Variability of parvovirus B19 genotype 2 in plasma products with different compositions in the inactivation sensitivity by liquid-heating.

BACKGROUND AND OBJECTIVES: Our previous report showed that parvovirus B19 genotype 1 in different solutions derived from plasma preparations showed different heat-sensitivity patterns during liquid-heating. In this study, we similarly examined B19 genotype 2. MATERIALS AND METHODS: Two plasma samples one containing B19 genotype 1 and the other genotype 2 DNA were used. Four process samples collected immediately before the heat treatment step in the manufacture of albumin, immunoglobulin, haptoglobin and antithrombin preparations were spiked with B19 and subsequently treated at 60°C for 10 h. A low pH immunoglobulin solution was also spiked with B19 and treated at room temperature for 14 days. Infectivity was then measured. RESULTS: B19 genotype 2, similar to genotype 1, showed three patterns of inactivation: (i) a rapid inactivation in the albumin and immunoglobulin preparations, (ii) a slow inactivation in the haptoglobin preparation and (iii) only limited inactivation in the antithrombin preparation. Its sensitivity in the low pH immunoglobulin solutions also resembled that of genotype 1. CONCLUSION: Both genotypes 1 and 2 of B19 varied in sensitivity to liquid-heating and low pH among different plasma preparations.

Extent of hepatitis E virus elimination is affected by stabilizers present in plasma products and pore size of nanofilters.

BACKGROUND AND OBJECTIVE: To investigate the physico-chemical properties of hepatitis E virus (HEV) with regard to inactivation/removal, we have studied four isolates with respect to sensitivity to heat during liquid/dry-heating as well as removal by nanofiltration. MATERIALS AND METHODS: Hepatitis E virus in an albumin solution or phosphate-buffered saline (PBS) was liquid-heated at 60 degrees C for a preset time. HEV in a freeze-dried fibrinogen containing stabilizers was also dry-heated at 60 or 80 degrees C for a preset time. In addition, to clarify the removal of HEV, the purified virus in PBS was filtered using several types of virus-removal filter (nanofilters) that have different pore sizes. HEV infectivity or genome equivalents before and after the treatments were assayed by a semiquantitative cell-based infectivity assay or quantitative polymerase chain reaction assay, respectively. RESULTS: Hepatitis E virus isolates in albumin solutions were inactivated slowly at 60 degrees C for 5 h and the resultant log reduction factor (LRF) was from 1.0 to > or = 2.2, whereas the virus in PBS was inactivated quickly to below the detection limit and the LRF was > or = 2.4 to > or = 3.7. The virus in a freeze dried fibrinogen containing trisodium citrate dihydrate and l-arginine hydrochloride as stabilizers was inactivated slowly and the LRF was 2.0 and 3.0, respectively, of the 72 h at 60 degrees C, but inactivated to below the detection limit within 24 h at 80 degrees C with an LRF of > or = 4.0. The virus in PBS was also confirmed as to be approximately 35 nm in diameter by nanofiltration. These results are useful for evaluating viral safety against HEV contamination in blood products. CONCLUSION: The sensitivity of HEV to heat was shown to vary greatly depending on the heating conditions. On the other hand, the HEV particles were completely removed using 20-nm nanofilters. However, each inactivation/removal step should be carefully evaluated with respect to the HEV inactivation/removal capacity, which may be influenced by processing conditions such as the stabilizers used for blood products.

Variability of parvovirus B19 to inactivation by liquid heating in plasma products.

BACKGROUND AND OBJECTIVES: Previously, we reported that although human parvovirus B19 in albumin and intravenous immunoglobulin preparations was rapidly inactivated during liquid heating, in contrast to other parvoviruses such as canine parvovirus, sensitivity to heat was highly dependent on the composition of the solution. In this study, we aimed to further elucidate the sensitivity to heat of B19 in haptoglobin and antithrombin (previously named antithrombin III) preparations during liquid heating. MATERIALS AND METHODS: Two different solutions collected immediately before heat treatment of haptoglobin and antithrombin preparations were spiked with B19 and subsequently treated at 60 degrees C for 10 h. B19 DNA-positive, anti-B19 IgG/IgM-negative plasma was used as a source of B19. The residual infectivity in each sample was measured using a B19 cell-based infectivity assay with an mRNA polymerase chain reaction. RESULTS: B19 in different plasma preparations showed different heat-sensitivity patterns during liquid heating: (i) slow inactivation in haptoglobin preparations, and (ii) only limited inactivation in antithrombin preparations. The kinetics of inactivation was greatly different from that in our previous studies in which the virus was shown to be rapidly inactivated in albumin and intravenous immunoglobulin preparations. CONCLUSION: B19 has unique properties in terms of heat sensitivity, depending on the composition of the solution during liquid heating. This finding may indicate the need for caution when interpreting the sensitivity of B19 to heat.

