IgG and IgA antibody titers against human heat-shock protein (hsp60) in sera of rheumatoid arthritis and osteoarthritis patients.

Abstract To learn whether heat-shock proteins (HSP) are involved in the pathogenesis of rheumatoid arthritis (RA), antirecombinant human heat-shock protein 60 (hsp60) IgG and IgA in sera of RA and osteoarthritis (OA) patients were investigated. Only the anti-hsp60 IgG titer of seropositive (RF-positive) patients was found to be elevated. Although RF titers of the sera of seropositive RA patients were increased, there was no correlation between the individual anti-hsp60 IgG titer and the corresponding RF titer. In contrast, all the anti-hsp60 IgA titers of the sera of OA, seronegative RA, and seropositive RA patients were found to be elevated. Among them, only the serum IgA concentration of seropositive RA patients was increased. Thus, it was suggested that the increased anti-hsp60 IgG reflects the pathogenesis of RA and its activity. It was also suggested that the increased anti-hsp60 IgA response reflects an involvement of hsp60 in the pathogenesis of arthritides rather than the pathogenesis of RA.

Cloning and characterization of a p53-related protein kinase expressed in interleukin-2-activated cytotoxic T-cells, epithelial tumor cell lines, and the testes.

A human protein kinase, p53-related protein kinase (PRPK), was cloned from an interleukin-2-activated cytotoxic T-cell subtraction library. PRPK appears to be a homologue of a growth-related yeast serine/threonine protein kinase, YGR262c. However, a complementation assay using YGR262c-disrupted yeast indicated that PRPK is not functionally identical to the yeast enzyme. PRPK expression was observed in interleukin-2-activated cytotoxic T-cells, some human epithelial tumor cell lines, and the testes. The intrinsic transcriptional activity of p53 was up-regulated by a transient transfection of PRPK to COS-7 cells. PRPK was shown to bind to p53 and to phosphorylate p53 at Ser-15. These results indicate that PRPK may play an important role in the cell cycle and cell apoptosis through phosphorylation of p53.

Mouse model of Bell's palsy induced by reactivation of herpes simplex virus type 1.

In order to investigate the mechanism of Bell's palsy, we developed an animal model of facial nerve paralysis induced by the reactivation of herpes simplex virus type 1 (HSV-1). Eight weeks after recovery from facial nerve paralysis caused by inoculation with HSV-1, the mice were treated with auricular skin scratch at the site of the previous inoculation, or with intraperitoneal injection of anti-CD3 monoclonal antibody (mAb), or combination of both procedures. No mice developed facial nerve paralysis when they were treated with either auricular scratch or mAb injection alone. In contrast, 20% of mice developed facial nerve paralysis with the combined treatment. With one exception, no mouse treated with either auricular scratch or mAb injection showed HSV-I DNA in their facial nerve tissue, whereas 4 out of 6 mice receiving both treatments showed HSV-1 DNA on day 10 after treatment. Histopathological findings showed neuronal degeneration in the geniculate ganglion and demyelination of the facial motor nerve in paralyzed mice. These findings suggest that a combination of stimuli, local skin irritation, and general immunosuppression is essential for successfully inducing facial nerve paralysis in mice with latent HSV-1 infection.

[Clinical significance of serum LpA-I levels measured by immunoturbidimetric assay in type 2 diabetes mellitus patients].

Previously, we developed an immunoturbidimetric assay method for lipoprotein A-I(LpA-I) on sera pre-absorbed with anti-apolipoprotein A-II. In the present study, correlations between serum lipoprotein A-I and other serum parameters levels were examined and LpA-I levels were studied in patients with type 2 diabetes mellitus. The serum levels of LpA-I did not correlate with those of diabetic markers such as fasted blood glucose, glycohemoglobin(HbA1c) and fructosamine, but correlated well with the levels of total cholesterol and HDL cholesterol, phospholipids, apolipoprotein A-I and seemed to correlate inversely with arteriosclerosis index. In patients with type 2 diabetes mellitus, LpA-I levels were significantly lower than those in normal subjects. Especially, LpA-I levels of patients with diabetic complications were significantly lower than those in normal subjects and non-complicated diabetic patients. Then, the measurement of LpA-I levels in patients with type 2 diabetes mellitus was considered to be useful for prevention and management of arteriosclerosis.

[Development of a serum lipoprotein A-I immunoassay method].

