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.

Teratogenicity and maternal effects of Lactobacillus brevis KB290 in rats and rabbits.

Lactobacillus brevis KB290 (KB290), a plant-derived probiotic lactic acid bacterium, improves gut health and stimulates immune function. Here we extensively investigated the teratogenicity of KB290 in rats and rabbits. We observed no adverse maternal or fetal effects and concluded that the no observable adverse effect level for maternal general toxicity, maintenance of pregnancy, and teratogenicity should be ≥ 10(10) cfu/kg/day. Our results suggest that KB290 would be safe for pregnant females and their offspring.

Safety assessment of Lactobacillus brevis KB290 as a probiotic strain.

Lactobacillus brevis KB290 (KB290), a plant-derived probiotic lactic acid bacterium, reportedly improves gut health and stimulates immune function. Here we extensively investigated the geno-, acute, subacute, and subchronic toxicity of KB290 and its bacterial translocation potential. KB290 was non-mutagenic in the bacterial reverse mutation assay by the preincubation method. In the single oral dose toxicity test, KB290 at 10(9) cfu/ml was nontoxic at maximum capacity (20 ml/kg). When 10(8), 10(9), or 10(10) cfu/kg was administered daily to rats by gavage for 2 weeks (subacute assay), we observed no clear treatment-related effect and no evidence of bacterial translocation from the gastrointestinal tract. When it was administered for 13 weeks (subchronic assay), we again observed no clear treatment-related effect and no significant toxicological effect. Based on those results, we consider 10(10) cfu/kg per day, the highest dose tested, to be the no observed adverse effect level (NOAEL). These results suggest that KB290 is safe for human consumption.

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.

Granulomatous prostatitis due to Cryptococcus neoformans: diagnostic usefulness of special stains and molecular analysis of 18S rDNA.

A 57-year-old Japanese man complained of pain on micturition. The prostate was of normal size but hard. Transrectal needle biopsy demonstrated granulomatous prostatitis with small focal abscesses. Staining with periodic acid-Schiff, Grocott's methenamine silver and Fontana-Masson revealed yeast-form fungus in the granulomas. The mucoid capsule of the fungus stained with mucicarmine. PCR specific for cryptococcal 18S rDNA using DNA extracted from the pathological specimen was positive, and the sequence was homologous to Cryptococcus neoformans. A diagnosis of cryptococcal granulomatous prostatitis was made. The patient was then found to suffer from meningitis and lung abscess, and was treated with amphotericin B and flucytosine. Careful histological and molecular studies are beneficial to reach the correct diagnosis and to prevent an unfavorable outcome of disseminated cryptococcosis.

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.

Vaccination of recurrent glioma patients with tumour lysate-pulsed dendritic cells elicits immune responses: results of a clinical phase I/II trial.

In this Phase I/II trial, the patient's peripheral blood dendritic cells were pulsed with an autologous tumour lysate of the glioma. Seven patients with glioblastoma and three patients with anaplastic glioma, ranging in age from 20 to 69 years, participated in this study. The mean numbers of vaccinations of tumour lysate-pulsed dendritic cells were 3.7 times intradermally close to a cervical lymph node, and 3.2 times intratumorally via an Ommaya reservoir. The percentage of CD56-positive cells in the peripheral blood lymphocytes increased after immunisation. There were two minor responses and four no-change cases evaluated by radiological findings. Dendritic cell vaccination elicited T-cell-mediated antitumour activity, as evaluated by the ELISPOT assay after vaccination in two of five tested patients. Three patients showed delayed-type hypersensitivity reactivity to the autologous tumour lysate, two of these had a minor clinical response, and two had an increased ELISPOT result. Intratumoral CD4+ and CD8+ T-cell infiltration was detected in two patients who underwent reoperation after vaccination. This study demonstrated the safety and antitumour effects of autologous tumour lysate-pulsed dendritic cell therapy for patients with malignant glioma.

Activated leucocytes express and secrete macrophage inflammatory protein-1alpha upon interaction with synovial fibroblasts of rheumatoid arthritis via a beta2-integrin/ICAM-1 mechanism.

