Fusion angle affects intervertebral adjacent spinal segment joint forces-Model-based analysis of patient specific alignment.

This study addresses the hypothesis that adjacent segment intervertebral joint loads are sensitive to the degree of lordosis that is surgically imposed during vertebral fusion. Adjacent segment degeneration is often observed after lumbar fusion, but a causative mechanism is not yet clearly evident. Altered kinematics of the adjacent segments and potentially nonphysiological mechanical joint loads have been implicated in this process. However, little is known of how altered alignment and kinematics influence loading of the adjacent intervertebral joints under consideration of active muscle forces. This study investigated these effects by simulating L4/5 fusions using kinematics-driven musculoskeletal models of one generic and eight sagittal alignment-specific models. Models featured different spinopelvic configurations but were normalized by body height, masses, and muscle properties. Fusion of the L4/5 segment was implemented in an in situ (22°), hyperlordotic (32°), and hypolordotic (8°) fashion and kinematic input parameters were changed accordingly based on findings of an in vitro investigation. Bending motion from upright standing to 45° forward flexion and back was simulated for all models in intact and fused conditions. Joint loads at adjacent levels and moment arms of spinal muscles experienced changes after all types of fusion. Hypolordotic configuration led to an increase of adjacent segment (L3/4) shear forces of 29% on average, whereas hyperlordotic fusion reduced shear by 39%. Overall, L4/5 in situ fusion resulted in intervertebral joint forces closest to intact loading conditions. An artificial decrease in lumbar lordosis (minus 14° on average) caused by an L4/5 fusion lead to adverse loading conditions, particularly at the cranial adjacent levels, and altered muscle moment arms, in particular for muscles in the vicinity of the fusion. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:131-139, 2017.

Analysis of enzyme-treated red blood cell surface and haemagglutination using a theory of soft-particle electrophoresis.

BACKGROUND AND OBJECTIVES: Enzymatic treatment of red blood cells is thought to reduce the cell zeta (zeta) potential, effectively decreasing the distance between cells to less than the length of an immunoglobulin G antibody binding site, and resulting in agglutination of cells. However, the zeta potential given by Smoluchowski's formula is based on theories of the electrophoresis of hard colloidal particles. A theory has recently been developed for the electrophoresis of colloidal particles covered with polyelectrolytes, which we call 'soft particles'. MATERIALS AND METHODS: The electrophoretic mobility of red blood cell treated with papain and neuraminidase was measured as the electrolyte concentration of the medium using phosphate buffer. The results were analysed via the formula for 'soft particles'. This mobility formula involved two parameters, the fixed charge density (ZN) and parameter 1/lambda characterizing the 'softness' of the cell surface layer. RESULTS: The best-fit curves of 0.1 units neuraminidase-treated red blood cells indicated that ZN decreased by 76% and 1/lambda decreased by 8% compared to intact red blood cells. In contrast, in 5 units of papain-treated red blood cells ZN decreased by 45% and 1/lambda decreased by 33% compared to intact red blood cells. CONCLUSION: The present study shows that the change in ZN for neuraminidase-treated cells was very large, but the cells did not become agglutinable. Papain-treated cells had changes in both ZN and 1/lambda, and the cells became agglutinable. 1/lambda is one of the important factors for agglutination.

Differences in ABO antibody levels among blood donors: a comparison between past and present Japanese, Laotian, and Thai populations.

Passively transfused blood group antibodies cause clinical problems. High titers of anti-A and anti-B seem to be one reason for hemolytic transfusion reactions and for ABO HDN. In Japan, anti-A and anti-B titers notably decreased in the 15 years between 1986 and 2001. At present, titers of more than 100,as measured using the saline method, are rare. Differences in the level of anti-A and anti-B among ethnic populations have been reported; these differences were found to be the result of environmental factors rather than hereditary factors. In the present study, the anti-A and anti-B titers of random donors in three Asian populations are compared. In Thailand, the IgM anti-A and anti-B titers are low and are similar to the Japanese titers reported in 2001, but the IgG anti-A and anti-B titers are high and are similar to the Japanese titers reported in 1986. In the Lao People's Democratic Republic, both the IgM and the IgG anti-A and anti-B titers are high and are similar to those reported in Japan in 1986. In addition, anti-A and anti-B titers of different sex donors and of various age groups were also compared. High titers were found in 8.8 percent of the female donors in the younger than 30 age group and in 36.7 percent of the female donors in the older than 50 age group.

Enzyme and DTT treatment of adherent RBCs for antibody identification by a solid-phase immunoassay system.

