Comparison of photodynamic therapy to transpupillary thermotherapy for polypoidal choroidal vasculopathy.

PURPOSE: To compare the therapeutic efficacy of photodynamic therapy (PDT) to that of transpupillary thermotherapy (TTT) for polypoidal choroidal vasculopathy (PCV). METHODS: PDT or TTT was performed on 46 eyes of 46 patients with PCV; 19 eyes were treated with TTT (TTT group) and 27 eyes with PDT (PDT group). PCV was diagnosed by fundus examination, fluorescein angiography (FA) , and indocyanine green angiography (ICGA) . The best-corrected visual acuity (BCVA) in logarithm of the minimum angle of resolution (logMAR) units and OCT-determined foveal thickness were evaluated before and after treatment. For statistical analyses, the Student's t-test and chi(2) test were used. RESULTS: The number of treatments during the 12-month follow-up period was significantly higher in the TTT group (1.7 times) than in the PDT group (1.3 times; P=0.0134). The difference in the BCVA between the TTT and PDT groups at the baseline was not significant (P=0.3150), but the BCVA in the PDT group was significantly better than that in the TTT group at 3, 6, and 12 months after treatment (P=0.0093, P=0.0074, P=0.0006, respectively). The foveal thickness decreased markedly at 6 months after treatment in the PDT group (P<0.0001) but not significantly in the TTT group (P=0.8982). A vitreous haemorrhage was observed after treatment in two eyes in the TTT group. CONCLUSIONS: BCVA was significantly better and the fovea was significantly thinner in the PDT group than in the TTT group after treatment. Thus, PDT may be more effective than TTT for the treatment of eyes with PCV.

Living-related partial liver transplantation for decompensated hepatitis B without reactivation of hepatitis B in the following 30 months.

We report a case of living-related partial liver transplantation for decompensated hepatitis B without reactivation of hepatitis B in the following 30 months, and we analyze the factors that indicate a favorable prognosis for transplantation. The 42-year-old female patient received continuously administered lamivudine before transplantation, and hepatitis B virus immunoglobulin (HBIG) from the anhepatic phase to the present. Currently, she shows a normal aminotransferase level and is negative for hepatitis B surface antigen and hepatitis B virus (HBV) DNA by polymerase chain reaction amplification. Sequence analysis was performed. The entire precore/core region and part of the polymerase region of HBV were sequenced by a direct sequencing method after polymerase chain reaction. No specific mutation was found in these regions. These observations show that the key factors in the long-term successful treatment of this patient appear to be the combination therapy of lamivudine and HBIG that the patient received from around the time of the transplantation. Furthermore, the lack of specific mutations, including lamivudine resistant-mutations, is likely to represent an additional factor in the effectiveness of this treatment.

Disease recurrence after living liver transplantation for primary biliary cirrhosis: a clinical and histological follow-up study.

We describe the recurrence of primary biliary cirrhosis (PBC) in recipients of living liver transplants. We are not aware of similar previous reports. Because most donors for living liver transplantation (LLT) are blood relatives with close HLA matches, the recurrence of PBC in transplant recipients might offer additional insights in the pathogenesis of the condition. We studied 6 women (age, 29 to 61 years) with PBC who survived LLT for at least 1 year. Tests for antimitochondrial autoantibody (AMA), antipyruvate dehydrogenase complex-E2, immunoglobulin G (IgG) anti-M2, and IgM anti-M2 had confirmed the diagnosis. Donors were blood relatives in 5 instances, and one donor who was not a blood relative still had multiple HLA matches with the recipient. After LLT, we observed a decrease in AMA titers, but within 1 year, these titers increased again in 5 of the 6 patients to pre-LLT levels or greater. Immunoblotting analysis of the anti-M2 protein profile failed to show loss of bands and showed new bands in 3 of 6 patients. Histologically, strong evidence of recurrent PBC was found in 2 patients, and findings compatible with PBC were present in 1 additional patient. All 6 patients are doing well, without symptoms of recurrent PBC (median time post-LLT, 35.5 months; range, 12 to 50 months).

Levels of interleukin-18 and its binding inhibitors in the blood circulation of patients with adult-onset Still's disease.

