Significance of glomerular activation of the alternative pathway and lectin pathway in lupus nephritis.

The objective of the present study was to elucidate the association between glomerular complement depositions belonging to the alternative (AP) and lectin (LP) pathways, and clinical findings of lupus nephritis (LN). Immunofluorescence (IF) was performed on 17 LN patients using antibodies against factor B, factor H, properdin, mannose-binding lectin (MBL) and L-ficolin. Compared with factor B/factor H negative patients (n = 9), positive patients (n = 8) showed longer duration of LN (p < 0.05) and more severe interstitial fibrosis (p < 0.05). Eleven patients had properdin deposition in glomeruli, and in three of them, with a duration of LN of less than 1 month, factor B was undetectable. Compared with properdin negative patients (n = 6), positive patients (n = 11) showed significantly higher urinary protein excretion (p < 0.01). MBL/L-ficolin positive patients (n = 11) also had significantly higher urinary protein excretion (p < 0.05) compared with negative patients (n = 6). An independent association was found between glomerular deposition of properdin and that of MBL/L-ficolin (p < 0.01) in addition to factor B/factor H. Traces of glomerular activation of AP and LP reflected the clinical status of LN. It appears that glomerular deposition of each complement component, especially properdin, may be an index of the histological activity of LN.

Multi-step lymphomagenesis deduced from DNA changes in thymic lymphomas and atrophic thymuses at various times after gamma-irradiation.

Whole-body gamma-irradiation to mice causes thymic atrophy where a population of precancerous cells with mutation can be found. Thus, clonal growth and DNA changes at Bcl11b, Ikaros, Pten, Notch1 and Myc were examined in not only thymic lymphomas but also in atrophic thymuses at various times after irradiation. Clonal expansion was detected from the distinct patterns of rearrangements at the TCRbeta receptor locus in a fraction of atrophic thymuses at as early as 30 days after irradiation. This expansion may be in part owing to the rearranged TCRbeta signaling because the transfer of bone marrow cells with the rearrangement and the wild-type locus into severe-combined immunodeficiency mice showed preferential growth of the rearranged thymocytes in atrophic thymus. Loss of heterozygosity (LOH) at Bcl11b and trisomy of Myc were found at high frequencies in both lymphomas and atrophic thymuses, and in contrast, LOH at Ikaros and Pten were rare in atrophic thymuses but prevalent in lymphomas. Notch1 activation was detected in lymphomas and in atrophic thymuses only at a late stage. Similar patterns of DNA changes were found in atrophic thymuses induced in Bcl11b(+/-) mice. These results suggest the order of genetic changes during lymphomagenesis, Bcl11b and Myc being at the early stage; whereas Ikaros, Pten and Notch1 at the late stage.

Leukocytapheresis (LCAP) for the treatment of rheumatoid arthritis on a maintenance hemodialysis patient.

A 57-year-old-woman, who was treated with regular maintenance hemodialysis (HD), newly contracted rheumatoid arthritis (RA). Oral predonisolone was effective for alleviating her arthralgia but the RA activity became steroid-dependent. For treatment of poorly controlled synovitis leukocytapheresis (LCAP) showed excellent efficacy in the treatment of her joint pain. No serious adverse effects were observed. Serological markers such as CRP, serum amyloid A, matrix metalloproteinase 3 and peripheral blood lymphocyte count fluctuated with her clinical symptoms. We recommend LCAP as candidate therapy for steroid-dependent patients with RA who are on maintenance HD.

Outcomes of grafting intrasynovial tendons of the toes to the hands in 10 patients: a preliminary report.

Ten patients had intrasynovial tendon grafting harvested from the toes for secondary flexor tendon reconstruction in nine fingers and one thumb in our institutes from 2009 to 2014. These patients were followed for a mean of 15 (range: 8-36) months. The ranges of total active motion of the proximal and distal interphalangeal joints of these nine fingers were 143° (range: 108-175°) and of the metacarpophalangeal and interphalangeal joints of one thumb were 110°. In conclusion, this technique is feasible and gives a good result when successful but with a high complication rate. Level of Evidence IV.

A new form of cytochrome P-450 responsible for mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline in human livers.