Treatment of RBCs with protease enzymes or dithiothreitol (DTT) causes denaturation of several RBC antigens and is regularly used in antibody identification. In this study,we have standardized enzyme and DTT treatment of adherent RBCs in the magnetic-mixed passive hemagglutination assay (M-MPHA) for antibody identification. We have also tried drying these treated RBCs. The optimal enzyme and DTT treatment conditions for intact adherent RBCs were determined, in addition to the optimal condition for drying RBCs. All tested agents with the exception of chymotrypsin could be applied to RBC drying, but the optimal condition for drying was different from that for intact RBCs. Enzyme or DTT treatment of adherent RBCs and their application in M-MPHA provide a simple and convenient method for antibody identification. Furthermore, the technique of drying treated RBCs provides an even stronger antibody identification tool, since dried RBCs can be stored for a long period.

Proteolytic activation and inactivation of the serine protease activity of plasma hyaluronan binding protein.

We prepared anti-plasma hyaluronan binding protein (PHBP) mouse monoclonal antibodies and studied the fragmentation profile of PHBP with them. PHBP is present in human plasma as a single polypeptide chain (70 kDa). During the purification, PHBP partially fragmentated into the 50-kDa N-terminal fragment and the 27-kDa C-terminal fragment. After the incubation of the purified PHBP, the 70-kDa precursor form was completely cleaved to the 50- and 27-kDa fragments, followed by the 50-kDa to the 26-kDa, and the 27-kDa to the 17-kDa plus the 8-kDa fragments, respectively. Because the purified PHBP contained no other detectable proteins and PHBP has a typical serine protease domain, we concluded that the fragmentation of PHBP was caused by own serine protease activity. PHBP cleaved the C-terminal side of Arg in the peptide effectively and that of Lys weakly. The results of the pre-incubation experiments of PHBP suggested that the single-chain form of PHBP is a precursor, the two-subunit structure is an active form and the three- or four-chain structure is an inactive form of a serine protease.

Evaluation of a new solid-phase immunoassay for alloantibody detection using bromelin-treated and untreated red blood cells.

The enzyme test is used to detect certain antibodies or facilitate antibody identification. This study compares antibody reactivity with bromelin-treated red blood cells (RBCs) and untreated RBCs using a newly developed solid-phase immunoassay. The reactivity of irregular antibodies was tested by a magnetic-mixed passive hemagglutination assay (M-MPHA). In addition, antibody reactivity was tested with dried stroma of bromelin-treated RBCs and untreated RBCs (M-MPHA-Dry). Rh antibodies were detected with enzyme-treated intact RBCs and untreated RBCs by M-MPHA. The slight increase in reactivity using M-MPHA was not seen using dried RBC stroma (M-MPHA-Dry). All donor-derived IgG alloantibodies, which were detected by either a conventional tube enzyme test or an indirect antiglobulin test, were detected by M-MPHA without using enzyme-treated RBCs. Both M-MPHA and M-MPHA-Dry can be used for antibody detection without using enzyme-treated RBCs and are also useful for antibody identification.

The novel acute phase protein, IHRP, inhibits actin polymerization and phagocytosis of polymorphonuclear cells.

OBJECTIVE AND DESIGN: In the present study, the involvement of the binding of IHRP (inter-alpha-trypsin inhibitor family heavy chain-related protein) and actin in phagocyte activity was investigated. MATERIALS AND METHODS: The actin polymerization and the phagocytic activity of the polymorphonuclear (PMN) cells were studied in the presence of IHRP. RESULTS: IHRP inhibited the polymerization of actin and the phagocytic activity of the PMN cells. CONCLUSION: 1) IHRP may bind to actin released from the damaged cells and suppress its toxic action by preventing the formation of actin fibril. 2) IHRP may bind to cell surface actin on PMN cells and inhibit their phagocytic activities. 3) From these results, IHRP may act as an anti-inflammatory protein.

A method for drying red blood cells for solid-phase immunoassay.

To develop a simpler and quicker alloantibody screening method, red cell stroma were bound and dried to microplate wells for use in magnetic-mixed passive haemagglutination (M-MPHA) tests. In the procedure of drying stroma, the Triton X-100-based haemolysing method gave lowest denaturation of red blood cells, and this method gave increased reactivity to Kidd and Rh antigens and clinically significant antibodies were detected as well as with the M-MPHA test. But long incubation with Triton X-100 and using high concentrations of Triton X-100 gave rise to some reduction in antigenicity, so the precise conditions for haemolysis are critical. This dried stroma-coated microplate can be stored for longer and more easily at room temperature than nondried intact red blood cells. The new system gave good sensitivity and the overall test time was shortened and should give a particular advantage for mass screening and for automation of this test.

