Circulating interleukin 10 and interleukin-6 serum levels in rheumatoid arthritis patients treated with methotrexate or gold salts: Preliminary report

In order to evaluate the relationship between serum concentrations of interleukin-10 (IL-10), IL-6, and acute phase proteins in rheumatoid arthritis (RA) patients treated with methotrexate (MTX) or intramuscular gold (IMG) we determined IL-10, IL-6, C-reactive protein (CRP), alpha-1-acid glycoprotein (AGP) and alpha-1-antichymotrypsin (ACT) in the sera of 35 RA patients. IL-10 and IL-6 levels were evaluated using an enzyme-linked immunoassay (ELISA). AGP and ACT level were measured using rocket immunoelectrophoresis. IL-10 serum level was not increased in RA patients as compared to controls (58.7 ± 18.1 pg/ml vs. 57.2 ± 11.9 pg/ml). IL-6 level was significantly elevated (91.6 ± 46.9 pg/ml vs. 45 ± 19 pg/ml, p < 0.05). CRP was significantly increased as compared to healthy controls (35 ± 19 mg/l vs. 3 ± 2 mg/l, p < 0.05). Patients treated with MTX or IMG presented an increased level of IL-10 and decreased amounts of IL-6, as compared to those treated with NSAID only. However, only changes between patients treated with IMG and NSAID were found to be statistically significant. A good negative correlation between IL-10 and IL-6 serum level was found (r = –0.75, p < 0.05). A positive significant correlation between IL-6 serum level and CRP (r = 0.62, p < 0.05), AGP (r = 0.78, p < 0.05) and ACT (r = 0.45, p < 0.05) was established. On the other hand, a negative correlation between IL-10 and serum level of CRP (r = –0.76, p < 0.05), AGP (r = –0.64, p < 0.05) and ACT (r = –0.38, p < 0.05) was also observed. Moreover, these relationships were maintained when patients treated with MTX, IMG, or NSAID were analyzed independently. According to the data thus far obtained, it seems that IL-10 decreases IL-6 production, and thereby indirectly affects the acute phase response, decreasing CRP, AGP, and ACT concentration in RA patients.Abbreviations ACT -1-antichymotrypsin - AGP ₁-acid glycoprotein - APP acute phase protein - CRP C-reactive protein - CSF colony stimulating factor - IFN interferon - IL interleukin - IMG intramuscular gold - MTX methotrexate - NSAID non-steroidal anti-inflammatory drug - RA rheumatoid arthritis     

BRCA2 germline mutations in male breast cancer patients in the Polish population

Breast cancer is a rare disease in men. Germ-line mutations in BRCA2 and androgen receptor (AR) genes are thought to be responsible for a proportion of male breast cancer cases. The present study was performed on a series of 37 consenting patients not selected for family history of breast/ovarian cancer. The entire coding region of the BRCA2 gene and two exons of the AR gene were analyzed for germ-line mutations to evaluate the association between BRCA2 and AR genes and male breast cancer in Poland. We identified four frameshift mutations (11%) in exons 10, 11, 17 and 18, two of them were novel: 6495del3insC and 8457insA. Three missense unclassified variants (8%) of the BRCA2 gene were also identified. The frequencies of missense alterations were examined in a set of 200 chromosomes. No alteration of the AR gene was found. We did not observe much difference in clinicopathological features between carriers and non-carriers of BRCA2 mutations. Five of 37 patients (14%) had a family history of breast cancer, in one first- or second-degree relative, among the latter was one mutation carrier. The results of this study suggest that germ-line BRCA2 mutations account for rather small proportion of male breast cancer in Poland.     

Interleukin 12-based immunotherapy improves the antitumor effectiveness of a low-dose 5-Aza-2'-deoxycitidine treatment in L1210 leukemia and B16F10 melanoma models in mice.

