Damage accrual related to pregnancies before and after diagnosis of systemic lupus erythematosus: a cross-sectional and nested case-control analysis from a lupus registry.

OBJECTIVE: The objective of this study was to evaluate the chronic damage associated with pregnancies before and after the diagnosis of systemic lupus erythematosus (SLE). METHODS: Using childbearing-aged female SLE patient data registered at the Okayama and Showa University Hospitals, a nested case-control analysis was performed to investigate the relationship between pregnancy and chronic damage using the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI). RESULTS: Pregnancy occurred in 22 patients before and 13 patients after the diagnosis of SLE in 104 eligible patients. Live births occurred in 82% (33/40) and 50% (9/18) of the pregnancies before and after the diagnosis of SLE, respectively. After matching age and disease duration, 33 case patients with chronic damage (SDI ≥ 1) and 33 control patients without chronic damage (SDI = 0) were selected. Hypertension was more frequent in cases than in controls (48% vs. 24%, p = 0.041). Pregnancies before and after the diagnosis of SLE were comparable between cases and controls (before the diagnosis: nine case patients and eight control patients; after the diagnosis: three case patients and five control patients; p = 1.00). Even after adjusting for hypertension using multivariate analysis, the pregnancies before and after the diagnosis were not significant predictors for chronic damage (odds ratio = 1.48 (95% confidence interval 0.33-6.65)), p = 0.60 of the pregnancy before the diagnosis; odds ratio = 0.78 (95% confidence interval 0.13-4.74), p = 0.78 of the pregnancy after the diagnosis). CONCLUSION: Pregnancies, either before or after the diagnosis of SLE, did not show any differences in chronic damage. Our results help alleviate fears regarding childbearing in female patients with SLE and their families.

Cell-bound exopolysaccharides of Lactobacillus brevis KB290 enhance cytotoxic activity of mouse splenocytes.

AIMS: This study aimed to identify the main active component of Lactobacillus brevis KB290 (KB290) that is responsible for enhanced cell-mediated cytotoxic activity of mouse splenocytes Live KB290, a probiotic strain derived from a Japanese traditional pickle, was previously reported to modulate innate immune responses as affecting on cell-mediated cytotoxic activity of mouse splenocytes. METHODS AND RESULTS: We used live KB290, heat-killed KB290, a derivative strain (Lact. brevis KB392) with different amounts of cell-bound exopolysaccharide (EPS-b), and a crude extract of EPS-b from KB290 cell surface. Female BALB/c mice were fed a diet containing 10(10) CFU live KB290, 10(10) CFU live KB392, 15 mg heat-killed KB290 or 600 µg crude extract of EPS-b for 1 day. Live KB290 (P < 0.01), heat-killed KB290 (P < 0.05) and crude EPS-b at 600 µg (P < 0.05) per mouse significantly enhanced cytotoxic activity; however, live KB392 had no effect. CONCLUSIONS: Both live and heat-killed KB290 and crude EPS-b significantly enhanced cytotoxic activity of mouse splenocytes. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrated that EPS-b produced by KB290 has a critical role in enhancing cell-mediated cytotoxic activity in mouse spleen.

Oral administration of Lactobacillus brevis KB290 to mice alleviates clinical symptoms following influenza virus infection.

