Human skeletal muscle-derived stem/progenitor cells modified with connexin-43 prevent arrhythmia in rat post-infarction hearts and influence gene expression in the myocardium.

Stem cell therapy in combination with genetic modification (e.g., transfection with the coding sequence for the connexion 43 gene, GJA1) may solve the problems associated with the occurrence of additional (secondary) stimulation in the post-infarcted heart (arrhythmia). Human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) were transfected with the pCiNeo-GJA1 plasmid at an efficiency of approximately 96%. Gene overexpression was assessed using qPCR, and subsequent analysis revealed that GJA1 expression increased more than 40-fold in SkMDS/PCs transfected with the appropriate coding sequence (SkMDS/PCsCX43) compared to that of the 'native' SkMDS/PCs control (SkMDS/PCsWT). Enhanced (4-fold) protein expression of connexin-43 was also confirmed by Western immunoblotting. Furthermore, using the arrhythmic score, we demonstrated the positive effects of SkMDS/PCsCX43 cell intervention in reducing additional secondary stimulations in rat post-infarcted hearts compared with that of wild-type cell delivery. Selected gene responses (Kcnq1, Cacna1c, Ncx1, Serca2a, and Tgfb1) showed significantly altered expression profiles in the rat myocardium upon intervention with SkMDS/PCsCX43. The genetic modification of human skeletal muscle-derived stem/progenitor cells with connexin-43 prevented the pro-arrhythmic effects of myogenic implanted stem cells on the host myocardium and positively influenced myocardial gene expression profiles in respect to myocardium conductivity.

Total and high molecular weight adiponectin levels in the rat model of post-myocardial infarction heart failure.

Adiponectin is a protein secreted primarily by adipose tissue. It has been suggested that adiponectin plays a protective role in the early phase following myocardial infarction. Our primary aim was to investigate the effects of post-myocardial infarction heart failure well-characterized by left ventricular hemodynamic parameters on the total and high molecular weight adiponectin concentrations in plasma, fat and cardiac tissue. Eight weeks after myocardial infarction or sham operation, total and high molecular weight adiponectin concentrations in plasma, fat, and cardiac tissues were assayed in rats. In addition, hemodynamic parameters and expression of the genes encoding atrial natriuretic peptide and brain natriuretic peptide in left ventricle were evaluated. Atrial natriuretic peptide and brain natriuretic peptide mRNA levels in left ventricle tissue were higher in rats with myocardial infarction-induced heart failure compared with the controls. Similarly, total adiponectin concentration was increased in left ventricle (but not in right ventricle) in rats with post-myocardial infarction heart failure. In contrast, adiponectin levels in plasma and cardiac adipose tissue in rats with post-myocardial infarction heart failure were lower than in sham-operated animals. Furthermore, there were no significant differences in levels of high molecular weight adiponectin in plasma, cardiac tissue or adipose tissue between these two groups. We conclude that in the rat model of post-myocardial infarction heart failure, adiponectin level is increased in left ventricle tissue. This is accompanied by decreased adiponectin levels in plasma and cardiac adipose tissue.

The non-neuronal heart's acetylcholine in health and disease.

In this review we describe the history of almost one century lasting investigations which eventually provided evidence convincing that cardiac myocytes possess all elements of the system of synthesis, intracellular transport and release of acetylcholine (ACh) independent of parasympathetic cholinergic innervation. The myocytes synthesis and release of ACh is tightly connected with their contractile activity. Moreover, it is necessary for maintaining the balance of autonomic control of the heart, particularly important in the heart failure. It has an antyhypertrophic activity and protects myocardium against ischemic/reperfusion injury which shows that it is involved in the regulation of the important cellular signaling pathways. These properties of the non-neuronal ACh system of the heart also rise the hope that it might be used for the therapeutic measures.

The effect of sarcoplasmic reticulum Ca2+ leak on contractile activity of guinea pig heart myocytes depends in activity of sarcoplasmic reticulum Ca2+-ATPase and Na+/Ca2+ exchanger.

