Study on CH50 levels in factor D-depleted serum

It is generally accepted that levels of serum whole complement activity (CH50) reflect the activities of complement (C) components of the classical C pathway (CP), since CH50 is assayed by use of sensitized sheep erythrocytes (EA). However, the alternative C pathway (AP) is considered to be also activated simultaneously in the process of activation of serum CP by EA. Thus, serum CH50 levels may possibly reflect not only CP but also AP activation in CH50 assay. We studied on the influence of AP activation during CH50 assay on CH50 levels, by comparison of CH50 levels in serum samples before and after treatment of factor D depletion. Polystyrene beads carrying polyanion, poly (2-acrylamide 2-methylpropane sulfonate) (PAMPS-beads), on the surface were prepared and used for preparation for factor D-depleted serum. After treatment of pooled normal human serum (NHS) with PAMPS-beads (2.5 mg/ml of serum), serum ACH50 level decreased to be undetectable, indicating that AP activation is prohibited in PAMPS-beads-treated serum. When isolated factor D was added to this PAMPS-beads-treated serum, ACH50 level recovered to that of before treatment. Immunoblot analysis revealed that factor D band observed in NHS disappeared completely after PAMPS-beads treatment. From these results, it is clear that factor-D deficient serum is prepared by PAMPS-beads treatment. Besides, since serum CH50 level was not decreased by PAMPS-beads treatment, it may be concluded that CH50 level is not affected by AP activation during CH50 assay.     

Pioglitazone improves the phenotype and molecular defects of a targeted Pkd1 mutant

Mutations of either PKD1 or PKD2 are associated with autosomal dominant polycystic kidney disease (ADPKD). The molecular function of the gene product of PKD1, polycystin-1, in vitro has been elucidated recently, but the molecular pathological consequences of the loss of polycystin-1 in vivo have remained unclear. We have generated a mouse with a targeted deletion of exons 2-6 of Pkd1 to study the molecular defects in Pkd1 mutants. Homozygote embryos (Pkd1(-/-)) developed hydrops, cardiac conotruncal defects and renal cystogenesis. Total protein levels of beta-catenin in heart and kidney and c-MYC in heart were decreased in Pkd1(-/-) embryos. In the kidneys of Pkd1(-/-), the expression of E-cadherin and PECAM in basolateral membranes of renal tubules was attenuated, and tyrosine phosphorylation of epidermal growth factor receptor and Gab1 were constitutively enhanced when cystogenesis started on embryonic day (E) 15.5-16.5. Maternally administered pioglitazone, a thiazolidinedione compound, resolved these molecular defects of Pkd1(-/-). Treatment with pioglitazone improved survival of Pkd1(-/-) embryos and ameliorated the cardiac defects and the degree of renal cystogenesis. Long-term treatment with pioglitazone improved the endothelial function of adult Pkd1(+/-). These data indicated that molecular defects observed in Pkd1(-/-) embryos contributed to the pathogenesis of ADPKD and that thiazolidinediones had a compensatory effect on the pathway affected by the loss of polycystin-1. Pathways activated by thiazolidinediones may provide new therapeutic targets in ADPKD.     

Interaction of poly(styrene sulfonic acid) with the alternative pathway of the serum complement system

Bioartificial pancreas, in which the islets of Langerhans are enclosed in artificial membrane to be protected from the host immune system, is expected to be a promising medical device to treat patients who suffer from insulin-dependent diabetes. Our strategy for preparation of a bioartificial pancreas involves utilizing a membrane including polymeric materials that can inhibit the complement reaction. In this study, we examined the effects of poly(styrene sulfonic acid) (PSSa) on the alternative pathway of the serum complement system to identify the mechanism(s) involved. PSSa was dissolved in pooled normal human serum (NHS), and the mixtures were incubated at 37 degrees C for 30 min. Complement activities in sera were determined by hemolytic assays. Amounts of complement activation products released were determined by ELISA. Interactions of PSSa with complement components and fragments were examined with electrophoresis and immunoblotting. From these examinations, it appeared that the manner of PSSa effects on the alternative pathway (AP) highly depends on its concentration. PSSa seemingly acted as an activator when its concentration was 0.005 g/dl to 0.05 g/dl, while it acted as an inhibitor when its concentration was more than 0.1 g/dl. In terms of activation or inhibition of the AP, forming complex of PSSa with factor H induced activation, and that with factor D induced inhibition.     