UNLABELLED: Lactobacillus brevis KB290 (KB290), isolated from a traditional Japanese pickle 'Suguki', has been reported to have immunomodulatory effects. We investigated whether oral administration of KB290 has protective effects against influenza virus (IFV) infection in mice. After 14 days of administration of lyophilized KB290 suspended in phosphate-buffered saline by oral gavage, BALB/c mice were intranasally infected with 2 × MLD50 (50% mouse lethal dose) of IFV A/PR/8/34 (H1N1). Prophylactically administered KB290 significantly alleviated the loss of body weight and the deterioration in observational physical conditions induced by the infection. In addition, 7 days after infection, the levels of IFV-specific immunoglobulin (Ig)A in bronchoalveolar lavage fluid were significantly increased in mice fed KB290 compared with controls. Moreover, there was a significant elevation of serum interferon (IFN)-α in KB290 group mice, even at three and 7 days after infection, despite the administration of KB290 being stopped before IFV infection. Our results demonstrated that oral administration of KB290 before infection could alleviate IFV-induced clinical symptoms. Alleviation of clinical symptoms by KB290 consumption may have been induced by long-lasting enhancement of IFN-α production and the augmentation of IFV-specific IgA production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that oral administration of Lactobacillus brevis KB290 (KB290), a probiotic strain derived from a Japanese traditional pickle, could protect against influenza virus (IFV) infection in mice. Our results demonstrated that continual intake of KB290 for 14 days prior to IFV infection alleviated clinical symptoms such as loss of body weight and deterioration in observational physical conditions induced by the infection. The beneficial effects of KB290 consumption may have been elicited by the long-lasting enhancement of interferon-α production and the augmentation of IFV-specific immunoglobulin A production.

Elevated serum levels of soluble CX3CL1 in patients with microscopic polyangiitis.

OBJECTIVES: To test the hypothesis that CX3CL1 contributes to the pathogenesis of microscopic polyangiitis. METHODS: Serum samples from 18 patients with microscopic polyangiitis (MPA), who fulfilled the revised criteria of the American College of Rheumatology (ACR), were collected during both the newly diagnosed, untreated active disease states and inactive disease states. Also serum was from patients with large vessel vasculitis (LVV), including giant cell arteritis (n=4) and Takayasu arteritis (n=3), and from 52 healthy individuals. Soluble (s)CX3CL1 levels in serum were measured using an enzyme-linked immunosorbent assay. Disease activity was assessed using Birmingham vasculitis activity scores (BVAS). Expression of CX3CR1 was examined by flow cytometry. RESULTS: Serum sCX3CL1 levels were significantly higher in MPA patients than in either LVV group or healthy individuals. The elevated sCX3CL1 levels seen in MPA patients correlated positively with BVAS, as well as with CRP levels and ESR, and similarly increased expression of cell-surface CX3CR1 was seen on peripheral blood CD4 and CD8 T cells from patients with MPA. Notably, sCX3CL1 levels and CX3CR1 expression were diminished during clinical remission following treatment. CONCLUSION: Our findings suggest that CX3CL1 may be involved in the pathogenesis of MPA, and may serve as a useful serologic marker of disease activity in systemic vasculitis.

Identification of expressed genes characterizing long-term survival in malignant glioma patients.

Better understanding of the underlying biology of malignant gliomas is critical for the development of early detection strategies and new therapeutics. This study aimed to define genes associated with survival. We investigated whether genes coupled with a class prediction model could be used to define subgroups of high-grade gliomas in a more objective manner than standard pathology. RNAs from 29 malignant gliomas were analysed using Agilent microarrays. We identified 21 genes whose expression was most strongly and consistently related to patient survival based on univariate proportional hazards models. In six out of 10 genes, changes in gene expression were validated by quantitative real-time PCR. After adjusting for clinical covariates based on a multivariate analysis, we finally obtained a statistical significance level for DDR1 (discoidin domain receptor family, member 1), DYRK3 (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 3) and KSP37 (Ksp37 protein). In independent samples, it was confirmed that DDR1 protein expression was also correlated to the prognosis of glioma patients detected by immunohistochemical staining. Furthermore, we analysed the efficacy of the short interfering RNA (siRNA)-mediated inhibition of DDR1 mRNA synthesis in glioma cell lines. Cell proliferation and invasion were significantly suppressed by siRNA against DDR1. Thus, DDR1 can be a novel molecular target of therapy as well as an important predictive marker for survival in patients with glioma. Our method was effective at classifying high-grade gliomas objectively, and provided a more accurate predictor of prognosis than histological grading.

Sequence analysis of Stx2-converting phage VT2-Sa shows a great divergence in early regulation and replication regions.

