Distribution of lactate dehydrogenase in healthy and degenerative canine stifle joint cartilage

In dogs, degenerative joint diseases (DJD) have been shown to be associated with increased lactate dehydrogenase (LDH) activity in the synovial fluid. The goal of this study was to examine healthy and degenerative stifle joints in order to clarify the origin of LDH in synovial fluid. In order to assess the distribution of LDH, cartilage samples from healthy and degenerative knee joints were investigated by means of light and transmission electron microscopy in conjunction with immunolabeling and enzyme cytochemistry. Morphological analysis confirmed DJD. All techniques used corroborated the presence of LDH in chondrocytes and in the interterritorial matrix of healthy and degenerative stifle joints. Although enzymatic activity of LDH was clearly demonstrated in the territorial matrix by means of the tetrazolium-formazan reaction, immunolabeling for LDH was missing in this region. With respect to the distribution of LDH in the interterritorial matrix, a striking decrease from superficial to deeper layers was present in healthy dogs but was missing in affected joints. These results support the contention that LDH in synovial fluid of degenerative joints originates from cartilage. Therefore, we suggest that (1) LDH is transferred from chondrocytes to ECM in both healthy dogs and dogs with degenerative joint disease and that (2) in degenerative joints, LDH is released from chondrocytes and the ECM into synovial fluid through abrasion of cartilage as well as through enhanced diffusion as a result of increased water content and degradation of collagen.

Measurements of C-reactive protein in serum and lactate dehydrogenase in serum and synovial fluid of patients with osteoarthritis

Diagnosis of osteoarthritis (OA) is based upon the clinical orthopaedic examination and the radiographic assessment, both of which can be non-specific and insensitive in early joint disease. The aim of our study was to investigate if there is an increase in serum levels of C-reactive protein (CRP) in degenerative joint disease (DJD) and if CRP could be used to help diagnose OA. We also wished to investigate whether it was possible to distinguish a joint with clinically and radiographically confirmed OA from a healthy joint by comparing lactate dehydrogenase (LDH) levels within the synovial fluid and the serum. We have shown a difference in synovial LDH levels between diseased and healthy joints (P<0.0001). There was also a significant difference between LDH in arthritic synovial fluid and serum, with no correlation between the values. Despite the fact that the values of our clinical patients tended to be higher than the values of our control group (P=0.05) all measured values were within the normal limits of previous publications. From these data, we conclude that single measurements of serum CRP do not permit detection of OA in clinical patients and that serum LDH is not a reliable marker for osteoarthritis. LDH levels in the synovial fluid could be of diagnostic value for identifying osteoarthritis.

Lactate dehydrogenase concentration in nasal wash fluid indicates severity of rhinovirus-induced wheezy bronchitis in preschool children.

The clinical course of rhinovirus (RV)-associated wheezing illnesses is difficult to predict. We measured lactate dehydrogenase concentrations, RV load, antiviral and proinflammatory cytokines in nasal washes obtained from 126 preschool children with RV wheezy bronchitis. lactate dehydrogenase values were inversely associated with subsequent need for oxygen therapy. lactate dehydrogenase may be a useful biomarker predicting disease severity in RV wheezy bronchitis.

The relationship between extravascular lung water and oxygenation in three patients with influenza A (H1N1)-induced respiratory failure

This case series reports the correlation between extravascular lung water (EVLW) and the partial arterial oxygen pressure/fractional inspiratory oxygen (PaO(2)/FiO(2)) ratio in three patients with severe influenza A (H1N1)-induced respiratory failure. All patients suffered from grave hypoxia (PaO(2), 26-42 mmHg) and were mechanically ventilated using biphasic airway pressure (PEEP, 12-15 mmHg; FiO(2), 0.8-1) in combination with prone positioning at 12 hourly intervals. All patients were monitored using the PICCO system for 8-11 days. During mechanical ventilation, a total of 62 simultaneous determinations of the PaO(2)/FiO(2) ratio and EVLW were performed. A significant correlation between EVLW and the PaO(2)/FiO(2) ratio (Spearman-rho correlation coefficient, -0.852; p < 0.001) was observed. In all patients, a decrease in EVLW was accompanied by an improvement in oxygenation. Serum lactate dehydrogenase levels were elevated in all patients and significantly correlated with EVLW during the intensive care unit stay (Spearman-rho correlation coefficient, 0.786; p < 0.001). In conclusion, EVLW seems increased in patients with severe H1N1-induced respiratory failure and appears to be closely correlated with impairments of oxygenatory function.

