CHO transfectants produce large amounts of recombinant protein in suspension culture.

Chinese hamster ovary (CHO) cells transfected with various genes are widely used as adherent cell monolayers to produce recombinant proteins. In this report we present a new culture technique for CHO cells transfected with the vector pPOL-DHFR-CD14 using a minifermenter (miniPERM, Heraeus) for the production of recombinant human endotoxin receptor CD14 (rCD14). The transfectants were cultured for 12-17 days under serum-free conditions and formed spheroids. From this system we harvested supernatants containing up to 3.1 mg/ml recombinant CD14 (rCD14). This represents a 200-fold increase of rCD14 yield compared to conventional adherent CHO cell culture.

[Glucocorticoid (prednisolone) effects on various blood, urine and liver parameters in cows in the second post partum week]. / Glukokortikoid-(Prednisolon-)Wirkungen auf einige Blut-, Harn- und Leberparameter bei Kühen in der zweiten Woche post partum.

Starting on the 7th day post partum, 200 mg prednisolone were given daily to four healthy cows over a period of five days in order to examine how fat metabolism and liver function were influenced. Samples of blood and urine were taken daily as well as liver biopsies on the third and fifth day. These findings in the cows of the experimental group were compared to those in the four untreated cows (control group). Prednisolone induced a significant increase in glucose and insulin serum concentrations and a decrease of FFA and bilirubin concentrations as well as lipid and rising glycogen concentrations in the liver. The ASAT and GLDH activities as well as the thyroxin concentration decreased tendentiously. The cortisol concentration decreased shortly insignificantly. Acid base status, calcium as well as anorganic phosphorus concentrations were not noticeably influenced. Signs of a higher risk of infection were not observed. The results show that the effect of prednisolone is indirect antilipolytic and protects the liver. They therefore do not support the idea that glucocorticoids lead to an increased lipolysis and fat accumulation in the liver of cows.

[The behavior of superoxide dismutase (SOD) in serum of cows with abomasal displacement (DA)]. / Verhalten der Superoxiddismutase (SOD) im Serum von Kühen mit Dislocatio abomasi (DA).

After surgical reposition of displaced organs (abomasum, uterus, intestines) restoration of blood flow and oxygen supply generates oxygen radicals and other reactive oxygen species. SOD indicates radical stress of the organism. Subject of the study was the question if SOD can be detected in blood serum samples of cows and if there are differences in SOD activity between healthy cows and cows with Dislocatio abomasi (DA). We also wanted to investigate the influence of breed "Schwarzbunte" with DA (16 left/5 rights). The samples were drawn before and 1, 3 and 24 post op. Ten healthy cows of the same breed were also examined (2 weeks and 4-6 weeks after calving). There are no significant differences between the SOD activity of healthy cows and cows with DA, but the SOD activity of cows with left DA is significant lower than the activity of cows with right DA. Post op. SOD activity decreases; 24 h after surgery cows with left but not with right DA show an increase of SOD activity similar to values before surgery. There is a close positive correlation between SOD activity and protein concentration as well as negative correlation to concentration of free fatty acids after surgery. The behaviour of SOD activity shows that the surgical replacement of the displaced abomasum can generate a depression of the antioxidative capacity of the organism.

[Etiology and prophylaxis of reperfusion injuries]. / Atiologie und Prophylaxe von Reperfusionsschäden.

In recent years the importance of oxyradicals in clinical veterinary medicine is incessantly grown. An important example in cattle breeding is the displaced abomasum in dairy cows. The antioxidative status of the animals is representable by means of the activity of superoxide dismutase (SOD) in the blood. Post operationem the activity of SOD decreased very fast. In cows with left abomasal displacement this activity increased within 24 hours to the starting level, but in cows with right abomasal displacement the SOD-activity in this period increased only insignificantly. Because of the low level of SOD-activity in blood serum the measuring should take place in erythrocyte lysate. Today there are many possibilities of therapeutical intervention of reperfusion injury, but their clinical efficiency has to be ascertained. The treatment of cows with displaced abomasum with ascorbate, tocopherole or prednisolone before the replacement of the abomasum shows first success in terms of the antioxidative and metabolic status.

Heparin-induced thrombocytopenia: towards standardization of platelet factor 4/heparin antigen tests.