OBJECTIVE: To examine the expression and regulation of chemotactic factor, macrophage inflammatory protein-1alpha (MIP-1alpha) by fibroblast-like synoviocytes (FLS), monocytes and polymorphonuclear neutrophils (PMN) isolated from the synovial fluid (SF) of rheumatoid arthritis (RA) patients. METHODS: Monocytes or PMN obtained from RA SF were co-cultured with unstimulated semiconfluent RA FLS. Culture supernatants were assayed for MIP-1alpha by enzyme-linked immunosorbent assay. The expression of MIP-1alpha mRNA and protein was also determined by Northern blot analyss and immunohistochemistry respectively. RESULTS: Interaction of activated leucocytes with FLS synergistically increased MIP-1alpha expression and secretion via a mechanism mediated by beta2-integrin/ intercellular adhesion molecule 1. CONCLUSION: MIP-1alpha expression within inflamed joints appears to be regulated not only by inflammatory cytokines but also by the physical interaction of activated leucocytes and FLS, and plays a crucial role in the progression and maintenance of RA synovitis.

Relationship between psychological factors and arthralgia in patients with rheumatoid arthritis.

Abstract Various factors were assessed in terms of their contribution to arthralgia in a rheumatoid arthritis patient. Eighty-two outpatients (62 women and 20 men) with rheumatoid arthritis (RA) were examined with respect to the subjective degree of arthralgia, age, disease duration, dysfunction, steroid dose, steroid period, depression, anxiety, extroversion, neurotic disorder, and number of caretakers. The results were explained on the basis of stepwise regression analysis and psychological and clinical data. We analyzed results of a correlation coefficient test on the mutual relationship between variables. Stepwise regression analysis was performed to assess factors of arthralgia in terms of "depression," "mean activity," "morning stiffness," and "steroid dose." Depression is a factor of arthralgia as shown in this study, but it is clear that other factors are also involved. Anxiety was a factor distinct from the activity of RA. The factor contributing most to arthralgia was found to be depression, whereas anxiety had no effect.

Vascular endothelial growth factor expression by activated synovial leukocytes in rheumatoid arthritis: critical involvement of the interaction with synovial fibroblasts.

OBJECTIVE: To examine the expression and regulation of the angiogenic factor, vascular endothelial growth factor (VEGF), by fibroblast-like synoviocytes (FLS), monocytes, and polymorphonuclear neutrophils (PMNs) isolated from the synovial fluid (SF) of rheumatoid arthritis (RA) patients. METHODS: Monocytes or PMNs obtained from RA SF were cocultured with unstimulated, semiconfluent RA FLS. Culture supernatants were assayed for the proliferation and in vitro tube formation of endothelial cells, and for the production of VEGF, by enzyme-linked immunosorbent assay. The expression of VEGF messenger RNA and protein was also determined by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. RESULTS: We found that the interaction of inflammatory, activated leukocytes with FLS resulted in synergistic increases in VEGF expression and secretion, which contributed to the proliferation of endothelial cells and to in vitro endothelial tube formation. The induction of VEGF was mediated via specific adhesion molecules, as indicated by the finding that anti-integrin antibodies significantly inhibited VEGF. Furthermore, the levels of VEGF secretion correlated with the expression of cell surface integrin (CD11b and CD18) on both monocytes and PMNs in the SF. CONCLUSION: VEGF expression within inflamed joints thus appears to be regulated not only by inflammatory cytokines, but also by the physical interaction of activated leukocytes and FLS. Once expressed, VEGF likely plays a crucial role in the neovascularization of the pannus and the progressive joint destruction associated with the synovial inflammation of RA.

Purification and properties of a galacturonic acid-releasing exopolygalacturonase from a strain of Bacillus.

