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Purpose: The incidence and patterns of isolated brain failure was examined in patients with stage III non-small-cell lung cancer (NSCLC) treated with concurrent chemoradiation (CCRT).Materials and Methods: Between 1996 and 2003, a total of 68 patients with stage III NSCLC were treated with radical CCRT. Among them, 63 patients were evaluable. Radiation therapy with a mean total dose of 61.4 Gy and chemotherapy (typically platinum-based) were administered concurrently.Results: Other than locoregional failure, isolated brain failure was the most common failure pattern as the initial failure, occurring 2-37 months (median 6.5 months) after radical CCRT. The isolated brain failure rates as the initial failure at 1, 3, and 4 years were 9%, 13%, and 25%, respectively. Isolated brain failure as the initial failure occurred more frequently in T4 cases (39% at 4 years) compared to T1-3 cases (14% at 4 years) in our series (P = 0.0099).Conclusion: Except for locoregional failure, isolated brain failure was the most common initial failure pattern of stage III NSCLCs treated with radical CCRT. Isolated brain failure as the initial failure occurred even after 3 years. Isolated brain failure as the initial failure occurred more frequently in T4 cases than in T1-3 cases. [ABSTRACT FROM AUTHOR]

Introduction: The retinal pigment epithelium (RPE) plays essential roles in maintaining retinal functions as well as choroidal capillaries and can lead to visual disorders if dysfunctional. Transplantation of human-induced pluripotent stem cell-derived RPE (hiPSC-RPE) is a promising therapy for such RPE impaired conditions including age-related macular degeneration. The challenge with cell suspension transplantation is targeted delivery of graft cells and undesired cell reflux. Gelatin hydrolysate, a soluble variant with specific molecular weight distribution, is examined in this study for its potential use in hiPSC-RPE suspension transplantation, particularly in reducing cell reflux and enhancing RPE engraftment., Methods: A retinal bleb model was created using polydimethylsiloxane (PDMS) soft lithography to quantify cellular reflux. We examined the effects of gelatin hydrolysate on the hiPSC-RPE of various aspects of cell behavior and performance such as cell viability, hypoxia reaction, morphology, induction of inflammation and immune responses., Results: Gelatin hydrolysate at 5 % concentration effectively mitigated cell reflux in vitro mimic, improved cell viability, reduced cell aggregation, and had an inhibitory effect on hypoxic reactions due to cell deposition with hiPSC-RPE. Additionally, gelatin hydrolysate did not affect cell adhesion and morphology, and decreased the expression of major histocompatibility complex class II molecules, which suggests reduced immunogenicity of hiPSC-RPE., Conclusion: Gelatin hydrolysate is considered a valuable and useful candidate for future regenerative therapies in hiPSC-RPE suspension transplantation., Competing Interests: The authors declare no conflict of interest., (© 2024 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Competing Interests: None.

Reactive oxygen species and nitric oxide (NO•) concomitantly play essential roles in guard cell signaling. Studies using catalase mutants have revealed that the inducible and constitutive elevations of intracellular hydrogen peroxide (H 2 O 2 ) have different roles: only the inducible H 2 O 2 production transduces the abscisic acid (ABA) signal leading stomatal closure. However, the involvement of inducible or constitutive NO• productions, if exists, in this process remains unknown. We studied H 2 O 2 and NO• mobilization in guard cells of catalase mutants. Constitutive H 2 O 2 level was higher in the mutants than that in wild type, but constitutive NO• level was not different among lines. Induced NO• and H 2 O 2 levels elicited by ABA showed a high correlation with each other in all lines. Furthermore, NO• levels increased by exogenous H 2 O 2 also showed a high correlation with stomatal aperture size. Our results demonstrate that ABA-induced intracellular H 2 O 2 accumulation triggers NO• production leading stomatal closure., Abbreviations: ABA: abscisic acid; CAT: catalase; cGMP: cyclic guanosine monophosphate; DAF-2DA: 4,5-diaminofluorescein-2 diacetate; H 2 DCF-DA: 2',7'-dichlorodihydrofluorescein diacetate; MeJA: methyljasmonate; NOS: nitric oxide synthetase; NR: nitrate reductase; POX: peroxidase; ROS: reactive oxygen species; SNAP: S -nitroso- N -acetyl-DL-penicillamine; SNP: sodium nitroprusside; NOX: NADP(H) oxidase.

