp 70 and various markers of inflammation (monocyte count, serum concentration of TNF-α, plasma concentrations of C-reactive protein, and fibrinogen), explaining the difference in Hsp70 serum concentrations in these subjects with various degrees of inflammation. We conclude that Hsp 70 is involved in inflammatory diseases and that the serum level of Hsp 70 is directly linked to the inflammatory status of the subject. However, the nature of this relationship remains to be elucidated.

Reversion of autoimmune lymphoproliferative syndrome with an antimalarial drug: preliminary results of a clinical cohort study and molecular observations

Summary. Autoimmune lymphoproliferative syndrome (ALPS) is a paediatric disease characterized by lymphoproliferation and autoimmunity. Most patients are known to carry heterozygous mutations of the TNFRSF6 gene leading to diminished Fas‐mediated apoptosis and failure of activated lymphocytes to undergo apoptosis. A subgroup of patients without the TNFRSF6 gene mutation has similar defective apoptosis and clinical features. No effective treatment has been reported so far. Glucocorticoids, intravenous immunoglobulin and/or immunosuppressive drugs have usually led to only transient clinical improvement. Seven ALPS patients (two type Ia and five type III) were treated with the antimalarial drug Fansidar. No toxicity was observed. An objective response was seen in six of them and, in two, the treatment was stopped without reappearance of the symptoms. Moreover, a marked decrease in interleukin‐10 levels was observed in two patients during the treatment. We found that the drug induced apoptosis in activated lymphocytes through activation of the mitochondrial apoptotic pathway.

Immune dysfunction associated with chronic professional stress in nurses

The relationship between chronic professional stress in nurses and immunity as well as the possible impact of psychopathology upon this relationship have been examined. Sixty subjects were selected on the basis of high/low scores on professional stress and psychopathology. Chronic professional stress appeared to be associated with immune dysfunction including signs of immune activation (increased numbers of cells expressing the interleukin-2 receptor, especially CD4+CD25+ cells) and possibly immune suppression (decrease in percentage of natural killer cells). The increase in activation markers, CD3+CD16CD56+ cells and serum neopterin was most pronounced in the group with high stress/low psychopathology whereas the decrease in CD8+CD11b+ cells was most pronounced in the group with high stress/high psychopathology. It is hypothesized that in the presence of chronic stress distinct psychological mechanisms are associated with specific immune dysfunctions.

Characterisation of pleural inflammation occurring after primary spontaneous pneumothorax

The aim of this study was to examine the inflammatory reaction occurring in the pleural space of patients suffering from primary spontaneous pneumothorax (PSP) using pleural lavage, which was performed in patients with PSP and in healthy control subjects (essential hyperhidrosis patients undergoing thoracoscopy for sympathicolysis treatment). Cellular and solute composition of lavage fluid, peripheral blood and parietal pleural biopsies were analysed. PSP lavage fluid showed an increase in all differentiated leucocytes, but most strikingly eosinophils and neutrophils. In the blood of patients with PSP, the total number of leucocytes and the absolute number of eosinophils, neutrophils and monocytes were also significantly increased. The time in which air was present in the pleural space was positively correlated with the increase of eosinophils in lavage fluid, parietal pleura and blood. Eosinophilic cationic protein was elevated after PSP and strongly correlated with the absolute number of lavage eosinophils. Chemo and cytokine analysis in lavage fluid showed differences in concentrations of interleukin (IL)‐5, IL‐6, IL‐8, IL‐12p40, tumour necrosis factor‐α and RANTES, but not of eotaxin. Surprisingly, high levels of lipopolysaccharide binding protein were also measured. Primary spontaneous pnumothorax is associated with a substantial pleural inflammatory reaction. The authors hypothesise that mechanical stretch factors, lipopolysaccharide binding protein/lipopolysaccharide complexes or other environmental components trigger pleural inflammation after primary spontaneous pnumothorax.

Enhanced assessment of allogeneic bone marrow transplant engraftment using automated fluorescent-based typing

Traditional qualitative gel electrophoresis approaches lack accurate and quantitative assessment of mixed chimerism in BMT patients. The likelihood of informative markers is greatly increased using simultaneous amplification of 10 highly polymorphic loci with fluorescent-labeled primers in an automated DNA sequencer. This allows for more precise interpretation of mixed chimerism with a detection level approximating 1%. To evaluate this approach to quantitative assessment of chimeric populations we mixed varying proportions of samples from two unrelated donors, by either mixing aliquots of DNA isolated from whole blood, or by first counting the white blood cells and mixing varying proportions of cells together prior to DNA isolation. The allelic-peak area ratios were identical to allelic-peak height ratios and corresponded to the proportion of mixed DNA, regardless of the method used to create the mixture. Formulas to provide routine, consistent and quantitative interpretation of mixed chimerism are presented. We analyzed 14 allograft recipients and one autologous BMT patient with transfusion-induced GVHD. In all cases, at least four out of nine markers were informative. Inter-laboratory concordance of results was also obtained with an eight marker panel using an automated Alf-Express. In conclusion, the automated DNA fluorescent-labeled primer approach using an eight to 10 marker panel is quantitative and informative in assessing chimerism.

The induction of heat shock protein 70 in peripheral mononuclear blood cells in elderly patients: A role for inflammatory markers

The induction of heat shock proteins (Hsp) is the response to a plethora of stress signals including hyperthermia, physical stress, and various disease states. Although changes in Hsp expression are associated with certain diseases, the question as to whether this is an adaptation to a particular pathophysiologic state or a reflection of the suboptimal cellular environment associated with the disease remains open. In this study we have investigated the effects of inflammatory mediators on the induction of Hsp 70 in human peripheral mononuclear blood cells using flow cytometry. We demonstrate that without heat shock, the levels of the inflammatory mediators are positively related to Hsp 70 production in monocytes. On the contrary, negative correlations were found between heat induced Hsp 70 production and interleukin-6 (IL-6), as well as various markers of inflammation. These observations are in agreement with the antagonistic effects between heat stress and the inflammatory mediators on the activation of Hsp promoter.