OBJECTIVE: Interleukin-18 (IL-18) is a proinflammatory cytokine that is involved in immunologically mediated tissue damage, but its bioactivity is regulated in vivo by its soluble decoy receptor, IL-18 binding protein (IL-18BP). This study was undertaken to determine levels of IL-18 and IL-18 binding inhibition in the blood of patients with adult-onset Still's disease (ASD). METHODS: Serum concentrations of IL-18 in ASD patients were compared by enzyme-linked immunosorbent assay (ELISA) with those in patients with other systemic rheumatic diseases and healthy controls. The biologically active mature protein of IL-18 was detected by Western blot analysis with anti-IL-18 antibody and its induction of interferon-gamma (IFNgamma) secretion from IL-18-responding human myelomonocytic KG-1 cells. The inhibitory activity on IL-18 binding to its receptor was determined by 125I-IL-18 binding inhibition assay using the Chinese hamster ovary cell line transfected with a murine IL-18 receptor (CHO-K1/mIL-18R). RESULTS: Concentrations of serum IL-18 were extremely elevated in patients with active ASD compared with those in patients with rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, polymyositis/dermatomyositis, Sjogren's syndrome, or healthy individuals. Levels of IL-18 were found to correlate with serum ferritin values and disease severity in ASD. Western blot analysis revealed that serum samples from patients with active ASD contained an 18-kd polypeptide of IL-18, corresponding in size to the mature form. Accordingly, the samples were able to induce IFNgamma secretion from KG-1 cells, which was largely abolished by neutralizing anti-IL-18 antibody. However, the level of IL-18 bioactivity was more than 10-fold weaker than the concentration of IL-18 protein measured by ELISA. Serum samples from patients with active ASD showed an inhibitory effect on the binding of 125I-IL-18 to CHO-K1/mIL-18R cells, and this activity was associated with elevation of IL-18. CONCLUSION: These data indicate that systemic overproduction of IL-18 may be closely related to the pathogenesis of ASD, despite the restriction on its inflammatory activity by IL-18 binding inhibitors such as IL-18BP. The disease activity appears to be determined on the basis of the relative levels of IL-18 and its specific inhibitors.

Interferon-gamma-inducing activity of interleukin-18 in the joint with rheumatoid arthritis.

OBJECTIVE: To examine the levels of interleukin-18 (IL-18) bioactivity within the rheumatoid arthritis (RA) joint, and the differential effects of IL-12 and IL-18 on interferon-gamma (IFNgamma) production by T cell infiltrates. METHODS: Expression of IL-18 protein and messenger RNA (mRNA) was determined by enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction, respectively. The biologic activity of IL-18 was detected on the basis of IFNgamma secretion from IL-18-responding human myelomonocytic KG-1 cells. To determine the extent of inhibitory activity on binding of IL-18 to its receptor, a [125I]-IL-18 binding inhibition assay was performed, using a Chinese hamster ovary cell line transfected with a murine IL-18 receptor. RESULTS: The amount of IL-18 protein detected in both the serum and synovial fluid of RA patients was markedly larger than that detected in the serum and synovial fluid ofosteoarthritis (OA) patients, and serum IL-18 levels correlated with the levels of serum C-reactive protein. IFNgamma production by KG-1 cells was more strongly stimulated in synovial fluid samples from RA patients than in samples from OA patients, and this activity was largely diminished in the presence of anti-IL-18 antibody. In contrast, the activity of IL-18 binding inhibition in the serum and synovial fluid of RA patients was not significantly elevated compared with that in OA patients. RA synovial tissues showed increased expression of IL-18 mRNA and increased IL-18 protein synthesis compared with that in OA tissues. Purified CD14+ macrophages, but not activated fibroblast cell lines, from RA synovium were able to release mature IL-18, although both cell types expressed its transcripts. IL-18 alone showed a negligible effect on IFNgamma production by RA synovial tissue cells, in contrast to IL-12, which was directly stimulatory. However, IL-12-induced IFNgamma production was synergistically enhanced by IL-18, and yet was >50% reduced by neutralization of endogenous IL-18 with anti-IL-18 antibody. CONCLUSION: These results indicate that IL-18, produced predominantly by tissue macrophages, primarily potentiates IL-12-induced IFNgamma production by T cell infiltrates in RA synovium. Detection of significant IL-18 bioactivity in the joints, despite the presence of IL-18 binding inhibitors, supports an integral role of this cytokine in perpetuating the IFNgamma-dominant T cell cytokine response in RA.

