Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro.

OBJECTIVE: Physical cues play a crucial role in skeletogenesis and osteochondral regeneration. Although human mesenchymal stem cells (hMSCs) offer considerable therapeutic potential, little is known about the molecular mechanisms that control their differentiation. We hypothesized that mechanical strain might be an inherent stimulus for chondrogenic and/or osteogenic differentiation in undifferentiated hMSCs, where c-Fos (FOS) might play a major role in mechanotransduction. METHOD: hMSCs from 10 donors were intermittently stimulated by cyclic tensile strain (CTS) at 3000 mustrain for a period of 3 days. Differential gene expression of strained and unstrained hMSCs was analysed by real-time RT-PCR for several marker genes, including the transcription factors FOS, RUNX2, SOX9, and others. Additionally, alkaline phosphatase activity (ALP) was determined kinetically. RESULTS: The application of CTS significantly stimulated the expression levels of the early chondrogenic and osteogenic marker genes (SOX9, LUM, DCN; RUNX2, SPARC, SPP1, ALPL); this was accompanied by stimulation of ALP activity (+38%+/-12 standard error of mean, P<0.05). Matrix analysis revealed that the osteo-chondrogenic response followed a coordinated expression pattern, in which FOS was attributed to early osteogenic but not chondrogenic differentiation. CONCLUSION: Undifferentiated hMSCs are highly sensitive to mechanical strain with a transcriptionally controlled osteo-chondrogenic differentiation response in vitro.

Transcriptional pattern analysis of adrenergic immunoregulation in mice. Twelve hours norepinephrine treatment alters the expression of a set of genes involved in monocyte activation and leukocyte trafficking.

We investigated in vivo effects of norepinephrine (NE) on the transcription of 200 immunologically relevant genes in the mouse. Balb/c mice were s.c. implanted with NE containing retard tablets. Twelve hours later, splenic mRNA was prepared and hybridized onto cDNA microarrays containing the sequences of the major cytokines, their receptors and all CD-antigens of the mouse. Consistent results were obtained with a set of five genes: in the NE-treated animals four genes (CXCR4, VCAM1, IL-1R2, CD 14) were found 2-8 fold upregulated as compared to sham treated animals, whereas the gene for CCR3 was downregulated (< 0.5 fold). The findings were confirmed using quantitative reverse transcriptase Real Time PCR. These first results prove the usefulness of gene microarray technology towards transcription pattern analysis in neuroimmune interactions. Furthermore, they support the relevance of catecholamines in the regulation of leukocyte migration and the inflammatory response.

Low grade inflammation in juvenile obesity and type 1 diabetes associated with early signs of atherosclerosis.

BACKGROUND: Subclinical inflammation has been implicated in the initiation and/or progression of atherosclerosis. Diabetes mellitus and obesity are risk factors for atherosclerosis, and asymptomatic low grade inflammation occurs prior to overt vascular lesions in these patients. In contrast to adults, little information exists concerning low grade inflammation in young type 1 diabetes and juvenile obesity. AIM: To investigate low grade inflammation and immune activation in juvenile diabetes mellitus and obesity. METHODS: hs-CRP, soluble interleukin-2 receptor (sIL-2R), C-peptide, insulin, cortisol, vitamin B12, folic acid, leptin, and homocysteine were determined in 148 patients with juvenile type 1 diabetes, 86 obese children and 142 normal weighted age-matched healthy controls. Intima-media thickness (IMT) and lumen diameter of both common carotid arteries (CCA) was measured by ultrasonography in 52 healthy pediatric controls, 10 diabetics, and 34 obese juveniles. RESULTS: Serum hs-CRP was significantly elevated in patients with type 1 diabetes (p < 0.0001), and obese children (p < 0.0001) as compared to the control group. The obese juveniles (p < 0.0001) and the diabetics (p < 0.0001) showed significantly increased values for IMT of CAAs. Levels of homocysteine, sIL-2R, insulin, cortisol, vitamin B12, and folic acid did not differ from the controls. The elevation of hs-CRP was more pronounced in obesity as compared to type 1 diabetes (p < 0.0001), and the hs-CRP values correlated significantly with body mass index standard deviation score (BMI-SDS) values. Furthermore, the IMT and the luminal diameter of CCAs showed significant correlations with BMI-SDS values. CONCLUSION: A low grade inflammation as determined by serum hs-CRP is significantly increased in children with type 1 diabetes, and even more pronounced in apparently healthy juveniles with obesity. The increased IMT of CCAs strongly argues for an association between this low grade inflammation and early atherosclerotic vessel injury.