BACKGROUND: Laboratory confirmation of heparin-induced thrombocytopenia (HIT) is based on detection of heparin-dependent platelet-activating antibodies. Platelet factor 4 (PF4)/heparin enzyme-immunoassays (EIA) are a widely available surrogate for platelet-activating antibodies. OBJECTIVE: Defining the optical density (OD) reactivity profiles of a PF4/heparin EIA in reference subject and patient populations and the correlation of the EIA results (expressed in OD units) with the prevalence of platelet-activating antibodies. PATIENTS/METHODS: Using quantile regression we determined the 97.5th percentile of PF4/heparin-immunoglobulin G (IgG) EIA reactivities in non-heparin-treated individuals [blood donors (n = 935)] and patients before heparin therapy (n = 1207). In patients with suspected HIT, we compared the correlation of EIA-IgG reactivities (Greifswald laboratory; n = 2821) and the heparin-induced platelet activation assay (HIPA) with the correlation of reactivities of another EIA-IgG (McMaster laboratory; n = 1956) with the serotonin-release assay (SRA). RESULTS: PF4/heparin-IgG EIA OD reactivities had a lower OD 97.5th percentile in blood donors compared with patient groups before heparin treatment (P < 0.001). The percentage of sera testing positive in the functional assays strongly correlated with PF4/heparin-IgG EIA OD reactivities in both laboratories with very similar results (correlation coefficient > 0.9) when normalized OD ranges (maximum OD divided by 10) were used instead of absolute OD values. CONCLUSIONS: Results of PF4/heparin-IgG EIA should not be reported as only positive or negative as there is no single acceptable cut-off value. Instead, reporting PF4/heparin-IgG EIA OD results in ranges allows for risk-stratified prediction for presence of platelet-activating antibodies. Use of normalized OD ranges permits a standardized approach for inter-laboratory comparisons.

Heparin-induced thrombocytopenia--therapeutic concentrations of danaparoid, unlike fondaparinux and direct thrombin inhibitors, inhibit formation of platelet factor 4-heparin complexes.

BACKGROUND: Treatment of heparin-induced thrombocytopenia (HIT), a disorder in which anti-platelet factor 4 (PF4)-heparin antibodies cause platelet activation and hypercoagulability, requires alternative (non-heparin) anticoagulation. Treatment options include direct thrombin inhibitors [lepirudin and argatroban (approved), and bivalirudin], danaparoid (approved) (mixture of anticoagulant glycosaminoglycans), or fondaparinux (synthetic heparin-mimicking pentasaccharide). PF4-heparin complexes form at optimal stoichiometric ratios. OBJECTIVES: To compare the effects of these various non-heparin anticoagulants in disrupting the formation of PF4-heparin complexes, and PF4-containing immune complexes. PATIENTS/METHODS: Sera were obtained from patients with serologically confirmed HIT. The effects of the alternative anticoagulants on PF4 and PF4-heparin complex interactions with platelets, as well as HIT antibody binding and platelet activation, were investigated. RESULTS: Danaparoid at very low concentrations increased PF4 binding to platelets. In therapeutic concentrations, however, it decreased PF4 binding to platelets (P = 0.0004), displaced PF4-heparin complexes from platelets (P = 0.0033) and PF4 from the surface of a PF4-transfected HEK-293 EBNA cell line expressing the PF4 receptor CXCR3-B (P = 0.0408), reduced PF4-heparin complex size (P = 0.025), inhibited HIT antibody binding to PF4-heparin complexes (P = 0.001), and prevented platelet activation by HIT antibodies (P = 0.046). Although fondaparinux also interfered with PF4 binding to platelets, HIT antibody binding to PF4-heparin complexes, and activation of platelets by HIT antibodies, these effects occurred only at supratherapeutic concentrations. The direct thrombin inhibitors had no effect at any concentrations. CONCLUSIONS: Danaparoid uniquely interferes with the pathogenesis of HIT by disrupting PF4-containing immune complexes at therapeutic dose concentrations. It is possible that these effects contribute to its therapeutic efficacy.

[Comparative studies of diagnostically significant enzymes in plasma, udder lymph and milk of healthy cows and cows with udder diseases]. / Vergleichende Untersuchungen diagnostisch bedeutsamer Enzyme in Blutplasma, Euterlymphe und Milch von gesunden und euterkranken Kühen.

Different secretions (colostrum, milk, dry udder secretion) of every quarter, peripheral lymph from superficial lymph vessels of the mammary gland and blood from the V. epigastrica superficialis were obtained in 43 cows at different stages of lactation. In these samples the activity of 5 enzymes (LDH, NAG, AP, LAP, GGT) was determined. Levels of LDH and NAG were highest in blood plasma and udder lymph. Levels of LAP, AP and GGT were highest in milk increasing in this order. LDH, NAG, AP and LAP were correlated in both compartments. Changes of the functional state (dry or colostral period) and tissue disturbances of the mammary gland were accompanied by marked changes of enzyme activity in the secretions, but were without obvious influence on enzyme levels in blood plasma and udder lymph.