Antibodies to P-450IA2 strongly inhibited the mutagenic activation of 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate but not aflatoxin B1 in human liver microsomes. The anti-rat P-450IA2 antibodies were capable of recognizing two proteins which show different mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of human liver microsomes. A new form of cytochrome P-450 (designated P-450-HM4) cross-reactive with anti-rat P-450IA2 antibodies showing that the smaller molecular weight was purified from human liver microsomes by means of the fast-performance liquid chromatography system. The molecular weight of P-450-HM4 was estimated to be 49,000, which was apparently different from that of P-450PA (human P-450IA2). The antibodies to P-450-HM4 did not cross-react with P-450PA (human P-450IA2) but inhibited to various extents the mutagenic activation of IQ in microsomes from human livers. In addition, P-450-HM4 showed significant mutagen-producing activity from IQ in a reconstituted system. Together with these and other results reported previously, it is concluded that at least two forms of cytochrome P-450 [P-450-HM4 and P-450PA (human P-450IA2)] are involved in the mutagenic activation of IQ in human liver.

Inhibition of in vitro ovarian cancer cell invasion by modulation of urokinase-type plasminogen activator and cathepsin B.

HOC-I ovarian cancer cells express the single-chain form of the urokinase-type plasminogen activator (uPA) and cathepsin B (cath B) on their cell surface. The significance of the expression of cell surface uPA/cath B activity to the invasive potential was examined by preincubating with uPA/cath B-modulating agents in in vitro invasion assay. The anti-uPA monoclonal antibody 394 effectively inhibited invasion in a dose-dependent manner. On the contrary, anti-cath B antibody did not affect the invasive potential of the cells. E-64, a specific inhibitor for cysteine proteases, blocked invasion as effectively as monoclonal antibody 394. The data reveal that the uPA and cysteine proteases contribute significantly to the invasive capacity of the cells. We suggest that the cysteine proteases facilitate the action of uPA, possibly by activating proenzyme uPA produced by cancer cells. Evidence for the role of a cathepsin-uPA activation cascade in HOC-I cell invasion is provided.

Effects of membrane-associated cathepsin B on the activation of receptor-bound prourokinase and subsequent invasion of reconstituted basement membranes.

The present study was undertaken to assess the role of membrane-associated cathepsin B as an activator of receptor-bound single-chain urokinase-type plasminogen activator (pro-uPA) and to determine the importance of receptor-bound uPA activity in the destruction of extracellular matrix by tumor cells with subsequent invasion through basement membranes. Ovarian cancer HOC-I cells express pro-uPA/HMW-uPA and cathepsin B on their surface. uPAs are bound to a specific surface receptor, about 30% of which is saturated. 60% of the receptor-bound uPA is pro-uPA. No reduction in the specific binding of biotinylated DFP-HMW-uPA was observed when cells were cultivated in the presence of E-64, a cysteine proteinase inhibitor, for 24 h. Inhibition of cell-surface cathepsin B activity was associated with a decrease in cell-bound uPA activity to undetectable levels, and > 95% of the membrane-associated uPA was pro-uPA in cells cultivated with E-64. This suggested that receptor-bound pro-uPA cannot be converted to HMW-uPA in the absence of enzymatically-active cathepsin B. The significance of the expression of cell-surface uPA activity regarding invasive potential was examined in an in-vitro Matrigel invasion assay. Decreased cell-surface uPA activity was associated with a decrease in invasive potential. These data support our hypothesis that membrane-associated cathepsin B may be important for the conversion of pro-uPA to HMW-uPA and that receptor-bound uPA activity constitutes an efficient mechanism which contributes to tumor cell invasion. As HOC-I cells produce both uPA and cathepsin B, the implications of tumor-cell-derived pro-uPA activation by cellular proteinase cathepsin B should be considered.

Purification of cytochrome P-450 from polychlorinated biphenyl-treated crab-eating monkeys: high homology to a form of human cytochrome P-450.

Cytochrome P-450, designated as P-450-MK2, was purified to an electrophoretic homogeneity from polychlorinated biphenyl (PCB)-treated female crab-eating monkeys. P-450-MK2 catalyzed nifedipine and nilvadipine oxidations, at a rate comparable to human P-450-HM1. The N-terminal amino acid sequence of P-450-MK2 was highly homologous to those of P-450-HM1 and NF 25. The antibodies to P-450-HM1 recognized P-450-MK2 and effectively inhibited the activity of testosterone 6 beta-hydroxylase in monkey liver microsomes. These results suggest that a form of cytochrome P-450 corresponding to human P-450-HM1 or P-450NF which belongs to the P450 III gene family is also present in liver microsomes of crab-eating monkeys.

Secretion of laminin alpha 2 chain in cerebrospinal fluid.

