Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

Oxidative Stress Level in Circulating Neutrophils Is Linked to Neurodegenerative Diseases

Alzheimers and Parkinsons diseases are the most common neurodegenerative conditions. Oxidative lesions are a hallmark of both diseases, but the respective roles of systemic and cerebral dysfunction are not elucidated. As circulating neutrophils are the most powerful sources of reactive oxygen species, we measured oxidative stress levels in resting neutrophils from 44 Alzheimers and Parkinsons disease patients and compared them to 40 healthy counterparts. Significantly increased oxidative stress levels were observed in patients groups, while control groups had very similar levels irrespective of age. One-third of the neurodegenerative patients presented with oxidative stress levels higher than those of any healthy donor. This increase was not due to an elevated production of reactive oxygen species during the neutrophil oxidative burst. Mitochondrial mass and activity were altered in neutrophils of the Parkinsonian group compared to controls, but not in those from Alzheimers disease group. To our knowledge, this is the first report linking oxidative stress and mitochondrial parameters in circulating neutrophils from neurodegenerative and normal donors. Our results indicate that oxidative stress levels in circulating neutrophils are of interest for further mechanistic studies of neurodegenerative diseases and might open the perspective of a diagnostic tool.     

A cyclin protein modulates mitosis in the budding yeast Saccharomyces cerevisiae

Summary For the budding yeast Saccharomyces cerevisiae the mitotic cell cycle is coordinated with cell mass at the regulatory step start. The threshold amount of cell mass (reflected as a critical size) necessary for start is proportional to nutrient quality. This relationship leads to a transient accumulation of cells at start, termed nutrient modulation, upon enrichment of nutrient conditions. Nutrient enrichment abruptly increases the critical size needed for start, causing the smaller cells, produced in the previous cell cycle, to be delayed at start while growing larger. Here we show that, in S. cerevisiae, a second cell-cycle step, at mitosis, also exhibits nutrient modulation, and is, therefore, another point of cell-cycle regulation. At both mitosis and start, nutrient modulation was found through mutation to be regulated by the activity of the cyclin-related WHI1 (CLN3) gene product.     

alpha1-acid glycoprotein possesses in vitro pro- and antiinflammatory activities

We studied the effects of ₁-acid glycoprotein on tumor necrosis factor- (TNF-) and interleukin-10 (IL-10) production and lymphocyte response to phytohemagglutinin in cultured peripheral blood mononuclear leukocytes from 6 healthy donors. We observed 2 opposite responses to ₁-acid glycoprotein: first, stimulation of TNF- and IL-10 production and inhibition of lymphocyte proliferation, and second, suppression of cytokine production and stimulation of lymphocyte proliferation. In cell cultures isolated from 4 of 6 donors, the TNF-/IL-10 ratio remained unchanged after addition of native ₁-acid glycoprotein, but some fractions isolated by chromatography on concanavalin A-Sepharose changed this parameter. These changes were most pronounced after treatment with fraction C enriched with molecules with incomplete (biantennary) carbohydrate chains. The mechanisms of ₁-acid glycoprotein-induced effects on peripheral blood mononuclear leukocytes are discussed.     

Neuronal activity in the flocculus of the alert monkey during sinusoidal optokinetic stimulation

Summary 1. Activity of single units was recorded in the flocculus of alert, behaving monkeys during sinusoidal optokinetic (0.02–5.0 Hz), constant velocity optokinetic, vestibular and visual-vestibular conflict stimulation. The maximal stimulus velocity for sinusoidal optokinetic stimulation at different frequencies was 40 deg/s or less (at frequencies above 1 Hz). For an amplitude series at 0.2 Hz, stimulus velocity was varied between ±10 to ±80 deg/s. In one trained monkey activity was also investigated during smooth pursuit eye movements and suppression of the vestibulo-ocular reflex by visual fixation (VOR-supp.). Only neurons which responded to 0.2 Hz (±40 deg/s) optokinetic stimulation were included in the study. 2. The majority of neurons (44 out of 59) were type I Purkinje cells (PCs), which increased their simple spike activity during optokinetic cylinder rotation to the ipsilateral recording side. The responses during other, vestibular related, paradigms allowed all these neurons to be classified as so called gaze velocity PCs. Three type II PCs were encountered, which responded similarly, but were only weakly modulated. 3. All type I PCs were modulated at frequencies of sinusoidal optokinetic stimulation between 0.05 and 2.5 Hz. PC's showed little or no modulation at 0.03 and 0.02 Hz. About half of the PC's still responded at 5.0 Hz. 4. Relative to eye velocity, the PC activity had a phase advance of about 30 deg between 0.1 and 2 Hz. It became larger at lower, and smaller at higher, frequencies. Eye velocity related sensitivity (imp/s/deg/s) was small at low stimulus frequencies and increased monotonically, on average from 0.16 at 0.02 Hz to 2.0 at 3.3 Hz. 5. Ten (out of 12) mossy fiber related input neurons were classified as visual neurons, since their activity could be related to the amount of retinal slip in all conditions. Neurons were clearly modulated at sinusoidal optokinetic stimulation up to 5 Hz. One input neuron, investigated during sinusoidal OKN, smooth pursuit eye movements, VOR and VOR-supp., behaved qualitatively like a gaze velocity PC. The remaining input neuron encoded eye velocity at 0.2 Hz optokinetic, vestibular and visual-vestibular conflict stimulation. 6. The results show that during sinusoidal and constant velocity optokinetic stimulation gaze velocity PC's do not encode eye velocity and/or eye acceleration. 7. The vestibular nuclei-flocculus complementary hypothesis (Waespe and Henn 1981) can explain PC responses to a large extent. However, a direct comparison shows that at low frequencies (particularly around 0.05 Hz) the complementary responses of most velocity storage encoding vestibular nuclei neurons and floccular PC's appears insufficient to account fully for the oculomotor response.Supported by Deutsche Forschungsgemeinschaft SFB 220, D7R.B. was a Alexander v. Humboldt fellow.     