Protective effects of dietary docosahexaenoic acid against kainate-induced retinal degeneration in rats.

PURPOSE: To investigate the role played by docosahexaenoic acid (DHA) in the retina, and more specifically, its ability to protect the retina from kainic acid (KA)-induced retinal damage. METHODS: Three-week-old female Wistar rats were used. DHA (1000 mg/kg per day) was fed to the rats for 7, 14, and 28 days, and the concentrations of DHA and arachidonic acid (AA) in the retina and serum were measured. In another group of rats, the right eyes were injected intravitreally with 3.12 nanomoles KA after DHA supplementation for 14 days. Electroretinograms (ERGs) elicited by different stimulus intensities were recorded before and on days 1, 7, and 14 after the KA injection. The amplitudes and implicit times of the a- and b-waves were compared. The number of cells in the ganglion cell layer (GCL) and inner nuclear layer (INL) were compared by histopathologic examination. RESULTS: The concentration of DHA in the serum and retina increased after DHA supplementation. The concentration of AA in serum decreased with DHA supplementation, but the concentration of AA in retina did not show any significant change. The b-waves of the ERGs recorded after KA injection were significantly attenuated in both groups of rats. However, the attenuation was significantly less in the DHA-supplemented rats than in gum arabic-supplemented control rats. The numbers of cells in the INL and GCL were significantly higher in DHA-supplemented rats. CONCLUSIONS: These results indicate that DHA supplementation can partially counteract KA neurotoxicity in the rat retina. DHA may play a role in modulating neuronal excitability by reducing KA-induced responses in the retina.

Cloning and expression of interleukin-18 binding protein.

Interleukin-18 binding protein is a novel glycoprotein that we successfully cloned and expressed. First, murine interleukin-18 binding protein was purified from the sera of mice with endotoxin shock using ligand affinity chromatography. The murine interleukin-18 binding protein cDNA was cloned after RT-PCR using mixed primer pair sequences based on partial murine interleukin-18 binding protein amino acid sequence analysis. Subsequently, human interleukin-18 binding protein cDNA was cloned from cDNA libraries of normal human liver using murine interleukin-18 binding protein cDNA as a probe. Next, we transiently expressed recombinant human and murine interleukin-18 binding proteins in COS-1 cells and purified them from culture supernatants. Both recombinant interleukin-18 binding proteins did not exhibit species specificity and prevented interleukin-18 binding to its receptor. In addition, they inhibited interleukine-18 dependent IFN-gamma production from KG-1 cells effectively. These results suggest that the interleukin-18 binding protein may possess interleukine-18 antagonist activity.

Purification and characterization of the human interleukin-18 receptor.

Interleukin (IL)-18 was identified as a molecule that induces IFN-gamma production and enhances NK cell cytotoxicity. In this paper, we report upon the purification and characterization of human IL-18 receptor (hIL-18R). We selected the Hodgkin's disease cell line, L428, as the most strongly hIL-18R-expressing cell line based on the results of binding assays. This binding was inhibited by IL-18 but not by IL-1beta. The dissociation constant (Kd) of 125I-IL-18 binding to L428 cells was about 18.5 nM, with 18,000 binding sites/cell. After immunizing mice with L428 cells and cloning, a single monoclonal antibody (mAb) against hIL-18R was obtained (mAb 117-10C). Sequentially, hIL-18R was purified from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-extracted L428 cells by wheat germ lectin-Sepharose 4B chromatography and mAb 117-10C-Sepharose chromatography. The internal amino acid sequences of hIL-18R all matched those of human IL-1 receptor-related protein (IL-1Rrp), the ligand of which was unknown to date. When expressed in COS-1 cells, the cDNA of IL-1Rrp conferred IL-18 binding properties on the cells and the capacity for signal transduction. From these results, we conclude that a functional IL-18 receptor component is IL-1Rrp.

