[Testing of medicinal products produced from pooled plasma]. / Prüfung von aus gepooltem Plasma hergestellten Produkten.

Medicinal products produced from human plasma fall under the administrative batch release procedure of the competent authority. In Germany, this has been carried out since 1995 by the Paul Ehrlich Institute (PEI), the responsible state control agency for blood products. Medicinal products released for the European and national market are tested for quality, efficacy and safety. Experimental testing of the final product and the starting materials, the plasma pools, as well as control of the production documentation guarantee a constantly high product safety. In the 28,000 batches tested since the beginning of the state controlled batch release testing of these blood products at the PEI, there has been no transmission of infectious viruses (HIV, HBV and HCV) to any patient. The batch release has made a contribution to the improvement of product quality. This procedure is still an important tool to ensure safety of blood products. The PEI is integrated in the batch release network of the European Directorate for the Quality of Medicines & Health Care (EDQM) in Strasbourg. Regulations and guidelines for official control authority batch release (OCABR) ensure harmonized procedures for mutual recognition of batch release on the European level. The EU certificates and German national certificates are requested and accepted in over 70 countries worldwide. Experimental testing in the EU and the requisite certificates have developed into a seal of quality for the world market.

Genomic organization and characterization of the promoter of the human ATP-binding cassette transporter-G1 (ABCG1) gene.

The ATP-binding cassette transporter G1 (ABCG1) was recently identified as a regulator of macrophage cholesterol and phospholipid transport. This transporter together with ABCA1 belongs to a group of sterol-sensitive ABC proteins which are induced by lipid loading or specific oxysterols. We report here the genomic structure of ABCG1 along with the 5' flanking sequence using library screening and BLAST search analysis. The ABCG1 gene spans more than 70 kb and contains 15 exons. The exon size is between 30 and 1081 bp and the introns range in size from 137 bp to more than 45 kb. All exon-intron boundaries display the canonical GT/AG sequences. Using promoter-luciferase reporter assays in the myeloid cell lines THP-1 and RAW246.7 and the hepatoma cell line HepG2 we could demonstrate the functionality of the ABCG1 promoter and the minimal sequence requirements for gene expression. The TATA-less proximal promoter contains multiple Sp1 binding sites and a consensus sequence for sterol regulatory element binding protein.

Normoxic cardiopulmonary bypass reduces oxidative myocardial damage and nitric oxide during cardiac operations in the adult.

OBJECTIVE: Hyperoxic cardiopulmonary bypass is widely used during cardiac operations in the adult. This management may cause oxygenation injury induced by oxygen-derived free radicals and nitric oxide. Oxidative damage may be significantly limited by maintaining a more physiologic oxygen tension strategy (normoxic cardiopulmonary bypass). METHODS: During elective coronary artery bypass grafting, 40 consecutive patients underwent either hyperoxic (oxygen tension = 400 mm Hg) or normoxic (oxygen tension = 140 mm Hg) cardiopulmonary bypass. At the beginning and the end of bypass this study assessed polymorphonuclear leukocyte elastase, nitrate, creatine kinase, and lactic dehydrogenase, antioxidant levels, and malondialdehyde in coronary sinus blood. Cardiac index was measured before and after cardiopulmonary bypass. RESULTS: There was no difference between groups with regard to age, sex, severity of disease, ejection fraction, number of grafts, duration of cardiopulmonary bypass, or ischemic time. Hyperoxic bypass resulted in higher levels of polymorphonuclear leukocyte elastase (377 +/- 34 vs 171 +/- 32 ng/ml, p = 0.0001), creatine kinase 672 +/- 130 vs 293 +/- 21 U/L, p = 0.002), lactic dehydrogenase (553 +/- 48 vs 301 +/- 12 U/L, p = 0.003), antioxidants (1.97 +/- 0.10 vs 1.41 +/- 0.11 mmol/L, p = 0.01), malondialdehyde (1.36 +/- 0.1 micromol/L,p = 0.005), and nitrate (19.3 +/- 2.9 vs 10.1 +/- 2.1 micromol/L, p = 0.002), as well as reduction in lung vital capacity (66% +/- 2% vs 81% +/- 1%,p = 0.01) and forced 1-second expiratory volume (63% +/- 10% vs 93% +/- 4%, p = 0.005) compared with normoxic management. Cardiac index after cardiopulmonary bypass at low filling pressure was similar between groups (3.1 +/- 0.2 vs 3.3 +/- 0.3 L/min per square meter). [Data are mean +/- standard error (analysis of variance), with p values compared with an oxygen tension of 400 mm Hg.] CONCLUSIONS: Hyperoxic cardiopulmonary bypass during cardiac operations in adults results in oxidative myocardial damage related to oxygen-derived free radicals and nitric oxide. These adverse effects can be markedly limited by reduced oxygen tension management. The concept of normoxic cardiopulmonary bypass may be applied to surgical advantage during cardiac operations.