Mens sana in corpore sano--and vice versa. The role of the autonomic nervous system in the immune-neuroendocrine dialogue.

Adrenergic and cholinergic transmitters of the autonomic nervous system have important roles in the mutual interrelationships between the brain and the immune system. Besides expressing functional adrenergic and cholinergic receptors, lymphocytes and other immune cells were found to synthesize and release catecholamines and acetylcholine pointing to a possible role of these mediators in the intrinsic regulation of the immune system. In this review we will summarize concepts of Psychoneuroimmunology on the basis of data as obtained in vitro and in experimental studies in animal models, and discuss their relevance to human clinical medicine.

Prolonged alpha-adrenergic stimulation causes changes in leukocyte distribution and lymphocyte apoptosis in the rat.

We have previously shown in the rat model that acutely or chronically increased peripheral catecholamines lead to suppression of lymphocyte responsiveness via alpha(2)-adrenoceptor activation. Here we investigated the effects of alpha-adrenergic treatment on total leukocyte numbers and proportions of leukocyte subsets in peripheral blood and lymphoid tissues. It was found that a 12-h treatment with subcutaneously implanted tablets, one containing norepinephrine (NE) and one propranolol, leads to an increase in total blood leukocyte counts, due to a pronounced increase in granulocytes. In contrast, the numbers of all classes of lymphocytes other than NK cells were decreased. This decrease in blood lymphocytes is apparently not due to redistribution, since in the thymus, spleen, mesenteric and peripheral lymph nodes, the total numbers of lymphocytes were decreased as well, without any changes in subpopulations. Analogous results were obtained with rats adrenalectomized before the catecholamine treatment. Animals that received the alpha-adrenergic treatment displayed significantly more apoptotic cells in the lymphoid organs, as determined by the TUNEL technique. In the spleen, the enhanced rate of apoptosis was confined to the white pulp; red pulp areas exhibited significantly fewer apoptotic cells. Thus, an increased alpha-adrenergic tone in rats led to a general loss of lymphocytes due to lymphocyte directed apoptosis that was independent of glucocorticoids.

Prooxidant activity of melatonin promotes fas-induced cell death in human leukemic Jurkat cells.

The antioxidant activity of melatonin (MEL) has been considered to constitute part of its physiological as well as pharmacological effects. However, as described herein we found a profound prooxidant activity of micro- to millimolar concentrations of MEL in the human leukemic Jurkat cell line. This prooxidant effect was increased in glutathione-depleted cells and counteracted by antioxidants. As a consequence MEL promoted fas-induced cell death. These data therefore indicate that MEL may be a modulator of the cellular redox status, but does not necessarily act as an intracellular antioxidant.

Serotonin and immune response: effect of the amine on in vitro proliferation of rat lymphocytes.

OBJECTIVE: The effect of serotonin (5-hydroxytryptamine; 5HT) on the in vitro proliferation of mitogen-stimulated lymphocytes was studied in primary cultures of rat spleen cells. METHODS: 5HT was added to the cultures 1 h prior to the mitogen, at final concentrations from 10(-13) up to 10(-2) M. T and B cell mitogens (concanavalin A, pokeweed mitogen and lipopolysaccharide) were used at suboptimal and optimal concentrations. The cell proliferation was measured 24-72 h after the addition of mitogen. The effect of each 5HT concentration was studied on a group of 6-12 animals and was expressed as a percentage of the control values obtained with mitogen alone. RESULTS: No significant effect of 5HT at concentrations from 10(-13) to 10(-5) M was found. At concentrations of > or =10(-4) M, a regular dose-dependent inhibition of the lymphocyte proliferation appeared, the concentration producing the half-maximal effect being 6 x 10(-4) M. The observed suppression was not due to 5HT cytotoxicity toward spleen cells. CONCLUSION: With the experimental system used, we failed to confirm an immunostimulatory effect of 5HT in the range of concentrations of its receptor sensitivities or lower, but found a clear-cut immunoinhibitory effect at higher concentrations.

Gene expression of the key enzymes of melatonin synthesis in extrapineal tissues of the rat.