Correlation Between Interferon Production and Clinical Disease Activity in Patients with Multiple Sclerosis

We determined the interferon (IFN) serum levels and in vitro activated IFN production in eight patients with relapsing/remitting multiple sclerosis (MS), using a whole-blood test system and the mitogen concanavalin A and the viral antigen Newcastle disease virus for induction of the IFN production. During the overall study period of 12 months we observed, in relation to clinical disease progression, a biphasic increase in the individual IFN and IFN production. While mitogen-induced IFN synthesis showed a significant augmentation prior to the onset of a new relapse (P < 0.05), virus-induced IFN production showed a temporal delayed increase which was related to clinical remission (P < 0.01). The observed fluctuations in the individual production of both IFN subtypes were not reflected in the sera of the patients. Although the reason for the temporal different imbalance in the production of both IFN subtypes remains unknown, the observed association between increased IFN production and clinical remission emphasizes a possible role for type 1 IFNs in the resolution of the MS relapse.     

Glucocorticoids modulate TGF-beta production

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGB- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFL-1) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TFG-1, -2, and -3. TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TGF-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and -2 production and reduced expression of the upregulated TGF-1 and -2 mRNA induced by exogenous TGF-1, -2 or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Potential roles for tumour necrosis factorα during embryonic development

This paper reviews the evidence indicating possible roles for tumour necrosis factor-alpha (TNF) in development. It is proposed that TNF may have essentially three major roles during embryonic development, which may be analogous to its roles in the immune system and during inflammation: a role in programmed cell death; a role as a cellular growth and differentiation factor; and also a role in the remodelling of extracellular matrix, and the regulation of cell adhesion molecules and integrins. The concept of the existence of a cytokine array during embryogenesis, analogous to that occurring in inflammation, is discussed, as well as potential roles for TNF in the induction of ubiquitin; protective mechanisms embryonic cells may employ against TNF-mediated cytotoxicity; and a consideration of the role TNF may play in a free radical theory of development.     

Defective NF-κB Signaling in Dedifferentiated Hepatoma Cells

Dedifferentiated rat hepatoma cells contain defects that result in the loss of hepatic gene expression, including the liver-enriched HNF4/HNF1 pathway. We examined induction of NF-B, a key mediator of the inflammatory response, in hepatoma and dedifferentiated hepatoma cells. We show that exposure of dedifferentiated hepatoma cells, but not rat and human hepatoma cell lines, to proinflammatory cytokines or lipopolysaccharide resulted in rapid and sustained NF-B induction. IB- levels, but not NF-B subunit p65 or IB- levels, were elevated compared with those for parental hepatoma cells. Interestingly, LPS-mediated activation of NF-B was found to be independent of degradation of IB- or IB-. Thus, these results suggest that loci responsible for maintaining hepatic gene expression also influence cellular responses to inflammatory agents.     