In enterohemorrhagic Escherichia coli, Shiga toxin is produced by lysogenic prophages. We have isolated the prophage VT2-Sa that is responsible for production of Shiga toxin type 2 protein, and determined the complete nucleotide sequence of this phage DNA. The entire DNA sequence consisted of 60,942 bp, exhibiting marked similarity to the 933W phage genome. However, several differences were observed in the immunity and replication regions, where cI, cII, cIII, N, cro, O, and P genes were present: Predicted amino acid sequences of N, cI, cro, O and P in the VT2-Sa genome did not show significant similarity to the counterparts of the 933W genome; however its cI showed higher similarity to lambda. Furthermore, O and P closely resembled those of phage HK022. These observations suggest that the various degrees of homology observed in the immunity and replication regions of VT2-Sa could have resulted from frequent recombination events among the lambdoid phages, and that these regions play a key role as a functional unit for phage propagation in competition with other lambdoid phages.

Protection of cultured gastric cells against tert-butyl hydroperoxide by glutathione isopropyl ester.

Oxidants are involved in the pathogenesis of a variety of gastrointestinal disorders. Intracellular GSH protects rat gastric cells against oxidants. We examined whether GSH isopropyl ester (GSH ester) can protect against oxidants and whether a system of GSH ester uptake is present in these cells; we also investigated a possible role of GSH in inhibiting lipid peroxidation. Cytotoxicity was quantified by 51Cr release. Lipid peroxidation was assessed by measuring malondialdehyde production. tert-Butyl hydroperoxide caused a dose-dependent increase in 51Cr release. Treatment with GSH ester attenuated the toxicity of tert-butyl hydroperoxide. Incubation with GSH ester enhanced the cellular GSH content, which was prevented by DL-buthionine-[S,R]-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase. GSH ester prevented tert-butyl hydroperoxide-induced lipid peroxidation, corresponding with the degree of protection. Therefore, we concluded that GSH isopropyl ester protects gastric cells against oxidants through the accumulation of intracellular GSH. This protection is mediated in part by the prevention of hydroperoxide-induced lipid peroxidation. However, the gastric epithelial system of GSH ester uptake appears distinctly different from those observed in hepatocytes, lymphoid cells and fibroblasts in terms of mediation of gamma-glutamylcysteine synthetase activity.

Efficient detection of deletions induced by a single treatment of mitomycin C in transgenic mouse gpt delta using the Spi(-) selection.

Transgenic rodent mutation assays permit the detection and molecular analysis of various types of gene mutations, such as base changes and frameshifts, in a number of tissues. It is reported, however, that deletion mutations are not efficiently detected by the assays, in particular those using lambda phage shuttle vectors. Recently, a new transgenic mouse model, i.e., gpt delta, has been developed to selectively detect some types of deletions by Spi(-) selection. Spi(-) selection has an advantage over the other selections to preferentially identify deletions because only lambda phages deficient in both the red and gam gene functions are allowed to form phage plaques. In this study, we examined whether in vivo deletions induced by the treatment of mitomycin C (MMC) are detectable by the Spi(-) assay in the mouse model. The mice were treated with MMC (0.5, 1.0, 2.0, and 4.0 mg/kg, single intraperitoneal injection) and sacrificed 14 days after the dosing. The treatment at 4.0 mg/kg approximately doubled the mutant frequency of Spi(-) in the bone marrow, i.e., 2.52 x 10(-6) vs. 1.31 x 10(-6). The molecular analyses using polymerase chain reaction (PCR) and DNA sequencing indicated that seven Spi(-) mutants at 4.0 mg/kg group had deletions with molecular sizes from 0.8 kilo basepairs (kb) to 8.5 kb, whereas no such deletions were observed in the Spi(-) mutants in the control group. The results suggest that deletions induced by MMC in the bone marrow are efficiently detectable by Spi(-) selection and also that the molecular analyses are useful to evaluate the significance of a marginal increase in mutant frequency in the transgenic rodent mutation assays.