Immunohistochemical characterization of hepatic lymphocytes in acute hepatitis A, B, and C.

The distinctive histologic findings in acute hepatitis A, B, and C suggest that different immunological mechanisms are involved in the pathogenesis of these diseases. This study was undertaken to define the immune response in each type of acute hepatitis by identification of the intrahepatic lymphocyte subsets. Thirty paraffin-embedded liver biopsy specimens from 10 patients with acute hepatitis A, 10 patients with acute hepatitis B, and 10 patients with acute hepatitis C were evaluated. Immunohistochemical staining was performed by the indirect immunoperoxidase technique using the following monoclonal or polyclonal antibodies: CD45RO, CD20-cy, CD57, and Mac387. Inflammatory infiltrates varied from specimen to specimen. However, CD45RO+ memory T cells were the predominant infiltrating mononuclear cells in all specimens. In the portal areas, CD45RO+ memory T cells were the most prominent in AHC, followed by AHA and AHB, and the difference between AHC and AHB was statistically significant. CD20-cy+ B cells were seen mainly in the portal areas, and were significantly less common in AHB than in AHA and AHC. In addition, the ratio of CD20-cy+ B cells to CD45RO+ memory was significantly lower in AHB than in the other types of acute hepatitis. The necrotic areas in all specimens contained mainly CD45RO+ memory cells in association with a few CD57+ NK and T cells or CD20-cy+ B cells. Our study revealed differences of the intrahepatic lymphocyte subsets among the various types of acute hepatitis, but the meaning of these differences is presently unknown. Therefore, further studies are required to fully elucidate the mechanism of the immune response in acute hepatitis.

Metastasis-promoting activity of a novel molecule, Ag 243-5, derived from mycoplasma, and the complete nucleotide sequence.

We found that mycoplasma-infected cells have a higher ability to metastasize in vivo than non-mycoplasma-infected cells. To investigate this phenomenon, we obtained a monoclonal antibody, MAb 243-5, by immunization with Mycoplasma arginini-infected RPMI 4788 cells. This MAb recognized a mycoplasmal protein with an MW of 47 kDa and completely inhibited the experimental metastasis of M. arginini-infected RPMI 4788 cells using a nude mouse model. Using this MAb, we purified a molecule called Ag 243-5 and determined the N-terminal amino acid sequence and clarified the entire nucleotide sequence of the Ag 243-5 gene. PCR analysis showed the existence of a homologous gene in Mycoplasma hyorhinis. Four sequential injections of Ag 243-5 (30 micrograms/shot) promoted the experimental metastasis of non-mycoplasma-infected RPMI 4788 cells more than 10-fold using a nude mouse model. Ag 243-5 also promoted the experimental metastasis of the non-mycoplasma-infected mouse colon cancer cell line colon 26. This metastasis-promoting effect was neutralized by MAb 243-5.

Immunohistochemical detection of proliferating cell nuclear antigen in hepatocellular carcinoma: relationship to histological grade.

Proliferating cell nuclear antigen (PCNA), also known as cyclin, is an auxiliary protein of DNA polymerase-delta and is found only in the nuclei of proliferating cells in the late G1 and S phases. The proliferation of hepatocellular carcinoma (HCC) by immunohistochemical staining for PCNA using paraffin sections of 20 surgically resected HCC specimens was analysed. The mean percentage of PCNA-positive nuclei in the HCC tissue was 10.3% in grade I of Edmondson and Steiner's classification, 25.5% in grade II, 28.4% in grade III and 41.5% in grade IV. In early HCC, we observed only a few PCNA-positive tumour cells. However, PCNA-positive nuclei were numerous in the tumour thrombi found in portal vein branches, in regions of extracapsular tumour growth, and in the inner nodules of tumours with a nodule-in-nodule formation. Proliferating cell nuclear antigen positivity was correlated with an increase of the nucleocytoplasmic ratio of tumour cells as determined by image analysis. Our findings showed that PCNA positivity was correlated with the histological grade and invasiveness of HCC, suggesting that this antigen may be used as an indicator to predict tumour invasion in patients with HCC.