Impact Statement: Cartilage particles derived from human induced pluripotent stem cells (hiPS-Carts) are one candidate source for transplants for treatment of articular cartilage damage. This study shows that hiPS-Carts integrate with each other in an in vitro model and analyzed the course of the integration. The integration starts at the perichondrium-like membrane at around 1 week and then progresses to the central cartilage within 4-8 weeks. The results indicate that FGF18 secreted from the perichondrium-like membrane accelerates the initial step of integration. The findings contribute to understanding how hiPS-Carts form repair tissue and provide clue to accelerate healing after transplantation.

The effects of the peroxisome proliferator, dehydroepiandrosterone sulfate (DHEAS), and the typical cytochrome P450 (CYP) inducers phenobarbital (PB) and 3-methylcholanthrene (3-MC) on fatty liver were examined in rats. Treating rats with orotic acid caused marked accumulation of lipid droplets in the liver. This effect of orotic acid was almost eradicated by co-treatment with DHEAS and PB. While DHEAS or PB alone also alleviated fatty liver, treatment with 3-MC caused little effect on a reduction in lipid droplets. Histopathological examinations revealed numerous peroxisomes in the liver of rats treated with DHEAS. In addition, a significant increase in the expression on hepatic CYPs was observed in rats the fatty liver of which was attenuated. Regarding other enzymes associated with hepatic fatty acid oxidation, the expression levels of sirtuin 1, sirtuin 6, and carnitine palmitoyltransferase 1 were also upregulated most markedly by treatment with DHEAS alone. Thus, the attenuation in fatty liver observed in the present study is likely due to peroxisome proliferation and the induction of fatty acid-metabolizing enzymes by DHEAS and typical CYP inducers.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic in central and northeastern China. This article describes the first identified patient with SFTS and a retrospective study on SFTS in Japan., Methods:  Virologic and pathologic examinations were performed on the patient's samples. Laboratory diagnosis of SFTS was made by isolation/genome amplification and/or the detection of anti-SFTSV immunoglobulin G antibody in sera. Physicians were alerted to the initial diagnosis and asked whether they had previously treated patients with symptoms similar to those of SFTS., Results: A female patient who died in 2012 received a diagnosis of SFTS. Ten additional patients with SFTS were then retrospectively identified. All patients were aged ≥50 years and lived in western Japan. Six cases were fatal. The ratio of males to females was 8:3. SFTSV was isolated from 8 patients. Phylogenetic analyses indicated that all of the Japanese SFTSV isolates formed a genotype independent to those from China. Most patients showed symptoms due to hemorrhage, possibly because of disseminated intravascular coagulation and/or hemophagocytosis., Conclusions: SFTS has been endemic to Japan, and SFTSV has been circulating naturally within the country.

Subclinical hypothyroidism (SCH) has been associated with type 2 diabetes mellitus. However, it is unknown whether common complications of type 2 diabetes, such as diabetic nephropathy, are also present with SCH. Here, we investigated the association between SCH and diabetic nephropathy among Japanese patients with type 2 diabetes mellitus. In this multicenter cross-sectional study, we recruited 414 such patients who had no previous history of thyroid disease. Serum thyroid hormone levels and the urinary albumin:creatinine ratio were measured. SCH was defined as an elevated thyroid-stimulating hormone (TSH) level (>4.0 mIU/L), and diabetic nephropathy was defined as urinary albumin/creatinine ratio ≥300 mg/g. The prevalence of SCH was 8.7% (n = 36) among patients with type 2 diabetes mellitus. The SCH group had a higher prevalence of dyslipidemia (p = 0.008) and diabetic nephropathy (p = 0.014) than the euthyroid group. Multivariate analysis identified significant positive associations between diabetic nephropathy and SCH (odds ratio [OR], 3.51; 95% confidence interval [CI], 1.10-10.0; p = 0.034), hypertension (OR, 4.56; 95% CI, 1.69-14.7; p = 0.001), and smoking (OR, 3.02; 95% CI, 1.14-7.91; p = 0.026). SCH may be independently associated with diabetic nephropathy in Japanese patients with type 2 diabetes mellitus.