Decreased expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA), overexpression of Na(+)/Ca(2+) exchanger (NCX) and diastolic SR Ca(2+) leak from the ryanodine receptors (RyRs) are believed to contribute to the decrease of myocyte contraction in failing heart. In this work we induced Ca(2+) leak through RyRs in isolated myocytes of guinea pig hearts by 20 microM FK-506. The SR Ca(2+) leak resulted in (1) decreased amplitude of cell shortening and of Ca(2+) transients, (2) decreased rate of Ca(2+) transients decay (3) enhanced diastolic Ca(2+) loss. The effect of FK-506 on amplitude of cell shortening was reversed and that on diastolic Ca(2+) loss blocked by partial inhibition of NCX due to lowering Na(+) concentration in superfusion solution from 144 mM to 100 mM. The amplitude of cell shortening and Ca(2+) transients decreased by FK-506 was significantly increased by 10(-7) M thapsigargin. In conclusion, the effect of SR Ca(2+) leak induced by FK-506 on myocyte contraction is strictly dependent on activity of SERCA and NCX.

Temperature dependent contribution of Ca2+ transporters to relaxation in cardiac myocytes: important role of sarcolemmal Ca2+-ATPase.

UNLABELLED: Activities of Ca(2+) -ATPase of sarcoplasmic reticulum (SERCA) and Na(+)/Ca(2+) exchanger (NCX) involved in cellular Ca(2+) turnover greatly change in hypertrophied and failing hearts. Unfortunately, contribution of these proteins as well as of the sarcolemmal Ca(2+)-ATPase (PMCA) to cellular Ca(2+) turnover has been investigated almost exclusively at room temperature. PMCA is of particular interest since it may affect activity of calcineurin and nNOS. Therefore the objective of this study was to reinvestigate contribution of SERCA, NCX and PMCA to cell relaxation and the effect of PMCA on cell contraction at 37 degrees C. Myocytes isolated from the ventricles of guinea pig and rat hearts and incubated with Indo-1 were field stimulated at the rate of 60/min. Contribution of SERCA, NCX and PMCA was calculated from the rate constants of the decaying components of electrically stimulated Ca(2+) transients or of the transients initiated by caffeine dissolved in normal Tyrode or in 0Na, 0Ca Tyrode. Increase in temperature from 24 to 37 degrees C increased the relative contribution of NCX from 6.1% to 7.5% in rat and from 21.3 to 51.9% in guinea pig at the expense of SERCA. The contribution of the PMCA to relaxation in both species increased upon rise in temperature from 24% to 37 degrees C from negligible values to 3.7%. In both species amplitude of Ca(2+) transients was at 24 degrees C nearly twice as high as at 37 degrees C. It was nearly doubled by carboxyeosine (CE), a PMCA blocker at 37 degrees C but was hardly affected at 24 degrees C. The effects of CE were concentration-dependent and conformed with the degree of inhibition of activity of PMCA. CONCLUSIONS: PMCA plays an important role in regulation of myocardial contraction despite its small contribution to relaxation. In guinea pig but not in rat relative contribution of SERCA and NCX to relaxation is highly temperature dependent.

Voltage dependent activation of tonic contraction in cardiac myocytes.