We developed an immunoturbidimetric assay method for lipoprotein A-I by treating sera with an anti-apo lipoprotein A-II and A-I antibody sequentially. The assay is sensitive to detect lipoprotein A-I as little as 155 mg/l with good precision. There was a good correlation in the level of serum lipoprotein A-I between the present assay and a conventional differential electroimmunoassay on ready-to-use plates (r = 0.947; p < 0.001). Reference intervals in normal healthy adults (n = 109) ranged from 284 to 736 mg/l. The values were significantly lower in patients with coronary artery disease than in normal subjects (p < 0.001). The present assay can be useful to analyze patho-physiologic events in various lipid disorders.

Inhibition of human and mouse complement-dependent hemolytic activity by mouse fibronectin.

Not only does mouse complement (C) have low hemolytic activity, but mouse serum has an inhibiting effect on hemolysis by human C. To purify and identify the putative mouse serum factor inhibiting human C activity, a sequential procedure of fractionated precipitation by PEG, followed by chromatographies with a heparin-Sepharose column, a phenyl-Sepharose column, a Protein G column, and a gel-filtration column was performed. The amino acid sequence analyses of two polypeptides obtained by digestion of the purified serum factor with TPCK-trypsin revealed that it was mouse fibronectin (FN). Highly purified mouse FN, but not human FN, has an inhibiting effect on human C-dependent hemolysis. Moreover, the hemolysis of sensitized rabbit erythrocytes by mouse C was also inhibited by the addition of mouse FN in a dose-dependent fashion, but not by the addition of human FN. These results suggest that FN is the putative internal C inhibitor in the mouse system.

Immunologic aspects of facial nerve paralysis induced by herpes simplex virus infection in mice.

This immunologic aspects of facial nerve paralysis due to herpes simplex virus type 1 (HSV-1) infection were investigated in a mouse model system. Half of the 4- to 5-week-old mice developed facial nerve paralysis, whereas none of the 6-week-old mice died or developed facial nerve paralysis on inoculation with HSV-1. Six-week-old mice showed significantly higher titers of anti-HSV-1 neutralizing antibody than did 4-week-old animals. Passive transfer of either anti-HSV-1 antibody or HSV-1-immunized splenic T cells into 4-week-old mice 3 hours after HSV-1 inoculation prevented development of facial nerve paralysis and death, whereas such transfers 48 or 96 hours after HSV-1 inoculation did not prevent or exacerbate facial nerve paralysis. These results demonstrate that the age and the immunologic potency of mice are closely related to the pathogenesis of facial nerve paralysis. That facial nerve paralysis developed even in 6-week-old mice whose T-cell function was suppressed with anti-CD3 antibody suggests that virus-induced cellular demyelination is unlikely as a cause of facial nerve paralysis in this animal model.

Two antigens on zygotes and ookinetes of Plasmodium yoelii and Plasmodium berghei that are distinct targets of transmission-blocking immunity.

We have developed transmission-blocking monoclonal antibodies (MAbs) against Plasmodium yoelii 21-kDa (Pys21) and 28-kDa (Pys25) ookinete surface proteins. These MAbs block infectivity of P. yoelii to Anopheles stephensi. One MAb, 14, cross-reacted by Western blotting with a 28-kDa surface protein (Pbs25) of P. berghei ookinetes and blocked oocyst development, as assayed by direct mosquito feeds on passively immunized P. berghei-infected mice. In total, we have identified two ookinete surface proteins in P. yoelii, one of which is also present in P. berghei. The transmission-blocking activity of the anti-Pys25 MAb 4 was complete and more potent than that of the anti-Pys21 MAb 2. Moreover, Fab fragments of MAb 4 had transmission-blocking activity in mice. In comparison, Fab fragments of MAb 2 did not have detectable transmission-blocking effect, although F(ab')2 did. Furthermore, MAb 2 and MAb 4 appeared to block the in vitro formation and development of zygotes as well.

The serum resistance of malaria-infected erythrocytes.

IgG and IgM antibodies were detected on non-parasitized as well as parasitized erythrocytes (E) from mice surviving over 15 days after infection with rodent malaria, Plasmodium berghei, whereas C3 was detected exclusively on parasitized E. Parasitized E, however, were quite resistant to the haemolytic activity of guinea pig complement and effectively inactivated human C3b to iC3b on their surface. Similarly, parasitized E were extremely resistant to homologous complement as assessed by haemolysis and C3 binding even when regulatory proteins (decay-accelerating factor, DAF; complement receptor related gene y, Crry; heat-stable antigen, HSA) were blocked with specific antibodies. DAF and Crry were equally expressed on both normal E and parasitized E from mice within a week post-infection; therefore, molecules that inhibit the haemolysis or C3 binding of parasitized E appear to be independent of DAF and Crry. Unexpectedly, the molecular forms of HSA and DAF in parasitized erythrocyte membranes were found to be different from those of normal erythrocyte membranes: DAF was detected as three bands (85,000, 64,000 and 30,000 MW) by immunoblotting. HSA was detected as more highly glycosylated forms than normal HSA. These alterations of DAF and HSA could be explained by the modification of membrane proteins and polysaccharides induced by parasitization, and we hypothesize that these changes of membranes or membrane proteins are involved in the resistance of parasitized E against homologous complement.