PURPOSE: Recent findings indicating that many genes related to cancer development are silenced by an aberrant DNA methylation suggest that inhibitors of this process may be effective cancer therapeutics. In this study we investigated the efficacy of low-dose 5-aza-2'-deoxycitydine (DAC), a methylation inhibitor, with interleukin (IL) 12, one of the most potent cytokines with antitumor activity. Experimental Design: Mice inoculated with L1210 leukemia cells or with B16F10 melanoma cells were treated with 7 daily injections of low-dose DAC (0.2 mg/kg) and/or 7 daily doses of IL-12 (100 ng/dose). Scid/scid mice as well as monoclonal antibodies against CD4, CD8, and NK1.1 were used to investigate the mechanisms of the antitumor effects of the combination treatment. The activity of murine lymphocytes was measured with enzyme-linked immunospot and (51)Cr release assays. RESULTS: Treatment with DAC or IL-12 given alone produced moderate antitumor effects. In both tumor models combined treatment resulted in potentiated antitumor effects and produced 70% long-term survivors among mice inoculated with L1210 cells. The antitumor efficacy of combined treatment was abrogated in scid/scid mice, and after depletion of CD4(+) and CD8(+) T cells. Mice inoculated with B16F10 melanoma cells had significantly delayed tumor growth after combined treatment with DAC and IL-12. Strong antitumor effect correlated with a significant activation of lymph node-derived CD8(+) and CD4(+) cells. Transient neutropenia was observed in mice under treatment of DAC alone, but remarkably this effect was not potentiated by IL-12. CONCLUSIONS: This study provides the first evidence that antitumor effects of DAC can be strongly potentiated by IL-12 and could be beneficial in an effective low-dose-based antitumor therapy.     

Simultaneous assessment of ecto- and cytosolic-5'-nucleotidase activities in brain micropunches

We propose a new methodology for simultaneous assessment of ecto- and cytosolic-5'-nucleotidase that can be utilized in brain to measure the activity of these enzymes in micropunches of tissues. It is based on the differential sensitivity of both enzymes to alpha,beta-methyleneadenosine 5'-diphosphate (AMP-CP) and the requirements for magnesium as a co-factor. The design of assay protocol contains an internal validation by allowing comparisons between total level of 5'-nucleotidase activity with that calculated from the sum of individual activities of the ecto- and cytosolic-5'-nucleotidases. We have applied this new approach to assess the activity of ecto- and cytosolic-5'-nucleotidase in the brain regions relevant to sleep regulation. The level of both enzymes was significantly lower in the cerebral cortex than other brain regions tested.     

Fetal membranes as a source of stem cells

In recent years, a constant growth of knowledge and clinical applications of stem cells have been observed. Mesenchymal stromal cells, also described as mesenchymal stem cells (MSCs) represent a particular cell type for research and therapy because of their ability to differentiate into mesodermal lineage cells. The most investigated source of MSCs is bone marrow (BM). Yet, collection of BM is an invasive procedure associated with significant discomfort to the patient. The procedure results in a relatively low number of these cells, which can decrease with donor's age. Therefore, it seems to be very important to find other sources of mesenchymal stem cells nowadays. A human placenta, which is routinely discarded postpartum, in spite of its natural aging process, is still a rich source of stem cells capable to proliferate and in vitro differentiate in many directions. Besides homing and differentiation in the area of injury, MSCs there elicit strong paracrine effects stimulating the processes of repair. In this review, we focus on the biology, characteristics and potential clinical applications of cells derived from human fetal membranes: amnion and chorion.     

The effect of intravenous cyclophosphamide pulse on peripheral blood lymphocytes in lupus erythematosus patients

In the present study we investigated the long-term effect of intravenous pulse cyclophosphamide (CY) on lymphocyte surface antigens in systemic lupus erythematosus (SLE) patients. Blood samples derived from 17 lupus erythematosus patients were analysed using two- and three-colour flow cytometry. During the CY therapy, the total number of T lymphocytes (CD3+) was reduced by 31.4%, B lymphocytes (CD19+) by 67.4% and NK cells (CD16+) by 27.4%. Six months after the end of the CY regimen, these values recovered to entry levels. At the onset of the study we observed increased percentages of CD3+ CD25+, CD3+ CD4– CD8–, CD4+ CD29+, CD19+ and CD19+ CD5+ cells. The CY treatment regimen decreased the CD3+ CD25+, CD3+ CD4– CD8–, CD19+ and CD19+ CD5+ cells, but increased the CD3+ CD8+ subpopulation. Taken together, a deficiency of CD8+ T cells associated with CD4+ CD29+ predominance may imply an immune regulatory imbalance leading to abnormal CD4+ cell activation and in consequence to autoimmunity. Depletion of CD19+ cells combined with an enlargement of CD8 cells as a result of CY therapy may reduce the enhanced immune response in SLE patients. Received: 13 December 1996 / Accepted: 10 March 1997     

On the source of Ca(2+) activating the tonic component of contraction of myocytes of guinea pig heart