The absence of laminin alpha 2 chain causes muscle cell degeneration and peripheral dysmyelination in congenital muscular dystrophy patients and dy mice, suggesting its role in the maintenance of sarcolemmal architecture and peripheral myelinogenesis. Here we demonstrate the secretion of laminin alpha 2 chain in cerebrospinal fluid (CSF). Laminin alpha 2 chain was detected as a minor component of the total CSF proteins or glycoproteins. Laminin alpha 2 chain was localized in the cytoplasm of epithelial cells of choroid plexus, suggesting active secretion. Our results suggest that immunochemical analysis of CSF laminin alpha 2 chain could be useful as an aid for the diagnosis of congenital muscular dystrophy.

Urinary trypsin inhibitor (UTI) and fragments derived from UTI by limited proteolysis efficiently inhibit tumor cell invasion.

We investigated the effects of purified human urinary trypsin inhibitor (UTI) and fragments derived from UTI by proteolysis on the invasive potential of ovarian cancer cells (HOC-I) and gestational choriocarcinoma cells (SMT-ccl) using an in vitro reconstituted basement membrane invasion assay. These cells express cell-associated plasmin and functional uPA receptors that are partially occupied by ligands. SMT-ccl cells, which express threefold higher levels of cell-associated plasmin activity than HOC-I cells, showed approximately twofold increase in their invasive potential. For the invasion assay, HOC-I cells were primed with exogenous plasminogen, but SMT-ccl cells were not. Human leukocyte elastase (HLE)-digested UTI (22 kDa fragment; UTI-22) inhibited plasmin practically with the same strength as native UTI. Trypsin-digested UTI (20 kDa fragment; UTI-20), however, did not inhibit plasmin significantly. Treatment of cells with UTI or UTI-22 reduced the incidence of tumor cell invasive capacity, whereas the inhibitory effect of UTI-20 was not remarkable. The inhibitory effect on tumor cell invasion was dose-dependent and non-toxic; moreover, it was not mediated by inhibition of the tumor cell chemotactic response or of cell attachment to matrigel. These results indicate that inhibition of the proteolytic enzyme plasmin specifically reduced the invasive capacity of tumor cells in vitro.

Detection of C3bBb-stabilizing activity (C3 nephritic factor) in the serum from patients with membranoproliferative glomerulonephritis.

It is known that membranoproliferative glomerulonephritis (MPGN), hypocomplementaemia and C3 nephritic factor (C3NeF) are closely related to each other, and the presence or absence of C3NeF in the serum is important for evaluating the nature of MPGN. However, some difficulties have been encountered in detecting this factor and therefore a new assay permitting the direct detection of C3NeF without purifying IgG from the patient's serum has been devised. Using this assay method, C3bBb-stabilizing activity was observed even in sera from MPGN patients who were non-hypocomplementaemic. Furthermore, among 98 cases with hypocomplementaemia. C3NeF was found to be absent in 66 cases.

Cellulose membranes suppress complement activation in patients after hemodialysis.

Membranes used for dialysis therapy activate complement. Complement activation is maximal after initiating dialysis and returns to predialysis values by the end of dialysis. No changes in C3 levels have been detected after dialysis. We hypothesized that although C3 levels were unchanged, C3 activity could be altered by dialysis. We measured complement activation in vitro in serum from patients randomized to dialysis treatments using different types of membranes. The classical pathway was activated with aggregated immunoglobulin G (IgG), and the alternative pathway was activated with inulin. Both the classical and alternative pathways were suppressed after dialysis using cellulose membranes (aggregate IgG, P < 0.01; inulin, P < 0.001). When polyacrylonitrile (PAN) or polyethylene glycol grafted cellulose membranes were used for dialysis, only minor suppression of complement pathways was measured. Levels of the control factor SP-40,40 increased at later times for dialysis using cellulose membranes (P < 0.05). Factor H levels were also greater after dialysis using cellulose membranes compared with PAN membranes (P < 0.05). In summary, cellulose membranes suppress complement activation in serum. One suppressing factor may be the complement control factor SP-40,40.

Complement activation through the lectin pathway in patients with Henoch-Schönlein purpura nephritis.