Enhancement of CD3-mediated thymocyte apoptosis by the cross-linkage of heat-stable antigen.

Heat-stable antigen (HSA) is a murine differentiating antigen that is expressed on both CD4-CD8- double-negative and CD4+CD8+ double-positive thymocytes but not CD4+ or CD8+ single-positive thymocytes. Effects of anti-HSA monoclonal antibody, R13, on thymocyte apoptosis induced by various stimulations were investigated by a single-cell suspension culture system. Immobilized R13 enhanced the CD3-mediated DNA fragmentation and killing of thymocytes but not the dexamethasone-induced or phorbol myristate acetate-induced killing of thymocytes. Immobilized R13 by itself could not induce thymocyte apoptosis. Soluble R13 enhanced CD3-mediated apoptosis when HSA and T-cell receptor (TCR)/CD3 were co-cross-linked by a cross-reactive secondary antibody. Even without the cross-reactive secondary antibody, soluble R13 enhanced CD3-mediated apoptosis, although a greater than 100-fold increase in the amount of R13 was needed to give a similar enhancement compared with immobilized R13. Neither R13 by itself nor R13 plus secondary antibody induced cytosolic calcium influx, whereas R13 enhanced CD3-mediated cytosolic calcium increase. These results suggest a functional role of HSA in promoting the activation-induced apoptosis of thymocytes and the involvement of HSA in negative selection.

Two mechanisms for platelet-mediated killing of tumour cells: one cyclo-oxygenase dependent and the other nitric oxide dependent.

We have tried to identify the cytotoxic effectors in platelet-mediated tumour cell killing, using two tumour cell lines K562 (a chronic myelogenic leukaemic cell line) and LU99A (a lung cancer cell line), which are both sensitive to platelet cytotoxicity. Cyclo-oxygenase inhibitors, acetylsalicylic acid (ASA) and indomethacin, effectively inhibited the platelet-mediated killing of K562 cells, but not that of LU99A cells. In contrast, inhibitors of the nitric oxide (NO) pathway. NG-nitro-1-arginine (L-NA), haemoglobin and methylene blue, reduced the cytotoxic activity of platelets against LU99A, but not against K562. Synthetic analogues of platelet cyclo-oxygenase products thromboxane A2/ prostaglandin H2(TXA2/PGH2) exerted cytotoxicity against K562 cells but not against LU99A cells. Electron microscopic study showed that TXA2/PGH2 analogues induced bleb formation and disruption of the plasma membrane of K562 cells. K562 cells enhanced the production of TXA2 by platelets, as inferred from the accumulation of thromboxane B2 (TXB2), a spontaneous hydrolysis product of TXA2. LU99A cells had no such effects. These results indicate that platelets kill these two tumour cell lines through different mechanisms. In K562, the cyclooxygenase products TXA2/PGH2 possibly play a significant role but in LU99A the NO pathway seems to be involved.

Attenuation of human chorionic gonadotropin release by nitric oxide in choriocarcinoma cell lines.

Nitric oxide (NO) is involved in the regulation of endocrine functions, but only a few studies have been reported about its role in placental hormone secretion. We investigated whether NO has any function in the release of human chorionic gonadotropin (hCG) in two different choriocarcinoma cell lines, JEG-3 and BeWo. First, nitric oxide synthase (NOS) was characterized in the choriocarcinoma cells. NOS activity was localized mainly in the particulate fraction and depended on calcium/calmodulin. Activity was inhibited by the presence of the L-arginine analog, NG-monomethyl-L-arginine (L-NMMA; 1 x 10(-4) M). Western blot analysis showed that the choriocarcinoma cells contained an endothelial isoform of NOS. The NO donor, sodium nitroprusside (SNP; 1 x 10(-5) and 1 x 10(-4) M), significantly inhibited hCG secretion in both choriocarcinoma cell lines. The suppression of hCG release by SNP (1 x 10(-5) M) was blocked by the addition of an NO scavenger, hemoglobin (1 x 10(-6) M). L-Arginine (1 x 10(-2) M), a NOS substrate, inhibited basal hCG secretion in JEG-3 cells. Incubation of the cells with L-NMMA (1 x 10(-4) and 1 x 10(-3) M) significantly increased hCG release. Exposure of both cell lines to increasing concentrations of a cyclic GMP analog (8-bromo-cyclic GMP; 1 x 10(-4) to 1 x 10(-2) M) caused a dose-dependent inhibition of hCG release. Cyclic GMP accumulation in response to SNP (1 x 10(-4) M), however, was not detected in either JEG-3 or BeWo cells. These data demonstrated that the endothelial isoform of NOS and a functional L-arginine-NO pathway are present in the choriocarcinoma cell lines. In addition, these findings support the hypothesis that NO produced in these cell lines is involved in the regulation of hCG secretion. We assume that although cyclic GMP is likely to play a role as a second messenger, a cyclic GMP-independent pathway cannot be excluded as a possible physiological mechanism in the attenuation of hCG release by NO.

