Comparative study of phagocytosis and intracellular bactericidal activity of human monocytes and polymorphonuclear cells

Simultaneous assessment of the total number of bacteria (TNB) ingested, phagocytosis (Ph), phagocytic index (PI), and intracellular bactericidal activity (ICBA) of human monocytes was done by applying the fluorochrome acridine orange technique. Living bacteria stained orthochromatically green, whereas the dead ones were metachromatically red. The stain of extracellular bacteria was completely quenched by crystal violet counterstain. Using the Hypaque-Ficoll separation method combined with glass adherence, the yield of monocytes was 84±11%, the purity 90±8%, and the viability 99±1%. After 60 min of incubation of monocytes withStaphylococcus aureus, phagocytosis was 94±4%, PI 10.0±0.5, ICBA 76 ±5%, and TNB ingested 946±67/100 cells.E. coli B₄ was equally ingested by PMNs and monocytes and killed intracellularly more efficiently by the latter type of cells. Over the ratios of bacteria to cells of 51 to 201, phagocytic activity of monocytes was equal or superior to that of PMNs. Phagocytic and bactericidal activities were enhanced by AB serum, more by the fresh one than by inactivated. Phagocytic activity of monocytes was markedly influenced by temperature of incubation. Room temperature (24°C) significantly suppressed phagocytosis. Contrary to the previous beliefs no significant quantitative differences were found between phagocytic and bactericidal functions of monocytes as compared to polymorphonuclear phagocytes. The acridine orange-crystal violet method is simple, reliable, reproducible, and can be used for assessment of functional capacity of human phagocytes.Supported in part by grant-in-aid of the Medical Research Council of Canada.     

Role of K+ channels in L-6 myoblast migration

Migration of myoblasts is an important component of the reparative response to muscle injury, and furthermore may be a key determinant of the success of myoblast transplantation for the treatment of genetic muscle diseases. The present study examined the hypothesis that K⁺ channels modulate myoblast migration. The migration of cultured L-6 myoblasts was assessed in vitro on confluent cultures with the razor wound method, in the absence and presence of the following agents: 3,4-diaminopyridine and tetraethylammonium (which block several types of K⁺ channels), apamin and charybdotoxin (which block Ca⁺⁺-activated K⁺ channels), glibenclamide (which blocks ATP-sensitive K⁺ channels), and -, -, -, and -dendrotoxin (which block voltage-gated K⁺ channels). Migration was assessed with respect to number of migrated cells, average distance migrated, and total distance migrated. Overall, myoblast migration was stimulated in response to low concentrations of tetraethylammonium, apamin, glibenclamide, and -, - and -dendrotoxin. With these agents, the number of migrated cells increased by 28–47%, the average distance migrated increased by 22–35%, and the total distance migrated increased by 60–85%. Conversely, migration was inhibited by high concentrations of 3,4-diaminopyridine, tetraethylammonium, and all dendrotoxins. These data indicate that in L-6 myoblasts migration is regulated by K⁺ channels, and that several types of K⁺ channels appear to participate in cell migration.     

β2-microglobulin and other proteins in serum and urine during preeclampsia

Summary Serum and urine samples of 5 patients with preeclampsia, an equal number with preeclampsia superimposed upon chronic pyelonephritis, and 20 normal pregnant women were analysed for fibrin split products (immunoelectrophoresis) and other proteins (Oudin-method) including ₂-microglobulin (radioimmunoassay).No fibrin split products could be detected in normal pregnant women or those with preeclampsia superimposed upon chronic pyelonephritis. Distinctly abnormal values were found, however, in the patients with preeclampsia (split D, 27.2±5.1 mg% (S.D.) in serum and 162±55mg/24 h (S.D.) in urine; split E, 0.3±0.1 mg% (S.D.) in serum and 4.2±3.1 mg%/24 h (S.D.) in the urine; fibrinogen in serum 532±146 mg% and in urine 340±78 mg/24 h (S.D.).Mean total protein excretion of patients with preeclampsia (1951±322 mg S.D./24 h) was not different from the value of patients with preeclampsia superimposed upon chronic pyelonephritis (1781±289 mg S.D./24 h).Urinary ₂-microglobulin excretion of patients with simple preeclampsia (glomerular filtration rate 100 ml/min) was 4 to 5-fold increased at term but more than 100-fold in patients whose preeclampsia was superimposed upon chronic pyelonephritis (glomerular filtration rate 30–70 ml/min).The transient urinary excretion of fibrin split products and other proteins in patients with preeclampsia and normal glomerular filtration rate is an indication of a reversible glomerular lesion, whereas the increased ₂-microglobulin excretion in this group of patients is due to a tubular lesion. In patients with preeclampsia superimposed upon chronic pyelonephritis the excretion of ₂-microglobulin is further increased which may be explained by an additional lesion of the already impaired tubular function during delivery.In serum, prealbumin was decreased to about 55% and albumin to 60% in the patients with preeclampsia and preeclampsia superimposed upon chronic pyelonephritis which cannot be explained by renal loss alone but is very likely due to an inhibition of protein synthesis in the liver cell.Contains parts of the Doctoral Thesis of D. Prüfer     