Objective: This study aimed to investigate the association between periodontal disease and peptic ulcers in a working population., Methods: Self-administered questionnaires were distributed to all employees of a large insurance company in Japan. The questionnaire asked about their health status and lifestyle habits. Peptic ulcer was defined as either stomach ulcer, duodenal ulcer, or both. For the evaluation of periodontal disease, three indices were used: (a) loss of five or more teeth, (b) having been told of having periodontitis, and (c) periodontal risk score., Results: Of the eligible 28 765 subjects analyzed, peptic ulcer was present in 397 (1.4%). The results of bivariate analyses showed that a significantly higher proportion of subjects with peptic ulcer reported that they lost five or more teeth (35.3 vs. 17.4%, p<0.001) or that they were told they had periodontitis (33.5 vs. 20.7%, p<0.001). Moreover, the periodontal risk score was higher for those with peptic ulcer than those without (mean 0.83 vs. 0.59, p<0.001). In multivariate logistic regression analyses, statistical associations were found between the presence of peptic ulcer and loss of five or more teeth (odds ratio (OR): 1.41, 95% confidence interval (CI): 1.13-1.76, p<0.01), having been told of having periodontitis (OR: 1.28, 95% CI: 1.03-1.59, p<0.05), and a 1-point increase in the periodontal risk score (OR: 1.17, 95% CI: 1.04-1.30, p<0.01), respectively., Conclusion: Modest but statistically significant associations were found between the self-reported measures of periodontal disease and peptic ulcers.

Purpose: In the present study, the nonclinical safety profile of tolvaptan was evaluated., Methods: A series of safety pharmacology and toxicology studies were performed in vitro and in mice, rats, dogs, rabbits and guinea pigs., Results: In safety pharmacological studies, tolvaptan had no adverse effects on the central nervous, somatic nervous, autonomic nervous, smooth muscle, respiratory and cardiovascular, or digestive systems. In general toxicity studies, a single dose of tolvaptan up to 2,000 mg/kg was not lethal in rats and dogs. Tolvaptan did not cause any target organ toxicity in rats after treatment for 26 weeks or in dogs after treatment for 52 weeks at oral doses of up to 1,000 mg/kg/day. The toxicities observed in the present studies were generally attributable to the exaggerated pharmacological action of tolvaptan. In reproductive and developmental toxicity studies in rats, fertility was not affected. Suppressed viability or growth observed in the prenatal and postnatal progeny occurred at the maternally toxic dose of 1,000 mg/kg/day. In rabbits, tolvaptan showed teratogenicity at 1,000 mg/kg/day, a dose that was maternally toxic causing abortion. Tolvaptan was not genotoxic or carcinogenic, and did not induce phototoxicity, antigenicity or immunotoxicity., Conclusion: Nonclinical toxicity that precludes the safe administration of tolvaptan to humans was not observed. However, appropriate cautions should be taken in women of childbearing potential.

Objective: To find vertebral metastases with high risk of symptomatic malignant spinal cord compression (MSCC), features of vertebral metastases caused motor deficits of the lower extremities were examined., Methods: From 2004 through 2006, 78 patients with metastases of the thoracic and/or the cervical spine were treated with radiation therapy (RT). Of these, 86 irradiated lesions in 73 patients were evaluable by magnetic resonance imaging and/or computed tomography at the initiation of RT and were reviewed retrospectively in this study. Twenty-eight patients (38%) had motor deficits at the initiation of RT. Assessed factors were age, sex, primary disease (lung, breast, digestive system and other cancer), lamina involvement, main level of tumor location and vertebral-body involvement., Results: Incidence of motor deficits at the initiation of RT was 55% for lesions with lamina involvement and 5% for lesions without lamina involvement (P < 0.0001). Incidence of motor deficits was 15% for lesions located mainly in the cervical spine and/or the upper thoracic spine (Th1-4), 54% for lesions located mainly in the middle thoracic spine (MTS) (Th5-8) and 30% for lesions located mainly in the lower thoracic spine (Th9-12) (P = 0.0095). Age, sex, primary disease and vertebral-body involvement were not statistically significant factors for incidence of motor deficits due to MSCC (P > 0.9999, P = 0.7798, P = 0.1702 and P = 0.366, respectively)., Conclusions: Vertebral metastases with lamina involvement tended to cause symptomatic MSCC. Latent development of MSCC occurred more frequently in the MTS compared with other levels of the thoracic and the cervical spine.

