Evaluation of risk factors for nephrotoxicity associated with high-dose vancomycin in Japanese patients.

Introduction: Considering the physique of the Japanese population, the standard daily vancomycin dose of 2 g/day and doses ≥ 3 g/day are high in terms of dose per body weight. Studies have reported that administering high-dose vancomycin to achieve a high target trough concentration has been associated with nephrotoxicity. The risk of high-dose vancomycin-associated nephrotoxicity is believed to be exceptionally high for Japanese patients because of their relatively low body weights, but data on the population is lacking. In this retrospective study, we aimed to evaluate risk factors associated with nephrotoxicity in Japanese patients treated with vancomycin. Methods: We examined the medical records of 107 Japanese patients who received vancomycin (3 to 4 g/day). They were divided into two groups based on the presence or absence of nephrotoxicity, and their demographics and clinical characteristics were compared. Results : The incidence of nephrotoxicity in patients receiving high-dose vancomycin was 13%. Age (≥ 60 years) and concurrent use of piperacillin/tazobactam were independent risk factors for vancomycin-associated nephrotoxicity (P = 0.027 and 0.017, respectively). Conclusions : We conclude that the nephrotoxicity risk of high-dose vancomycin in Japanese patients is not excessively high when administered within the confines of a therapeutic drug-monitoring program. However, special care must be taken with patients who are older or on concurrent piperacillin/tazobactam therapy.

Evaluation of the extent of ground-glass opacity on high-resolution CT in patients with interstitial pneumonia associated with systemic sclerosis: comparison between quantitative and qualitative analysis.

AIM: To verify whether quantitative analysis of the extent of ground-glass opacity (GGO) on high-resolution computed tomography (HRCT) could show a stronger correlation with the therapeutic response of interstitial pneumonia (IP) associated with systemic sclerosis (SSc) compared with qualitative analysis. MATERIALS AND METHODS: Seventeen patients with IP associated with SSc received autologous peripheral blood stem cell transplantation (auto-PBSCT) and were followed up using HRCT and pulmonary function tests. Two thoracic radiologists assessed the extent of GGO on HRCT using a workstation. Therapeutic effect was assessed using the change of vital capacity (VC) and diffusing capacity of the lung for carbon monoxide (DLco) before and 12 months after PBSCT. Interobserver agreement was assessed using Spearman's rank correlation coefficient and the Bland-Altman method. Correlation with the therapeutic response between quantitative and qualitative analysis was assessed with Pearson's correlation coefficients. RESULTS: Spearman's rank correlation coefficient showed good agreement, but Bland-Altman plots showed that proportional error could be suspected. Quantitative analysis showed stronger correlation than the qualitative analysis based on the relationships between the change in extent of GGO and VC, and change in extent of GGO and DLco. CONCLUSION: Quantitative analysis of the change in extent of GGO showed stronger correlation with the therapeutic response of IP with SSc after auto-PBSCT than with the qualitative analysis.

Phenotypes of CCAAT/enhancer-binding protein beta deficiency: hyperdontia and elongated coronoid process.

OBJECTIVES: This investigation aimed to conduct a case-control study of mandibular morphology and dental anomalies to propose a relationship between mandibular/dental phenotypes and deficiency of CCAAT/enhancer-binding protein beta (CEBPB). MATERIALS AND METHODS: Skulls of CEBPB(-/-), CEBPB(+/-) and CEBPB(+/+) mice were inspected with micro-computed tomography. Mandibular morphology was assessed with a method of Euclidean distance matrix analysis. RESULTS: Elongation of the coronoid process was identified in CEBPB(+/-) (P ≤ 0.046) and CEBPB(-/-) 12-month-olds (P ≤ 0.028) but not in 14-day-olds (P ≥ 0.217) and 0-day-olds (P ≥ 0.189) of either genotype. Formation of supernumerary teeth in CEBPB(-/-) adult mice was demonstrated (χ(2) = 6.00, df = 1, P = 0.014). CONCLUSIONS: CEBPB deficiency was related to elongation of the coronoid process and formation of supernumerary teeth. The mandibular and dental phenotypes of CEBPB deficiency were unseen by the 14th day after birth. Future investigations into the influence of CEBPB on mandibular and dental development are needed.