UNLABELLED: Contractions of isolated single myocytes of guinea pig heart stimulated by rectangular depolarizing pulses consist of a phasic component and a voltage dependent tonic component. In this study we analyzed the mechanism of activation of the graded, sustained contractions elicited by slow ramp depolarization and their relation to the components of contractions elicited by rectangular depolarizing pulses. Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses from the holding potential of -40 to +5 mV or by ramp depolarization shifting voltage within this range within 6 s. [Ca2+]i was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. Myocytes responded to the ramp depolarization between -25 and -6 mV by the slow, sustained increase in [Ca2+]i and shortening, the maximal amplitude of which was in each cell similar to that of the tonic component of Ca2+ transient and contraction. The contractile responses to ramp depolarization were blocked by 200 microM ryanodine and Ca2+-free solution, but were not blocked by 20 microM nifedipine or 100-200 microM Cd2+ and potentiated by 5 mM Ni2+. The responses to ramp depolarization were with this respect similar to the tonic but not to the phasic component of contraction: both components were blocked by 200 microM ryanodine, and were not blocked by Cd2+ or Ni2+ despite complete inhibition of the phasic Ca2+ current. However, the phasic component but not the tonic component of contraction in cells superfused with Ni2+ was inhibited by nifedipine. Both components of contraction were inhibited by Ca2+-free solution superfused 15 s prior to stimulation. CONCLUSIONS: In myocytes of guinea pig heart the contractile response to ramp depolarization is equivalent to the tonic component of contraction. It is activated by Ca2+ released from the sarcoplasmic reticulum by the ryanodine receptors. Their activation and inactivation is voltage dependent and it does not depend on the Ca2+ influx by the Ca2+ channels or reverse mode Na+/Ca2+ exchange, however, it may depend on Ca2+ influx by some other, not yet defined route.

Agonist of dihydropyridine receptors, BayK8644 depresses excitation-contraction coupling in myocytes of guinea pig heart.

BayK8644(-)(BayK), an agonist of L-type Ca2+ channels has been recently shown to impair excitation-contraction coupling in cardiac myocytes by increasing Ca2+ leak from the sarcoplasmic reticulum (SR) and by decreasing the gain factor of calcium induced release of calcium. It has been proposed that BayK affects the properties of ryanodine receptors (RyRs) of SR by binding to the sarcolemmal dihydropyridine receptors (DHPRs). This would suggest that the linkage between these receptors is more direct than currently sought. However, it has been recently found that BayK may also directly affect the RyRs increasing their open probability. In this paper we tested the effect of BayK on excitation-contraction coupling in single ventricular myocytes of guinea-pig heart superfused with 5 mM Ni2+ which blocks the L-type Ca2+ current and Na+/Ca2+ exchange. We have previously shown that it is possible to activate in these cells nearly normal Ca2+ transients and contractions despite total inhibition of ICa. This eliminated the effect of ICa increased by BayK on excitation contraction coupling thus simplifying the studied system. 0.5 microM BayK increased the diastolic [Ca2+]i and decreased the diastolic length in stimulated or rested cells superfused with Ni2+, decreased by approximately 50% amplitude of Ca2+ transients and contractions and decreased by approximately 70% the responses of cells to rapid superfusion of 15mM caffeine used as an indirect index of the SR Ca2+ content. The effects on diastolic length and [Ca2+]i in rested cells were not affected by 20 microM nifedipine. We conclude that under our experimental conditions the dominating mechanism of suppression of excitation-contraction coupling by BayK was depletion of the SR Ca2+ by the direct effect on the RyRs.

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.

Dihydropyridine receptors functioning as voltage sensors in cardiac myocytes.