In vivo transgenic mutation assays.

Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage-based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3 x 10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two-thirds the MTD, and one-third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control.

Evaluation of the rodent micronucleus assay by a 28-day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)-Mammalian Mutagenicity Study Group (MMS).

To examine whether micronucleus tests can be incorporated into general toxicology assays, we performed micronucleus tests applying the treatment protocols typically used in such assays. In this 13th Collaborative Study of the CSGMT, both rats and mice were tested, although rats were used in the majority of the studies. Fifteen mutagens were tested in rats, mainly by oral (p.o.) administration. Micronucleus induction was evaluated 2, 3, and 4 days, and 1, 2, 3, and 28 days after the beginning of the treatment in the peripheral blood, and at 28 days in the bone marrow. Of the 15 chemicals that induced micronuclei in rats in short-term assays, two chemicals (1,2-dimethylhydrazine.2HCl and mitomycin C) were negative in all our experiments, possibly because of insufficient dose levels. The remaining 13 were positive within the estimated dose range of a general toxicology assay, suggesting the possibility of integrating the micronucleus assay into general toxicology assays. Three patterns were observed in micronucleus induction during the period of repeated treatment: (1) gradual increases in micronucleus frequency with sequential doses, (2) a peak at 3-5 days followed by gradual decreases in micronucleus frequency with sequential doses, and (3) a rapid increase in micronucleus frequency followed by a plateau. We evaluated factors that might have been involved in those patterns, such as the spleen function, target organ exposure, extramedullary hematopoiesis, hypothermia, and hypoxia. Another factor we considered was dosage. Because the dosages employed in a general toxicity assay are usually lower than those used in short-term micronucleus assays, this discrepancy was considered the greatest potential problem for integrating the micronucleus assay into general toxicology assays. Our results indicate that the integration of the micronucleus assay into a 28-day toxicological assay is feasible. To serve this purpose, blood samples collected 4 days after the beginning of treatment and blood and bone marrow samples collected at autopsy should be examined. Furthermore, although it is recognized that mice may be suitable for performing independent micronucleus assays, we propose that rats can provide biologically important and relevant information regarding potential chemical mutagens that can be evaluated under conditions used in the conduct of general toxicology studies.

Reverse mutation tests in Salmonella typhimurium and chromosomal aberration tests in mammalian cells in culture on fluorinated pyrimidine derivatives.

Reverse mutation (Ames) tests with Salmonella typhimurium TA98, TA100 and TA1537, and chromosomal aberration tests in vitro with a Chinese hamster fibroblast cell line (CHL), were carried out on fluorinated pyrimidine derivatives, such as 5-fluorouracil (5-FU), 1-(2-tetrahydrofuryl)-5-fluorouracil (FT), 5-fluorodeoxyuridine (FUdR), 1,3-bis(2-tetrahydrofuryl)-5-fluorouracil (FD-1) and a mixture of uracil and FT in the molar ratio 4 : 1 (UFT) (Fujii et al., 1978). For comparison, similar tests were also carried out on 4 anti-metabolic agents, a metabolite of FD-1 and a component of UFT, such as cytosine-1-beta-D-arabinofuranoside (AraC), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), 8-azaguanine (8-AG), 3-(2-tetrahydrofuryl)-5-fluorouracil (3-FT) and uracil. The anti-bacterial action of 4 fluorinated pyrimidine derivatives such as 5-FU, FT, FD-1 and UFT to TA100 was tested under the condition that buffer, S9 mix, S9 and albumin were present. 6-MP was only positive in the Ames test with TA100 in the system without S9 mix, while all others failed to show mutagenic activity. On the other hand, all compounds tested, except uracil, induced chromosomal aberrations on CHL cells in the system without metabolic activation. FT was degraded by S9, but there was no significant difference in the killing activity of FT among with buffer, S9 mix and albumin. The killing activity of 5-FU was the strongest with buffer, and it was slightly binding to albumin. The killing activity of 5-FU was mostly decreased by S9 mix. FD-1 showed the strongest anti-bacterial action when S9 mix was present but it was degraded by S9. UFT showed no anti-bacterial action in any conditions.