Heat sensitivity of a SARS-associated coronavirus introduced into plasma products.

BACKGROUND AND OBJECTIVES: Various measures to inactivate/remove viruses have been implemented for manufacturing plasma-derived products. Here, we examined the heat inactivation ability of an agent of the severe acute respiratory syndrome (SARS), SARS coronavirus (CoV). MATERIALS AND METHODS: The Frankfurt-1 strain of SARS-CoV was incorporated in manufacturing processes of several products by using samples collected immediately before liquid heat treatment at 60 degrees C. RESULTS: SARS-CoV was easily inactivated by this treatment for 60 min in all in-process samples. However, the different composition of the tested samples affected the heat sensitivity of the virus strain: the infectivity of the virus in Antithrombin III preparation still remained after heating for 30 min at 60 degrees C. CONCLUSION: If by rare chance SARS-CoV contaminates source plasma, there should be no or only minor risk of this virus infection, due to sufficient inactivation by the 60 degrees C 10 h liquid heating step, although we must pay attention to the composition used for blood product preparation.

Heat sensitivity of human parvovirus B19.

BACKGROUND AND OBJECTIVES: To date there has been no published report on a systematic evaluation of the heat sensitivity of human parvovirus B19 (B19) and the related safety of the plasma-derived fractionated products. In this study, we examined the heat sensitivity of B19 by using the infectivity assay with cultured cells. MATERIALS AND METHODS: The heat sensitivity of B19 was examined by measuring the reduction in viral infectivity titres after heating liquid containing B19 at 60 degrees C. Viral infectivity was assayed by detection of viral antigens or viral mRNA in infected cells. As a control, canine parvovirus (CPV) was also heat-treated. RESULTS: B19 displayed quite different inactivation kinetics to CPV when both were heated in liquid at 60 degrees C. In sharp contrast to the latter, B19 was rapidly inactivated within 1 h when the virus was suspended in 5% or 25% human serum albumin solution, phosphate-buffered saline, or complete medium. However, B19 appeared to be resistant to heat inactivation in liquid containing 60% sucrose. CONCLUSIONS: The heat sensitivity of B19 in liquid was clearly different from that of CPV. Significantly, the efficiency to inactivate B19 and reduce its infectivity following heating in liquid was mainly affected by the composition of the solutions used for virus suspension.

An automated assay for measuring serum ascorbic acid with use of 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical and o-phenylenediamine.

We developed a novel, cost-effective, and automated assay for ascorbic acid (AsA) in serum using a COBAS MIRA S analyzer (Roche Diagnostic System). Our method has a wide dynamic range and covers AsA concentrations from well below the lower reference interval to well above it. AsA is oxidized by 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy, free radical (TEMPO) to dehydroascorbic acid (DAsA). The latter condenses with o-phenylenediamine (OPDA) to form a quinoxaline derivative that absorbs light at 340 nm. The change in absorbance at 340 nm is proportional to the concentration of AsA in the specimen. The automated system permitted the assay of 65 specimens per hour at a cost of approximately US$ 0.01 per specimen for reagents. The assay can be applied directly to serum specimens (direct method) and also to sera with a prior deproteinization step with metaphosphoric acid. The detection limit for the direct serum assays is 0.8 vs. 0.4 mg/l with the deproteinization method. The recovery of AsA from a supplemented serum pool was of >95% for both procedures. We used four distinct methods on 66 patients sera. The direct method for AsA correlated well with an HPLC method (r=0.964, P<0.001); the direct method also correlated well with a method that uses AsA oxidase (r=0.975, P<0. 001). The deproteinization method correlated well with HPLC (r=0.981, P<0.001), and with the AsA oxidase procedure (r=0.994, P<0.001). Ten within-day determinations on a serum pool gave a C.V. <4.3% for both the direct and deproteinization procedures. The between-day assays of the same serum pool over 10 days gave a C.V. of <6.7% by both methods.