Isolated, superfused with [Ca2+]0 = 2.5-3 mM (T = 37 degrees C) and voltage clamped ventricular myocytes of guinea-pig hearts were stimulated by pulses from a holding potential -40 mV to +5 mV (duration 300 ms). They activated L-type Ca2+ current and a biphasic contractile response: a phasic component of amplitude approximately 7% of resting cell length (duration approximately 150 ms) and a tonic component of amplitude approximately 3% of resting cell length. The phasic component was inhibited by 10(-6) M thapsigargin (Tg). Pulses from -40 mV to +5 mV stimulated a similar bi-phasic contractile response in 74% of cells (n = 126) superfused from the beginning of a 30 s period of rest with 5-10 mM Ni2+ which blocked the Ca2+ current and Na+ -Ca2+ exchanger (Ni2+-contractions). Thus, the Ni2+-contractions could be activated only by intracellular Ca2+ release. The phasic component of control contractions showed the bell-shaped voltage relation at [Ca]0 = 2 mM and sigmoid relation at [Ca]0 = 3 mM. The phasic component o Ni2+-contractions showed a sigmoid relation at voltages from -40mV to +100 mV and could not be activated at [Ca]0 = 2 mM. It was inhibited by 20 microM nifedipine, a blocker of dihydropyridine receptors, even when activated by the pulses to +70 mV, during which the Ca2+ current does not flow. We proved that nifedipine does not affect Na+-Ca2+ exchange. The phasic component of Ni2+-contractions was also inhibited by 2 nM indolizinesulphone SR33557, another dihydropyridine receptor blocker, which halved the phasic component of contractions in sontrol cells without any significant effect on the Ca2+ current. Stimulation did not activate contraction in any of 19 cells in which 20 microM nifedipine was superfused from the beginning of 30 s rest instead of 5 mM Ni2+. These cells were depolarized to +5 mV over the rest period in order to prevent intracellular Ca2+ loss by Na+ -Ca2+ exchange. Residual Ca2+ currents were much stronger in cells superfused with nifedipine than residual currents in cells superfused with Ni2+ (hardly visible in the records). Our results suggest that a vestigial remnant of a voltage-sensing mechanism similar to that in the skeletal muscle may trigger the Ca2+ release from the SR of cardiac myocytes under specific experimental conditions. In normal cells it may be complementary to calcium induced calcium release (CICR).

Properties of ventricular myocytes isolated from the hypertrophied and failing hearts of spontaneously hypertensive rats.

OBJECTIVE: To investigate how the morphological and physiological properties of single myocytes isolated from the hypertrophied, failing left ventricles (LV) differ from those of normal or hypertrophied not failing ventricles. METHOD: Single myocytes were isolated separately from right (RV) and left ventricles (LV) of male spontaneously hypertensive rats (SHR) or Wistar-Kyoto (WKY) rats at the age of 6 and 12 months and of SHRs which developed or not developed heart failure at the age of 20-24 months. We measured cells dimensions, range and kinetics of electrically stimulated or initiated by caffeine contractions and Ca2+ transients, and investigated the response of cells to thapsigargin. RESULTS: The transversal dimensions of the LV myocytes of 6 months old SHRs showed approximately 20% increase with respect to transversal dimensions of their RV myocytes and LV and RV myocytes of WKY rats. The difference did not change with progressing age and in the heart failure. The LV myocytes of 6 or 12 months old SHRs showed slowed kinetics of the Ca2+ transients and of contraction and relaxation and decreased contractile response to 2 s superfusion with 15 mM caffeine preceded by 5 mM Ni2+ used as an index of the sarcoplasmic reticulum (SR) Ca2+ content. Despite of this the range of shortening and relative contribution of the SR to contraction (assessed by measuring of the residual contractile response to electrical stimulation in cells poisoned with thapsigargin) or relaxation (assessed by calculation of the ratio of rate constants of the electrically stimulated and stimulated by 30 s superfusion with caffeine Ca2+ transients) was not altered in the hypertrophied myocytes. Properties of the LV myocytes of the 20-24 old SHRs with or without heart failure did not differ from those of LV myocytes of younger SHRs. The contractile response to caffeine of their RV myocytes dropped to the level of that in the LV myocytes. CONCLUSION: Our results suggest that transition from the compensated hypertrophy to the heart failure in 20-24 months old SHRs did not result from the further changes in properties of the surviving myocytes. Data from literature suggest that myocyte apoptosis and remodeling of the extramyocyte space is the more likely reason.

Effect of thyroid hormone on the myosin heavy chain isoforms in slow and fast muscles of the rat.