IL-6-STAT3 controls intracellular MHC class II alphabeta dimer level through cathepsin S activity in dendritic cells

We found IL-6-STAT3 pathway suppresses MHC class II (MHCII) expression on dendritic cells (DCs) and attenuates T cell activation. Here, we showed that IL-6-STAT3 signaling reduced intracellular MHCII alphabeta dimmer, Ii, and H2-DM levels in DCs. IL-6-mediated STAT3 activation decreased cystatin C level, an endogenous inhibitor of cathepsins, and enhanced cathepsin activities. Importantly, cathepsin S inhibitors blocked reduction of MHCII alphabeta dimer, Ii, and H2-DM in the IL-6-treated DCs. Overexpression of cystatin C suppressed IL-6-STAT3-mediated increase of cathepsin S activity and reduction of MHCII alphabeta dimer, Ii, and H2-DM levels in DCs. Cathepsin S overexpression in DCs decreased intracellular MHCII alphabeta dimer, Ii, and H2-DM levels, LPS-mediated surface expression of MHCII and suppressed CD4(+) T cell activation. IL-6-gp130-STAT3 signaling in vivo decreased cystatin C expression and MHCII alphabeta dimer level in DCs. Thus, IL-6-STAT3-mediated increase of cathepsin S activity reduces the MHCII alphabeta dimer, Ii, and H2-DM levels in DCs, and suppresses CD4(+) T cell-mediated immune responses.     

Hepatic changes in the acute phase of streptozotocin (SZ)-induced diabetes in mice

We have reported the streptozotocin (SZ)-induced hepatic lesions in the subacute phase (4 to 12 weeks after the treatment), which are characterized by appearance of oncocytic hepatocytes, cytomegalic hepatocytes and bile duct hyperplasia. In this study, we focused on the acute phase (6 to 48 hours after the treatment) of the SZ-induced hepatic lesions of mice to clarify the onset of the hepatic alterations, especially before the induction of hyperglycemia. Livers were taken from 8-week-old Crj:CD-1 (ICR) male mice at 6,12, 24, 36 and 48 hours after the 200 mg/kg b.w. of SZ-injection. SZ-induced hyperglycemia was noted at 36 and 48 hours after the treatment, but the hepatic changes including lipid peroxidation, mitochondrial swelling, peroxisome proliferation and inhibition of hepatocyte proliferation occurred before the elevation of the serum glucose levels. The present findings indicate the direct effects of SZ on hepatocytes rather than the secondary effects of diabetes, and certain correlations between the hepatocytic changes in the acute phase and those in the subacute one. In addition, ulcer and submucosal edema of the gallbladder were observed at 36 or 48 hours after the SZ-treatment, which can be a novel finding in SZ-treated animal.     

Momentary changes in the cardiovascular autonomic system during mental loading in patients with panic disorder: a new physiological index "rho(max)"

BACKGROUND: Although panic disorder (PD) is suggestive of autonomic nervous system dysfunction, especially in the cardiovascular autonomic system (CAS), the results in many previous studies are still controversial. Using a new physiological index which could well reflect emotional reaction to visual stimuli (Yoshizawa, M., Sugita, N., Tanaka, A., Abe, K., Yambe, T., Nitta, S., 2001. Quantiatative Physioligical Evaluation of Three Dimensional Images. The Seventh International Conference on Virtual Systems and Multumedia, 25-27.), we studied momentary changes in the CAS in patients with PD during audiovisual stimulation (AS) as mental loading. METHODS: During AS, exposed to a video of imaginary experiences such as driving a motor vehicle or diving into the sea, blood pressure (BP) and heart rate (HR) were measured in 12 remitted patients with PD and 19 age- and sex-matched normal controls (NC). We used the maximum cross-correlation coefficient (rho(max)) from the BP to the HR, whose frequency components were limited to around 0.1 Hz. RESULTS: The rho(max) was an available index which could detect the momentary changes in the CAS during AS in both groups. The two-way ANOVA disclosed significant group and time effects on the rho(max). The momentary response to emotional stimuli in the PD patients was slower than that in the NC subjects. LIMITATIONS: Antidepressants have a potential impact on the autonomic variables in this study. CONCLUSIONS: These findings suggest that there may be a dysfunction of the CAS in remitted PD patients and that the dysfunction may be one of the trait markers of PD. To confirm these findings, however, further studies with a large sample size are required.     