Effect of long-term exogenous hyperinsulinemia and fructose or glucose supplementation on triglyceride turnover in rats.

We examined the effect of long-term (6 months) hyperinsulinemia on VLDL-triglyceride turnover in male Wistar rats. Hyperinsulinemia was induced in rats by daily s.c. injection of Ultralente insulin (6 U/day at 19:00). Fructose (F) or glucose (G) was supplied in the drinking water (10%) in order to prevent hypoglycemia. The rats were divided into 5 groups: (1) hyperinsulinemia with F water: group F + I; (2) hyperinsulinemia with G water: group G + I; (3) F water alone: group F; (4) G water alone: group G; and (5) control rats without sugar water group C. After 6 months of daily insulin injection triglyceride secretion rate (TGSR) was estimated using Triton WR1339 in all the rats. Groups F + I and G + I were obese and hypoglycemic compared to the other groups. Fasting plasma glucose level of group F was higher than any other group value. TGSR of group F + I was significantly higher than that of the control group, while that of group G + I was not, indicating that long-term hyperinsulinemia can stimulate hepatic triglyceride production when the rats were supplemented only with fructose. On the other hand, the rats in group G + I showed the lowest plasma free fatty acid level of all and their postheparin lipolytic activity was significantly elevated compared to that of the control rats. Moreover, they had suppressed plasma triglyceride levels and its fractional catabolic rate was significantly increased, suggesting that hyperinsulinemia can still stimulate triglyceride removal from the circulation of glucose supplemented rats even at month 6. In conclusion, exogenous hyperinsulinemia can stimulate hepatic triglyceride secretion even after 6 months duration when supplemented with fructose, while its stimulating effect on triglyceride removal from the circulation can be seen only with glucose supplementation. Thus, the effect of long-term hyperinsulinemia on plasma triglyceride turnover differs depending on the supplemented monosaccharides.

Organization and nucleotide sequences of ten ribosomal protein genes from the region equivalent to the S10 operon in the archaebacterium, Halobacterium halobium.

A determination was made of the nucleotide sequence of the 7340-bp region of a ribosomal protein gene cluster of Halobacterium halobium, which is equivalent to the S10 operon of Escherichia coli. The sequence was analyzed with the codonpreference program deduced from the halobacterial codon usage table that showed a very high GC content of the third codon position. The sequence was comprised of a string of 13 tightly linked ORFs. Most of the ORFs were homologous with ribosomal protein genes (ORF1-ORF2-rpl3-rpl4-rpl23--rpl2- rps19-rpl22-rps3-rpl29-ORF11-rps17-r pl14). The 13-gene string was preceded by three putative AT-rich promoter sequences. The order of the genes in H. halobium essentially agreed with that of the corresponding genes of E. coli (S10-operon), except for certain deletions or insertions of additional protein genes.

Thrombospondin modulates adhesion, proliferation and production of extracellular matrix in mesangial cells.

Thrombospondin (TSP) is produced by glomerular mesangial cells and one of the extracellular matrix in the mesangium, whereas the physiological role of TSP in mesangial cells is poorly understood. In order to know whether TSP modulates mesangial cell functions, we investigated the effects of TSP on cell adhesion, proliferation, synthesis of extracellular matrix and serine proteinases in cultured human mesangial cells. The substratum of TSP inhibited cell attachment and spreading in a TSP-dose-dependent manner in mesangial cells. Soluble TSP (50 micrograms/ml) also caused the detachment of fully adherent mesangial cells, whereas TSP less than 10 micrograms/ml did not. [3H]-thymidine incorporation into mesangial cells was dose-dependently reduced by TSP. On the other hand, the production of both fibronectin and type IV collagen from mesangial cells was enhanced by TSP. The incubation of mesangial cells with TSP increased the secretion of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), while plasminogen activator inhibitor-type 1 (PAI-1) decreased. These observations indicate that TSP inhibits cell adhesion and proliferation in cultured human mesangial cells. It is also suggested that TSP influences the metabolism of mesangial matrix by modulating both synthesis and degradation of matrix components. Thus, TSP, may be an important mediator of mesangial cell functions in an autocrine fashion.