Beta-glucuronidase release from leukocytes in children

Summary The release of -glucuronidase from polymorphonuclear leukocytes (PMNs) is important in the killing of bacteria and in producing tissue damage in acute inflammation. To investigate the effects of various diseases or drugs on degranulation, we studied the kinetics of -glucuronidase release from PMNs exposed to opsonized zymosan. PMNs of children with bacterial infections demonstrated increased degranulation. Within 5, 15, and 30 min the PMNs released 19±3%, 23±3%, and 26±3% of total -glucuronidase compared to 12±2%, 15±2%, and 16±2% of total -glucuronidase of control PMNs. Viral infections induced a significant delay of -glucuronidase release from PMNs. Maintenance therapy of acute lymphoblastic leukemia with 6-mercaptopurine and methotrexate, as well as administration of vincristine, diminished the degranulation. After 5, 15, and 30 min the PMNs released 8±1%, 10±1%, and 11±1%, as well as 6±3%, 8±2%, and 9±2% of total -glucuronidase. This study demonstrated that bacterial infections stimulate -glucuronidase release by PMNs. In contrast, cytostatic drugs inhibit lysosomal enzyme release, increasing the susceptibility to bacterial infections. The total enzyme activities were unchanged.Abbreviations c-AMP cyclic Adenosinemonophosphate - c-GMP cyclic Guanosinemonophosphate - PMNs polymorphonuclear Leukocytes Supported by DFG Ri 275/6-1Dedicated to Prof. Dr. E. Gladtke on the occasion of his 60^th birthday     

Role of elevated monocyte transforming growth factorβ (TGFβ) production in posttrauma immunosuppression

We previously reported that increased production of prostaglandin E₂ by monocytes is a pivotal mechanism in posttrauma immunopathology. Here we characterize monocyte levels of transforming growth factor and examine the effects of elevated transforming growth factor on prostaglandin E₂ release by patients' monocytes. Trauma patients' and normals' monocyte supernates (± stimulation with muramyl dipeptide) were acid treated and assayed for transforming growth factor using the mink lung-cell bioassay. Alternatively, human transforming growth factor was added to patients' and normals' monocytes and prostaglandin E₂ production assayed. Significantly elevated transforming growth factor levels (median=181.7 pmol/10⁶ monocytes) were detected in immunosuppressed patients' monocytes but not immuno-competent trauma patients' (median=32.0 pM) or normals' (median=20.4 pM) monocytes. Adding transforming growth factor to monocytes resulted in a significant elevation of prostaglandin E₂ levels. Elevated monocyte transforming growth factor levels in trauma patients could be both suppressing T-lymphocyte functions and maintaining elevated monocyte prostaglandin E₂ synthesis.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Candida albicans induces the release of inflammatory mediators from human peripheral blood monocytes

Candida albicans (C. albicans) is a major nosocomial pathogen. We examined arachidonic acid (AA) and cytokine production by monocytes stimulated with C. albicans. [¹⁴C]-AA labeled monocytes released 8.9 ±2.3% of the incorporated AA following stimulation with live C. albicans (C. albicans: monocyte of 161) (P=0.0002). Prior studies indicate that soluble-mannans and-glucans antagonize mannose and-glucan receptors, respectively. Preincubation of monocytes with-mannan (100g/ml) caused 45.8 ±5.7% inhibition of [¹⁴C]-AA release, whereas-glucan (100g/ml) yielded 43.7 ±6.0% inhibition (P<0.05 for each compared to control). Additionally, monocytes stimulated with C. albicans also released interleukin-1 (IL-1), tumor necrosis factor- (TNF), interleukin-6 (IL-6) and interleukin-8 (IL-8). However, a-mannan or-glucan failed to inhibit IL-1 release. These data indicate that C. albicans induces monocytes to release AA and inflammatory cytokines. Furthermore, AA, but not cytokine liberation, is partially mediated by a-mannan and-glucan components of the fungus.     