The myosin heavy chain (MHC) was studied by biochemical methods in the slow-twitch (soleus) and two fast-twitch leg muscles of the triiodothyronine treated (hyperthyroid), thyroidectomized (hypothyroid) and euthyroid (control) rats. The changes in the contents of individual MHC isoforms(MHC-1, MHC-2A, MHC-2B and MHC-2X) were evaluated in relation to the muscle mass and the total MHC content. The MHC-1 content decreased in hyperthyreosis, while it increased in hypothyreosis in the soleus and in the fast muscles. The MHC-2A content increased in hyperthyreosis and it decreased in hypothyreosis in the soleus muscle. In the fast muscles hyperthyreosis did not affect the MHC-2A content, whereas hypothyreosis caused an increase in this MHC isoform content. The MHC-2X, present only in traces or undetected in the control soleus muscle, was synthesised in considerable amount in hyperthyreosis; in hypothyreosis the MHC-2X was not detected in the soleus. In the fast muscles the content of MHC-2X was not affected by any changes in the thyroid hormone level. The MHC-2B seemed to be not influenced by hyperthyreosis in the fast muscles, whereas the hypothyreosis caused a decrease of its content. In the soleus muscle the MHC-2B was not detected in any groups of rats. The results suggest that the amount of each of the four MHC isoforms expressed in the mature rat leg muscles is influenced by the thyroid hormone in a different way. The MHC-2A and the MHC-2X are differently regulated in the soleus and in the fast muscles; thyroid hormone seems to be necessary for expression of those isoforms in the soleus muscle.

Effects of mibefradil, a blocker of T-type Ca2+ channels, in single myocytes and intact muscle of guinea-pig heart.

We investigated the effects of a relatively selective blocker of the T-type Ca2+ channels, mibefradil (MBF), in the isovolumic left ventricles of the isolated, perfused hearts of guinea-pigs and single myocytes isolated from the ventricles of this species. In the myocytes superfused with 0 Na+ solution containing 200 microM lidocaine and pulsed from -90 mV to -40 mV to +5 mV, MBF proved to be about 3 times more potent inhibitor of the T-type than of the L-type Ca2+ current. The effect on the L-type current was strongly voltage and use dependent. In the ventricles and in the myocytes contraction was reduced by 50% by about 1 microM MBF, the concentration 12 times higher than this increasing the coronary flow by 50%. In myocytes the decrease in unloaded shortening paralleled inhibition of the T-type rather, than of the L-type Ca2+ current. Inhibition of electrically stimulated contraction of the myocytes was three times stronger than inhibition of the caffeine contractures regarded as an index of sarcoplasmic reticulum (SR) Ca2+ content. These findings are consistent with the hypothesis that the T-type Ca2+ channels may contribute to release of Ca2+ from the SR. It is concluded that MBF has a definite negative inotropic effect in the ventricular myocardium of guinea-pig heart at the concentrations found in the blood of the patients submitted to the clinical trials.

The differences in the expression of CD45 isoforms on peripheral blood lymphocytes derived from patients with seasonal or perennial atopic allergy.

In order to determine the role of memory/naive T cells in atopic allergy patients we analyzed peripheral blood mononuclear cells before and during the grass pollen season. The study comprised 28 patients with seasonal symptoms of atopic allergy and 18 with perennial symptoms. Flow cytometry was employed to detect the expression of CD3, CD4, CD4CD45RA, CD4CD45RO, CD8, CD16, and CD19 molecules on peripheral blood lymphocytes. Allergic patients showed a decreased proportion of memory (CD4+CD45RO+) T cells compared with healthy subject (p < 0.05). The proportion of naive (CD4+CD45RA+) helper T cells did not differ between allergic patients and controls. The percentage of CD4+CD45RO+ cells increased during natural antigen exposure (grass pollen season) in allergic patients with seasonal symptoms. The results show at least two important observations. A potential homing tendency to nasal, bronchial and conjunctival mucosa of memory T cells (CD45RO) in atopic allergy patients may explain their deficiency in peripheral blood. Secondly, the grass pollen season may switch their phenotype from naive into memory T cells causing the increase of CD45RO cells. These events do not occur in non-allergic individuals and may thus constitute new insight into the basic mechanism of atopic allergy.