International reference reagents: antihuman globulin. An ISBT/ICSH joint working party report. International Society of Blood Transfusion. International Committee for Standardization in Haematology.

An international working party has conducted a study designed to select a suitable reference reagent for antihuman globulin, to replace those first made available in 1987. The chosen preparation contains levels of anti-IgG and anti-C3 (anti-C3c and anti-C3d) potency that are considered suitable to serve for reference when evaluating either polyspecific antihuman globulin reagents or those containing their separate monospecific components. The reference material is available in 2-ml freeze-dried aliquots from seven assigned distribution centres.

A new solid-phase assay system using magnetic force on blood group serology.

A new solid-phase assay system has been developed for the detection of irregular antibodies in plasma and serum specimens. By means of a magnetic indicator cell, we have established a sensitive and easy-to-handle antihuman globulin (AHG) test involving magnetic-mixed passive hemagglutination (M-MPHA). Comparative studies of M-MPHA with conventional AHG tests demonstrated that this is sufficiently sensitive and specific to detect irregular antibodies to red blood cells. Elimination of centrifugation in this test seems to open the way toward new standardization of solid-phase systems.

Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma.

By use of its affinity to gelatin-Cellulofine, a novel protein, GBP28 (gelatin-binding protein of 28 kDa), was obtained from human plasma. GBP28 bound to gelatin-Cellulofine could be eluted with 1 M NaCl. By analysis of its amino-terminal amino acid sequences and the peptides obtained by protease digestion, GBP28 was identified as a novel protein. After repeated gel chromatography of the 1 M NaCl eluate from gelatin-Cellulofine, about 50 micrograms of GBP28 was purified from 500 ml of human plasma. On gel chromatography, the protein migrated as a molecule of about 420 kDa. On SDS-PAGE, its molecular mass was 28 kDa under reducing conditions and 68 kDa under nonreducing conditions. Recently, human mRNA specific to adipose tissue, cDNA clone apM1, has been registered [Maeda, K., Okubo, K., Shimomura, I., Funahashi, T., Matsuzawa, Y., and Matsubara, K. (1996) Biochem. Biophys. Res. Commun. 221, 286-289]. The assumed amino acid sequence of cDNA clone apM1 contained all the sequences of GBP28 and its peptides. Therefore, it is evident that the cDNA clone apM1 encodes GBP28 and the protein is specific to adipose tissue. The clone encodes a polypeptide of 244 amino acids with a secretory signal sequence at the amino terminus, a small non-helical region, a stretch of 22 collagen repeats and a globular domain. Thus, GBP28 appears to belong to a family of proteins possessing a collagen-like domain through which they form homo-trimers, which further combine to make oligomeric complexes. Although its biological function is presently unclear, its adipocyte-specific expression suggests that GBP28 may function as an endogenous factor involved in lipid catabolism and storage or whole body metabolism.

Purification and characterization of a novel hyaluronan-binding protein (PHBP) from human plasma: it has three EGF, a kringle and a serine protease domain, similar to hepatocyte growth factor activator.

A novel hyaluronan-binding protein (PHBP) was purified from human plasma by affinity chromatography on hyaluronan-conjugated Sepharose. The contaminating IgM and albumin in the partially purified preparation were removed with anti-IgG antibody-conjugated Sepharose and anti-albumin antibody-conjugated Sepharose, respectively, and no other contaminant was observed. Finally, 800 micrograms of PHBP was isolated from 500 ml of human plasma. PHBP gave a single 70-kDa band on SDS-PAGE under non-reducing conditions, and 50-kDa and 17-kDa bands under reducing conditions. Thus, PHBP was a heterodimer composed of 50-kDa and 17-kDa subunits, bridged by a disulfide linkage. Both subunits had novel N-terminal amino acid sequences, indicating that PHBP was a novel hyaluronan-binding protein in human plasma. The amino acid sequence deduced from the nucleotide sequence of the cloned PHBP cDNA exhibited significant homology to that of hepatocyte growth factor activator (HGFA). The results of Northern blot analysis indicated that liver, kidney, and pancreas expressed PHBP mRNA. The predicted structure of PHBP showed three epidermal growth factor (EGF) domains, a kringle domain and a serine protease domain, from its N-terminus, although HGFA has a fibronectin type II domain, an EGF domain, a fibronectin type I domain, an EGF domain, a kringle domain, and a serine protease domain, from its N-terminus.

Use of standardized protease enzymes for antibody screening of blood donor samples with the microplate system AutoAnalyzer.