Naloxone in treatment of circulatory shock resistant to conventional therapy

Summary The effect of naloxone (4.4–5.9 mg i.v.) was evaluated in 10 patients with circulatory shock (sepsis,n=7; intoxication,n=1; cardiogenic shock,n=2) not responding to full conventional therapy. In addition, we measured plasma ACTH and immunoreactive -endorphin before and 60 min after administration of naloxone and compared the results with hormone concentrations in 10 intensive care patients without shock. Only in two patient with septic shock a transient increase (duration 15 min and 60 min, respectively) of systolic blood pressure was observed, while naloxone was ineffective in the remaining eight patients. No adverse effects of naloxone were found. Plasma ACTH and immunoreactive -endorphin concentrations in patients with shock were not different from those in controls (ACTH, 79±28 vs 120±60 pg/ml; immunoreactive -endorphin, 952±262 vs 1,070±378 pg/ml).Our findings suggest that naloxone in a single dose of 4.4–5.9 mg i.v. does not improve the management of circulatory shock unresponsive to conventional treatment. -endorphin seems to play no major role in the hypotension of shock.Abbreviations ACTH Adrenocorticotrophic hormone - HD intermittent hemodialysis - HF heart rate - ir immunoreactive - RR_syst systolic blood pressure Supported by Landesamt für Forschung, NRW     

Elevation of Transforming Growth Factor-β1 Is Associated with Recurrent Miscarriage

To investigate the significance of transforming growth factor-1 (TGF-1) in reproduction we have compared plasma levels in normal pregnant women and patients suffering miscarriages. We examined 188 normal pregnant women and 12 pregnant women with miscarriages. Eight women with severe recurrent miscarriages (mean ± SD of previous number of miscarriages; 10.4 ± 2.4 times) were also examined before conception; 34 nonpregnant women served as controls. Plasma TGF-1 level increased with the gestational week and returned within the normal range 1 month after delivery. The levels among pregnant women with miscarriages (mean ± SD; 2.44 ± 0.83 ng/ml) were significantly higher than those of pregnant controls (1.74 ± 0.95 ng/ml) of matched gestational weeks; levels among nonpregnant women with severe recurrent miscarriages were extremely elevated (4.1 ± 3.04 ng/ml) compared to the control value (1.34 ± 0.59 ng/ml). These data suggest that TGF-1 may be necessary to maintain pregnancy but also may be a risk factor for recurrent miscarriages.     

Cathepsin D activity in human peripheral blood mononuclear leukocytes

To investigate the cellular origins of cathepsin D (CD) in inflammatory lesions, the CD content of lymphocyte subsets, monocytes, and macrophages were compared. Human monocytes, B lymphocytes, CD4+ T lymphocytes, and CDS + T lymphocytes were separated from peripheral blood of normal donors. CD content was 0.13±01g equivalents of CD per million cells and significant differences between different cell types were not found. To determine the CD content of macrophages, differentiation of peripheral blood monocytes was induced by either in vitro culture or treatment with 4-phorbol-12-myristate-13-acetate (PMA). Macrophages induced by five-day culture contained four times more CD than unstimulated monocytes, and macrophages induced by 18-h treatment with 20 mg/ml 4-PMA contained nine times more CD than monocytes treated with 4-PMA, an inactive stereoisomer of 4-PMA. These results suggest that macrophages are one of the enriched sources of CD in inflammatory lesions.     