In order to ensure a widespread distribution in the lung and to avoid the effect of anesthesia, bleomycin at a total dose of 4.5 or 6.0 mg/kg was administered in four divided doses (0.5 ml/kg/time) at intervals of 2 h to male rats via a catheter (tracheotomy tube) without anesthesia. In comparison to vehicle (saline) controls, bleomycin-treated rats showed a significant suppression of body weight gain that was observed transiently at 4.5 mg/kg and continuously (throughout the 3-week observation period) at 6.0 mg/kg. Histopathologically, interstitial pneumonitis, thickening of alveolar walls, thickening of pulmonary arterial walls, foamy cells in alveoli, and hemorrhage were observed in both 4.5 and 6 mg/kg groups, and also emphysema in the 6 mg/kg group. Both groups exhibited a significant decrease in the partial pressure of arterial oxygen (PaO(2)) and a significant increase in alveolar-arterial oxygen tension difference (AaDO(2)), and a significant increase in erythrocyte count was observed in the 6 mg/kg group. Furthermore, both treated groups showed a significant increase in the ratio of the right ventricular weight versus left ventricle plus septum weights. The significant increase in erythrocyte count might have been caused by diffusion disturbance and ventilation-perfusion imbalance due to the pulmonary damage. These findings suggest that the present experimental method will be useful for clarification of the pulmonary damage induced by bleomycin in rats.

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

Twenty-eight patients with Japanese spotted fever were clinically investigated. The diagnosis was determined by confirming an increase of specific antibody. All patients were treated with minocycline, and all recovered, excluding one patient with a fulminant course. Fever and exanthema were observed in all patients, and an eschar was pointed out in 20 (71%) patients. The platelet count was 10 x 10(4)/microl or lower in 8 (28%) patients. The fibrin degradation product (FDP)-level was abnormally high, 10 microg/ml or more, in 16 (57%) patients. The creatine kinase (CK) value was high in 14 of 22 patients, suggesting the presence of myositis. The leukocyte count, FDP, C-reactive protein, and soluble interleukin 2 receptor (sIL2-R) levels were significantly higher in severe cases. In the group without concomitant steroid therapy, mean times of 54.7 h and 101.4 h were required to reduce the temperature to 38 degrees C and 37 degrees C or lower, respectively, after the initiation of tetracycline treatment. There were 6 severe cases: 1 with disseminated intravascular coagulation, 2 with multiorgan failure, 1 with acute respiratory distress syndrome, and 2 with meningoencephalitis. These severe cases formed a group that required 6 or more days to initiate therapy after the onset (P < 0.005 vs non-severe group), showing that delay in diagnosis and therapy is the major cause of aggravation. In the 2 patients complicated by multiorgan failure, the sIL2-R level, produced by activated lymphocytes, was 10,000 U/ml or higher, suggesting that an sIL2-R level of more than 10,000 U/ml can be used as a marker of poor prognosis. It may be better that moderate to severe cases are treated with minocycline plus short-term steroid therapy.

Objective: The aim of this study was to assess the utility of 201Tl SPECT in the differential diagnosis of intracranial tumors and to determine the relationship between 201Tl uptake and histological types., Methods: Thirty-eight patients (19 males and 19 females) with thirty-eight brain tumors were evaluated with 201Tl-SPECT. The early and delayed 201Tl uptake ratio was calculated, and the retention index (RI) was applied as follows; RI = delayed uptake ratio/early uptake ratio., Results: The RI of malignant tumors was higher (0.72 +/- 0.18) than that of benign tumors (0.50 +/- 0.16) and the difference was statistically significant (p = 0.00045). The difference between high-grade glioma (0.80 +/- 0.15) and metastatic tumors (0.64 +/- 0.19) was statistically significant (p = 0.039)., Conclusion: 201Tl-SPECT may add useful biochemical information and could differentiate malignant brain tumors from benign lesions, but the RI of metastatic tumors varied depending on the organs with the primary lesion and histological types.