[An adhesion dependent injury of target cell membrane by NK cell surface associated metaloprotease].

Natural killer (NK) cytotoxicity was assayed with P3-X63-Ag8-U1 (P3U-1) target cells which had been previously demonstrated to release endogenous alkaline phosphatase (AlP) on the attack of lymphocyte-activated killer cells). P3U-1 cells showed a definite sensitivity to the AlP-release test, but no response in the Cr-release test at all. The AlP-release was not inhibited by anti-perforin antibody, benzoate, phenyl-methyl-sulfonyl-fluoride, soybean trypsin inhibitor, Succinyl-Gly-Pro-Leu-Gly-Pro-amino-methyl-coumarin, or gamma-radiation to effector cells, but was inhibited by o-phenanthroline, anti-CD13 antibody, and anti-LFA-1 alpha antibody. The AlP-release from P3U-1, therefore, did not appear to be brought on by the NK cell-derived perforin, hydroxy-radical, granzymes or cytosolic proteases. The inhibition by o-phenanthroline and the antibody for CD13 (aminopeptidase N) or the adhesion factor in NK cells, however, indicated that the membrane of such cells with adhesion ligand to NK cells was probably susceptible to NK cell surface-associated metaloprotease in an adhesion dependent manner to the extent of some injury without complete perforation through the membrane.

[Cytotoxicity test based on luminescent assay of alkaline phosphatase released from target cells].

Assay of 51Cr release from target cells has been commonly used in various methods of examining the cytotoxic properties of lymphocytes. In this paper a non-isotopic assay of cytotoxicity based on the leak of endogenous alkaline phosphatase (AIP) in target cells, is described. Enzyme activities were assayed by the luminescence on hydrolysis of the lumigen-PPD substrate. P3-X63-Ag8-U1 (P3U-1) cells were demonstrated to contain AIP and proved sensitive to the IL-2-induced killer lymphocytes, while no AIP activity was detected in human effector lymphocytes. Comparative studies of the test with 51Cr- and AIP-release in P3U-1 target cells were carried out, and the results obtained suggested that the AIP release test is useful as a new, simple lymphocyte cytotoxicity test.

Hepatic cirrhosis showing false-positive serum C-reactive protein reaction.

A 58-year-old male who had been diagnosed as hepatic cirrhosis four years previously was admitted to our hospital because his serum C-reactive protein (CRP) level had gradually risen, reaching 139 mg/dl. No inflammation findings were observed subjectively or objectively. Close examination revealed his CRP reaction to be false positive. His serum CRP showed positive only in a latex agglutination method using goat anti-CRP IgG. This false-positive reaction was thought to be owing to the abnormally glycosylated IgM, which has an affinity for the goat serum IgG.

Discrete phase of calcium excess in the process of deterioration in an individual rat myocyte.

While there have been many reports of the significant role of cytoplasmic free calcium ion in myocardial injury, these have been carried out in multicellular preparations. Since cell injury may occur inhomogeneously, it is necessary to observe the 'history' of an individual myocyte in order to investigate the detailed role of the calcium ion in the process of myocardial injury. We have observed the natural history of individual myocytes isolated from the left ventricle of rats with respect to changes in shape and cytoplasmic free calcium concentration ([Ca2+]i) measured with fura-2. We can discriminate four phases in the time course of cell deterioration. In the first phase (phase O), the myocyte is rod shaped, quiescent and responsive to electrical stimulation. The [Ca2+]i is stable. In the next phase (Phase 1), once initiated, the myocyte exhibits an asynchronous wavy contraction and gradually decreases in length. The [Ca2+]i gradually increases with some fluctuation. Phase 2 is characterized by rapid development of contracture with a marked increase in [Ca2+]i. In the period following establishment of contracture (Phase 3), changes in [Ca2+]i vary from cell to cell, possibly because of leakage of the dye caused by loss of cell membrane integrity. Our results indicate that, during naturally occurring cell deterioration, loss of [Ca2+]i control at the membrane of the sarcoplasmic reticulum precedes contracture and catastrophic increase in [Ca2+]i.