Clotrimazole is an Inhibitor of Inflammatory Angiogenesis and the Metabolic Activity in Sponge Granuloma

Clotrimazole (CLT), clinically used as an antifungal drug, inhibited sponge-induced angiogenesis and granulation tissue metabolic activity when administered systemically (120 mg/kg) in rats. We have used functional, biochemical and histological parameters to assess neovascularization and fibrovascular tissue infiltration of the rat sponge granuloma. The sequential development of local blood flow as determined by the outflow rate of sodium fluorescein applied intraimplant, showed that the t 1/2 values for the fluorescence peak in the bloodstream decreased in the control group from an initial value of 11 ± 0.87 min (avascular implants, day 1) to 7.6 ±1.5 min at day 7 postimplantation. By contrast t 1/2 values in the CLT-treated group remained stable during the 7-day period. The hemoglobin content extracted from the control implants was 2.7 ± 0.14 gHb/w.w vs. 1.8 ± 0.18 gHb/w.w in the treated group. The functional and biochemical parameters correlated well with the histological study. Furthermore, the metabolic activity of the sponge-induced granulomas was inhibited by CLT. Because CLT is an inhibitor of signal transduction interfering with the ionic fluxes across the cell membranes, our results suggest that the onset and maintenance of inflammatory angiogenesis induced by subcutaneous implantation of sponge matrix may be regulated by ionic fluxes.     

Enhanced drug metabolism after sulfinpyrazone treatment in patients aged 50 to 60 years

Summary The induction of liver drug metabolism was investigated in five patients before and after the administration of 800 mg sulfinpyrazone daily for 4 weeks, by using antipyrine plasma-pharmacokinetics and by determining urinary excretion of 6--OH-cortisol and serum gamma-glutamyl-transpeptidase (GGT) activity. Antipyrine half-life was shortened in all patients from a mean value of 12.3±3.9 h to 7.8±2.0 h and antipyrine clearance was increased from 39.0±16.0 ml/min to 57.6±13.7 ml/min. In contrast the volume of distribution of antipyrine was unaffected; the values being 38.0±8.6 liters and 37.4±5.7 liters, respectively. In all patients the excretion of 6--OH-cortisol in the urine went up from 65.0±25.7 µg/24 h to 346.8±193.4 µg/24 h. The ratio 6--OH-cortisol/free cortisol changed from 4.1 to 15.8. After 21 days of treatment the GGT increased from 17.4±4.9 units/liter to 32.6±12.5 units/liter The data presented confirm that sulfinpyrazone induces drug metabolism in patients of the older age group. Interactions between sulfinpyrazone and other drugs given simultaneously must be borne in mind.     

Über die Bedeutung von Acetacetat und β-Hydroxybutyrat im Stoffwechsel des menschlichen Herzens

Zusammenfassung Das Verhalten von Acetacetat und -Hydroxybutyrat im Stoffwechsel des menschlichen Herzens wurde im Rahmen diagnostischer Herzkatheterisationen an 55 stoffwechselgesunden Patienten untersucht. Außerdem erfolgte die Bestimmung der arterio-coronarvenösen Differenzen für Glucose, Lactat, Pyruvat, nicht veresterten Fettsäuren, Aminosäuren und Sauerstoff, um einen vollständigen Einblick in die Substratversorgung des Myokards zu erhalten.Bei physiologisch stark schwankenden arteriellen Konzentrationen von Acetacetat (7,2±0,66 µMol/100) und -Hydroxybutyrat (21,3±2,6 µMol/100) extrahiert der Herzmuskel vom Angebot durchschnittlich 50% Acetacetat (3,6±0,36 µMol/100) und 35,2% -Hydroxybutyrat (7,5±1,2 µMol/100). Für beide Substrate konnte zwischen arterieller Konzentration und myokardialer Extraktion eine statistisch gesicherte Abhängigkeit nachgewiesen werden.Der Quotient -Hydroxybutyrat/Acetacetat beträgt arteriell 2,87±0,25 und coronarvenös 3,69±0,31.Der Anteil von Acetacetat am oxydativen Stoff-wechsel des menschlichen Herzens erreicht trotz hoher prozentualer Extraktion nur 2,6±0,3%. Der Wert für -Hydroxybutyrat liegt mit 6,0±1,0% wesentlich höher, so daß sich für die Gesamtketonkörper ein Sauerstoffextraktionsquotient von 8,6% ergibt.Für die übrigen am Myokardstoffwechsel beteiligten Substrate errechnen sich Sauerstoffextraktionsquotienten, die in den von uns bereits früher angegebenen Bereich fallen:Glucose 14,5±1,9%, Lactat 12,9±1,1%, Pyruvat 1,0±0,1%, nicht veresterte Fettsäuren 69,7±3,0%.Bei überdurchschnittlich hohen arteriellen Ketonkörperkonzentrationen nimmt der Anteil der Ketonkörper am gesamten oxydativen Stoffwechsel des Herzens beträchtlich zu, während die Kohlenhydrate in etwa gleicher Größenordnung abnehmen. Die nicht veresterten Fettsäuren zeigen keine Änderung ihrer prozentualen Extraktion. Die Ketonkörper stellen somit gegenüber den Kohlenhydraten ein vom Herzmuskel bevorzugtes Substrat dar.Die Redoxpotentiale der Substratpaare ß-Hydroxybutyrat/Acetacetat und Lactat/Pyruvat werden bei der Herzmuskelpassage im Prinzip in gegensätzlicher Weise verändert, und zwar erfährt das -Hydroxybutyrat/Acetacetat-System eine Negativierung (E=–3,1±0,6 mV) und das Lactat/Pyruvat-System eine angedeutete Positivierung (E=+0,9±0,63 mV).