A 72-year-old man was admitted to the emergency ward in our hospital on July 20, 2001, because of consciousness disturbance, fever, generalized skin eruption, and severe general weakness beginning 7 days previously. Physical examination on admission revealed marked systemic cyanosis, erythema, and purpura. Laboratory findings indicated disseminated intravascular coagulation (DIC) and multiorgan failure (platelet count, 0.9 x 10(4)/micro l; fibrin degradation product, 110 micro g/ml; C-reactive protein, 22.6 mg/dl). Soluble interleukin 2-receptor (sIL-2R) was markedly increased to 14 710 U/ml. Blood gas analysis demonstrated severe metabolic acidosis. He was diagnosed with multiorgan failure due to DIC. Administration of heparin and sodium bicarbonate was started immediately, but respiratory failure was exacerbated and systemic spasm caused by encephalitis was noted. Although he was supported by an artificial ventilator, deterioration of metabolic acidosis occurred, and the blood pressure decreased to less than 60 mm Hg. He died 5.5 h after admission. The serological test showed no positive antibody titers against Orientia tsutsugamushi, Rickettsia japonica, or Rickettsia typhi. However, a specific DNA band derived from R. japonica was detected by the polymerase chain reaction (PCR) method using a primer from a blood clot. Therefore, he was definitively diagnosed as having Japanese spotted fever. The PCR method may be markedly useful for establishing a definitive diagnosis of Japanese spotted fever during the critical stage.

Hydrogen peroxide (H2O2)-induced deacetylation of non-fluorescent acetyl resorufin (1) to fluorescent resorufin (2) as a novel indicator reaction for fluorometric detection of glucose using only glucose oxidase (GOD) is described. When a 1:1:1 mixture of 1 (in CH3CN), glucose, and GOD (each in pH 7.4 phosphate buffer) was incubated at 25 degrees C under aerobic conditions, the resulting solution turned yellow to fluorescent pink due to 2. The formation of 2 was markedly retarded on incubation under anaerobic conditions. When a mixture of 1 and H2O2 was incubated under aerobic conditions, the formation of 2 was noted as in the case of the enzymatic reaction of 1. These results demonstrated that the observed color change is brought about through deacetylation of 1 to 2 induced by H2O2 generated in GOD-catalyzed oxidation of glucose. With regard to the fluorometric traces of the enzymatic reaction with 1 (0.2 mM), GOD (0.5 mg/ml), and glucose at 25 degrees C, fluorescence intensity exhibited a linear relationship against glucose concentration between 0.2 and 2.0 mm, with a correlation coefficient of 0.997. Neither ascorbic acid, uric acid, nor bilirubin significantly interfered with the transformation of 1 to 2 through GOD-catalyzed oxidation of glucose.

Resorufin (1) has been found to act as an electron acceptor in glucose oxidase (GOD)-catalyzed oxidation of glucose. When a 1: 1: 1 mixture of solutions of 1 (5.0 microM), glucose, and GOD (4.0 mg/ml) in phosphate buffer (pH 7.4, 0.1 M) was incubated at 36 degrees C under aerobic conditions and the reaction was followed by a measurement of changes in fluorescence intensity due to 1, only two types of fluorometric traces were observed: (1) when a glucose solution of less than 0.7 mM was subjected to the enzymatic reaction, no consumption of 1 was observed; (2) the reaction with glucose at more than 1.0 mM always consumed 1, affording a regression fluorometric curve, and yet the obtained fluorometric traces could be almost superimposed on one another with no dependence on the glucose concentration. The reasons for the observed phenomena are discussed.

Background: The nonspecific cross-reacting antigen-50/90 (NCA-50/90) is a glycoprotein antigen which shares some antigenic determinants with carcinoembryonic antigen (CEA). No definite clinical value has been established for the measurement of NCA-50/90 in cancer patients., Methods: We established and evaluated a chemiluminescent enzyme immunoassay (CLEIA) specific for NCA-50/90 using two monoclonal antibodies., Results: No significant reactivity with 4 purified related antigens including CEA was found in the NCA-50/90 CLEIA. Serum samples (n = 572) from patients with malignant (n = 326) as well as from healthy individuals (n = 246) were analyzed by the NCA-50/90 CLEIA and by the established ACCESS CEA assay. The average sensitivity of NCA-50/90 for malignant disease was 40.8%, compared to 45.4% of CEA. Relatively high positive rates of NCA-50/90 were observed in sera from patients with lung cancer (72.0%), hepatoma (62.5%), pancreatic cancer (47.6%), breast cancer (35.6%), and colorectal cancer (34.5%). About 15% of patients with malignant disease were positive only for NCA-50/90. The levels of NCA-50/90 and CEA in sera from patients with malignant disease correlated only poorly., Conclusions: The present study suggests that increases in blood levels of NCA-50/90 occur in a population of cancer patients which is different from those with elevated levels of CEA and that NCA-50/90 might be useful for NCA-50/90-positive, but CEA-negative patients.