Mutation of DNASE1 in people with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a highly prevalent human autoimmune diseases that causes progressive glomerulonephritis, arthritis and an erythematoid rash. Mice deficient in deoxyribonuclease I (Dnase1) develop an SLE-like syndrome. Here we describe two patients with a heterozygous nonsense mutation in exon 2 of DNASE1, decreased DNASE1 activity and an extremely high immunoglobulin G titer against nucleosomal antigens. These data are consistent with the hypothesis that a direct connection exists between low activity of DNASE1 and progression of human SLE.

Tacrolimus is effective for lupus nephritis patients with persistent proteinuria.

OBJECTIVES: To evaluate the safety and potential efficacy of tacrolimus for the treatment of patients with lupus nephritis and persistent proteinuria. METHODS: A total of 23 Japanese patients with lupus nephritis (21 females/2 males) were enrolled in this study. Patients were administered tacrolimus at a dose of 2-3 mg once daily after the evening meal for 6 months. The dose of tacrolimus was unchanged throughout the study period. Concomitant prednisolone therapy was unchanged or gradually tapered, while other immunosuppressants were stopped at the start of tacrolimus treatment. RESULTS: Tacrolimus was well tolerated, and none of the patients developed adverse drug reactions that required discontinuation of the study. Daily urinary protein loss, the U-prot/U-creat ratio, and serum albumin were significantly improved after 4 months, 3 months, and 1 month of treatment with tacrolimus (p<0.05), respectively, and the improvement persisted until 6 months. The serum complement hemolytic activity (CH50), complement C3 level, and CRP level were also significantly improved after treatment with tacrolimus (p<0.05). Improvement of the U-prot/U-creat ratio was most prominent for patients who were in WHO class IV. CONCLUSIONS: Tacrolimus is safe and effective as maintenance therapy for patients with lupus nephritis, at least for 6 months. A larger randomised, controlled trial over a longer period is needed to confirm these results.

Infectious complications in patients receiving autologous CD34-selected hematopoietic stem cell transplantation for severe autoimmune diseases.

Long-term analysis of infectious complication after high-dose immunosuppressive therapy with CD34-selected autologous hematopoietic stem cell transplantation for patients with severe autoimmune diseases (AD) was performed. Theoretically, CD34 selection can reduce the risk of reinfusion of autoreactive lymphocytes. However, it is also associated with a significant reduction in T cells, natural killer cells, and monocytes, which in turn may compromise immune reconstitution, thereby increasing the risk of infection. Moreover, AD compromises host immunity and causes organ damage resulting in dysfunction of the cutaneous or mucosal barrier. In this study, the incidence rate of infections is reported in 14 patients who underwent high-dose (200 mg/kg) cyclophosphamide therapy followed by reinfusion of CD34-selected autologous peripheral blood stem cells. Bacterial complication occurred in 3 of 14 (21%) patients. Cytomegalovirus reactivation and adenovirus hemorrhagic cystitis were observed in 9 (64%) and 2 (14%) patients, respectively. As for late infectious complications, 7 patients (50%) developed dermatomal varicella zoster virus infection. No infection-related mortality was seen in this case series. Because the risk for infections approaches that seen in allogeneic transplant recipients, infection surveillance, diagnostic workup, and prophylactic strategies similar to those applicable to allogeneic recipients are warranted.

A dose-escalation study of rituximab for treatment of systemic lupus erythematosus and Evans' syndrome: immunological analysis of B cells, T cells and cytokines.

OBJECTIVE: Accumulating evidence suggests that B-cell depletion therapy by rituximab may be effective for autoimmune disorders. However, an optimal dose of rituximab and a mechanism of its action remain to be established. We performed a dose-escalation study for treatment of Japanese patients with autoimmune diseases including eight with SLE and one with Evans' syndrome. METHODS: Rituximab was infused intravenously, weekly 4 times in a dose-escalating fashion at three different doses of 100, 250 or 375 mg/m(2) to three patients each. Immunological parameters were monitored at certain points until 12 months after the treatment. RESULTS: Rituximab was well tolerated and safe in these patients. Seven out of eight SLE patients and one with Evans' syndrome clinically responded completely or partially to the treatment. Four patients achieved long-term remission (18-30 months) without any additional treatment. In these patients, a significant decrease in circulating B cells continued for 6 months after the treatment. The mean fluorescence intensities of CD19, CD21, CD40 and BR3 on the residual B cells as well as the percentage of CD69+ CD4+ T cells decreased significantly. Serum TNF-alpha levels decreased significantly on day 2. The Th1/Th2 balance of CD4+ T cells gradually shifted towards a Th1 type by 6 months. CONCLUSION: In addition to B-cell depletion, modification of B-cell and T-cell phenotypes as well as cytokine profiles may be involved in the action of rituximab.