UVB radiation and its role in the treatment of postmenopausal women with osteoporosis.

In humans, the serum concentration of parathyroid hormone (PTH) is higher in winter than in summer. The increase of PTH can be suppressed by oral vitamin D supplements, which is considered beneficial to those with osteoporosis. The present study investigates whether this effect can also be achieved by serial ultraviolet (UV) irradiation of the skin. In total, 34 women suffering from postmenopausal osteoporosis were included in the open trial. In late winter, 20 patients were irradiated with a spectrum containing UVB, eight times over a period of 4 weeks. The serum concentrations of 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2D], PTH, osteocalcin, alkaline phosphatase (AP), calcium and phosphorus were measured before the first, and 2 days after the last, dose of radiation. The data were compared to the controls (n = 14, no UV exposure), who were evaluated once at the start of the study and then again 4 weeks later. After UV irradiation the level of 25(OH)D was increased, whilst that of PTH remained unchanged. The serum level of osteocalcin decreased in the control group, but did not change in the group of women who had been exposed to UV radiation. The present study of osteoporotic women does not confirm previous findings in studies of healthy volunteers i.e. that PTH can be suppressed by exposure to UVB radiation in winter. Further studies are required to specify whether there are subgroups of osteoporotic people who may benefit from exposure to UVB radiation during winter.

Experimental application of controlled limb reperfusion after incomplete ischaemia.

Severe local and systemic complications may occur after revascularization of extremities exposed to prolonged complete or incomplete ischaemia. These complications may be reduced by controlling the reperfusate and the circumstances of the reperfusion period. Ten adult German domestic pigs were exposed to 6 h of incomplete limb ischaemia by occlusion of the left iliac artery. To simulate the clinical situation of embolectomy, the occlusive snares were released after the ischaemic period in five pigs and normal blood flow developed with systemic pressure (uncontrolled reperfusion). In the other five pigs, a controlled reperfusate was delivered at controlled pressure before establishing normal blood reperfusion (controlled reperfusion). At the end of the observation period (90 min after start of reperfusion), the group with controlled reperfusion had a lower mean(s.e.m.) tissue water content (81.8(0.7) versus 84.3(0.7) per cent, P < 0.05, a greater increase in tissue adenosine 5'-triphosphate compared with values at the end of ischaemia (6.2(1.5) versus -2.5(1.8) mumol per g protein, P < 0.03), a higher tissue pH (7.2(0.1) versus 6.8(0.1), P < 0.03), a smaller temperature decrease (0.3(0.2) versus 1.2(0.3) degrees C, P < 0.05), lower concentrations of creatine kinase (355.0(87.5) versus 624.4(73.4) units/l, P < 0.05) and lactate dehydrogenase (LDH) (369.5(42.5) versus 538.4(39.2 units/l, P < 0.03) in the femoral vein blood and lower LDH concentrations (356.5(48.9) versus 546.0(37.8 units/l, P < 0.03) in central venous blood. These data indicate that severe local and systemic damage occurs with uncontrolled (normal blood) reperfusion even after incomplete limb ischaemia, and that these changes can be reduced by delivering a controlled reperfusate under controlled conditions.

[Controlled reperfusion of the extremities for preventing local and systemic damage after prolonged ischemia. An experimental study with the swine model]. / Kontrollierte Extremitäten-Reperfusion zur Verminderung lokaler und systemischer Schäden nach mehrstündiger Ischämie. Eine experimentelle Studie am Schweinemodell.