Mouse monoclonal antibody FU-MK-1, raised against a human gastric adenocarcinoma, recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas and seems to be valuable in immunodiagnosis and immunotherapy of various cancers. In a recent study, we constructed a mouse/human chimeric antibody, designated Ch FU-MK-1, by fusing the FU-MK-1 V(H) and Vkappa genes to the human Cgamma1 and Ckappa genes, respectively. In the present study, we tested combination immunotherapy of Ch FU-MK-1 with human lymphokine-activated killer (LAK) cells in vitro and in mice with severe combined immunodeficiency (SCID) bearing human MK-1-expressing tumors. In in vitro experiments, Ch FU-MK-1 effectively mediated antibody-dependent cell-mediated cytotoxicity (ADCC) against MK-1-expressing MKN-74 cells, which was completely blocked by an anti-FcR antibody. Since the apoptotic pathway as well as the necrotic pathway have been shown to be utilized in various cytotoxic effector mechanisms, we investigated the role of apoptosis in ADCC mediated by LAK cells and Ch FU-MK-1 against MKN-74 cells. The implication of the apoptosis during ADCC was demonstrated by means of both a terminal-deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling assay and a propidium iodide staining method. In vivo antitumor activity of combination treatment with LAK cells and Ch FU-MK-1 was estimated using SCID mice inoculated s.c. with MKN-74 cells. The i.v. administration of LAK cells and i.p. administration of Ch FU-MK-1 and interleukin-2 (IL-2) produced a marked growth inhibition of MKN-74 tumors in SCID mice. When the actual tumor weights were measured 16 days after initiation of treatment, more than 70% reduction was observed in the group receiving LAK cells plus Ch FU-MK-1 plus IL-2 as compared to the control untreated group. Together these results suggest that Ch FU-MK-1 may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors.

The mouse/human chimeric antibody Ch F11-39, recently generated by ourselves, shows the same high specificity and affinity for carcinoembryonic antigen (CEA) as those of its parental mouse monoclonal antibody. Ch F11-39 is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) with human peripheral blood lymphocytes (PBL). Interleukin-2 (IL-2) modulates the function of immunocytes, in particular inducing lymphokine-activated killer (LAK) cells and enhancing ADCC. In the present study, we therefore tested the combination immunotherapy of Ch F11-39 with LAK cells in vitro and in severe combined immunodeficiency (SCID) mice bearing human CEA-producing tumors. In vitro experiments using human gastric tumor cell lines, Ch F11-39 effectively mediated ADCC against CEA-positive MKN-45 cells, but not against CEA-negative cells. The specificity of ADCC for Ch F11-39 was demonstrated by experiments with irrelevant target cells or irrelevant antibody. ADCC activity of PBL with Ch F11-39 was enhanced by double after preincubation with IL-2 at 10 U/ml. The concentration of Ch F11-39 required for 50% maximal cell killing was about 0.25 microgram/ml at 10 U/ml of IL-2. Increasing ADCC was triggered by IL-2 earlier (1 day) than the generation of LAK cells (3 days). Control human IgG blocked the ADCC, suggesting that the enhancement of ADCC by IL-2 may be caused by activation of effector cells expressing Fc receptors. In vivo anti-tumor activity of combined immunotherapy was estimated using SCID mice inoculated s.c. with 1 x 10(7) MKN-45 cells. The i.v. administration of LAK cells and i.p. administration of Ch F11-39 and IL-2 produced a marked growth inhibition of MKN-45 tumors in SCID mice (about 50% reduction in tumor size as compared to the control untreated group, measured 15 days after treatment). In summary, the enhanced antitumor activity of Ch F11-39 with LAK cells suggests that it might be a useful immunotherapeutic reagent for CEA-expressing tumors.