[Behavior of various metabolites of carbohydrate metabolism in the liver of young pigs following inhibition of C-11 hydroxylase and adrenalectomy]. / Das Verhalten einiger Metabolite des Kohlenhydratstoffwechsels in der Leber von Läuferschweinen nach C-11 Hydroxylase-Nemmung und Adrenalektomie

The effects of glucocorticoid depression by C-11 hydroxylase inhibition and total adrenalectomy upon certain metabolites of the plasma, liver, and muscles that are related to gluconeogenesis were studied on ten store pigs. Metopiron caused decline of glucose and pyruvate concentrations in plasma and drop of liver glycogen. Total adrenalectomy caused changes of the same kind but much stronger in the agonal phase, yet without rendering the animals hypoglycaemic. Muscular glycogen dropped to one third of the normal. The results are discussed with reference to earlier studies into store pig.

[Gluconeogenetic capacity of suckling piglets and young pigs]. / Die glukoneogenetische Kapazität von Saugferkeln und Läuferschweinen

No marked gluconeogenetic performance was recordable from nursed piglets aged, between one and five days, in response to ACTH nor glucocorticosteroid application. Store pigs, aged twelve weeks, however, exhibited glucose rises of 34 per cent one hour after injection or 55 per cent three hours from ACTH application. The point was made, in an attempt to elucidate the above findings, that in newborn piglets, few days after birth, the gluconeogenetic capacity is insufficient because of functional immaturity of the liver. The behaviours of several metabolites in the liver and muscles of store pigs were examined under differentiated model conditions to check up and verify that view.

Histamine affects growth of sheep ruminal epithelial cells kept in primary culture.

Ten, 100 and 1000 microM histamine were applied to ruminal epithelial cells in primary culture. Each of these concentrations diminished cell count as well as the protein and DNA contents of cultures by approximately 20%. On Pappenheim staining, reduced cell count was predominantly attributable to a decrease in relatively chromophobic cells. It is concluded that histamine either induces apoptosis, or increases cell shedding, or interferes with mitosis and cell maturation. Each of these three possibilities implies that histamine may disturb epithelial regeneration after ruminal lactic acidosis.

CELISA for rapid screening of monoclonal islet cell surface antibodies using living rat insulinoma cells as target.

An improved rapid cell enzyme-linked immunosorbent assay (CELISA) is described which is suitable for the large scale screening of monoclonal antibodies to islet cell surface antigens. 5 x 10(4) insulin-producing rat insulinoma (RIN) cells were seeded per well in a 96-well flat-bottomed polystyrene plate coated one day before a 0.01% poly-D-lysine solution in PBS. After culture for 4 days in 200 microliters/well RPMI 1640 supplemented with 7.5% heat-inactivated fetal calf serum, the cell number per well was up to 2.1 x 10(5). These monolayer RIN cell cultures were used as a target for the detection of islet cell surface antibodies (ICSA) in the supernatants of hybridomas. The cells were used without fixation to avoid modification of sensitive surface antigens. Poly-D-lysine did not cause non-specific binding of immunoglobulins to the plastic wells as tested with irrelevant monoclonals. The specificity and sensitivity of the method is comparable to indirect immunofluorescence. All mc-ICSA primary screened by indirect immunofluorescence using viable RIN cell suspensions were positive in this CELISA. There was a correlation (r = 0.7; n = 44) between the antibody binding measured by CELISA and the indirect immunofluorescence technique. The advantage of this CELISA is that cell surface structures are well preserved in a viable cell monolayer used as target without chemical fixation. This assay procedure should be generally suitable for the initial screening of monoclonal antibodies to cell surface antigens of cells growing under culture conditions.

Different multiple reactivity of monoclonal islet cell binding antibodies using indirect immunofluorescence technique on viable cells or cellular ELISA on desiccated cells as target.