Association of polymorphisms in complement component C3 gene with susceptibility to systemic lupus erythematosus.

OBJECTIVE: Identification of the genes responsible for systemic lupus erythematosus (SLE). METHODS: All the exons and putative promoter regions of 53 candidate genes (TNFRSF6/Fas, TNFSF6/FasL, Fli1, TNFSF10/TRAIL, TNFSF12/TWEAK, Bcl-2, PTEN, FADD, TRADD, CDKN1A, TNFRSF1A/TNFR1, TNFRSF4/OX40, TNFSF4/OX40L, TNFSF5/CD40L, TNFSF13B/BAFF, ICOS, CTLA4, CD28, FYN, G2A, CR2, PTPRC/CD45, CD22, CD19, Lyn, PDCD1, PTPN6, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, TGFBR3, CD3Z, DNASE1, APCS, MERTK, C3, C1QA, C1QB, C1QG, C2, MBL2, IGHM, IL-2, IL-4, IL-10, IFNG, TNFA, MAN2A1, TNFRSF11A/RANK, TNFRSF11B/OPG, TNFSF11/OPGL) were screened for single nucleotide polymorphisms (SNPs) and their association with SLE was assessed by case-control studies. A total of 509 cases and 964 controls of Japanese descent were enrolled. RESULTS: A total of 316 SNPs was identified. When analysed in the Japanese population, the allele frequencies of T at rs7951 and G at rs2230201 of the C3 gene were 0.110 and 0.626, respectively, in SLE patients; significantly higher than the frequencies of 0.081 and 0.584, respectively, in controls [odds ratio (OR) = 1.40, 95% confidence interval (CI) = 1.05-1.86, P = 0.016 and OR=1.19, 95% CI = 1.01-1.41, P = 0.038, respectively]. The mean serum C3 level of carriers of the rs7951 T allele was significantly lower than that of non-carriers of the T allele in 87 SLE patients whose medical records were available (P = 0.0018). CONCLUSION: rs7951 T allele of the C3 gene was significantly associated with SLE, and decreased serum level of C3 seems to be correlated with this allele.

Clinical significance of topical instillation technique in Japanese glaucoma patients.

The clinical significance of a proper eye drop application technique was evaluated in Japanese glaucoma patients. Patients diagnosed with primary open-angle glaucoma having intraocular pressure (IOP) greater than 21 mmHg were treated with eye drops at home. In some patients, however, the topical treatment was ineffective. They returned to the hospital to receive surgical treatment. On admission, 56% of these patients had IOP greater than 21 mmHg. Patient instillation technique was evaluated based on the proximity of the eyedropper tip to the eyes, application position, eyelid closure, treatment (removal) of excess fluid, and nasolacrimal occlusion. In addition, pharmacists interviewed patients to determine the level of understanding of glaucoma, knowledge of prescribed drugs, home application technique, and sensation after application. Multivariate analysis revealed that the key factors influencing the control of IOP to less than 21 mmHg with topical medication were: application of drops in the center of the eye and removal of excessive fluid, in addition to gender and age. Proper topical application at home was dependent on the patient's understanding of the disease, knowledge of prescribed drugs, patient education on the use of drugs, the competence of the instructor, and knowledge of correct application technique. This study indicates that easily comprehensible patient education on the use of eye drops, the nature of glaucoma and the proper use of prescribed drugs is vital to improving the clinical efficacy of topical ophthalmic medication of glaucoma in adult patients.

Differential expression in glioblastoma multiforme and cerebral hemangioblastoma of cytoplasmic proteins that bind two different domains within the 3'-untranslated region of the human glucose transporter 1 (GLUT1) messenger RNA.