We have established a new chemiluminescent enzyme immunoassay for carcinoembryonic antigen (CEA), designated ACCESS CEA, which is adapted to the fully automated ACCESS immunoassay analyzer. The assay is based on a one step sandwich-type method using two monoclonal antibodies, one of which is immobilized on micrometer-size paramagnetic particles and the other is conjugated to alkaline phosphatase. Ten microliters of calibrators or sera are incubated for 5 minutes at 37 degrees C with the particles and with the alkaline phosphatase conjugate. The particles are then magnetically separated and washed to remove unbound components. Time needed to obtain the first result is less than 15 minutes. The assay range was 0.04-1000 micrograms/l of CEA, and the possible high-dose hook effect was prevented at CEA concentrations up to 100000 micrograms/l in this working range. The coefficient of variation (CV) for intra-assay precision was 3.0 to 4.7%, and inter-assay CV was 3.4 to 5.6%. The sample carryover was less than 0.001%. The analytical recovery ranged from 98 to 104% and a dilution linearity was demonstrated. No interference was detected in any sample with levels up to 300 mg/l for bilirubin, 12000 mg/l for haemoglobin, 50000 mg/l for human serum albumin, 8500 mg/l for triacylglycerol, and 500000 IU/l for rheumatoid factor. The ACCESS CEA assay also showed very homogeneous reactivity with purified CEA preparations from different tumours and could discriminate CEA from four CEA-related normal antigens tested. Serum samples (n = 362) from patients with malignant or non-malignant disease, as well as from healthy individuals, were analyzed by the ACCESS CEA assay and by the established IMx CEA assay. The CEA values determined by the ACCESS CEA assay were in good agreement with those determined by the IMx CEA assay, and the ACCESS CEA assay significantly increased the sensitivity and specificity of tumour diagnosis as compared with the IMx CEA assay.

A mouse/human-chimeric bispecific antibody, designated CBA-CEACD3, with dual specificities for carcinoembryonic antigen (CEA) and CD3, was generated by chemical cross-linking of a chimeric antibody specific for CEA to another chimeric antibody against CD3. Flow cytometric analysis showed that CBA-CEACD3 can bind specifically to cells expressing CEA and to normal human peripheral blood mononuclear cells (HPBMCs) bearing CD3, respectively. Furthermore, a cell to cell adhesion analysis by a colorimetric assay using the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) demonstrated that CBA-CEACD3 is able to bind CEA-producing cells to CD3-expressing cells, suggesting that both arms of CBA-CEACD3 are simultaneously working and can retarget T-cells to the tumor. In an additional colorimetric assay using MTT, this antibody was shown to effectively mediate CEA-expressing tumor cell killing by freshly isolated HPBMCs. Together these results demonstrate that this chimeric bispecific antibody may serve as a potentially useful immunotherapeutic reagent for human CEA-producing cancers.

Mouse monoclonal antibodies against CD3 on human T lymphocytes have been used for therapy in organ-transplant patients as a potent immunosuppressive agent or for treatment of cancer as a potent T cell activating agent. However, an inherent problem in their in vivo application is the human anti-mouse antibody response. In this study, we cloned and sequenced the variable region genes of the heavy and light chains (VH and V kappa) of a mouse anti-human CD3 monoclonal antibody (OKT3) using the reverse transcription-polymerase chain reaction method. Then, we constructed a mouse/human chimeric antibody, designated as Ch OKT3, by fusing the OKT3 VH and V kappa genes to the human heavy and light chain constant region genes (C gamma 1 and C kappa) derived from a human plasma cell leukemia line (ARH77), respectively. The chimeric gene constructs were sequentially co-transfected into mouse non-Ig-producing hybridoma cells (Sp2/0) by electroporation. The Ch OKT3 antibody thus prepared bound to human peripheral blood mononuclear cells and competitively inhibited the binding of the parental MAb OKT3 to the blood mononuclear cells, indicating that this chimeric antibody seems to be suitable for in vivo therapeutic approaches.

Whole-body autoradiography (WBAR) was used to study the biodistribution of 125I-labeled mouse-human chimeric antibody (Ch F11-39) to carcinoembryonic antigen (CEA) in athymic nude mice bearing the CEA-producing MKN-45 human gastric carcinoma xenografts. Significantly high uptake of 125I-Ch F11-39 in the tumors obtained by tissue-counting technique was confirmed by WBAR of mice of 12, 24, 48, and 96 h postinjection of 125I-Ch F11-39. When compared with histochemical or immunohistochemical staining results of the tumor tissue sections, imaging profiles of 125I-Ch F11-39 obtained by WBARs were topographically correlated with histopathological findings of tissues and immunohistochemical localization of CEA in the tumor tissues, indicating that the accumulation of 125I-Ch F11-39 at the tumor site is based on its specificity for CEA. These results demonstrate that this chimeric antibody may serve as a potential useful diagnostic and/or therapeutic reagent for human CEA-producing cancers.