Henoch-Schönlein purpura nephritis (HSPN) is considered a form of systemic vasculitis of the small blood vessels with immune pathogenesis. In this disorder, the complement system is recognized as an important mechanism of glomerular injury. The aim of this study is to determine whether the lectin pathway, a novel pathway of complement activation, is related to the pathogenesis of HSPN. Renal biopsy material from 10 patients with HSPN was studied immunohistochemically and examined for a clinicopathologic correlation. Serum levels of complement components, including mannose-binding lectin (MBL), and plasma levels of complement activation products were also evaluated in these patients and compared with levels in patients with immunoglobulin A (IgA) nephropathy or mesangial proliferative glomerulonephritis (GN) without IgA deposition (non-IgA GN). Glomerular deposition of components of the pathway, MBL and MBL-associated serine protease (MASP-1), as well as C3b/C3c, C5b-9, and C4-binding protein (C4-bp), was detected in 8 of 10 patients. Although no significant correlation was found between glomerular deposition of MBL/MASP-1 and histological or clinical findings, the biopsies on all patients with MBL/MASP-1 deposits were performed within 20 weeks from the onset of disease. Levels of plasma C4d, the activation fragment of C4, and C4-bp, a soluble regulatory protein of the pathway, were greater in patients with HSPN than in those with non-IgA GN. However, there was no difference in serum MBL levels between the three groups of patients (HSPN, IgA nephropathy, and non-IgA GN). These results suggest that complement activation through the lectin pathway was involved at the onset of HSPN, and this mechanism might be important in the disease pathogenesis.

Glomerular and extraglomerular immune complex deposits in a bone marrow transplant recipient.

A 44-year-old man developed nephrotic syndrome 9 months after HLA-identical sibling bone marrow transplantation. Membranous changes consisted mainly of alterations of glomeruli, which were interpreted as chronic graft-versus-host disease (GVHD) caused by lodging of the circulating immune complex. In the tubules, a lumpy deposition of IgG and complement breakdown products was distributed along the tubular basement membrane, which coincided with the peculiar deposits ascertained by electron microscopy. These findings suggest that an extraglomerular reaction should be considered in evaluating renal involvement of GVHD.

Comparative study of cytochrome P-450 in liver microsomes. A form of monkey cytochrome P-450, P-450-MK1, immunochemically cross-reactive with antibodies to rat P-450-male.

Cytochrome P-450, designated as P-450-MK1, which is cross-reactive with antibodies to rat P-450-male, was purified to an electrophoretical homogeneity from liver microsomes of the untreated male crab-eating monkey. The molecular weight of P-450-MK1 was estimated to be 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The oxidized form of P-450-MK1 showed a peak at 418 nm, indicating that this cytochrome is in a low spin state. The carbon monoxide-bound reduced form showed a peak at 451 nm. The first 22 amino acid residues of the NH2-terminal sequence of P-450-MK1 was fairly homologous to those of P-450-male (75% identity, not including unidentified amino acid residues). Unlike the P-450-male, P-450-MK1 did not exhibit catalytic activities for testosterone 2 alpha- and 16 alpha-hydroxylations and catalyzed testosterone 6 beta-hydroxylation. It is, therefore, suggested that although the spectral and immunochemical properties and the N-terminal amino acid sequence of P-450-MK1 were similar to those of P-450-male, the physiological functions of P-450-MK1 may be somewhat different from those of P-450-male. Comparison of the physico-chemical properties of P-450-MK1 with those of P-450-D1 and P-450-HM2, which are cross-reactive with anti-P-450-male antibodies, purified from liver microsomes of dogs and humans, respectively, are also discussed.

Effects of serum testosterone level with buserelin on the activities of drug and testosterone hydroxylase and on the content of a male-specific form of cytochrome P-450 in male rats.

The effects of administration of buserelin, a synthetic agonist of lutenizing hormone-releasing hormone (LH-RH), on the content of P-450-male, a male specific form of cytochrome P-450, and the activities of drug and testosterone hydroxylases were examined in liver microsomes of male rats. Administration of buserelin resulted in a decrease in the serum level of testosterone in a dose-dependent manner. Similar decreases were seen in the activities of aminopyrine N-demethylase, 7-propoxycoumarin O-depropylase and testosterone 2 alpha- and 16 alpha-hydroxylases, but not in the activity of aniline p-hydroxylase. Thus, correlations were observed between the serum level of testosterone and the activities of drug and testosterone hydroxylases except for aniline p-hydroxylase. These results indicate that the drug and testosterone hydroxylases are affected sensitively and sequentially by the changes in the testosterone levels in the body.

Murine fetal resorption and experimental pre-eclampsia are induced by both excessive Th1 and Th2 activation.