DNA breaks and repair in the mouse leukemia L1210 cells exposed to three different types of interstrand DNA cross-linkers.

DNA breaks and repair in mouse leukemia L1210 cells treated with 3 different types of cross-linkers, mitomycin C (MMC), 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride (ACNU) and SN-07 (a macromolecular antibiotic), were studied. Measured in D37 values, MMC gave the highest number of cross-links per lethal 'hit' directly after the 1-h treatment in the alkaline elution assay, followed by ACNU and SN-07. A good dose-response increase in induced interstrand DNA cross-linking frequency was observed in cells treated with 2.5-10 micrograms/ml MMC and with 10-100 micrograms/ml ACNU for 1 h with and without 24-h post-incubation. After 6-h post-incubation, the highest frequency of cross-linking was observed in cells treated with 2.5 micrograms/ml MMC and 30 micrograms/ml ACNU, while cross-link production continued in the cells treated with SN-07 for 12-h post-incubation. No significant increase in DNA breaks was observed in cells treated with MMC throughout 24-h post-incubation. The highest frequency of single-strand DNA breaks in cells treated with ACNU was observed immediately after the treatment and they disappeared after 6-h post-incubation. After 24-h post-incubation, a marked enhancement of the DNA breaks was observed in cells treated with SN-07 and the cells contained double-strand DNA breaks also. RNA synthesis was not affected in the cells treated with 10 micrograms/ml MMC and slightly inhibited to 70% of control in those treated with 100 micrograms/ml ACNU, while DNA synthesis in both cells was significantly inhibited after 24-h post-incubation. By contrast, both RNA and DNA synthesis were completely inhibited in cells treated with 8.0 micrograms/ml SN-07.

Modes of genotoxicity of a macromolecular antibiotic, SN-07, a novel type of interstrand DNA cross-linker.

The modes of genotoxicity of a novel macromolecular antitumor antibiotic (SN-07) were examined using both prokaryotic and eukaryotic cells in vitro. The antibiotic induced a frameshift-type reverse mutation in Ames Salmonella typhimurium TA98 at 1.6-400 ng/plate with and without S9 mix. SN-07 also induced chromosomal aberrations and a forward mutation (6-TGr) in Chinese hamster V79 cells after 1 h treatment at 12.5-100 ng/ml without metabolic activation. The alkaline elution technique revealed that SN-07 induced interstrand DNA cross-linking dose-dependently after treatment with 2.5-10 micrograms/ml for 1 h followed by elution at pH 12.1, but it did not induce the dose-dependent cross-linking after the same treatment followed by elution at pH 12.6. It was also found that SN-07 induced single-strand DNA breaks (pH 12.1) and alkali-labile (pH 12.6) sites after treatment with 0.1-10 micrograms/ml for 1 h followed by 24-h post-incubation.

Ethyl nitrosourea and methyl methanesulfonate mutagenicity in sperm and testicular germ cells of lacZ transgenic mice (Muta Mouse).

The germ cell mutagens ethyl nitrosourea (ENU) and methyl methanesulfonate (MMS), were tested for their genotoxicity in sperm cells and testicular germ cells using lacZ transgenic mice (Muta Mouse). Eight- to 10-week-old Muta mice were treated with ENU (150 mg/kg) or MMS (40 mg/kg) by intraperitoneal injection. Three and 14 days after treatment, testes and sperm were collected for lacZ mutation analysis. Sperm were isolated from the epididymis and vas deferens by washing out the minced tissue. Germ cell DNA was isolated from testicular germ cells and sperm with the help of 2-mercaptoethanol, and the target lacZ gene, which is integrated into a lambda shuttle vector, was recovered by in vitro packaging. The resultant phages were allowed to infect to E. coli C (galE), and the lacZ mutant plaques were dominantly selected on a plate containing phenyl-beta-D-galactoside. Spontaneous mutant frequencies (MF) in vehicle-treated control mice were approximately 1 x 10(-5) and 3 x 10(-5) in testicular germ cells and sperm, respectively, at both sampling times. ENU treatment increased the MF in the testicular germ cells to 5 x 10(-5) on days 3 and 14, but did not affect sperm MF. MMS was not mutagenic in either tissue. The peripheral blood micronucleus assay was performed on the same animals 48 h after treatment, and strong inductions of micronucleated reticulocytes (MNRETs) were observed in both ENU- and MMS-treated mice. These data suggest that agents mutagenic to premeiotic germ cells, e.g., ENU, can be detected by transgenic mutation assay system using germ cells isolated from the testis. On the other hand, those mutagenic to postmeiotic cells, e.g., MMS, are insensitive in the assay system.