Binding reactivities of 62 anti-CEA MAbs from 10 different research groups with cell membrane-bound CEA and with free CEA in solution were compared by inhibition of MAb binding to CEA-expressing tumor cells by free CEA. Bindings of 30 MAbs to the cell membrane-bound CEA (280 ng CEA/2 x 10(5) cells) were inhibited by approximately equal amounts of free CEA, indicating that binding affinities of about half the MAbs for cell membrane-bound CEA are similar to those for free CEA, respectively. Bindings of 15 MAbs to the cell membrane-bound CEA were easily inhibited by free CEA of less than half the amount of the cell membrane-bound CEA, while inhibition of bindings of the remaining 17 MAbs required twice more free CEA than the amount of cell membrane-bound CEA, showing that about one-fourth of the MAbs have higher affinities for free CEA and the remaining about one-fourth of the MAbs possess higher affinities for cell membrane-bound CEA. These results help form the basis for selecting the anti-CEA MAbs for use in clinical applications, such as serum CEA assay, tumor imaging and immunotherapy.

To reduce heterophilic antibody interference in a two-site immunoassay for carcinoembryonic antigen (CEA), we utilized a human/mouse chimeric antibody to CEA as the tracer. One mouse monoclonal antibody (MAb), F82-61, which reacts with an epitope present on the domain N of CEA, was immobilized on 96-well polystyrene microtiter plates. A human/mouse chimeric antibody (Ch F11-39), which recognizes an epitope present on the domain B3 of CEA, was biotinylated for the tracer (Ch F11-39 system). Another MAb F11-39, the parental MAb of Ch F11-39, was also biotinylated and used as the control tracer (F11-39 system). For a fair comparison, the same 503 serum samples from healthy individuals were simultaneously assayed in the present study. When a tentative common reference limit of 5 ng/ml was used, the false positive rate with the Ch F11-39 system was only 2.8% (14/503) and that with the F11-39 system was 29.0% (146/503). Adding normal mouse serum (NMS; 1%) or a mixture of purified mouse IgG subclasses (heterophilic blocking reagent (HBR, 15 micrograms/test)) to the F11-39 system reduced the false positive rate from 29.0% to 6.2% (31/503) or 4.8% (24/503), respectively, suggesting that heterophilic antibodies reactive with mouse IgG gave rise to the high positive rate in normal populations with the F11-39 system. On the other hand, the false positive rate with the Ch F11-39 system was only slightly reduced from 2.8% to 2.6% (13/503) or to 2.0% (10/503) by adding NMS or HBR to the Ch F11-39 system. The false positive rates with two commercially available assay systems, CEA Roche EIA.DM or Abbott IMx CEA, were 5.4% (27/503) and 5.8% (29/503), respectively, which both corresponded roughly to that with the F11-39 system including NMS or HBR. These results indicate that the application of human/mouse chimeric antibodies in two-site immunoassays is more effective for reducing interference from heterophilic antibodies than the adding of NMS or purified mouse IgG in the assay using conventional MAbs.

We have found a new compound from thermophile extracts which inhibits antigen presentation on mouse macrophages. The substance inhibits the expression of the class II major histocompatibility molecule (Ia). It was extracted from Bacillus stearothermophilus UBT8038 and purified by silica gel column chromatography. The isolated inhibitor, Fr. 8-A, was a phosphatidylethanolamine with isofatty acids and chemically different from any of the natural or synthetic products which have been reported to modify Ia expression. Fraction 8-A inhibits Ia expression by mouse peritoneal macrophages induced by the supernatant from concanavalin A stimulated spleen cell cultures in a dose-dependent fashion over the range 0.1-10 micrograms/ml. This fraction also exhibited inhibitory effects on antigen presentation by splenic macrophages in vitro and on the mixed leukocyte reaction. The present results show that Fr. 8-A has a unique inhibitory effect on antigen presenting cells.

The common (and definitive) intracranial tumors in patients with one form of neurofibromatosis (NF), bilateral acoustic NF (NF-2), are bilateral acoustic neuromas. In this study, we present 2 cases with what appears to be another form of NF, namely, NF-1, who showed bilateral enlargement of the internal auditory canals. Bilateral caloric responses were remarkably impaired in both cases; a unilateral sensorineural hearing loss was observed in 1. No tumor was demonstrated in the enlarged internal auditory canals in either case. Although the pathophysiology remains uncertain, some patients with NF-1 show abnormal cochleovestibular function in association with bilateral internal auditory canal enlargement without an acoustic tumor.