An exopolygalacturonase [exo-PGase; poly (1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67] was found to be extracellularly produced by Bacillus sp. strain KSM-P443. The exo-PGase was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, through sequential column chromatographies. The enzyme had a molecular weight of approximately 45,000 and an isoelectric point of pH 5.8. The N-terminal sequence was Ser-Met-Gln-Lys-Ile-Lys-Asp-Glu-Ile-Leu-Lys-Thr-Leu-Lys-Val-Pro-Val-Phe and had no sequence similarity to those of other pectinolytic enzymes reported to date. Maximum activity toward polygalacturonic acid (PGA) was observed at 60 degrees C and at pH 7.0 in 100 mM Tris-HCl buffer without requiring any metal ions. When the chain length of oligogalacturonic acids increased, the apparent Km for them decreased, but the kcat values increased. This is the first bacterial exo-PGase that releases exclusively mono-galacturonic acid from PGA, di-, tri-, tetra-, and penta-galacturonic acids.

MST, a physiological caspase substrate, highly sensitizes apoptosis both upstream and downstream of caspase activation.

The human serine/threonine kinase, mammalian STE20-like kinase (MST), is considerably homologous to the budding yeast kinases, SPS1 and STE20, throughout their kinase domains. The cellular function and physiological activation mechanism of MST is unknown except for the proteolytic cleavage-induced activation in apoptosis. In this study, we show that MST1 and MST2 are direct substrates of caspase-3 both in vivo and in vitro. cDNA cloning of MST homologues in mouse and nematode shows that caspase-cleaved sequences are evolutionarily conserved. Human MST1 has two caspase-cleavable sites, which generate biochemically distinct catalytic fragments. Staurosporine activates MST either caspase-dependently or independently, whereas Fas ligation activates it only caspase-dependently. Immunohistochemical analysis reveals that MST is localized in the cytoplasm. During Fas-mediated apoptosis, cleaved MST translocates into the nucleus before nuclear fragmentation is initiated, suggesting it functions in the nucleus. Transiently expressed MST1 induces striking morphological changes characteristic of apoptosis in both nucleus and cytoplasm, which is independent of caspase activation. Furthermore, when stably expressed in HeLa cells, MST highly sensitizes the cells to death receptor-mediated apoptosis by accelerating caspase-3 activation. These findings suggest that MST1 and MST2 play a role in apoptosis both upstream and downstream of caspase activation.

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.

Expression of vascular endothelial growth factor by synovial fluid neutrophils in rheumatoid arthritis (RA).

Most of the leucocytes infiltrating rheumatoid synovial fluid (SF) are neutrophils capable of producing a variety of inflammatory mediators known to contribute significantly to the disease process during active RA. In the present study, we investigated the contribution made by SF neutrophils to the elevated levels of vascular endothelial growth factor (VEGF) seen in rheumatoid SF. Rheumatoid SF neutrophils were found to contain significantly larger amounts of both VEGF protein and its mRNA than peripheral blood neutrophils from either RA patients or healthy controls. Levels of cell-associated VEGF were well correlated with free VEGF in SF, which was significantly higher than in SF from osteoarthritis patients. Levels of SF neutrophil-associated VEGF also correlated with RA disease activity and cell surface integrin expression. Thus, SF neutrophil-associated VEGF may be considered an indicator of both local and systemic inflammation of RA, contributing to the neovascularization seen during RA synovitis.

Reduction of thymocyte numbers in transgenic mice expressing viral FLICE-inhibitory protein in a Fas-independent manner.

A viral FLIP (FLICE/caspase-8-Inhibitory Protein), equine herpesvirus type 2 E8 protein, has been shown to inhibit Death receptor-induced apoptosis by suppressing the activation of FLICE/caspase-8. We generated transgenic mice specifically expressing E8 in thymocytes under the control of lck-proximal promoter. Although E8-expressing thymocytes were resistant to Fas-mediated apoptosis, the total number of thymocytes in 4-8-week-old E8 transgenic mice was more than 3-fold less than that in control littermates. This reduction was also observed in E8 transgenic mice with a Fas-/- background suggesting the reduction to be independent of Fas. The thymocytes of the transgenic mice, however, could similarly respond to CD3-mediated stimulation, indicating that the reduction of thymocyte numbers might be independent of T cell receptor complex-mediated stimulation. Thus, the Death receptor-mediated signaling pathway is too complex to be regarded as only an executor for apoptosis.