Papain, bromelin and ficin can be standardized by the casein method for use in red blood cell antibody screening tests. The minimal and optimal enzyme activity for detecting blood group antibodies in donors, using the new Automated Pre-Transfusion Blood Testing System Olympus PK7200 by the two-stage method for all three enzymes, is one casein unit. One casein unit of proteinase activity changed the red blood cell surface charge to a low plateau value as measured by electrophoretic mobility and sialic acid content, and removed sterically inhibiting structures of surface protein as detected by SDS-PAGE.

Discrimination of human HLA-DRB1 alleles by PCR-SSCP (single-strand conformation polymorphism) method.

A single-strand conformation polymorphism (PCR-SSCP) method has been adopted for discrimination of human HLA-DRB1 alleles. This method enabled the detection of DNA polymorphisms including point mutations at a variety of positions in the DNA fragments of the HLA-DRB1 gene. A total of 27 HLA-DRB1 alleles from 172 healthy donors were analysed using a combination of PCR-SSCP with group-specific amplifications. Application of a small amount of amplified and denatured DNA to non-denaturing electrophoresis followed by silver staining resulted in distinct banding patterns. Samples possessing a single allele in each amplification group showed two-band patterns which correspond to the sense and antisense strands, while heterozygotes in the same group or a mixture of two single-type samples showed four-band patterns. All of the analysed alleles were discriminated in each DRB1 group. The method described here may be somewhat complicated for routine typing of HLA-DRB1 alleles. However, it is useful in the screening of "new' alleles as well as the donor-recipient molecular matching of HLA class II genes for various purposes, e.g. selection of bone marrow transplant donors.

Follow-up study of anti-hepatitis C virus antibodies in blood donors implicated in post-transfusion non-A, non-B hepatitis.

We retrospectively examined the antibodies to p22, a hepatitis C virus (HCV) nucleocapsid protein, and to c100-3, a HCV nonstructural protein, in donors whose blood was transfused to patients who later developed post-transfusion non-A, non-B hepatitis. Of 13 such blood donors, three seroconverted and three seroreverted with the anti-c100-3 test. In contrast, 12 of the 13 blood donors showed the same results at transfusion and follow-up, and one donor showed seroconversion with the anti-p22 assay. The follow-up study shows that the anti-p22 antibody test provides consistent results and is far more suitable for screening blood than the anti-c100-3 test.

A comparative study of urinary xanthopterin and neopterin in liver diseases.

By adsorption to activated charcoal, various pteridine derivatives in human urine are oxidized to xanthopterin. Following this oxidation, xanthopterin in urine from healthy subjects and from patients with liver diseases was assayed by high performance liquid chromatography. The mean values for xanthopterin in healthy subjects were 532 +/- 116 mumol/mol creatinine (mean +/- SD) in males and 585 +/- 153 mumol/mol creatinine in females; the difference was statistically significant (p < 0.01). Xanthopterin concentrations in patients with liver disease were significantly higher than those in normal subjects. When compared with urinary neopterin, which is a marker of activated cell immunity, xanthopterin was significantly increased even in fatty liver disease. These findings suggest that increased concentrations of urinary xanthopterin in liver diseases reflect not only the status of activated cell-mediated immunity, but also injury to liver cells.

Beta-endorphin binding activity of SP-40,40.

SP-40,40 bound to beta-endorphin via C-terminal non-opioid portion of beta-endorphin as well as S-protein (vitronectin) bound. Beta-endorphin bound mainly to SP-40,40, but not to S-protein, in the soluble membrane attack complex (SMAC, SC5b-9) of complement, because the results of autoradiography of the cross-linking experiment of SMAC with [125I] beta-endorphin revealed only a typical band of SP-40,40. The binding of SP-40,40 to beta-endorphin inhibited the binding of beta-endorphin to its receptor of rat brain; thus SP-40,40 might inhibit the biological action of beta-endorphin.

The effect of prior transfusion history on blood donor anti-hepatitis C virus antibody.

In Japan, the major transfusion-associated disease is non-A, non-B hepatitis. We studied the relationship between transfusion history and blood donor antibodies to hepatitis C virus (HCV). The positive rate of antibodies to the HCV nonstructural protein (c100-3) depended on age and the time elapsed since transfusion. The anti-c100-3 ratio for subjects with transfusions made prior to 20 years ago was high. One quarter century ago, a change occurred in national blood policy from paid to non-paid voluntary donations. We also have studied the anti-HCV positive rate among donors with prior transfusion using a second generation HCV test kit which includes anti-HCV core antibody detection. The anti-HCV positive rate for the second generation test was higher than that for the anti-c100-3 test. Introduction of the second generation test is therefore more useful in screening than the anti-c100-3 test for blood programs.