Suppression of cytoplasmic Ca2+ response and protein secretion by oxidizing agents.

The influence of the oxidizing agents (H2O2, KO2 and Vitamin K) on the action of vasopressin to guinea pig hepatocytes was investigated from the view-point of cytoplasmic Ca2+ concentration and protein secretion. 10 nmol/l vasopressin brought about increase in prothrombin secretion along with increase in the cytoplasmic Ca2+ concentration compared to the non-stimulation level. The pretreatment of the cells with 1 mumol/l of the oxidizing agents, however, led to suppression of Ca2+ elevation and inhibited the vasopressin-induced prothrombin secretion completely, while no leak if lactic dehydrogenase (LDH), Na+ and K+ were detected. The same results of the inhibition in fibrinogen and albumin secretion were observed. These results suggested a possibility that the oxidizing agents such as the peroxides act on some site of cellular signal transduction system in cell membrane to reduce the cytoplasmic Ca2+ level and to suppress the vasopressin-induced secretion.

The effect of oxidizing agents on cell division and shape.

Certain oxidizing agents such as vitaminK(VK) and lipid peroxides were found to suppress an increase in cytoplasmic Ca2+ concentration by growth factors, and inhibit on cell proliferation. These oxidizing agents induced a marked change in cell shape. In a detailed analysis of each phase in the cell cycle, the inhibition of an increase in cytoplasmic Ca2+ and cell division occurred only when the agents were added at G0/G1 phase. The addition to S or M phase cells did not influence in cytoplasmic Ca2+ and cell division. These experimental results suggest that these oxidizing agents may inhibit the transfer of stimulation signals from growth factors by acting on cell membrane sites and suppress subsequent DNA replication and mitotic division.

Propagation and collision characteristics of calcium waves in rat myocytes.

In myocytes, local contractions occur spontaneously and propagate as traveling waves. We observed the waves in myocytes as local changes in fura-2 fluorescence and determined some characteristics of the wave. Myocytes were enzymatically isolated from rat left ventricles and incubated with 2 microM fura-2/AM for 60 min. Microscopic fluorescence images of myocytes were recorded with a high-sensitivity video camera. The images were digitally analyzed, frame by frame, and temporal changes in local fluorescence were displayed. With the excitation wavelength at 380 nm, the darker band propagates as the traveling wave. With the excitation wavelength at 340 nm, the wave appears brighter. With the isosbestic wavelength at 360 nm, the wave is not discernible. The waves are thus considered to be traveling waves of change in local cytoplasmic calcium ion concentration (calcium wave). Velocity, amplitude, and width of the calcium waves appeared to be fairly constant during their propagation. When two waves propagating in opposite directions collided, summation of the waves did not occur. After the collision both waves disappeared. These observations support the idea that the waves propagate by inducing calcium release from adjacent sarcoplasmic reticulum. Phenomena observed during the collision indicate that there is a refractory period after the calcium transient; spatially, a refractory zone exists in the wake of the wave.

The third nationwide survey in Japan of vitamin K deficiency in infancy.

The occurrence of hemorrhagic disease due to vitamin K (VK) deficiency beyond the neonatal period has come under investigation in Japan. In 1980 the 1st nationwide survey was conducted in Japan by Nakayama and others, and was followed by the 2nd nationwide survey in 1985 by Hanawa. The present survey was designed to further monitor the incidence of this disease in Japan during the 3-year period from July 1985 to June 1988. Questionnaires were sent to 1,315 hospitals having more than 200 beds, located throughout Japan. Responses were received from 775 hospitals, for an answer rate of 58.9%. The total number of reported cases was 175, including 129 idiopathic type, 28 secondary type and 18 near-miss type. In this survey it was revealed that the incidence rate of the idiopathic type of vitamin K deficiency in infancy (VKDI) has decreased remarkably, to about one-fourth that reported in the first survey. The declining incidence rate of VK deficiency in Japan is considered to be the result of ever more widespread prophylactic administration of VK during the neonatal period, as most occurrences of VK deficiency in infancy are preventable by prophylactic administration of VK from the neonatal period. However, in 16 cases of the idiopathic type of VK deficiency found in the present survey, VK had been administered at least once during or after the neonatal period. This shows the heterogeneity of this condition.