OBJECTIVE: Contractions of isolated, single myocytes of guinea pig heart stimulated at 37 degrees C consist of a phasic component and a voltage dependent tonic component. In this study we investigated the source of Ca(2+) activating the tonic component. METHODS: Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped cells were stimulated by the pulses from the holding potential of -40 to +5 mV. [Ca(2+)](i) was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. RESULTS: Superfusion of 5 mmol/l Ni(2+) during 30 s pause did not inhibit subsequent biphasic Ca(2+) transients and contractions despite inhibition of Ca(2+) current and Na(+)/Ca(2+) exchange. KB-R7943 (5 micromol/l) or intracellular dialysis with 0 Na(+) solution, both of which inhibit reversed Na(+)/Ca(2+) exchange, decreased amplitude of Ca(2+) transients and contractions by approximately 40%. The ratio of amplitudes of tonic to phasic component was increased by Ni(2+) and was not changed by KB-R7943 or 0 Na(+)(i). Ryanodine (200 micromol/l) inhibited both components of contractions in cells superfused with Ni(2+). The phasic component but not the tonic component was inhibited by 20 micromol/l nifedipine in cells superfused with Ni(2+). CONCLUSIONS: Tonic component of contraction of single myocytes of guinea pig heart is not activated by Ca(2+) current or by the reverse mode Na(+)/Ca(2+) exchange as currently proposed in literature. Rather, it is activated by Ca(2+) released from the sarcoplasmic reticulum. However, kinetics and mechanism of release seem to be quite different from those of Ca(2+) fraction activating the phasic component of contraction.     

The Impact of Water and Road Salt with Anti-Caking Agent on the Stiffness of Select Mixes Used for the Road Surface

An original experimental method was used to investigate the influence of water and road salt with anti-caking agent on the material used in pavement construction layers. This method allowed for monitoring material changes resulting from the influence of water and road salt with anti-caking agent over time. The experiment used five different mineral road mixes, which were soaked separately in water and brine for two time intervals (2 days and 21 days). Then, each sample of the mix was subjected to tests of the complex module using the four-point bending (4PB-PR) method. The increase in mass of the soaked samples and the change in value of the stiffness modulus were analyzed. Exemplary tomographic (X-ray) imaging was performed to confirm the reaction of the road salt and anti-caking agent (lead agent) with the material. Based on measurements of the stiffness modulus and absorption, the correlations of the mass change and the value of the stiffness modulus were determined, which may be useful in estimating the sensitivity of mixes to the use of winter maintenance agents—e.g., road salt with anti-caking agent (sodium chloride). It was found that the greatest changes occur for mixes intended for base course layers (mineral cement mix with foamed asphalt (MCAS) and mineral-cement-emulsion mixes (MCE)) and that the smallest changes occur for mixes containing highly modified asphalt (HIMA).     

Serum IgA, acute phase proteins, and glycosylation of alpha 1-acid glycoprotein in ankylosing spondylitis.

Several investigators have suggested that gastrointestinal inflammation has a role in the pathogenesis of ankylosing spondylitis. To test this hypothesis markers of gastrointestinal immunostimulation, as manifested by serum IgA concentrations, were compared with serum markers of inflammation, as manifested by acute phase proteins. Serum samples from 45 unrelated Caucasian patients with ankylosing spondylitis (AS) were tested for correlation of serum IgA and six acute phase proteins: C reactive protein (CRP), alpha 1-antitrypsin, alpha 1-antichymotrypsin, caeruloplasmin, alpha 1-acid glycoprotein (AGP), and haptoglobin. Serum IgA was shown to be significantly positively correlated with four of these six acute phase proteins: CRP (r = 0.58, p less than 0.001), alpha 1-antitrypsin (r = 0.29, p less than 0.05), AGP (r = 0.61, p less than 0.01), and haptoglobin (r = 0.58, p less than 0.001), suggesting that gastrointestinal immunostimulation does have a role in the pathogenesis of inflammation in AS. In addition, the microheterogeneity of the pattern of glycosylation of AGP, expressed as reactivity coefficients, was examined. The AGP reactivity coefficient has been shown to increase in infection, remain the same in systemic lupus erythematosus, and decrease in rheumatoid arthritis. It was found that the AGP reactivity coefficient was significantly decreased in patients with AS as compared with healthy controls (p less than 0.006). As recent studies have indicated that patterns of glycosylation reflect intrahepatocellular biosynthetic processes induced by cytokines our data suggest that cytokine-hepatocellular mechanisms in AS may be similar to those occurring in rheumatoid arthritis, but different from those in systemic lupus erythematosus or infection.