Expression and intracellular localization of FKHRL1 in mammary gland neoplasms

FKHRL1 (FOXO3a), a member of the Forkhead family of genes, has been considered to be involved in the development of breast tumors; however, the in vivo expression and activation status of FKHRL1 in breast tumors still remains unclear. We immunohistochemically demonstrated the expression and intracellular localization of FKHRL1 in human breast tumors by the novel anti-FKHRL1 antibody which is available for formalin-fixed paraffin-embedded specimens. In a total of 51 cases of benign tumors, FKHRL1 was diffusely expressed in all cases, and its intracellular localization was revealed to be cytoplasmic (inactive form) in 94% of cases of intraductal papillomas (16/17) and 91% cases of fibroadenomas (31/34), with a similar pattern to normal glandular epithelium. In invasive ductal carcinomas, 83% of the cases (93/112) diffusely expressed FKHRL1; however, unlike benign tumors, 71% of the cases (66/93) showed the nuclear-targeted, active form of FKHRL1. Moreover, activated FKHRL1 was predominantly observed in scirrhous (29/36, 81% of the cases) and papillotubular (30/38, 79% of the cases) subtypes, compared to the solid-tubular subtype (7/19, 37% of the cases). Furthermore, the cases with nuclear-targeted FKHRL1 showed a tendency to have lymph nodal metastasis with statistical significance (P < 0.0001). Thus, the activation of FKHRL1 seems to be recognized as one of the specific features of invasive ductal carcinoma of the breast.     

Deposition of complement protein C3b on mixed self-assembled monolayers carrying surface hydroxyl and methyl groups studied by surface plasmon resonance

Since complement activation is recognized as a common response of the host defense system when an artificial medical device is applied to a patient, great effort has been devoted to studies on the interaction of the complement system with artificial materials. However, some uncertainties remain, partially because of the lack of well characterized surfaces and suitable analytic methods for study of the surface phenomena that occur on artificial materials under physiologic conditions. In this study, we employed self-assembled monolayers (SAMs) and the surface plasmon resonance (SPR) technique to study interactions of the serum complement with well characterized surfaces. Self-assembled monolayers carrying various concentrations of hydroxyl groups were prepared using 11-mercapto-1-undecanol (C11-OH) and one of n-nonanethiol, n-dodecanethiol, and n-hexadecanethiol. The amount of NHS deposition on the SAMs increased with increasing C11-OH content of the SAMs, and the amount of anti-C3b antibody immobilization formed on the NHS deposition layers increased with increasing C11-OH content of the SAMs. These results clearly demonstrate that a large amount of C3b, produced through the activation of the complement system, binds covalently to and is adsorbed by hydroxyl-group-rich surfaces. The combination of SAMs and the SPR technique is suitable for studying the interaction of the complement system with solid surfaces, and the results should give basic information needed for a rational design of biocompatible surfaces on synthetic materials.     

Exacerbation of rheumatoid arthritis following Helicobacter pylori eradication: disruption of established oral tolerance against heat shock protein?

A 62-year-old Japanese woman with RA received an eradication therapy against Helicobacter pylori in November 1999. Eight weeks later, successful eradication was confirmed by negative results for rapid urease test, pathologic findings, and a fall in anti-H. pylori IgG antibody titer. During the course, parameters for RA activity were exacerbated: C-reactive protein 1.1-4.2 mg/dL, rheumatoid arthritis precipitation antigen 2560-5120 dils., erythrocyte sedimentation rate 52-123 mm/h, and complements CH50 50 to over 60 U/mL. Lansbury index increased from 70% to 105%. Two more weeks later, the patient noticed right shoulder pain. She also complained of bilateral gonalgia two months later, and physical examination revealed increased fluid in the knee joints. Prednisolone was required to control the disease activity. The results of this case suggested that RA patients might experience a deleterious effect on the disease activity following H. pylori eradication possibly through disruption of the established oral tolerance against stress protein such as mycobacterial heat shock protein 65.     

Improved affinity of a human anti-Entamoeba histolytica Gal/GalNAc lectin Fab fragment by a single amino acid modification of the light chain

We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose- and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica. To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser91 and Arg96 in the third complementarity-determining region of the light chain with other amino acids. The screening of 200 clones of each exchange by an indirect fluorescent antibody test showed that 14 clones for Ser91 and nine clones for Arg96 reacted strongly with E. histolytica trophozoites. Sequence analyses revealed that the substituted amino acids at Ser91 were Ala in five clones, Gly in three clones, Pro in two clones, and Val in two clones, while the amino acid at position 96 was substituted with Leu in three clones. The remaining eight clones exhibited no amino acid change at position 91 or 96. These mutant Fab fragments were purified and subjected to a surface plasmon resonance assay to measure the affinity of these proteins to the cysteine-rich domain of lectin. Pro or Gly substitution for Ser91 caused an increased affinity of the Fab, but substitution with Ala or Val did not. The replacement of Arg96 with Leu did not affect affinity. These results demonstrate that modification of antibody genes by recombination PCR is a useful method for affinity maturation and that amino acid substitution at position 91 yields Fabs with increased affinity for the lectin.