The glucose transporter 1 (GLUT1) protein is underexpressed in human glioblastoma multiforme and is overexpressed in human cerebral hemangioblastoma. To gain in-sight into possible posttranscriptional mechanisms regulating the expression of the GLUT1 protein in human brain tumors, cytosolic proteins were prepared from these two tumors and used in RNase T1 protection assays that employed [32P]human GLUT1 synthetic RNA prepared from transcription plasmids. Gel shift mobility assays and ultra-violet light cross-linking studies demonstrated the formation of specific RNA/protein complexes that migrated with a mol mass of 120, 44, and 41 kD. RNase T1 mapping and oligodeoxynucleotide competition studies showed that the 120 kD complex was comprised of an RNA fragment that localized to nucleotides 2186-2203 of the GLUT1 mRNA. The 44 kD complex contained an adenosine-uridine-rich RNA fragment that localized to nucleotides 1885-1906 of the human GLUT1 mRNA, and the formation of this complex was inhibited by synthetic RNA enriched in adenosine-uridine sequences. The 44 kD complex was selectively downregulated in hemangioblastoma as compared to glioblastoma multiforme. These studies demonstrate that human brain tumors have differential regulation of cytosolic proteins that specifically interact with two different domains in the 3'-untranslated region of the GLUT1 mRNA, which may serve to mediate the posttranscriptional regulation of GLUT1 gene expression in these tumors.

Experimental liver cirrhosis induced by alcohol and iron.

To determine if alcoholic liver fibrogenesis is exacerbated by dietary iron supplementation, carbonyl iron (0.25% wt/vol) was intragastrically infused with or without ethanol to rats for 16 wk. Carbonyl iron had no effect on blood alcohol concentration, hepatic biochemical measurements, or liver histology in control animals. In both ethanol-fed and control rats, the supplementation produced a two- to threefold increase in the mean hepatic non-heme iron concentration but it remained within or near the range found in normal human subjects. As previously shown, the concentrations of liver malondialdehyde (MDA), liver 4-hydroxynonenal (4HNE), and serum aminotransferases (ALT, AST) were significantly elevated by ethanol infusion alone. The addition of iron supplementation to ethanol resulted in a further twofold increment in mean MDA, 4HNE, ALT, and AST. On histological examination, focal fibrosis was found < 30% of the rats fed ethanol alone. In animals given both ethanol and iron, fibrosis was present in all, with a diffuse central-central bridging pattern in 60%, and two animals (17%) developed micronodular cirrhosis. The iron-potentiated alcoholic liver fibrogenesis was closely associated with intense and diffuse immunostaining for MDA and 4HNE adduct epitopes in the livers. Furthermore, in these animals, accentuated increases in procollagen alpha 1(I) and TGF beta 1 mRNA levels were found in both liver tissues and freshly isolated hepatic stellate cells, perisinusoidal cells believed to be a major source of extracellular matrices in liver fibrosis. The dietary iron supplementation to intragastric ethanol infusion exacerbates hepatocyte damage, promotes liver fibrogenesis, and produces evident cirrhosis in some animals. These results provide evidence for a critical role of iron and iron-catalyzed oxidant stress in progression of alcoholic liver disease.

Inhibitory effects of tumor invasion-inhibiting factor 2 and its conjugate on disseminating tumor cells.