Deficient CEBPA DNA binding function in normal karyotype AML patients is associated with favorable prognosis

CCAAT/enhancer binding protein-α (CEBPA) mutations in acute myeloid leukemia (AML) patients with a normal karyotype (NK) confer favorable prognosis, whereas NK-AML patients per se are of intermediate risk. This suggests that blocked CEBPA function characterizes NK-AML with favorable outcome. We determined the prognostic significance of CEBPA DNA binding function by enzyme-linked immunosorbent assay in 105 NK-AML patients. Suppressed CEBPA DNA binding was defined by 21 good-risk AML patients with inv(16) or t(8;21) (both abnormalities targeting CEBPA) and 8 NK-AML patients with dominant-negative CEBPA mutations. NK-AML patients with suppressed CEBPA function showed a better overall survival (P = .0231) and disease-free survival (P = .0069) than patients with conserved CEBPA function. Suppressed CEBPA DNA binding was an independent marker for better overall survival and disease-free survival in a multivariable analysis that included FLT3-ITD, NPM1 and CEBPA mutation status, white blood cell count, age and lactate dehydrogenase. These data indicate that suppressed CEBPA function is associated with favorable prognosis in NK-AML patients.

Characterization of major zinc containing myonecrotic and procoagulant metalloprotease 'malabarin' from non lethal trimeresurus malabaricus snake venom with thrombin like activity: its neutralization by chelating agents

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes A? followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

Early reperfusion hemodynamics predict recovery in rat hearts: a potential approach towards evaluating cardiac grafts from non-heart-beating donors

Aims Cardiac grafts from non-heartbeating donors (NHBDs) could significantly increase organ availability and reduce waiting-list mortality. Reluctance to exploit hearts from NHBDs arises from obligatory delays in procurement leading to periods of warm ischemia and possible subsequent contractile dysfunction. Means for early prediction of graft suitability prior to transplantation are thus required for development of heart transplantation programs with NHBDs. Methods and Results Hearts (n = 31) isolated from male Wistar rats were perfused with modified Krebs-Henseleit buffer aerobically for 20 min, followed by global, no-flow ischemia (32°C) for 30, 50, 55 or 60 min. Reperfusion was unloaded for 20 min, and then loaded, in working-mode, for 40 min. Left ventricular (LV) pressure was monitored using a micro-tip pressure catheter introduced via the mitral valve. Several hemodynamic parameters measured during early, unloaded reperfusion correlated significantly with LV work after 60 min reperfusion (p<0.001). Coronary flow and the production of lactate and lactate dehydrogenase (LDH) also correlated significantly with outcomes after 60 min reperfusion (p<0.05). Based on early reperfusion hemodynamic measures, a composite, weighted predictive parameter, incorporating heart rate (HR), developed pressure (DP) and end-diastolic pressure, was generated and evaluated against the HR-DP product after 60 min of reperfusion. Effective discriminating ability for this novel parameter was observed for four HR*DP cut-off values, particularly for ≥20 *103 mmHg*beats*min−1 (p<0.01). Conclusion Upon reperfusion of a NHBD heart, early evaluation, at the time of organ procurement, of cardiac hemodynamic parameters, as well as easily accessible markers of metabolism and necrosis seem to accurately predict subsequent contractile recovery and could thus potentially be of use in guiding the decision of accepting the ischemic heart for transplantation.

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 mug of LPS or 20 mug of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12h relative to the challenge to measure mRNA expression of tumor necrosis factor-alpha (TNFalpha), IL-1beta, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFalpha. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFalpha in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFalpha, IL-1beta, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.

Immune response of bovine milk somatic cells to endotoxin in healthy quarters with normal and very low cell counts

Low somatic cell count (SCC) is a reliable indicator of high-quality milk free of pathogenic microorganisms. Thus, an important goal in dairy practice is to produce milk with low SCC. Selection for cows with low SCC can sometimes lead to extremely low SCC in single quarters. The cells in milk are, however, predominantly immune cells with important immune functions. To investigate the mammary immune competence of quarters with very low SCC, healthy udder quarters of cows with normal SCC of (40-100) x 10(3) cells/ml and very low SCC of < 20 x 10(3) cells/ml were challenged with lipopolysaccharide (LPS) from Escherichia coli. In the first experiment, SCC and cell viability after a challenge with 50 ng of LPS/quarter was investigated. In the second experiment, tumour necrosis factor alpha (TNF-alpha) concentration and lactate dehydrogenase (LDH) activity in milk, and mRNA expression of various innate immune factors in milk cells were measured after a challenge with 100 mug LPS/quarter. LPS challenge induced an increase of SCC. SCC levels reached were higher in quarters with normal SCC and maximum SCC was reached 1 h earlier than in very low SCC quarters. The increase of TNF-alpha concentrations in milk in response to LPS challenge was lower in quarters with very low SCC than in quarters with normal SCC. The viability of cells and the LDH activity in milk increased in response to LPS challenge, however, without a difference between the groups. The mRNA expression of IL-1beta and IL-8 was increased in milk cells at 12 h after LPS challenge, whereas that of TNF-alpha and lactoferrin was not increased at the measured time points (12, 24 and 36 h after LPS challenge). No differences of mRNA expression of measured immune factors between normal and very low SCC samples were detected. The study showed that udder quarters with very low SCC responded with a less marked increase of SCC compared with quarters with normal SCC. This difference corresponded with simultaneously lower TNF-alpha concentrations in milk. However, the immune competence of the cells themselves based on mRNA expression of TNF-alpha, IL-8, IL-1beta, and lactoferrin, did not differ. The results may indicate that very low SCC can impair the immune competence of udder quarters, because the immune response in udder quarters with lower SCC is less efficient as fewer cells contribute to the production of immunoregulators.