Lipase activities in post-heparin plasma and tissues, and susceptibilities of lipoproteins in experimental diabetic rats.

Heparin administration to diabetic rats caused no change in VLDL, an increase in IDL and a decrease in LDL on electrophoretic analysis of plasma lipoproteins, while the administration to control rats markedly decreased VLDL and increased IDL and LDL. Both hepatic triglyceride lipase (HTGL) and lipoprotein lipase (LPL) activities in the postheparin plasma were lower in the diabetic rats than in the controls, and the reduction of HTGL activity was greater than that of LPL activity in the diabetic rats. The LPL activity in the adipose tissue was lower in the diabetic rats than in the controls, but the activities in the cardiac and skeletal muscles were similar in the two rats. The HTGL-catalyzed fatty acid (FA) releases from the diabetic VLDL and IDL were lower than those from the normal rat VLDL and IDL, while the LPL-catalyzed FA release in the diabetic rats was not different from those in the controls. The decreases in LPL and HTGL activities and the markedly impaired susceptibility of IDL to HTGL coincide well with the postheparin changes in plasma lipoproteins in diabetic rats, an increase in IDL and a decrease in LDL.

Expression of mammalian 60-kD heat shock protein in the joints of mice with pristane-induced arthritis.

Previous work has indicated that autoimmunity to the mammalian 60-kD heat shock protein (hsp60) may be necessary for the development of pristane-induced arthritis (PIA), a murine model of rheumatoid arthritis. To characterize the expression of hsp60 in murine joints, immunoblots of joint extracts and frozen histological sections prepared from normal or arthritic mice were probed with the hsp60-specific MoAb 4B989. Hsp60 could be detected in the joints of mice with PIA by both techniques, and was seen to be localized within the inflamed pannus using immunhistochemistry. Immunoblotting revealed that lower concentrations of hsp60 are also present in normal mouse joints, and that the level of expression increases with age, in parallel with greater susceptibility to PIA. In other studies, it was demonstrated that the titres of serum IgG antibodies reactive with the related mycobacterial hsp65, and the in vitro responsiveness of splenic T cells to hsp65, are both elevated in older mice. It is considered that the results are consistent with the hypothesis that PIA develops following environmental priming with mycobacterial hsp65, and the targeting of cross-reactive T cells to self-hsp60 in the joints.

Murine complement reduces infectivity of Plasmodium yoelii to mosquitoes.

The alternative pathway of complement in the mouse serum significantly reduced, but did not eliminate, the infectivity of Plasmodium yoelii to Anopheles stephensi. The reduction of the infectivity is mainly due to the inability of the zygote to transform into the ookinete in the mosquito midgut.

Compositions of very low density lipoprotein subfractions from patients with polydisperse low density lipoproteins.

In some hyperlipidemic patients, low density lipoprotein (LDL) shows several peaks (polydisperse) on polyacrylamide gel disc electrophoreses, though LDL usually shows a single peak (monodisperse). In order to clarify the relationship between the LDL polydispersion and VLDL heterogeneity, LDL and VLDL were prepared from hyperlipidemic patients sera with mono- and polydisperse LDL by sequential ultracentrifugation and fractionated by gradient ultracentrifugation and their compositions were analyzed. Polydisperse LDL was rich in triacylglycerol (TG) and poor in esterified cholesterol (CE) as compared with monodisperse LDL and consisted of the lowest and the medium density subfractions when the LDL was separated into six subfractions. The monodisperse LDL was composed of a single major subfraction of a medium density. VLDL from the patients with polydisperse LDL was relatively rich in the dense and poor in the buoyant subfractions as compared with that from the patients with monodisperse LDL. The subfractions in the former contained more CE and less TG than the corresponding subfractions in the latter. There were no significant differences in the apolipoprotein compositions between those VLDLs. The results suggest that polydisperse LDL might be originated from VLDL that differs in particle sizes, densities and compositions from ordinary VLDL.