Tumor invasion-inhibiting factor 2 (IIF-2) is a polypeptide of 21 amino acids which binds to the surface of tumor cells and inhibits experimental invasion in vitro. An albumin conjugate of IIF-2 was used to examine its potential as an antimetastatic compound. The conjugate inhibits in vivo lung metastasis of various highly metastatic tumor cells, including murine melanoma, colon adenocarcinoma, squamous cell carcinoma, forestomach carcinoma, and human fibrosarcoma. In addition to the anti-lung metastasis activity of this compound, it also showed the inhibitory effects on liver and spleen metastasis of murine T-lymphoma cells. A single administration of the conjugate with melanoma cells resulted in prolonged survival times, and their lung colonization was also inhibited when the conjugate was administrated i.v. at times ranging from 6 h before to 1 h after tumor cell inoculation. Similarly, i.p. administration 1 h prior to melanoma cell injection suppressed lung colonization. Pharmacokinetic analysis revealed that the conjugate was more stable than IIF-2 peptide alone. Approximately 10% of the conjugate remained circulating 2 h postinjection and persisted 20 h without degradation, compared with rapid clearing of the unconjugated IIF-2 peptide within 5 min. Furthermore, spontaneous lung metastasis of murine melanoma and colon adenocarcinoma cell was inhibited by successive i.p. administration of the conjugate before the removal of the primary site, with no effect on primary tumor growth. The conjugate significantly reduced tumor cell arrest in the lung and both the IIF-2 peptide and its conjugate demonstrated potent inhibition of basal as well as cytokine-induced-stimulated tumor cell motility. These results suggest that one mode of IIF-2 action may be inhibition of the extravasation of metastasizing cells which have arrested in a target organ, and that the IIF-2-albumin conjugate may be a potent antimetastatic substance with utility in the prevention of artificial seeding of tumor cells during surgical removal of the primary lesions as well as inhibiting metastasis from established metastases.

Anti-tumor immunity elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1.

Interleukin (IL)-28A/interferon (IFN)-λ2 and IL-29/IFN-λ1 have been demonstrated to elicit direct and indirect anti-tumor actions. In this study, we constructed an adenovirus vector expressing either IL-28A/IFN-λ2 (AdIL-28A) or IL-29/IFN-λ1 (AdIL-29) to evaluate the therapeutic properties of intratumoral injection of recombinant adenovirus to apply for the clinical implementation of cancer gene therapy. Despite the lack of an anti-proliferative effect on MCA205 and B16-F10 cells, a retarded growth of established subcutaneous tumors was observed following multiple injections of either AdIL-28A or AdIL-29 when compared with AdNull. In vivo cell depletion experiments displayed that both NK cells and CD8(+) T cells have a major role in AdIL-28A-mediated tumor growth suppression. A significant increase in the number of infiltrating CD8(+) T cells into the tumors treated with either AdIL-28A or AdIL-29 was observed. Moreover, specific anti-tumor cytotoxic T lymphocyte reactivity was detected in spleen cells from animals treated with either AdIL-28A or AdIL-29. In IFN-γ-deficient mice, anti-tumor activities of AdIL-28A were completely impaired, indicating that IFN-γ is critically involved in the tumor growth inhibition triggered by AdIL-28A. IL-12 provided a synergistic anti-tumor effect when combined with AdIL-28A. These results indicate that AdIL-28A and AdIL-29 could be successfully utilized as an alternative cancer immunogene therapy.

Time dependent conversion of creatine kinase MM isoforms in man.

Subforms of creatine kinase MM isoenzyme (isoforms, pI: MMA = 7.95, MMB = 7.76, MMC = 7.54) in human myocardium and serum were quantified by chromatofocusing. Creatine kinase MMA was a dominant isoform (greater than 94% of MM) in normal (n = 3) and in both infarcted and non-infarcted myocardium (n = 2). To investigate isoform conversion in vitro partially purified MMA was incubated with human plasma at 37 degrees C for 24 h (n = 5). Creatine kinase MMB (0%, 47%, 44% of MM at 0, 12, 24 h respectively) and MMC (0%, 22%, 42%) sequentially appeared in incubation media whereas MMA (100%, 31%, 14%) disappeared rapidly with a mean disappearance rate of -0.00169(0.00021)(SD) min-1. Individual differences in conversion velocity were small (SD less than 5%). To investigate isoform conversion in vivo serum isoforms were analysed in patients with acute myocardial infarction (n = 7). MMA was first dominant (A:B:C = 54:34:12%) in the early stage (6-9 h after the onset of chest pain) followed by MMB dominant (19:42:39%) in the middle stage (24-35 h), and MMC dominant (6:22:72%) in the late stage (54-60 h). Changes in isoform proportion were time dependent regardless of serum creatine kinase activity. These findings are consistent with canine isoform conversion reported previously except that in man the velocity of conversion was slower than in the dog. Thus analysis of serum creatine kinase MM isoforms may allow the onset of acute myocardial infarction to be precisely dated. Moreover, determination of MMA, the isoform native to myocardium with a short serum half life, may be useful in the prompt diagnosis of myocardial infarction.