---

Summary The myocardial extraction of acetoacetate and -hydroxybutyrate was determined in 55 metabolically normal patients during diagnostic heart catheterizations. In order to obtain a complete insight into the substrate supply of the myocardium, arterio-coronary-venous differences of glucose, lactate, pyruvate, non esterified fatty acids, amino acids and oxygen were also measured.From the considerably varying arterial concentrations of acetoacetate (7,2±0,66 µMol/100) and -hydroxybutyrate (21,3±2,6 µMol/100) the human heart muscle extracts an average of 50% acetoacetate (3,6±0,36 µMol/100) and 35,2% -hydroxybutyrate (7,5±1,2 µMol/100). Both substrates showed a statistically significant correlation between arterial concentration and myocardial extraction.The -hydroxybutyrate/acetoacetate quotient was found to be 2,87±0,25 in the arterial and 3,69±0,31 in the coronaryvenous blood.Although a high percentage of the arterial level of acetoacetate is extracted by the heart, this substrate accounts for only 2,6±0,3% of the total oxydative myocardial metabolism. The value for -hydroxybutyrate at 6,0±1,0% is considerably higher, thus obtaining an oxygen extraction ratio of 8,6% for the total ketone-bodies.The other substrates involved in the myocardial metabolism showed oxygen extraction ratios similar to those previously reported by our group: glucose 14,5±1,9%, lactate 12,9±1,1%, pyruvate 1,0±0,1%, non esterified fatty acids 69,7±3,0%.With higher arterial levels of ketone-bodies an increase of their oxygen extraction ratio was found, whereas the uptake of carbohydrates was proportionally decreased. The percentual extraction of non esterified fatty acids did not change. These results indicate that the myocardium oxidizes ketonebodies in preference to carbohydrates.The redox-potentials of the -hydroxybutyrate/acetoacetate and the lactate/pyruvate systems are changed across the heart in an opposite way. The -hydroxybutyrate/acetoacetate system becomes more negative (E=–3,1±0,6 mV), the lactate/pyruvate system tends to be slightly positive (E=+0,9±0,63 mV).

---

---

Mit UnterstÜtzung der Deutschen Forschungsgemeinschaft.     

In vitro effects of bone- and platelet-derived transforming growth factor-β on the growth of Walker 256 carcinosarcoma cells

Conditioned media from fetal rat calvarial cultures has previously been shown to stimulate the growth of the bone-metastasizing Walker 256 carcinosarcoma cell line. In the current investigation we looked at the possibility that transforming growth factor- (TGF-), present in conditioned media, and positively correlated with resorption in vitro, may be responsible for the enhanced proliferation of Walker cells cultured in these conditioned media. Purified platelet-derived TGF- produced a dose-dependent growth response in Walker cells with an ED₅₀ equal to 0·05 ng/ml. Bone-derived TGF- activity in conditioned media, measured by NRK fibroblast colony formation, correlated well with percentage resorption in bone cultures, and growth activity in Walker cell culture. In addition to this, the growth response normally seen with conditioned media cultures of Walker cells was significantly inhibited by the addition of anti-TGF-1 neutralizing antibody. We conclude that TGF- is an important growth stimulatory component from fetal rat calvaria.     