Two commonly used methods for screening hybridoma supernatants secreting monoclonal islet cell reactive antibodies (mc-ICRA) were performed to investigate the specificity of the monoclonals established. For this, endothelial, neuroblastoma, murine subcutis and two myeloma cell lines were used as targets in comparison to the insulin-producing rat insulinoma cell line (RIN), either immobilized and permeabilized in cellular enzyme linked immunosorbent assay (CELISA) or in suspension of viable cells in the indirect immunofluorescence test. In addition, rat splenocytes were used for estimating multireactivity of mc-ICRA in ELISA. Using permeabilized target cells, we obtained a high multireactivity of the monoclonal antibodies (mab) tested, indicating a high incidence of molecular mimicry between cytoplasmic antigens of different cell lines. In contrast to CELISA, if only cell surface antigens of viable cells are accessible, detected by the immunofluorescence technique, the high multireactivity is not observed. For investigating the specificity of monoclonals, the complexity of target antigens used must be taken into consideration.

[Thiocyanate content in blood plasma, udder lymph and milk from healthy cows and cows with mastitis]. / Thiocyanatgehalt in Blutplasma, Euterlymphe und Milch von gesunden und euterkranken Kühen.

After storage for more than one year the SCN(-)-content of udder lymph was not changed seriously, but in blood serum and mainly in milk, there is an evident decrease. In lactational cows with healthy udders the SCN(-)-content of blood plasma is higher than in milk (in average twice). There is no difference of the SCN(-)-content of the four udder parts of healthy cows. Decrease of milk production (10. to 13. lactational month) causes an increase of the SCN(-)-content of the milk, it is not changed in subclinical mastitis. In lactational cows with healthy udders the SCN(-)-content of udder lymph is nearly triple higher in average than in milk of the corresponding quarter of the udder. The existence of a common compartment of SCN- in blood and udder lymph is assumed, which is separated from them of the milk by the blood udder barrier.

Induction of islet cell surface and insulin antibodies in Balb/c mice by application of porcine insulin and Freund's adjuvant is not associated with insulitis.

To investigate whether insulin antibody (IAB) formation is associated with the appearance of islet cell cytoplasmic antibodies (ICA), islet cell surface antibodies (ICSA) and insulitis Balb/c Bln mice were immunized with porcine insulin in combination with or without Freund's adjuvant. The animals received 8 i.p. injections and were followed up to 150 days for the development of antibodies and insulitis. Mice immunized with insulin in CFA developed IAB as well as ICSA. Mice only receiving Freund's adjuvant emulsified in saline also developed ICSA. ICA were not detectable. Inflammatory infiltrates were found in the exocrine pancreatic parenchyma but not in islets. The results show that nonspecific stimulation of the immune system and the application of insulin as antigen leads to both the formation of ICSA and IAB, while insulitis was not detectable.

Different efficacy of soluble CD14 treatment in high- and low-dose LPS models.

BACKGROUND: About 50% of septic shock cases are attributed to Gram-negative bacteria or their cell wall compound lipopolysaccharide (LPS, endotoxin). An attractive therapeutic strategy could target the binding of LPS to its cellular receptors. In vitro the soluble form of the endotoxin receptor CD14 (sCD14) competitively prevents binding of LPS to membrane-bound CD14 and inhibits LPS-stimulated macrophage responses. METHODS: We tested the in vivo endotoxin-neutralizing capacity of human recombinant sCD14 using a mouse model of shock induced by 8 micrograms g-1 of LPS from Salmonella abortus equi. RESULTS: In this model, treatment with sCD14 reduced mortality if administered before or simultaneously with LPS. However, application of sCD14 had no effect on the secretion of early proinflammatory cytokines and did not protect the animals against the development of apparent shock symptoms and liver injury. sCD14 also failed to prevent LPS-inducible (7.5 ng g-1) liver injury in galactosamine-sensitized mice. CONCLUSION: In line with these findings, sCD14 did not block LPS-induced activation of Kupffer cells in vitro, which might explain why the compound only partially protected in vivo.

Lipopolysaccharide-binding protein is required to combat a murine gram-negative bacterial infection.

An invading pathogen must be held in check by the innate immune system until a specific immune response can be mounted. In the case of Gram-negative bacteria, the principal stimulator of the innate immune system is lipopolysaccharide (LPS), a component of the bacterial outer membrane. In vitro, LPS is bound by lipopolysaccharide-binding protein (LBP) and transferred to CD14--the LPS receptor on the macrophage surface--or to high-density lipoprotein (HDL) particles. Transfer to CD14 triggers an inflammatory response which is crucial for keeping an infection under control. Here we investigate how LBP functions in vivo by using LBP-deficient mice. Surprisingly, we find that LBP is not required in vivo for the clearance of LPS from the circulation, but is essential for the rapid induction of an inflammatory response by small amounts of LPS or Gram-negative bacteria and for survival of an intraperitoneal Salmonella infection.