Changes in agalactosyl IgG levels correlate with radiological progression in early rheumatoid arthritis.

Immunoglobulin G-lacking galactose (Gal[0]) appears to be helpful in differential diagnosis of early synovitis, and correlates with disease activity in rheumatoid arthritis (RA). Its utility for RA monitoring and prognosis has been evaluated in the present study. Forty-eight patients with early RA were observed for 3 years. Hand radiographs were assessed according to Larsen and results were expressed as damage score (DS) and progression of damage score (PDS). Gal[0], DS, and PDS were evaluated at the onset of the study and after 1 and 3 years. The average values of Gal[0] in RA patients at the onset of the observation were significantly higher as compared to healthy controls (0.43 +/- 0.22 vs. -0.03 +/- 0.09, P < 0.05). The findings of Gal[0] after a 3-year follow-up were also higher as compared to healthy controls (0.37 +/- 0.21 vs. -0.03 +/- 0.09, P < 0.05). Radiological progression (PDS > 15) was observed in 16 patients. This group was characterized by a constantly high level of Gal[0]. The level of Gal[0] in patients without or with moderate radiological progression (PDS < 15) was significantly lower at the onset of the study and remained low during the observation. The relationship between Gal[0] and radiological progression was shown. The data thus far obtained suggest that Gal[0] may serve as an indicator for the disease course in patients with RA. Secondly, we cannot exclude the possibility that the constantly elevated level of Gal[0] causes erosions.

Acute phase response in rheumatoid arthritis patients treated with immunosuppressive drugs.

We sought to investigate an influence of immunosuppressive drugs on acute phase response (APR) in rheumatoid arthritis (RA). Ninety-six patients (pts) were treated with methotrexate (MTX), or with cyclophosphamide (CTX) (9-intravenously, 19-orally), or with cyclosporin A (CSA). C-reactive protein (CRP), alpha-1-acid glycoprotein (AGP), and alpha-1 antichymotrypsin (ACT) serum levels were measured by rocket immunoelectrophoresis. AGP and ACT microheterogenities evaluated using immunoelectrophoresis were expressed as reactivity coefficient (RC). Clinical improvement was observed in 71.4% MTX pts, 77.8% CTX intravenously pts, 36.8% CTX orally pts, 60.0% CSA pts. The number of side effects was the highest in CTX oral group (57.9% left the study). CRP, AGP, and ACT serum levels were increased in all groups of RA pts as compared to healthy controls. CRP level decreased only after MTX and CTX intravenous treatment. Moreover, a decrease in ACT was observed in CTX intravenously treated pts. AGP-RC was lower in the initial population of RA pts as compared to healthy control. After 6 months of treatment RC became significantly higher in MTX pts only. In opposite ACT-RC in RA pts was found to be elevated as compared to controls. After the treatment it fell down. The decrease was found to be significant only in pts treated with MTX. From our study we can conclude that MTX is the safest and the most effective agent among immunosuppressive drugs applied in RA. CTX given orally causes a number of adverse reactions, which frequently make continuous and effective treatment impossible. CTX intravenously and CSA are attractive in the treatment of the patients with severe and refractory RA. A lack of clinical benefit is reflected in the absence of acute markers changes.

Do changes in clinical improvement in rheumatoid arthritis patients treated with immunosuppressive drugs reflect the changes in acute phase proteins?