Besides the pineal gland, melatonin is reported to be produced in a number of extrapineal sites, where it could act as an intracellular mediator or paracrine signal in addition to its endocrine effects. In view of the suggested immunoregulatory role of melatonin, we compared lymphoid organs and several other tissues of the rat for their potential to synthesize melatonin. Using the reverse transcription-polymerase chain reaction (RT-PCR) method, we determined the tissue-specific expression of mRNAs encoding two key enzymes of the melatonin biosynthesis: serotonin-N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). The minimal number of PCR cycles required to obtain a positive signal served as a measure for the abundance of a given mRNA. NAT and HIOMT mRNAs were detected in all tested tissues at high numbers of PCR cycles (40 and 45, respectively). At 35 cycles, only gut, testis, spinal cord, raphe nuclei, stomach fundus and striatum yielded positive signals for both enzymes. In conclusion, the presence of NAT and HIOMT mRNAs in a wide range of tissues corroborates and extends the notion of extrapineal melatonin synthesis. Comparatively low levels of the HIOMT messages in lymphoid organs, however, indicate a limited significance of melatonin synthesis within the immune system.

mRNA expression of serotonin receptors in cells of the immune tissues of the rat.

Serotonin (5-hydroxytryptamine, 5-HT) has been shown to play a role in immunoregulation; however, little is known about specific subtypes of 5-HT receptors involved in peripheral immunomodulation. In the present study we used RT-PCR methods to examine the mRNA expression of 5-HT receptors in the cells of lymphoid tissues of the rat. All 13 rat 5-HT receptor genes cloned so far were examined in ex vivo isolated spleen, thymus, and peripheral blood lymphocytes, as well as in mitogen-stimulated spleen cells. Positive signals were obtained for 5-HT1B, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT6, and 5-HT7 receptor mRNAs in all three compartments. Mitogen (ConA and PWM) stimulated cells additionally expressed mRNA corresponding to the 5HT-3 receptor subtype. In contrast, 5-HT1A, 5-HT1D, 5-HT2C, 5-HT4, 5-HT5A, and 5-HT5B mRNAs were not detected in any of the examined cell populations. These results may be useful as a starting point for future functional studies on immunomodulatory effects of 5-HT and may help to understand conflicting serotonergic effects on immune functions as found in the literature.

Cystatin C, an early indicator for incipient renal disease in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic disease requiring potential nephrotoxic therapy with nonsteroidal antiinflammatory drugs (NSAIDs) and disease modifying antirheumatic drugs (DMARDs). The rationale of our study was to examine the renal status of patients suffering from prolonged RA by means of plasma cystatin C, a new parameter of renal function. Fifty-six patients affected with RA for more than 5 years, and treated with NSAIDs for more than 50 months, were included in the study. Besides conventional markers of renal function (i.e. plasma creatinine, estimated glomerular filtration rate, creatinine clearance), we analysed plasma cystatin C by an automated, nephelometric immunoassay on a Behring nephelometer. Sixty percent of the RA patients exhibited elevated levels of plasma cystatin C, whereas only three out of 56 patients showed an elevated plasma creatinine, even though the creatinine clearance was decreased in 57% of these patients. Cystatin C exhibited a by far better correlation with creatinine clearance than plasma creatinine. In conclusion, patients with prolonged RA for more than 50 months, show a disturbed renal function despite normal plasma creatinine. Elevated cystatin C indicates such incipient renal disease, and is, not least because of a simple, well reproducible technique, more recommendable for screening purposes than tedious clearance determinations.

The in vivo effects of beta-3-receptor agonist CGP-12177 on thyroxine deiodination in cold-exposed, sympathectomized rat brown fat.

OBJECTIVE: The effects of the beta-3-receptor agonist CGP-12177 on thyroxine (T4) deiodination in sympathectomized (SX) interscapular brown adipose tissue (BAT) were assessed in 300 g body weight (BW) Wistar rats. DESIGN: Seven days after SX, groups of rats were implanted s.c. with pellets containing 5mg CGP-12177 or 5mg norepinephrine (NE) and were immediately placed at 4 degrees C for 24h. Other SX groups were injected with CGP-12177 or NE 1mg/kg BW i. p. and placed in the cold for 4h. The latter group was injected, in addition, with prazosin 0.4 mg/100g BW i.p. or propranolol 0.5mg/100g BW i.p. 15 min before and 2h after the administration of CGP-12177 or NE. METHODS: Two hours after the last injection of prazosin or propranolol, animals were killed and BAT was removed, homogenized and centrifuged at 500 g for 10 min at 4 degrees C. The infranatants were incubated during 60 min in the presence of dithiothreitol and 1 microCi [(125)I]T4. Aliquots were chromatographed on paper for the measurement of [(125)I]T4 and its deiodinated subproducts. RESULTS: CGP-12177 restored normal T4 deiodination in SX BAT from both groups, but NE was slightly more effective. Propranolol, although not prazosin, blocked the CGP-12177 effects. Contrariwise, the NE-induced rise in deiodination was blocked by prazosin and to a lesser extent by propranolol. CONCLUSIONS: The results indicate that CGP-12177 stimulated the in vivo activation of 5'-deiodinase type II activity predominantly via beta-3-receptor, without participation of alpha-1-receptors.