Induction of lacZ mutation by 7,12-dimethylbenz[a]anthracene in various tissues of transgenic mice.

The induction of gene mutations was examined in MutaMouse after an intraperitoneal injection of 7, 8-dimethylbenz[a]anthracene (DMBA) at 20 mg/kg in a collaborative study participated by four laboratories. Although the DMBA dose used was lower than the level that has been reported to induce micronucleated erythrocytes maximally in several mouse strains, a killing effect appeared after day 9 of the post-treatment interval. Mutations in lacZ transgene were detected by the positive selection assay following in vitro packaging of phage lambda from the genomic DNA of the transgenic animals that survived. The mutant induction was evaluated in the bone marrow, liver, skin, colon, kidney, thymus, and testis 7 to 28 days after the treatment. In the bone marrow, the mutant frequency reached a maximum, approximately a 30-fold increase, 14 days after the treatment and the increased frequency persisted at least up to day 28 of the post-treatment. Induction of mutants was detected in the liver, colon, thymus, and skin to lesser extents. Marginal responses were obtained in the kidney and testis. The slight increases in the mutant frequencies in the kidney and testis observed in some laboratories were within laboratory-to-laboratory or animal-to-animal variations. In contrast to the gene mutation induction in the bone marrow, the frequency of micronucleated reticulocytes increased transiently 3 days after the treatment and returned to a control level before day 8 of the post-treatment. It was suggested that DMBA induced gene mutation is fixed in stem cells depending on cell proliferation while DNA damages responsible for chromosome breakage are not transmitted to progeny cells.

A novel macromolecular antibiotic, SN-07. Taxonomy of producing organism, isolation, characterization and biological activities.

A novel macromolecular antibiotic SN-07 was obtained from the cultural supernatant of Actinomadura roseoviolacea var. miuraensis nov. var. The antibiotic was soluble in water, had a molecular weight of 18,000-22,000 daltons in 50 mM Tris-HCl buffer, pH 7.0, containing 2.0 M KCl as compared with authentic proteins. Its major constituents were nucleic acids. The substance had antibacterial activity against Gram-positive bacteria. It was also effective against lymphocytic leukemia P388 in vivo.

Anomalous artery directly connecting the external and internal carotid arteries.

An anomalous artery directly connecting the external with the internal carotid artery was encountered on the right side of a 68-year-old Japanese female cadaver. This anomalous artery (5 mm in diameter, 12 mm in length) branched out from the posterior aspect of the external carotid at the level of the origin of the lingual artery, ran obliquely upward posteriorly along the course of the hypoglossal nerve, and was confluent with the anterior aspect of the internal carotid artery. No other variations were found in the morphological aspects of, or in the anatomical relationships between, the carotid arteries and their surrounding structures on either side. The carotid body-like structure was observed at the carotid bifurcation and was innervated by small branches of the glossopharyngeal, the vagus and the sympathetic trunk. Embryologically, it is conceivable that this anomalous artery may have derived from the right second branchial arch artery, although there is no abnormality in other derivative structures of the second pharyngeal arch. There may have been no effect from this anomaly on the functions of the arterial blood flow and blood supply under normal circumstances in the present case, but this report may be of embryological significance and contribute some insight into the mechanisms of the formation of the carotid circulation systems.