Closure of apical access site after transapical, transcatheter paravalvular leak closure

The safety of percutaneous transapical mitral paravalvular leak (PVL) closure could potentially be enhanced by device closure of the ventricular access site. Percutaneous transapical PVL closure was performed. The 9F delivery sheath was pulled back, and a 6-mm Amplatzer muscular ventricular septal defect occluder was deployed at the apical puncture site. Immediate hemostasis was achieved. Total hospitalization was 9 days. New York Heart Association functional class was improved, hemoglobin and haptoglobin rose, while lactate dehydrogenase fell. Follow-up fluoroscopy and transthoracic echocardiography revealed a good functional result. Closure of the apical access site by means of an Amplatzer muscular ventricular septal defect occluder is feasible.

Cardiac transplantation with hearts from donors after circulatory declaration of death: haemodynamic and biochemical parameters at procurement predict recovery following cardioplegic storage in a rat model

OBJECTIVES: Donation after circulatory declaration of death (DCDD) could significantly improve the number of cardiac grafts for transplantation. Graft evaluation is particularly important in the setting of DCDD given that conditions of cardio-circulatory arrest and warm ischaemia differ, leading to variable tissue injury. The aim of this study was to identify, at the time of heart procurement, means to predict contractile recovery following cardioplegic storage and reperfusion using an isolated rat heart model. Identification of reliable approaches to evaluate cardiac grafts is key in the development of protocols for heart transplantation with DCDD. METHODS: Hearts isolated from anaesthetized male Wistar rats (n = 34) were exposed to various perfusion protocols. To simulate DCDD conditions, rats were exsanguinated and maintained at 37°C for 15-25 min (warm ischaemia). Isolated hearts were perfused with modified Krebs-Henseleit buffer for 10 min (unloaded), arrested with cardioplegia, stored for 3 h at 4°C and then reperfused for 120 min (unloaded for 60 min, then loaded for 60 min). Left ventricular (LV) function was assessed using an intraventricular micro-tip pressure catheter. Statistical significance was determined using the non-parametric Spearman rho correlation analysis. RESULTS: After 120 min of reperfusion, recovery of LV work measured as developed pressure (DP)-heart rate (HR) product ranged from 0 to 15 ± 6.1 mmHg beats min(-1) 10(-3) following warm ischaemia of 15-25 min. Several haemodynamic parameters measured during early, unloaded perfusion at the time of heart procurement, including HR and the peak systolic pressure-HR product, correlated significantly with contractile recovery after cardioplegic storage and 120 min of reperfusion (P < 0.001). Coronary flow, oxygen consumption and lactate dehydrogenase release also correlated significantly with contractile recovery following cardioplegic storage and 120 min of reperfusion (P < 0.05). CONCLUSIONS: Haemodynamic and biochemical parameters measured at the time of organ procurement could serve as predictive indicators of contractile recovery. We believe that evaluation of graft suitability is feasible prior to transplantation with DCDD, and may, consequently, increase donor heart availability.

Macrophage activating syndrome is associated with lobular hepatitis and severe bile duct injury with cholestasis

Macrophage activating syndrome (MAS) is a rare hematological disorder associated with uncontrolled systemic T-cell activation. Persistent fever, fatigue and hepatosplenomegaly are frequent clinical manifestations, whereas hyperferritinemia, elevated serum lactate dehydrogenase levels and cytopenia are key criteria for the diagnosis of MAS. The nature of liver pathology in MAS has been partially elucidated but destructive biliary lesions have been rarely described. This report illustrates four cases of MAS developing marked cholestasis, leading to one case of biliary cirrhosis necessitating liver transplantation. Histologically, liver involvement was characterized in all cases by acute lobular hepatitis, marked hepatocyte apoptosis and small bile duct injury similar to the vanishing bile duct syndrome. Immuno-histological studies showed that the inflammatory changes and bile duct lesions were dominated by the presence of activated macrophages and T-cells, in particular CD8+ lymphocytes, and in part NK-cells. These findings suggest that in MAS, various T-cell triggers such as infection, autoimmune disease and malignancy might result in the release of cytokines, which in turn activate macrophages to trigger a systemic acute phase response and local tissue damage. This communication suggests that a macrophage, T- and NK-cell network is operational in the pathogenesis of the cholangiocyte, hepatocyte and sinus endothelial cell damage in MAS.