Hepatitis C virus antibody in patients with primary liver cancer (hepatocellular carcinoma, cholangiocarcinoma, and combined hepatocellular-cholangiocarcinoma) in Japan.

BACKGROUND: In hepatocellular carcinoma (HCC), a high prevalence of hepatitis C virus antibody (anti-HCV) has been reported, indicating that it may be an important etiologic factor in the pathogenesis of HCC. In this study, the authors investigated the prevalence of anti-HCV in HCC patients, as well as the same prevalence in patients with cholangiocarcinoma (CC) and combined hepatocellular-cholangiocarcinoma (combined HCC-CC), to study the clinicopathologic features of anti-HCV-positive cases. METHODS: The authors examined 141 patients with primary liver cancer who were pathologically diagnosed as having HCC (121 cases), CC (13 cases), or combined HCC-CC (7 cases). Hepatitis B surface antigen (HBsAg) and anti-HCV were measured in these patients. RESULTS: Of 121 HCC cases, 85 (70.3%) were found to be anti-HCV positive, 16 (13.2%) were HBsAg positive, and 5 (4.1%) were both anti-HCV and HBsAg positive. In 13 cases with CC and in 7 with combined HCC-CC examined, 4 (30.8%) and 5 (71.4%), respectively, were anti-HCV positive. CONCLUSIONS: The anti-HCV-positive rate was high in combined HCC-CC as well as in HCC. These three types of primary liver cancer, which were anti-HCV positive, shared two common features: male dominance and high incidences of complication with liver cirrhosis.

Prevalence of primary sclerosing cholangitis and other liver diseases in Japanese patients with chronic ulcerative colitis.

An association between primary sclerosing cholangitis (PSC) and chronic ulcerative colitis (CUC) is well known in Western countries, but there have been no reports on this association in Japan. We reviewed 163 consecutive CUC patients (91 males and 72 females) diagnosed from 1984 to 1990 at Tokyo Women's Medical College. Abnormal liver function tests were found in 42 patients with CUC (25.8%), but chronic liver disease was only diagnosed in seven patients (4.3%). Among these seven patients, there were four with PSC, one with small-duct PSC, one with transfusion-associated chronic hepatitis and one with Type B liver cirrhosis. No relationship was found between the documented colonic manifestations of CUC and the presence of PSC. The four PSC patients did not have a longer history of CUC at the time of diagnosis of PSC than CUC patients without PSC. At the time of PSC diagnosis, two patients were asymptomatic, one presented with right upper quadrant pain, and the other had fatigue. Three patients were diagnosed as having CUC before the onset of PSC (range 2-13 years), and the other patient had both diseases simultaneously. All four had a good prognosis. Thus PSC was the most common chronic liver disease associated with CUC in our series, and it was present in all our CUC patients with alkaline phosphatase levels exceeding twice the upper limit of normal and mild transaminase elevation.

Epitopes on Cry j I and Cry j II for the human IgE antibodies cross-reactive between Cupressus sempervirens and Cryptomeria japonica pollen.

Forty sea from French patients allergic to Cupressus sempervirens pollen were tested for cross-reactivities against Cry j I, Cry j II (major allergens of Cryptomeria japonica pollen) and other pollen allergens from botanically related plants. Seventy-three per cent of the sera reacted with either Cry j I or Cry j II, or with both of them. These IgE cross-reactions were blocked effectively by mAb 046 (anti-Cry j I) or N26, T27 (anti-Cry j II), and weakly by mAbs 052, 027 and 026 (anti-Cry j I). Furthermore, the IgE antibodies in two sera, #40 and #11, bound to peptide fractions obtained from enzyme-digested Cry j I, and mAb 027 could also bind to the fractions. Analyses of the amino acid sequences of the peptides revealed that reactive peptides contained "NGNATPQLTKNAGVLTCSLSKR" sequence and the third residue N3 was glycosylated, however, when the N3 was not glycosylated, the IgE antibodies did not react, but mAb 027 could. The glycosylation of the N3 might be required for IgE-binding to the peptides. Sugar component on the N3 residue was found to be 0.4 mol galactose, 1.3 mol mannose, 0.8 mol fucose and 2.0 mol N-acetyl-glucosamine. Cross-reactivities against other pollen allergens from botanically related plants were found in most of the sera. However, many of these reactivities were detected by sandwich ELISA but not by an ELISA using allergen-coated plates, indicating that it is important to select an appropriate ELISA procedure in order to detect an allergen or an IgE antibody to an allergen.