Unrelated peripheral blood stem cell transplantation with 'megadoses' of purified CD34+ cells in three children with refractory severe aplastic anemia.

Three children with refractory severe aplastic anemia were transfused with high numbers of unrelated matched (n = 2) or C-locus haploidentical mismatched (n = 1) CD34-selected peripheral blood stem cells in the absence of an HLA-identical family donor. Two leukaphereses of the donors yielded a median number of 10.1 x 10(10) nucleated cells (range 9.7-15.4) with a median number of 9.89 x 10(8) CD34+ cells (range 7.46-26.1) and a median percentage of CD34+cells of 0.98% (range 0.77-1.7). After positive selection by magnetic cell sorting the patients received a median of 14.3 x 10(6) CD34+ cells/kg (range 11.7-24.3) and of 1.3 x 10(4) CD3+ cells/kg (range 0.57-5.8). Median time to ANC >/=0.5 x 10(9)/l was 7 days (range 7-12) and to platelets >/=20 x 10(9)/l 13 days (range 13-27). Chimerism analysis of peripheral blood after transplantation revealed permanent 100% donor hematopoiesis in all patients. The patient with the C-locus haploidentical mismatch presented with acute GVHD (grade III-IV) of the skin, liver and lower gastrointestinal tract (onset day +40) and died despite intensive immunosuppressive treatment on day +238. The two survivors developed lymphopoietic recovery of B and T lymphocytes within 3 months after transplantation. To our knowledge this experience represents the first report of transplantation with unrelated CD34+ enriched peripheral blood stem cell in children with refractory severe aplastic anemia. Bone Marrow Transplantation (2000) 25, 513-517.

The tumor-associated shift in immunoglobulin G1/G2 is expressed at the messenger RNA level of peripheral blood B lymphocytes in patients with gynecologic malignancies.

BACKGROUND: Previously, it could be demonstrated that human patients with malignant diseases of various tissues exhibited characteristic and highly significant changes in the serum patterns of immunoglobulin (Ig)G subclasses, consisting of a decrease in IgG1 and an increase in IgG2 relative to total IgG. The aim of the current study was to determine whether this phenomenon was detectable at the level of IgG-producing B lymphocytes. METHODS: Using a competitive reverse transcriptase polymerase chain reaction specific to IgG1 and IgG2, the gene expression of these 2 IgG subclasses in peripheral B cells from 10 patients with carcinomas of various sites within the female reproductive tract and 10 healthy controls was quantitatively determined, in parallel with the concentrations of the respective serum proteins. RESULTS: Absolute levels of IgG subclass messenger ribonucleic acid (mRNA) showed a slight but not significant decrease in IgG1 and an increase in IgG2 in patients with gynecologic malignancies. However, the ratio of IgG1 to IgG2 expression showed a highly significant (P < 0.001) decrease in tumor patients compared with healthy controls, and corresponded to the change in the ratio of IgG1 to IgG2 serum proteins. CONCLUSIONS: These data suggest that the shifts in the serum patterns of IgG1 and IgG2 observed in patients with gynecologic malignancies are due to irregular biosynthesis of these IgG subclasses at the B-cell level.

The dialogue between the brain and immune system involves not only the HPA-axis.

Starting out from our previous observations that defects in the immune system-brain feedback predispose to pathogenic immune responses, our interest focuses at the roles of adrenergic/cholinergic neurotransmitters in brain-immune interactions. We have shown in rodent models that 1) both catecholamines and acetylcholine are potent modulators of peripheral immune functions, 2) cholinergic signals are involved in the afferent signalling of the immune system, and 3) lymphocytes not only express functional adrenergic and cholinergic receptors, but synthesize and release neurotransmitters, such as acetylcholine, in quantitative dependence of differentiation and activation. Studies are presently being initiated to investigate the role(s) of these non-neuronal neurotransmitters within immune tissues, and to explore the relevance of excitatory amino acids as important central neurotransmitters in the brain-immune system dialogue.