In vitro studies on the Fc-receptor function of mononuclear phagocytes in rheumatoid arthritis: Relation between the Fc-receptor blockade and the concanavalin A-binding capacity of autologous immunoglobulin G

The Fc-receptor (Fc-R) function of monocytes isolated from 19 control subjects and from 30 patients presenting with a rheumatoid arthritis (RA) was assessedin vitro by a classical rosette assay using IgG-coated sheep red blood cells. In RA patients, the percentage of monocytes forming rosettes was significantly lower than in controls (34.4±20.4 versus 67.4±4.5%;P<0.001). The blockade observed was reversed by a prior trypsin treatment of RA monocytes, the percentage of recovery being correlated with the IgG plasma levels. Besides, IgG purified from the serum of four RA patients bound a mean of 7.3, 5.2, 1.6, and 1.6 times more than normal IgG did onto concanavalin A (Con A), peanut agglutinin (PNA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM), respectively. Although similar amounts of¹²⁵I-labeled normal and RA IgG were bound to normal monocytes, RA IgG inhibited more efficiently than normal IgG the Fc-R function of normal monocytes, for all concentrations tested (10 to 100 µg/100 µl). A prior treatment of RA IgG by -mannosidase, but not by -galactosidase, significantly reduced their inhibitory properties. The incubation of monocytes withD-mannose or mannan reduced their capacity to form rosettes. The percentage of monocytes forming rosettes in the presence of both mannan and normal IgG was significantly lower than that measured in the presence of normal IgG only. On the contrary, the rosetting capacity of monocytes in the presence of both RA IgG and mannan was the same as that calculated in the presence of RA IgG only. The inhibitory effect of RA IgG was not related to their abnormal circular dichroism. Our data suggest that the greater ability of RA IgG to block the Fc-R function of monocytes probably depends on the presence of a greater number of accessible mannosyl residues on the glycosidic side chains located in the Fc domain of the molecules.     

Circannual variation in the expression of β2-adrenoceptors on human peripheral mononuclear leukocytes (MNLs)

Summary Peripheral mononuclear leukocytes (MNLs) are widely used as a tissue model in studies of -adrenoceptor disturbances in hypertension and asthmatic diseases. The ₂-adrenoceptor density (B_max), however, depends not only on the gender of the person under study and on the time of day the blood specimens are obtained. Evidence is now reported for a circannual variation in the expression of ₂-adrenoceptor sites on peripheral MNLs. In male volunteers the 24-h mean was found to be highest in the men studied in April/May (1135±10 sites/cell) and decreased to 891±16 sites/cell in August and to 712±90 sites/cell in December (±SE,P<0.01 April/May compared to December). Concomitantly the circadian amplitude increased from 17.3%±6.4% of 24-h mean in April/May to 28.2%±1.4% of 24-h mean in August and to 34.2%±4.2% of 24-h mean in December (±SE,P<0.05, April/May compared to December). The circadian acrophase remained constant (190°±30° equivalent to 12 h 40 min±2 h 00 min, ±SE).Abbreviations MNLs Peripheral mononuclear leucocytes - B_{max 2}-Adrenoceptor density - ¹²⁵ICYP ¹²⁵Iodo-cyanopindolol - PEF Peak expiratory flow - SE Standard error - ANOVA Analysis of variance - M Circadian mesor - A Circadian amplitude - Circadian acrophase     

The in vitro motility activity of β-cardiac myosin depends on the nature of the β-myosin heavy chain gene mutation in hypertrophic cardiomyopathy

Several mutations in the -myosin heavy chain gene cause hypertrophic cardiomyopathy. This study investigates (1) the in vitro velocities of translocation of fluorescently-labelled actin by -myosin purified from soleus muscle of 30 hypertrophic cardiomyopathy patients with seven distinct -myosin heavy chain gene mutations: Thr124Ile, Tyr162Cys, Gly256Glu, Arg403Gln, Val606Met, Arg870His, and Leu908Val mutations; and (2) motility activity of -myosin purified from cardiac and soleus muscle biopsies in the same patients. The velocity of translocation of actin by -myosin purified from soleus or cardiac muscle of 22 normal controls was 0.48 ± 0.09 m s–1. By comparison, the motility activity was reduced in all 30 patients with -myosin heavy chain gene mutations (range, 0.112 ± 0.041 to 0.292 ± 0.066 m s–1). Notably, the Tyr162Cys and Arg403Gln mutations demonstrated significantly lower actin sliding velocities: 0.123 ± 0.044, and 0.112 ± 0.041 m s–1, respectively. -myosin purified from soleus muscle from four patients with the Arg403Gln mutation had a similar actomyosin motility activity compared to -myosin purified from their cardiac biopsies (0.127 ± 0.045 m s–1 versus 0.119 ± 0.068 m s–1, respectively). Since these seven mutations lie in several distinct functional domains, it is likely that the mechanisms of their inhibitions of motility are different     