An Interlaboratory Validation Study of the Improved Transformation Assay Employing Balb/c 3T3 Cells: Results of a Collaborative Study on the Two-stage Cell Transformation Assay by the Non-genotoxic Carcinogen Study Group.

The Non-genotoxic Carcinogen Study Group of the Environmental Mutagen Society of Japan organised the first step of an interlaboratory validation study on an improved cell transformation assay employing Balb/c 3T3 A31-1-1 cells. Nineteen laboratories participated in this study. The modified transformation assay was evaluated for its responsiveness, its interlaboratory reproducibility and its transferability. In this study, a mixture of Dulbecco's modified Eagle's medium and nutrient mixture F12, supplemented with insulin-transferrin-ethanolamine-sodium selenite and 2% fetal bovine serum (FBS) was used during the period of expression of transformed foci, intead of the usual minimum essential medium with 10% FBS. 20-Methylcholanthrene (MCA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) were selected as a prototype initiator and a tumour promoter, respectively. Two series of experiments were conducted. In the first series, the transformation activity of MCA was examined at various concentrations. In the absence of the promoting treatment with TPA, exposure to MCA only weakly induced transformed foci. In the presence of 0.1µg/ml TPA, all laboratories observed significant dose-dependent increases in the number of transformed foci with increasing MCA concentrations. In the second series of experiments, various concentrations of TPA were tested. In the absence of initiating treatment with MCA, exposure to TPA weakly induced transformed foci in about half of the laboratories. In the presence of 0.2µg/ml MCA, all the laboratories observed significant dose-dependent increases in the number of transformed foci with increasing TPA concentrations. The results from this study support the usefulness of this modified two-stage transformation assay with Balb/c 3T3 cells.

[Gastric mucosal congestion following endoscopic variceal ligation--analysis using reflectance spectrophotometry].

UNLABELLED: Endoscopic variceal ligation (EVL) has been accepted as a new treatment for esophageal varices in cirrhotic patients, and evaluated to have a lower incidence of complications compared to sclerotherapy. Sclerotherapy may increase the risk of portal hypertensive gastropathy (PHG), which is recognized to be gastric mucosal congestion due to portal hypertension in cirrhotics. However, the gastric mucosal hemodynamics after EVL has not been established yet. The aim of this study is to assess whether EVL could affect the hemodynamics of the gastric mucosa. PATIENTS AND METHODS: Eleven cirrhotic patients with severe esophageal varices, who underwent prophylactic EVL were enrolled in the trial. Age and sex-matched non-cirrhotic patients who had only gastric mucosal atrophy were entered as control to compare the mucosal hemodynamics to that of cirrhotic patient. EVL was performed as described by Stiegmann et al. The gastric mucosal hemodynamics was evaluated with both gastric mucosal blood volume (IHB) and hemoglobin O2 saturation (ISO2), which were measured by reflectance spectrophotometry during endoscopy. These parameters were measured in the three points of the stomach (gastric antrum, lower corpus and upper corpus) just before and after EVL. RESULT: In all patients with cirrhosis, mild PHG was observed endoscopically. ISO2 in cirrhotic patients was significantly lower in all points of the stomach compared with control. IHB in cirrhotic patients was not significantly different from control. ISO2 of post-EVL was significantly lower than that of pre-EVL, whereas IHB of post-EVL revealed significantly higher than that of pre-EVL. However, endoscopic grade of PHG remained mild. The hemodynamics of the mucosa two weeks after the initial EVL showed improvement of the congestion. CONCLUSIONS: The gastric mucosal hemodynamics showed increment of the gastric blood volume and decreased hemoglobin O2 saturation in cirrhotic patients, indicating that cirrhotic gastric mucosa is in congestive condition. EVL for esophageal varices makes the gastric mucosa more congestive soon after the procedure in spite of the same grade of endoscopic PHG. However, the worsened congestion improves within a few weeks.