Disappearance and appearance of isoenzymes of creatine kinase, lactate dehydrogenase and aspartate aminotransferase in the myocardium undergoing infarction.

Recent investigations have shown that cardiac isoenzymes change with mechanical overload and possibly with myocardial ischaemia. This complicates the interpretation of serum enzyme changes in acute myocardial infarction. We have therefore investigated the rate of release of isoenzymes from necrosing myocardium and the effect of ischaemia per se. Discrete myocardial infarction was produced in 35 male Wistar rats by ligation of left coronary artery. Six (n = 7), 12 (n = 6), 24 (n = 9), 72 (n = 7) h and 3 weeks (n = 6) after surgery, total and isoenzyme activities of creatine kinase (CK), lactate dehydrogenase (LD) and aspartate aminotransferase (AST) were measured in the infarcted myocardium. Untreated rats (n = 12) were used as the control (time 0). Sham operation was performed in 36 rats. During the early period (0 to 12 or 24 h) of infarction, each (iso)enzyme disappeared monoexponentially from the myocardium (mean r = 0.88) with different disappearance rates. Cytosolic isoenzyme fractions decreased more rapidly than mitochondrial fractions. CK MB and the LD-H subunit decreased faster than CK MM and the LD-M subunit. Such differences in the disappearance rate may be related to subcellular localisation of each isoenzyme. In the late period (72 h and 3 weeks), CK BB and the LD-M subunit showed significant reaccumulation in the infarcted myocardium. Although inflammatory cells can be responsible for the reaccumulation of LD-M subunit, the origin of CK BB is unknown.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-smooth muscle actin expression in tumor and stromal cells of benign and malignant human pigment cell tumors.

We examined the altered expression of alpha-smooth muscle actin (alpha-Sm) in human benign, pre-malignant, and malignant pigment cell tumors by immunohistochemical as well as biochemical (Western blot) analysis using anti-alpha-Sm monoclonal antibody (anti-alpha-Sm MoAb). The expression of alpha-Sm has been revealed immunohistochemically to be associated with mesodermal cells rather than with pigment cells. Western blot analysis using anti-alpha-Sm MoAb detected alpha-Sm expression as a 43-kD band in the extracts from normal papillary dermis, nevus cell nevus, and metastatic melanoma with stromal tissues, but not from primary melanoma with stromal tissues examined. The above findings of alpha-Sm expression by Western blot analysis were further characterized immunohistochemically in terms of the localization at the cellular level as follows. 1) In normal papillary dermis, pericytes encircling capillary vessels showed only positive staining with anti-alpha-Sm MoAb. 2) In nevus tissues, nevus cells were not shown to be positively stained, despite similar positivity of pericytes in normal papillary dermis. 3) In melanoma tissues, alpha-Sm expression of metastatic melanoma detected by Western blot analysis was found to be derived from fibroblasts with smooth-muscle differentiation (myofibroblasts), but not from melanoma cells. Such myofibroblastic stromal changes could not be found on primary melanoma tissue sections, which showed no reactivity in Western blot analysis. We conclude that the major sources of alpha-Sm in benign and pre-malignant pigment cell tumors are capillary pericytes, whereas alpha-Sm found in malignant melanoma tissue is primarily from melanoma-surrounding stromal fibroblasts that were changed to myofibroblasts by some cytokine factor(s), presumably secreted from melanoma cells.

Comparison of tyrosine phosphorylation of proteins by membrane fractions from mouse liver, Ehrlich ascites tumor and MH134 hepatoma.

When membrane fractions from mouse liver, Ehrlich ascites tumor and MH134 hepatoma were incubated with [gamma-32P]ATP at 0 degree C in the presence of MnCl2, ZnCl2 and NaVO3, proteins were phosphorylated on tyrosines to a larger extent in liver membranes than in tumor membranes. Separation of labelled proteins by SDS-gel electrophoresis showed phosphorylated alkali-resistant bands of 170, 140, 130, 80, 56, 53 and 46 kDa proteins in Ehrlich ascites tumor membranes; liver membranes exhibited more strongly phosphorylated bands of 170, 56, 53 and 46 kDa proteins. Epidermal growth factor stimulated the tyrosine phosphorylation of only a 170 kDa protein, which was more significant in liver membranes. Liver membranes exhibited slightly higher levels of tyrosine protein kinase activity compared to tumor membranes.