It has been proposed that immune responses in mammalian normal pregnancy are Th2-like, thereby protecting the fetus and placenta from being rejected. Administration of exogenous Th1 cytokines into pregnant mice is reported to induce feto-placental resorption. However, the effects of exogenous Th2 cytokines and Th2 directed responses in pregnant animals have not been well studied. In this study, we examined IL-4 and IL-12, which play decisive roles in the development of Th2 and Th1 responses, respectively, in the induction of fetal resorption and development of experimental pre-eclampsia. Transfer of either IL-4 and/or IL-12 stimulated splenocytes from BALB/C virgin female mice into BALB/C pregnant mice mated with either C57BL/6 or BALB/C male mice resulted in fetal resorption and glomerular nephritis associated with hypertension and proteinuria. In mice treated with IL-12 stimulated splenocytes, fatty liver degeneration associated with bile retention was observed. These results indicate that both excessive Th1 and Th2 activation contribute to the development of fetal resorption and pre-eclampsia, but that Th1 is critical to the development of liver degeneration.

Molecular cloning and sequence analysis of cDNA containing the entire coding region for human fetal liver cytochrome P-450.

From a human fetal liver cDNA library, a cDNA clone (lambda HFL33) containing the entire coding region for a form of cytochrome P-450 related to P-450 HFLa was obtained. The clone was 1,971 bp long and had an open reading frame of 1,509 nucleotides coding for a 503 amino acid polypeptide. The nucleotide and the deduced amino acid sequences of lambda HFL33 were very similar to but clearly distinct from those of NF25 and HLp cDNAs, which code for forms of cytochrome P-450 in adult human liver. The deduced N-terminal amino acid sequence of the HFL33 protein was identical to that of P-450 HFLa.

Cytochrome P-450 in human liver microsomes: high-performance liquid chromatographic isolation of three forms and their characterization.

Three forms of cytochrome P-450, designated as P-450-HM1, P-450-HM2, and P-450-HM3, were isolated from human liver microsomes using high-performance liquid chromatography (HPLC) techniques. Each purified preparation showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the results of SDS-PAGE, the molecular weights of P-450-HM1, P-450-HM2, and P-450-HM3 were estimated to be 51,000, 54,000, and 52,000, respectively. The oxidized absolute spectra of these three forms of cytochrome P-450 showed Soret absorption peaks at around 417 nm, indicating that these forms were in the low spin state. In a reconstituted system, P-450-HM1 showed the highest catalytic activities of nifedipine and (S)- or (R)-nilvadipine oxidases. The same form showed higher activities of testosterone 6 beta-hydroxylase and progesterone 6 beta- and 16 alpha-hydroxylases. P-450-HM2 showed high N-demethylase activities for benz-phetamine and aminopyrine, and also showed the highest activity of testosterone 16 beta-hydroxylase among the three forms, while it did not show detectable activities of testosterone 6 beta-hydroxylase and progesterone 6 beta- and 16 alpha-hydroxylases. Anti-P-450-HM1 immunoglobulin G (IgG), but not anti-P-450-HM2 IgG, inhibited the activities of testosterone 6 beta-hydroxylase and nifedipine and nilvadipine oxidases in human liver microsomes. Anti-P-450-HM1 IgG was also inhibitory against progesterone 6 beta- and 16 alpha-hydroxylases.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretory expression of the human serum albumin gene in the yeast, Saccharomyces cerevisiae.

We have fused a cDNA gene encoding mature human serum albumin (HSA) to several secretory leader-encoding sequences. The hybrid genes were cloned into an episomal vector under the control of several yeast promoters and then introduced into yeast cells. The GAL1 promoter in combination with either the native HSA pre-sequence or a modified HSA pre-sequence gave the highest production of immunoreactive HSA, 90 mg/liter being reached in a shake flask culture. The invertase pre-sequence, the mating factor alpha 1 prepro-sequence, and the modified HSA pre-sequence directed accurate processing. In contrast, the chicken lysozyme pre-sequence and the native HSA pre-sequence directed incorrect processing. Episomal vectors were unstable within the host cells under non-selective culture conditions. To improve the plasmid stability, the hybrid genes were incorporated into an integrative vector. Transformants carrying multicopies of the plasmid integrated at the LEU2 locus stably secreted HSA. The highest yield of 65 mg/liter in a shake flask culture was obtained with the combination of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter and the modified HSA pre-sequence. By constructing transformed strains containing multicopies of plasmids integrated at both the chromosome LEU2 and HIS4 loci, we have obtained a stable strain that continuously secretes as much as 85 mg HSA per liter of culture medium.