Our previous studies in isolated rat hindlimbs using crystalloid perfusion solutions have shown that control of the initial reperfusion reduces postischemic complications. However, no experimental study has been undertaken to evaluate the concept of controlled limb reperfusion experimentally in an in-vivo blood-perfused model and to assess the local as well as systemic effects of normal blood reperfusion and controlled limb reperfusion. Of twenty pigs undergoing preparation of the infrarenal aorta and iliac arteries, six were observed for 7.5 hours and served as controls. Fourteen other pigs underwent 6 hours of complete infrarenal occlusion. Thereafter, embolectomy was stimulated in 8 pigs by removing the aortic clamp and establishing normal blood reperfusion at systemic pressure. In 6 other pigs, control of the composition of the reperfusate and control of the conditions of reperfusion was done during the first 30 min, followed by normal blood reperfusion. Six hours of infrarenal aortic occlusion lead to a severe decrease in high energy phosphates and muscle temperature and a slight increase in creating kinase (CK) and potassium in the systemic circulation. Normal blood reperfusion resulted in severe reperfusion injury: massive edema developed (80.6% vs. 76.6%, p < 0.0009), the tissue showed a marked decrease in oxygen consumption (7.3 +/- 1.1 vs. 14.3 +/- 2.5 mL )2/100 g/min, p < 0.02), glucose consumption (0.19 +/- 0.06 vs. 0.51 +/- 0.03 mg/100 g/min, p < 0.06), tissue ATP (18.3 +/- 1.9 vs. 36.1 +/- 0.9 mumol/g protein, p < 0.000001), total adenine nucleotides (26.3 +/- 2.6 vs. 45.8 +/- 1.5 mumol/g protein, p < 0.00001), muscle pH (5.9 +/- 0.1 vs. 7.3 +/- 0.1, p < 0.000006) and total calcium in the femoral vein (2. +/- 0.1 vs. 2.7 +/- 0.1 mmol/L, p < 0.002). Furthermore, a massive increase was seen in CK concentration (12,743 +/- 2,562 vs. 513 +/- 80 U/L, p < 0.0003), potassium (7.9 +/- 0.3 vs. 4.4 +/- 0.2 mmol/L, p < 0.000001) and muscle rigidity (60 +/- 11 vs. 122 +/- 1 degree, p < 0.00008). In sharp contrast, initial treatment of the ischemic skeletal muscle by controlled limb reperfusion resulted in normal water content (77.6 +/- 0.4 vs. 76.8 +/- 0.3%), oxygen consumption (13.2 +/- 1.6 vs. 14.9 +/- 3.2 mL O2/100 g/min), glucose consumption (0.58 +/- 0.18 vs. 0.46 +/- 0.11 mg/100 g/min), flow (5.4 +/- 1.1 vs. 4.6 +/- 4.6 +/- 0.5 mL/100 g/min) and muscle rigidity (106 +/- 4 vs. 122 +/- 1 degree). Furthermore, controlled limb reperfusion resulted in higher total adenine nucleotides content (78% vs. 57% of control), less tissue acidosis (6.6 +/- 0.2 vs. 5.9 +/- 0.1, p < 0.002), severely reduced CK release (2,618 +/- 702 vs. 12,743 +/- 2.562, p < 0.02) and potassium release (5.1 +/- 0.3 vs. 7.9 +/- 0.3 mmol/L, p < 0.0002) as compared to normal blood reperfusion. In conclusion this study shows that 6 hours of acute infrarenal aortic occlusion will result in a severe reperfusion injury (postischemic syndrome) if normal blood at systemic pressure is given in the initial reperfusion phase. In contrast, initial treatment of the ischemic skeletal muscle by controlled limb reperfusion reduces the metabolic, functional and biochemical alterations.

Reperfusion injury in skeletal muscle: controlled limb reperfusion reduces local and systemic complications after prolonged ischaemia.

Previous studies in isolated limbs using crystalloid perfusion solutions have shown that control of the initial reperfusion reduces postischaemic complications. However, no experimental study has been undertaken to evaluate the concept of controlled limb reperfusion experimentally in an in vivo blood-perfused model and to assess the local as well as systemic effects of normal blood reperfusion and controlled limb reperfusion. Of 20 pigs undergoing preparation of the infrarenal aorta and iliac arteries, six were observed for 7.5 h and served as controls; 14 others underwent 6 h of complete infrarenal occlusion. Thereafter, embolectomy was simulated in eight pigs by removing the aortic clamp and establishing normal blood reperfusion at systemic pressure. In six other pigs, the composition of the reperfusate and the conditions of reperfusion were controlled during the first 30 min, followed by normal blood reperfusion. Some 6 h of infrarenal aortic occlusion leads to a severe decrease in high-energy phosphates and muscle temperature, together with a slight increase in creatine kinase and potassium in the systemic circulation. Normal blood reperfusion resulted in severe reperfusion injury: massive oedema developed, the tissue showed a marked decrease in oxygen consumption, glucose consumption, tissue ATP, total adenine nucleotides, muscle pH and total calcium in the femoral vein. Furthermore, a massive increase was seen in plasma creatine kinase concentration and potassium, together with the development of muscle rigidity. In sharp contrast, initial treatment of the ischaemic skeletal muscle by controlled limb reperfusion resulted in normal water content, oxygen consumption, glucose consumption, flow and muscle rigidity. Furthermore, controlled limb reperfusion resulted in higher total adenine nucleotides content, less tissue acidosis, markedly reduced creatine kinase release, and potassium release as compared with that of normal blood reperfusion. This study shows that 6 h of acute infrarenal aortic occlusion will result in severe reperfusion injury (postischaemic syndrome) if normal blood at systemic pressure is given in the initial reperfusion phase. In contrast, initial treatment of the ischaemic skeletal muscle by controlled limb reperfusion reduces the metabolic, functional and biochemical alterations.

Bone metabolism before and after irradiation with ultraviolet light.