We sought to investigate whether clinical improvement after immunosuppressive treatment reflects changes in acute phase response (APR) in rheumatoid arthritis (RA). Fifty-eight patients (pts) were treated with methotrexate (MTX), nineteen with intravenous cyclophosphamide (CTX), and fifteen with cyclosporin A (CSA). C-reactive protein (CRP), alpha-1-acid glycoprotein (AGP), alpha-1 antichymotrypsin (ACT), and alpha-1 antitrypsin (AT) serum levels were measured by nephelometry or rocket immunoelectrophoresis. Clinical improvement was observed in 67% MTX pts, 53% CTX pts, and 47% CSA pts. Baseline serum levels of CRP, AGP, ACT, and AT were significantly higher as compared to healthy controls. After MTX and CTX therapy CRP level significantly decreased. The decrease in serum level of ACT and AT in CTX treated patients was also observed. All analyzed acute phase proteins remained substantially elevated after CSA therapy despite a clear reduction in disease activity. We established a correlation between changes in disease activity and all acute phase proteins (APP) in MTX and CTX pts. From our study we can conclude that clinical improvement after immunosuppressive treatment correlated with quantitative changes in all APR markers in MTX and CTX treated pts, and none in CSA pts. Although measurement of APP remains the best marker for monitoring RA pts, not always they properly reflect changes in disease activity.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Does IgA-alpha1 antitrypsin complex correlate with IgM, IgG and IgA rheumatoid factor in early rheumatoid arthritis?

This study was undertaken to determine whether the rheumatoid factor (RF) isotypes affect the level of IgA-AT complex in early rheumatoid arthritis. IgA-AT complex and IgM, IgG, and IgA RF were evaluated using a double antibody enzyme-linked immunosorbent assay (ELISA) in sera of 83 (61 women and 22 men) patients. The mean level of the complex was higher in RA patients as compared with the control group (0.60 +/- 0.41 U vs 0.19 +/- 0.11 U, p < 0.05). The IgA-AT complex values exceeding the norm were found in 19 of 83 RA cases (23%), mainly in patients non-treated with DMARDs. IgM RF values judged as positive were present in 53 (64%), IgG RF in 55 (66%), and IgA RF in 56 (67%) patients. Higher RF values of IgM and IgG, but not those of IgA class, were found more frequently in patients with elevated IgA-AT levels. A good correlation was established between IgA and IgM RF (r = 0.61, p < 0.01), but not between IgA and IgG, or IgM and IgG RF in the group of patients under study. However, we did not find any significant correlation between isotypes of RF and parameters of the disease activity.

Immunological studies in patients with rheumatoid arthritis treated with methotrexate or cyclophosphamide.

One-hundred-and-two-patients (pts) with rheumatoid arthritis (RA) were observed for 12 months. Forty-eight pts were treated with a weekly low-dose of methotrexate (MTX), 23 pts with cyclophosphamide (CTX) (eight pts with one single intravenous dose, and 15 pts orally with a single daily dose), and 31 pts with nonsteroidal antiinflammatory drugs (NSAID) only. In all individuals acute phase response, i.e., C-reactive protein (CRP) and alpha-1-acid glycoprotein (AGP) serum levels, and AGP microheterogeneity using affinoimmunoelectrophoresis with concanavalin A were evaluated. The phenotype of lymphocytes isolated from peripheral blood was characterized using immunofluorescence technique. Following treatment the increased level of CRP significantly decreased whereas AGP serum level remained unchanged. Among the patients, microheterogeneity of AGP expressed as reactivity coefficient (RC) was lower before treatment when compared with 17 controls (0.95 +/- 0.23 vs. 1.35 +/- 0.15, p < 0.01). After 12 months of MTX therapy AGP-RC rose significantly (1.19 +/- 0.13, p < 0.01). No changes were observed in AGP-RC levels in CTX and NSAID treated individuals. No significant differences were observed in the percentage of CD3+, CD4+, and CD8+ cells in all the patient groups, except in CTX intravenously treated patients. In this group of patients a decrease of CD4+ cells was noticed (60.1 +/- 11.5% and 43.8 +/- 12.5% before and after treatment respectively -p < 0.01). The percentage of CD19 positive cells decreased significantly during 12 months of treatment with MTX and CTX. The percentage of activated T cells (CD25+ cells and HLA-DR+ cells) remained unchanged in MTX treated patients and was reduced in both CTX groups.(ABSTRACT TRUNCATED AT 250 WORDS)