Molecular Mechanisms of Changes in TNF-α Production by Blood Mononuclears in Chronic Viral Hepatitis C

Chronic viral hepatitis C is associated with decreased production of TNF- by the peripheral blood mononuclear leukocytes irrespective of virus genotype and degree of the morphological activity of the process in the liver. This process positively correlates with the increase in the content of TNF- soluble receptor (molecular weight 55 kDa), which can play a role in the mechanisms of immunopathogenesis of long persistence of hepatitis C virus in the body.__________Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 139, No. 2, pp. 191–195, February, 2005     

Antitumor effects of liposomal IL1α and TNFα against the pulmonary metastases of the B16F10 murine melanoma in syngeneic mice

Interleukin 1 alpha (IL1) and tumor necrosis factor alpha (TNF) have been successfully incorporated into specific phosphatidylcholine (PC) and phosphatidylserine (PS) multilamellar vesicle (MLV) liposomes by modifying the concentration of calcium ion and pH of the encapsulation buffer. Under these conditions, some of the cytokines may attach to the exterior surface of the MLV and therefore be readily accessible to target cells for receptor binding and signal transduction. These cytokine-associated liposomes are stable for up to 2 weeks in serum-free buffer, and leakage of cytokines into medium containing 10% fetal bovine serum was about 50% at the end of a 3-day incubation period at 37°C. The biological activities mediated by liposomal IL1 and TNF were specific: the stimulation of thymidine uptake in T-helper D10 lymphocytes and the cytolysis of TNF-sensitive L929 target cells could be blocked by specific neutralizing antibodies in a dose-dependent fashion. When administered intravenously into C57BL/6 mice bearing the syngeneic B16F10 murine melanoma cells, dual entrapment of liposomal IL1 and TNF significantly reduced the number of metastatic tumor nodules in the lungs and prolonged the life span of the animals. Thus, liposomal IL1 and TNF displayed significant in vivo antitumor activity against the IL1- and TNF-resistant B16F10 metastatic murine melanoma.     

Expression of transforming growth factor-alpha and its receptor during human liver development and maturation

We investigated the expression of transforming growth factor-alpha (TGF-) and its receptor during human liver development and maturation, using immunohistochemistry. In the fetal liver, strong immunoreactivity for TGF- and its receptor was noted in intrahepatic bile duct cells of various developmental stages; moderate immunoreactivity for TGF- and mild immunoreactivity for TGF- receptor were found in immature hepatocytes. In the postnatal liver, reactivity for TGF- in hepatocytes decreased gradually and was negative or only weakly positive in the adult liver, while reactivity for TGF- receptor in hepatocytes increased gradually and was strongly positive in the adult liver. In contrast, immunoreactivity of TGF- and its receptor in intrahepatic bile duct cells persisted in the postnatal liver and was positive in the adult liver. These data suggest that the system of TGF- and its receptor has an important role in the proliferation and differentiation of intrahepatic biliary cells and hepatocytes in the fetal liver. The decreasing expression of TGF- in hepatocytes in the postnatal liver may indicate that proliferative activity of hepatocytes gradually decreases with liver maturation. The presence of TGF- and its receptor in intrahepatic bile ducts in the postnatal liver suggests that the system of TGF- and TGF- receptor is operative postnatally.     

Synchronous appearance of fibronectin,integrin α5β1, vinculin and actin in epithelial cells and fibroblasts during rat tracheal wound healing

The distribution of integrin 51 (51) and associated components during wound healing was investigated in the rat trachea following mechanical injury. Under anesthesia, the ventral surface of the trachea was scratched, and tissue specimens were obtained from 6 h to 3 weeks after injury and studied using light and electron microscopy and immunohistochemistry. 51, vinculin and actin in regenerating epithelial cells and extracellular fibronectin appear virtually simultaneously after injury (from 12 h to 7 days) as do 51, vinculin and -smooth muscle actin in fibroblasts and cellular fibronectin in granulation tissue (from 3 to 10 days). Immunoelectron microscopy 2 days after injury showed that 51 and vinculin were localized on the basal and lateral surfaces of regenerating epithelial cells and fibroblast surfaces, and fibronectin was localized just under the regenerating epithelial cells, around collagen fibrils and sporadically around fibroblasts. Bromodeoxyuridine labeling showed that the appearance of these components was associated with the period of cell proliferation. The appearances of fibronectin, 51, vinculin and actin in regenerating epithelial cells and fibroblasts during tracheal wound healing are well coordinated. During the initial cell migration phase, plasma fibronectin may stimulate cell migration before cellular fibronectin is produced in situ, and regenerating epithelial cells appear to begin to migrate into the wound before cell proliferation starts.     

Occult hepatosplenic T-γδ lymphoma

We report on a patient with a rare hepatosplenic T-cell lymphoma ( TCL) presenting clinically with B-symptoms, hepatosplenomegaly and pancytopenia. During the initial stage of the disease the sparse malignant cells could not be detected histologically. Furthermore, their identification was obscured by massive macrophage proliferation with haemophagocytosis in the spleen. Diagnosis was established by detection of a clonal T-cell receptor (TcR) rearrangement and, retrospectively, by demonstration of rare cells expressing an aberrant T-cell phenotype. The findings in this patient emphasize that minimal neoplastic T-cell infiltrates can lead to severe clinical symptoms. Initial biopsy findings may be misinterpreted as benign. TCL may elaborate lymphokines that suppress haematopoiesis, leading to pancytopenia and macrophage proliferation.     