The beneficial effects of ultraviolet light on cutaneous vitamin D synthesis, calcium metabolism, and bone formation are well known. Regarding the increasing fear of side effects from ultraviolet B (UV-B), lamps with less energy in the UV-B range have been developed. Two spectra with differences in the emission of UV-B have therefore been evaluated for their influence on calcium metabolism. A group of 24 healthy male volunteers was subdivided into two treatment groups. Group 1 was exposed to lamps with higher energy of total UV-B but less energy at the wavelengths below 300 nm than the lamps used in group 2. All subjects were irradiated ten times within 12 days. Exposure time was 3 min in the first session and time of exposure was increased by 10% in every following irradiation (suberythematous doses only). Before the first irradiation, 3 days after the last exposure, and after 4 more weeks, the serum parameters of bone metabolism were determined by standard laboratory methods. Significantly increased levels of 25-hydroxyvitamin D3 were found in both groups. There was only a slight increase of 1,25-dihydroxyvitamin D3. Parathyroid hormone decreased significantly in group 2 only. The data would suggest beneficial effects on bone metabolism for both regimens. The observed effects were more pronounced when shorter wavelengths (group 2) were applied, although the total energy of UV-B was lower in these lamps.

Cyclosporin treatment in rheumatoid arthritis is associated with an increased serum activity of beta-glucuronidase.

The serum activity of beta-glucuronidase (beta-gluc) has been presumed to indicate the disease activity in rheumatoid arthritis (RA). In 10 patients with RA the serum beta-gluc was repeatedly determined after the initiation of a treatment with cyclosporin for one year. A significant increase of beta-gluc was found after 8, 12 and 16 weeks compared to the values before treatment, while the concentration of the soluble interleukin 2-receptor decreased. The data reveal, that beta-gluc is not a useful indicator of the disease activity during cyclosporin treatment.

[Serum level of beta-glucuronidase as potential indicator of disease activity in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE)]. / beta-Glucuronidase-Konzentration im Serum als potentieller Indikator der Krankheitsaktivität bei rheumatoider Arthritis (RA) und systemischem Lupus erythematodes (sLE).

The lysosomal enzyme beta-glucuronidase is involved in the degradation of glycosaminoglycans to monosaccharides. beta-glucuronidase-activity has been found to correlate well with histomorphological changes in active arthritis. By use of a new--enzyme kinetic--assay serum activity of beta-glucuronidase was measured in 32 patients with rheumatoid arthritis (RA) and 23 patients with systemic lupus erythematodes (sLE) and compared to 120 healthy volunteers. In 72% of the patients with RA and 65% of the patients with sLE increased beta-glucuronidase-activity was found (greater than 97.5. percentile of healthy volunteers). There was no correlation with an estimated score of disease activity, duration of morning stiffness, or blood sedimentation rate. The determination of beta-glucuronidase revealed increased serum levels in most of the patients with RA and sLE. The role of beta-glucuronidase as a predictor of joint destruction remains to be evaluated in a prospective study. For this evaluation the use of the enzyme-kinetic method described here is recommendable.

Anti-human immunodeficiency virus (HIV) drug HOE/BAY946 increases membrane hydrophobicity of human lymphocytes and specifically suppresses HIV-protein synthesis.

The polysulfated polyxylan HOE/BAY946, which has been tested in two pilot studies in ARC/AIDS patients and in asymptomatic HIV carries in Germany, was believed to act by inhibiting virus attachment to the cell. However, the drug was also found to reduce the amount of HIV particles released from infected peripheral blood mononuclear cells (PBMC) in vitro. Furthermore, preincubation of PBMC with the drug led to a partial inhibition of a following HIV infection, suggesting that the drug also affects virus entry. Electron Paramagnetic Resonance (EPR) measurements on uninfected human lymphocytes using 5-proxyl-nonane as spin label demonstrated smaller hyperfine coupling constant (aN) values in the presence of HOE/BAY946 or dextran sulfate 5000. Accordingly, h-1p/h-1H ratios were decreased, indicating increased plasma membrane hydrophobicity and a membrane-stabilizing effect of the drugs. Culture of the chronically HIV-infected monocytic cell line U937/HIV-2D194 in the presence of HOE/BAY946 specifically and drastically reduced the release of virions and the intracellular synthesis of viral proteins as determined by radioimmunoprecipitation and reverse transcriptase assays. In conclusion, although the EPR studies showed a physico-chemical effect on membrane polarity, HOE/BAY946 and dextran sulfate clearly affect processes beyond the cell membrane. Thus, in contrast to previous reports suggesting that polysulfated sugars affect HIV only by inhibiting virus binding to uninfected cells, they clearly inhibit HIV in infected cells as well and appear to have a pleiotropic mode of action. Such drugs may be less likely to result in viral resistance after prolonged application than substances acting only on one step in the life cycle of the virus.