An improved method for the quantitation of cellular migration: Role of αvβ3 integrin in endothelial and smooth muscle cell migration

The present study was undertaken to develop a simple and improvedmethod for the accurate quantitation of cellular migration and to examinethe role of v3 integrins in different cellular migration. Usingour newly developed micro-volume chemotaxis assay, we developed an improvedquantitative method to measure in vitro chemotaxis of smooth muscle orendothelial cells toward different extracellular matrix proteins. Theconvenience in setup and counting of migrated cells using this methodallows for large capacity screening and for various research applicationswith other cells as well. The signal. to noise ratios were in the range of10/1, along with about 10–20% intra- or inter-assayvariabilities. Using this method, we have determined that eithervitronectin at 0.4 µg/well or osteopontin at 0.4 µg/well areselective v3 chemoattractants for endothelial or smooth musclecells (0.5 × 105 cells/well). Additionally, a selective v3small molecule peptiddomimetic, monoclonal antibody LM609, or an anti-3 (v3/II3) anti-body, c7E3 demonstratedmaximal inhibition of cellular migration toward vitronectin or osteopontin.These data suggest the potential utility of this method in assessing therole of various mechanisms in cellular migration and also suggests the potential implication of an v3 antagonist in blocking pathologicalprocesses involving endothelial or smooth muscle cell adhesion/migration.     

Analysis of synovial fluid from clinically healthy Iranian fat-tailed sheep

In this study synovial fluid from the radiocarpal joints of 100 clinically healthy Iranian sheep (Lori-Bakhtiari) were analyzed. Total nucleated cell count (TNCC) of the synovial fluid was 178.9±75 cells/l (mean±SD). Lymphocytes were the predominant cell type composing 48.34±17.2% of the cells found in the synovial fluid, whereas monocytes, macrophages and neutrophils composed 36.52±3.5%, 12.75±5.9% and 2.28±1.18% of the cells found in the synovial fluid, respectively. The glucose concentration of synovial fluid was 44.9±9 mg/dl. The concentration of total protein, albumin and globulin of the synovial fluid were 2.31±0.55, 1.49±0.38, 0.81±0.28 g/dl, respectively. The albumin to globulin ratio (A/G) was 2.02±0.61. Age and sex had no significant effects on TNCC, percentage of lymphocytes, monocytes, macrophages and neutrophils, concentration of total protein, albumin, globulin, and A/G ratio of fluid from the radiocarpal joint. However, glucose concentration in radiocarpal fluid in sheep less than 1-year-old was significantly (P0.05) higher (48.27±1.4 mg/dl) than 1- to 2-years-old (42.1±1.4 mg/dl) and more than 2-years-old sheep (43.9±1.8 mg/dl). No significant differences were found between right and left limbs for any parameters evaluated in this study.     

Integrin-Dependent Human Macrophage Migration Induced by Oscillatory Electrical Stimulation

Electrical stimulation has been used to promote wound healing. The mechanisms by which such stimulation could interact with biological systems to accelerate healing have not been elucidated. One potential mechanism could involve stimulation of macrophage migration to the site of a wound. Here we report that oscillatory electric fields induce human macrophage migration. Macrophages exposed to a 1 Hz, 2 V/cm field show an induced migration velocity of 5.2±0.4 ×10^-2 m/min and a random motility coefficient of 4.8±1.4 ×10^-2 m²/min on a glass substrate. Electric field exposure induces reorganization of microfilaments from ring-like structures at the cell periphery to podosomes that are confined to the contact sites between cell and substrate, suggesting that the cells are crawling on glass. Treatment of cells with monoclonal antibodies directed against ₂-integrins prior to field exposure prevents cell migration, indicating that integrin-dependent signaling pathways are involved. Electric fields cause macrophage migration on laminin or fibronectin coated substrates without inducing podosome formation or changes in cellular morphology. The migration velocity is not significantly altered but the random movement is suppressed, suggesting that cell movements on a laminin- or fibronectin-coated surface are not mediated by cell crawling. It is suggested that electric field-induced macrophage migration utilizes several modes of cell movement, including cell crawling and possibly cell rolling. ©