Complement activation by cross-linked B cell-membrane IgM.

The B cell membrane IgM (mIgM) occurs in a monomeric form incapable of activating C. However, when cross-linked by a polyvalent ligand, mIgM may activate C by assuming a polymeric structure like secreted IgM. This possibility was tested with CR2- lymphoma cells, which did not activate C spontaneously. When CR2-deficient mIgM(lambda)+ Ramos cells were treated with F(ab')2 goat anti-lambda, then exposed to human serum, a marked C3 deposition took place, as examined by the flow cytometry. Similarly, C3 deposition on mIgM(kappa)+, mIgD(kappa)+ P32 cells was induced by F(ab')2 of either anti-kappa or anti-mu. Anti-delta was without effect, but the C3 deposition resulting from anti-kappa was markedly enhanced after mIgD was modulated by anti-delta. The mIgM-cross-linked cells bound C1q, and C3 deposition on these cells was abrogated by depletion of C1q, but not Factor B nor D, from serum. The C1-binding step of the mIgM-mediated C activation was inhibited by monomeric Fab' of polyclonal anti-mu containing a blocking Ab to the hemolytic activity of human IgM Forssman Ab. A large proportion of C3 deposits on mIgM-cross-linked cells was found to be associated with mIgM in the form of C3dg or C3d. These results demonstrate that cross-linked mIgM indeed triggers the classical pathway of C.

Isolation of mouse complement component C7.

Mouse complement component C7 was purified from serum by a sequential procedure of fractionation precipitation by ammonium sulfate, followed by DE-52 anion exchange chromatography. Protein G affinity column chromatography, Mono S cation exchange chromatography and Superdex 200 gel filtration. The final product contained a highly purified mouse C7 component showing a single band on SDS-PAGE at the apparent Mrs of 90 kDa and 100 kDa under non-reduced and reduced conditions respectively. The yield of C7, which was measured by the biological activity, was 7.0%

Role of membrane-associated lymphotoxin (mLT) in the killing activity of lymphokine-activated killer (LAK) cells towards various tumour cell lines.

Human lymphokine-activated killer (LAK) cells developed by an incubation of peripheral mononuclear cells with IL-2 express the membrane-associated lymphotoxin (LT)-related molecule (mLT). By a further cultivation of mLT expressing (mLT-positive) LAK cells for 24 h without IL-2, mLT disappears (mLT-negative LAK cells). Cytotoxicities of various tumour cell lines by either mLT-positive or -negative LAK cells were compared. Eight out of 12 tumour cell lines, less susceptible to mLT-negative LAK cells than mLT-positive LAK cells, were categorized as group A. Two tumour cells (K562 and Molt-4) had the same susceptibility to both kinds of LAK cells. The others (Daudi and Jurkat) had less susceptibilities only when they were assessed at E:T ratios of less than 5. The four tumour cell lines in the latter two cases, containing K562, Molt-4, Daudi and Jurkat cells, were categorized as group B. The cytotoxicities of group A tumour cells, but not group B tumour cells, by LAK cells were significantly suppressed by the presence of anti-LT antibody. Group A tumour cells had higher LT-binding ability (2.82-16.44 fmol/10(6) cells) than group B tumour cells (less than 1.46 fmol/10(6) cells). Both mLT-positive and -negative LAK cells had similar perforin activities and tumour cell-binding capacities. These results suggest that the mLT-mediated killing mechanism is involved in tumour cell killing by LAK cells. Further, various tumour cell lines can be classified into two large groups according to their susceptibilities to the mLT-mediated killing by LAK cells.

Decreased susceptibility of glycated very low density lipoproteins to lipoprotein lipase in vitro and prevention by glutathione.

In vitro glycation of very low density lipoprotein (VLDL) reduced the susceptibility to lipoprotein lipase (LPL) as the level of glycation increased. Addition of reduced glutathione to an incubation medium of serum and glucose interfered with glycation of serum proteins when the concentration of reduced glutathione was higher than 10 mM. At concentrations higher than 25 mM, it also significantly prevented the glycation induced reduction of fatty acid releases from VLDL by LPL. There were no such effects on glycation of serum protein and the fatty acid release from the addition of aminoguanidine. By contrast, addition of D-lysine enhanced glycation of serum proteins by glucose and further decreased fatty acid release from VLDL by LPL. From these results, it is suggested that glycation of VLDL decreases the susceptibility of VLDL to LPL. Delayed catabolism of VLDL in diabetic patients is considered partly caused by glycation of apoproteins, which renders VLDL less sensitive to LPL, in addition to the decreased LPL activity in diabetes mellitus.