Arachidonate 12-lipoxygenase is localized in neurons, glial cells, and endothelial cells of the canine brain.

12-Lipoxygenase oxygenates the 12 position of arachidonic acid, producing its 12S-hydroperoxy derivative. We have been interested in the distribution of 12-lipoxygenase in the central nervous system. Previously, by the use of a monoclonal anti-12-lipoxygenase antibody, we proved that one of the arachidonate 12-lipoxygenases existed in canine brain. The present study was therefore designed to elucidate the exact immunohistochemical localization of arachidonate 12-lipoxygenase in canine brain, using a polyclonal anti-12-lipoxygenase antibody. Canine brains were irrigated thoroughly with ice-cold heparinized saline and phosphate-buffered saline to remove blood cells, and then were fixed in 4% periodate-lysine-paraformaldehyde at 4 degrees C for 3 hr. Immunostaining by the indirect method with the peroxidase-labeled antibody was performed for light microscopic observation. The immunohistochemical study demonstrated that many neurons and glial cells in the cerebrum, basal ganglia, and hippocampus were positively stained. Biochemical results as described previously and those of immunohistochemistry indicate that 12-lipoxygenase is definitely localized in various brain parenchymal cells.

Genetic characterization of protein C deficiency in Japanese subjects using a rapid and nonradioactive method for single-stand conformational polymorphism analysis and a model building.

We studied the molecular basis of protein C deficiency in 28 Japanese families including 4 asymptomatic families. Two showed a decreased level of function with a normal antigen concentration consistent with type II protein C deficiency and the remaining 26 showed type I deficiency with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analysis. The PCR-amplified fragments with aberrant migration on SSCP analysis were sequenced. We identified 11 missense mutations, 1 nonsense mutation, 2 neutral polymorphisms, 1 frameshift deletion, 1 inframe deletion, and 1 splice site mutation. We also identified two different rare mutations in the 5'-untranslated region in the protein C gene that may be responsible for the phenotype. Of these molecular defects, ten were novel. From the results of genetic analysis of 47 Japanese families with protein C deficiency reported in this and previous studies, Phe139Val and Met364Ile substitutions and a G8857 deletion were only found in Japanese subjects and seem to be a founder effect. In contrast, Arg169Trp and Val297Met substitutions, both occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These molecular defects were found in 22 families in total, accounting for 47% of Japanese families with protein C deficiency. The structural models of the second EGF and protease domains of activated wild-type and mutant human protein C suggest a possible substrate binding exosite on two loops; one from amino acid position 349 to 357 and the other from position 385 to 388, both of which are close to each other in the three-dimensional model.

Tumor-associated glycoantigen, sialyl Lewis(a) as a target for bispecific antibody-directed adoptive tumor immunotherapy.

The KM231 mAb recognizing sialyl Lewis(a) (sLe(a)) epitope of glycoprotein or glycolipid expressed on various human cancers was used to prepare bispecific antibody (BSAb) containing anti-CD3 x anti-sLe(a) mAb. The effect of anti-CD3 x anti-sLe(a) BSAb on the induction of cytotoxicity by activated T cells was investigated. The activated CD3+ T cells expressing CD8 or CD4 were induced from human peripheral blood mononuclear cells by culture with recombinant IL-2 plus immobilized anti-CD3 mAb. The activated CD8+ and CD4+ T cells showed marginal cytotoxicity against tumor cells by themselves. However, addition of anti-CD3 x anti-sLe(a) BSAb resulted in a great augmentation of their cytotoxicity against gastrointestinal tumor cells. The BSAb also triggered IL-2 production of CD4+ helper/killer T cells during lysis of tumor cells. Moreover, the BSAb was demonstrated to have a potent in vivo antitumor activity against human colon cancer implanted in nude mice by combination with CD4+ helper/killer cells. These results demonstrated that sLe(a) antigen might be a good target molecule for BSAb-directed adoptive tumor immunotherapy.