Antigenic analyses of Sugi basic protein by monoclonal antibodies: I. Distribution and characterization of B-cell-tropic epitopes of Cry j I molecules.

Using 23 monoclonal antibodies raised against Sugi basic protein (SBP, major allergen of Japanese cedar pollen), composed of Cry j I and Cry j II, analyses of B-cell-tropic epitopes of Cry j I were performed. The following results were obtained. (1) As far as the mAbs were used, no major cross-reactive determinants were detected between Cry j I and Cry j II molecules. (2) 21 of the 23 mAbs were specific for Cry j I, and the anti-Cry j II mAbs were classified into four groups by their fine specificities, suggesting that Cry j I bears at least four antigenic determinant regions. (3) Cry j I molecules were found to take a monomeric form in solution and to display no repeating antigenic epitopes on their surfaces. (4) Some of the determinants seemed to be located in the interior of a Cry j I molecule, and when the Ag is coated on a plastic plate, the determinants become exposed on its surface. (5) Binding of human IgE antibodies to Cry j I and Cry j II was blocked by some of the obtained mAbs, suggesting that these epitopes recognized by the mAbs might have an important role in human allergic response against the cedar pollen.

Antigenic analyses of Sugi basic protein by monoclonal antibodies: II. Detection of immunoreactive fragments in enzyme-cleaved Cry j I.

The 4 anti-Cry j I mAbs showing an epitope specificity different from each other, 046, 029, 026 and 027, were selected to analyze the structure of the antigenic determinant for each mAb on a Cry j I molecule. Immunoreactive fragments in enzyme-cleaved Cry j I were detected by means of the adsorption on the mAb column and of the binding to the mAbs on Elisa. The mAb 026 was found to be reactive to the fragments containing a Cry j I N-terminal region obtained by V8 protease or pepsin digestion, but not to those by lysylendopeptidase digestion. The mAb 027 was found to be capable of binding to the fragments containing a linear structure of Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser-Leu-Ser-Lys, which were generated by V8 protease, lysylendopeptidase or pepsin digestion. Furthermore, the synthetic peptide Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser- Leu-Ser-Lys-Arg could bind to 027, but not to 026, and could inhibit the binding of 027 to Cry j I or to its immunoreactive fragments. No fragments capable of reacting to the mAbs 046 and 029 could be found in this study, suggesting that 046 and 029 recognize a conformationally constituted epitope of Cry j I molecule which is destroyed by enzymatic cleavage. The epitope recognized by the mAbs 027 or 026 was found to be located in conformationally hidden parts of the molecule which was exposed to react to the mAbs only after the physicochemical or enzymatic treatment.

Cloning and nucleotide sequence of the gene encoding arginine deiminase of Mycoplasma arginini.

The existence of a mycoplasmal arginine deiminase which catalyzes the conversion of L-arginine to L-citrulline has been postulated. Here we show the partial amino acid sequence of arginine deiminase of Mycoplasma arginini and the complete nucleotide sequence of the arginine deiminase gene of M. arginini. The open reading frame deduced from this sequence consists of 1,230 bp encoding 410 amino acids. The mature form of this enzyme contains 409 amino acids after the deletion of the first methionine. In this open reading frame, TGA nonsense codons are used as tryptophan codons; this usage was verified by determination of the amino acid sequence. The molecular weight of the enzyme calculated from the deduced amino acid sequence is 46,372. Recently, the nucleotide sequence of the arginine deiminase gene of M. arginini was reported by Kondo et al. (K. Kondo, H. Sone, H. Yoshida, T. Toida, K. Kanatani, Y.-M. Hong N. Nishino, and J. Tanaka, Mol. Gen. Genet. 221:81-86, 1990). However, their sequence differed from ours in several places and especially at the C terminus.