Selective decrease of serum immunoglobulin G1 as marker for early stages of invasive breast cancer.

The diagnostic value of the decrease in percentage of immunoglobulin G1 (%lgG1) in breast cancer was analyzed with special emphasis on early tumor stages. IgG1 and total IgG were preoperatively measured in the sera of a total of 801 individuals using a modified quantitative affinity chromatography. Group A consisted of 174 healthy individuals of both sexes, group B of 324 female patients with benign breast disease, and group C of 303 patients with invasive and non-invasive breast cancer. Within group C, 13 patients presented with intraductal carcinoma, and 22 patients with a pT1a-tumour (diameter less than 0.5 cm). The %IgG1 values were compared among groups A, B and C. In addition, correlations were sought between %IgG1 values of group C and tumor size, stage (UICC), histopathological grade and oestrogen (ER) and progesteron receptor (PR) expression. The mean value of %IgG1 in group A was 63.3 +/- 0.5 s.e.m., in group B 57.75 +/- 0.4 s.e.m. and in group C 52.37 +/- 0.5 s.e.m. The differences of mean values were highly significant between all three groups. Sensitivity and specificity of %IgG1 to discriminate between group A and C were 75% and 87%, and between group B and C 62% and 63%, respectively. The significant decrease of %IgG1 in total serum IgG is able to distinguish patients with breast cancer of more than 5 mm in diameter from healthy controls and patients with benign breast diseases. Finally, calculated posterior probabilities revealed that within certain concentration limits %lgG1 may provide predictive information with high probabilities.

In vivo immunomodulation by peripheral adrenergic and cholinergic agonists/antagonists in rat and mouse models.

Our work is devoted to defining relationships between the immune system and the adrenergic and cholinergic systems in vivo. In the rat model, we have shown that the cells of different immune compartments express the genes of a defined set of adrenergic/cholinergic receptors, and it was shown that lymphocytes are a site of non-neuronal production of norepinephrine and acetylcholine. Furthermore, using implantable slow-release tablets containing adrenergic or cholinergic agonists/antagonists, distinct and partly opposite effects were observed on peripheral immune functions. Concerning sympathetic immunoregulation, our data--in contrast to those of other studies--suggest that an enhanced adrenergic tonus leads to immunosuppression primarily via alpha 2-receptor-mediated mechanisms. Beta-blockade strongly enhances this effect, most likely by inhibition of pineal melatonin synthesis. In recent experiments on the kinetics it was found that the continuous alpha-adrenergic treatment entails a strong suppression of cellular responsiveness during the first few hours, which is increasingly followed by a general loss of lymphocytes in blood and lymphoid organs most likely due to enhanced apoptosis. More recently, we have extended our studies to the mouse model. First data obtained with RNAse protection assays suggest a biphasic effect on the gene expression of several cytokines in spleen cells due to adrenergic in vivo treatment.

Long-term follow-up of cytokines and soluble cytokine receptors in peripheral blood of patients with juvenile rheumatoid arthritis.

Plasma levels of interleukin-1beta (IL-1beta), IL-2, soluble IL-2 receptor (sIL-2R), IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and the p60 soluble TNF receptor (sTNFR) were repeatedly determined by enzyme-linked immunosorbent assays (ELISA) in 35 patients with different subtypes of juvenile rheumatoid arthritis (JRA) during an observation period of up to 36 months. The data were related to conventional inflammatory parameters and disease activity. Patients with systemic disease showed the most pronounced elevations of plasma cytokines, followed by polyarticular and pauciarticular JRA. Soluble receptors sIL-2R and sTNFR were consistently elevated in patients of all JRA subtypes and indicated disease activity even in patients with normal C-reactive protein (CRP). In contrast, the determination of IL-1beta, IL-2, IL-8, and TNF-alpha revealed strikingly different individual profiles in patients of the same clinical subtype of JRA and irrespective of disease activity. It is concluded that the determination of sIL-2R and sTNFR may be relevant for monitoring JRA, as they indicate disease activity also in cases with unaltered conventional inflammatory parameters. The different individual cytokine profiles of patients within identical subtypes of disease suggest JRA to be even more heterogeneous than hitherto assumed. The data should be considered in attempts to develop anticytokine strategies in the therapy of JRA.