Effect of monoterpenes on the formation and activation of osteoclasts in vitro

Monoterpenes, present in aromatic plants, are known to inhibit bone resorption in vivo. In this in vitro study, they inhibited the activation of osteoclasts only at high concentrations but inhibited the formation at much lower concentrations. Therefore, monoterpenes may act in vivo directly on osteoclastogenesis. INTRODUCTION: Monoterpenes are the major components of essential oils, which are formed in many plants. Typically, they are found in herbs and certain fruits. When fed to rats, they inhibit bone resorption by an unknown mechanism. In this study, their effect on the activity and formation of osteoclasts in vitro was studied. MATERIALS AND METHODS: The effect of monoterpenes on the development of osteoclasts was studied in co-cultures of bone marrow cells and osteoblasts and in cultures of spleen cells grown with colony stimulating factor (CSF)-1 and RANKL. In cultures of primary osteoblasts, alkaline phosphatase activity and levels of mRNA encoding RANKL and osteoprotegerin (OPG) mRNA (RT-PCR), and in osteoblast and spleen cell cultures, lactate dehydrogenase activity, a measure of toxicity, were determined. The activity of isolated rat osteoclasts was determined by counting the osteoclasts with actin rings using histofluorometry. RESULTS: The monoterpenes inhibited the formation of osteoclasts more strongly in co-cultures (> or = 1 microM) than in cultures of spleen cells (> or = 10 microM). They had a minor effect on osteoblasts. Toxic effects were not observed. The inhibition of the formation of osteoclasts was not reversed by the addition of farnesol and geranylgeraniol, excluding an effect of the monoterpenes through the mevalonate pathway. A high concentration of 1 mM was required to inhibit the activation of osteoclasts. This effect, shown for menthol and borneol, was reversible. CONCLUSIONS: The results suggest that the monoterpenes inhibit bone resorption in vivo through a direct effect on the formation of osteoclasts acting mainly on the hemopoietic cells.

Different vascular smooth muscle cell apoptosis in the human internal mammary artery and the saphenous vein. Implications for bypass graft disease

BACKGROUND: The remarkable patency of internal mammary artery (MA) grafts compared to saphenous vein (SV) grafts has been related to different biological properties of the two blood vessels. We examined whether proliferation and apoptosis of vascular smooth muscle cells (VSMC) from human coronary artery bypass vessels differ according to patency rates. METHODS AND RESULTS: Proliferation rates to serum or platelet-derived growth factor (PDGF)-BB were lower in VSMC from MA than SV. Surface expression of PDGF beta-receptor was slightly lower, while that of alpha-receptor was slightly higher in MA than SV. Cell cycle distribution, expression of cyclin E, cdk2, p21, p27, p57, and cdk2 kinase activity were identical in PDGF-BB-stimulated cells from MA and SV. However, apoptosis rates were higher in MA than SV determined by lactate dehydrogenase release, DNA fragmentation, and Hoechst 33258 staining. Moreover, caspase inhibitors (Z-VAD-fmk, Boc-D-fmk) abrogated the different proliferation rates of VSMC from MA versus SV. Western blotting and GSK3-beta kinase assay revealed lower Akt activity in VSMC from MA versus SV, while total Akt expression was identical. Adenoviral transduction of a constitutively active Akt mutant abrogated the different proliferation rates of VSMC from MA versus SV. CONCLUSIONS: Higher apoptosis rates due to lower Akt activity rather than different cell cycle regulation account for the lower proliferation of VSMC from MA as compared to SV. VSMC apoptosis may protect MA from bypass graft disease.

Toxic effects of lipid-mediated gene transfer in ventral mesencephalic explant cultures

Adverse effects of cDNA and oligonucleotide delivery methods have not yet been systematically analyzed. We introduce a protocol to monitor toxic effects of two non-viral lipid-based gene delivery protocols using CNS primary tissue. Cell membrane damage was monitored by quantifying cellular uptake of propidium iodide and release of cytosolic lactate dehydrogenase to the culture medium. Using a liposomal transfection reagent, cell membrane damage was already seen 24 hr after transfection. Nestin-positive target cells, which were used as morphological correlate, were severely diminished in some areas of the cultures after liposomal transfection. In contrast, the non-liposomal transfection reagent revealed no signs of toxicity. This approach provides easily accessible information of transfection-associated toxicity and appears suitable for prescreening of transfection reagents.