Statin treatment and a disease-specific pattern of β-amyloid peptides in Alzheimer’s disease

According to the amyloid cascade hypothesis, sporadic Alzheimers disease (AD) is caused by the production and aggregation of -amyloid (A), and the production of A has recently been linked to the metabolism of cholesterol. We have previously published clinical studies where the effect of statin treatment on A production has been investigated. No effect on A was found, which is in disagreement with cell and animal studies. In the present study we investigated the effect of statin treatment on a disease-specific pattern consisting of a C-terminally-truncated quintet of A peptides. Nineteen patients with AD were treated with simvastatin for 12 months and the quintet of A peptides were analysed in cerebrospinal fluid before and after treatment. Also included was a group of 15 untreated patients with AD. We found that the A peptide pattern at baseline was in agreement with earlier findings; however, we did not find any change in the A peptide pattern after statin treatment. We suggest that clinical studies with extended treatment periods are performed where higher dosages of statins are used. We also believe that the pleiotropic effects of statins should be investigated further in order to elucidate the connection between Alzheimers disease and statin treatment.     

Temporal and mechanistic effects of heat shock on LPS-mediated degradation of IkappaBalpha in macrophages

Previous studies demonstrated important interactions between the heat shock response and the IB/NF-B pathway when these two pathways are induced sequentially. One such interaction involves the ability of heat shock to inhibit subsequent degradation of IB in response to a proinflammatory signal. Herein we investigated the temporal relationship between recovery from heat shock and inhibition of IB degradation, and the proximal mechanisms by which heat shock inhibits degradation of IB in macrophages. In RAW 264.7 murine macrophages, prior heat shock inhibited LPS-mediated IB degradation up to 4 h after recovery from heat shock, and this effect correlated with inhibition of LPS-mediated activation of NF-B. Beyond these recovery periods, heat shock did not inhibit IB degradation. IB kinase (IKK) assays demonstrated that heat shock inhibited LPS-mediated activation of IKK up to 1 h after recovery from heat shock. Heat shock also increased intracellular phosphatase activity, and inhibition of intracellular phosphatase activity partially reversed the ability of heat shock to inhibit both LPS-mediated degradation of IB and LPS-mediated activation of IKK. These data demonstrate that the ability of heat shock to inhibit degradation of IB is dependent on the recovery period between the heat shock stimulus and the proinflammatory stimulus. The mechanism by which heat shock inhibits degradation of IB involves dual modulation of IKK and intracellular phosphatase activity.     

Expression of estrogen receptors-α and -β in primary breast neoplasms and tumors exposed to neoadjuvant hormonal therapy

We studied the content and expression of mRNA for estrogen receptors receptors- and - in breast tumors before and after 3-month neoadjuvant hormone therapy with antiestrogen tamoxifen and/or aromatase inhibitors. Expression of estrogen receptors- and - was most often detected in ER⁺PR⁺ tumors and most significantly decreased in these neoplasms after exemestane therapy. Immunocytochemical and radioligand assays showed that tamoxifen and anastrozole have little effect on the number of estrogen receptors- The number of progesterone receptors in tumors decreased by the end of anastrozole therapy. Estrogen receptors- were immunocytochemically revealed in 50% primary breast tumors. Anastrozole slightly decreased, while tamoxifen increased the incidence of these receptors. Interruption of signaling through estrogen receptors and suppression of estrogen biosynthesis had different effects on the receptor status of neoplasms and distribution of estrogen receptors- and -.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 11, pp. 559–562, November, 2004     

Analysis of γδ+ T cells in peripheral blood of children with perinatal human immunodeficiency virus (HIV) infection

The present study examined CD8 antigen expression and variable (V) gene segment usage by T cell receptor (TCR)-⁺ lymphocytes in peripheral blood of symptomatic children with perinatal HIV infection. The relative number of ⁺, CD8⁺ T cells in most of the infected children was higher than that in uninfected children from HIV⁺ or HIV^– mothers and correlated with the immunodeficiency status of the patients. Infected infants and children over 1 year old also showed an increased proportion of V1-J1⁺ T lymphocytes. CD8 expression on those cells was higher in infected than in uninfected infants and children. Sequence analysis of the gene rearrangement of the predominant V1 family in peripheral blood of three HIV⁺ donors revealed extensive junctional diversity. These results suggest that the V skewing in the majority of HIV⁺ children reflects peripheral expansion of V1-J1⁺ T lymphocytes early in life, which might be involved in the mechanisms of HIV-induced immunodeficiency.