Survival study by organ disorders in 306 Japanese patients with systemic lupus erythematosus: results from a single center.

Survival rate and causes of death according to the period of diagnosis and four accompanying organ disorders were analyzed in 306 Japanese patients with systemic lupus erythematosus. The survival rate was gradually improved, and the survival rate during 5- and 10-year periods of the patients diagnosed in 1990-2004 was 94 and 92%, 20-year period of those in 1980-1989 was 77%, 30-year period of those in 1975-1979 was 71%, respectively. Survival rate of those with serositis, pulmonary hypertension, and positive family history tended to be reduced, while that of the cases with neuropsychiatric disorder and renal disorder was significantly reduced. Overlapping of these organ disorders was an important factor for a poor prognosis. Bronchopneumonia and cerebrovascular accidents were frequent causes of death, and treatment for anti-phospholipid antibody syndrome and life-style diseases such as hypertension and arteriosclerosis was thought to be important for a good outcome.

The beneficial effects of treatment with all-trans-retinoic acid plus corticosteroid on autoimmune nephritis in NZB/WF mice.

Corticosteroids are highly effective anti-inflammatory or immunosuppressive drugs used commonly to treat human systemic lupus erythematosus (SLE). All-trans-retinoic acid (ATRA), which belongs to a class of retinoids that exert immunomodulatory and anti-inflammatory functions, can also suppress the development of lupus nephritis in an animal model. However, both agents can inflict serious adverse effects. Here, we have asked whether ATRA can serve as a steroid-sparing drug in the treatment of lupus nephritis. To examine the efficacy of combining predonisolone (PSL) with ATRA, we treated intraperitoneally New Zealand black/white F1 (NZB/W F1) mice with PSL, ATRA or both agents. Survival rate and proteinuria were determined once a month. Cytokine and anti-DNA antibody production were determined by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Renal histopathology was observed by haematoxylin and periodic acid Schiff (PAS), immunoperoxidase and immunohistochemical assay. Survival rate and proteinuria were improved in all experimental groups, and were much improved in the mice receiving the combination of ATRA and PSL (P <0.05). A single administration of ATRA reduced the Th1 [interleukin (IL)-2, interferon (IFN)-gamma and IL-12], and a Th2 (IL-4) cytokine level, as effectively as administration of PSL. ATRA also suppressed the expression of inducible nitric oxide synthetase (iNOS) and monocyte chemoattractant protein-1 (MCP-1) in the kidney. The combination of PSL and ATRA significantly reduced IgG2 (especially IgG2b)-specific anti-DNA antibody levels in comparison with administration of either agent alone. These data suggest that ATRA might have the potential to act as a new therapeutic and steroid-sparing drug against lupus nephritis.

Predominant inhibition of Th1 cytokines in New Zealand black/white F1 mice treated with FK506.

The T-helper 1/T-helper 2 (Th1/Th2) cell balance was examined in 6-month-old New Zealand black/white F1 (B/WF1) mice treated with an immunosuppressive agent, FK506. The survival rate of mice treated with 10 mg/kg/day of FK506 was 7/8, while that of those treated with 2.5 mg/kg/day was 5/8, and 4/8 after treatment for 8 weeks with placebo. Proteinuria, which was already positive in all mice before the treatment, in the seven of eight mice treated with 10 mg/kg/day remained mildly positive (< or = 1+), while seven of eight mice treated with 2.5 mg/kg/day and six of eight mice treated with the placebo showed severe proteinuria (> or = 2+). Pathological changes in the kidneys of mice treated with 10 mg/kg/day of FK506 were less severe than in mice treated with the placebo or 2.5 mg/kg/day of FK506. Expression of mRNA was unchanged for all cytokines determined in the groups treated with 2.5 mg/kg/day of FK506 or placebo. In contrast, expression of mRNA for interleukin (IL)-2, and interferon (IFN)-gamma was suppressed, while that for IL-4 and IL-10 was not suppressed in the group treated with 10 mg/kg of FK506. The serum levels of IgG-class anti-DNA antibodies, which had been elevated before the treatment, were suppressed--especially in the IgG2a subclass--and the deposition of IgG2a and IgG2b in the glomeruli was reduced in the group treated with 10 mg/kg/day of FK506 compared with the other groups. These findings suggest that an improvement in the lupus nephritis of 6-month-old B/WF1 mice induced by FK506 might be associated with a predominant inhibition of Th1 cytokine.

A possible role of apoptosis for regulating autoreactive responses in systemic lupus erythematosus.

It has been reported that apoptotic cells are increased in the peripheral blood from patients with systemic lupus erythematosus (SLE), where dysfunctions of T helper 1 (Th1) cells are known. In order to study whether apoptosis of Th1 cells is associated with the pathogenesis of SLE, early apoptotic cells in various T-cell subsets were detected using fluorescence-labeled annexin V (AnV). AnV binding was most frequently observed in CD4+CCR5+ T cells, and AnV binding rate (%) in this subset was higher in SLE than in normal controls (14.7 +/- 2.6), although that in active SLE (43.6 +/- 7.3) tended to be lower than that in inactive SLE (48.0 +/- 6.8). CD95/Fas expression was also increased in both active and inactive SLE. In some SLE patients, AnV binding rate changed in inverse proportion to titer of the serum anti-DNA antibody and in proportion to serum complement activity. These data suggest that apoptosis in Th1 cells is important in the pathogenesis of SLE and might play a role in regulating over-activation or autoreactive responses by T cells.

A case of progressive systemic sclerosis complicated by massive pleural effusion with elevated CA125.

A tumor marker, CA125, is known to increase in the serum or other body fluids in various malignancies such as ovarian cancer. Here we present a case of progressive systemic sclerosis (PSS) with massive pleural effusion, in which CA125 in the serum and pleural fluid were elevated. The serum level of CA125 decreased in accordance with the change of the pleural effusion. CA125 level may be an indicator for the activity of serositis in some cases with collagen vascular diseases.

Amidase and peptidase activities of polyclonal immunoglobulin G present in the sera of patients with rheumatoid arthritis.

Polyclonal Immunoglobulin (Ig) G from patients with rheumatoid arthritis (RA) and healthy subjects hydrolyzed carbobenzoxy-Val-Gly-Arg p-nitroanilide and D-Pro-Phe-Arg p-nitroanilide. RA IgG exhibited higher activity against the former substrate, but not the latter. On the other hand, RA IgG showed reduced activity against D-Pro-Phe-Arg methylcoumarinamide, when compared with those of the healthy controls. These results suggest that RA IgGs differ from normal IgGs in the substrate specificity of amidase activity. Preliminary studies have shown that two out of three RA IgG samples cleaved a pentapeptide--Gln-Arg-Arg-Ala-Ala--which is assumed to be associated with the risk of developing RA (Gregersen, P. K. et al. (1987), Arthritis Rheum. 30, 1205-1213). By contrast, virtually no cleavage of the same peptide was observed with IgG from healthy controls. A peptide analog, Gln-Arg-Arg-Trp-Ala, was not cleaved at all by any IgGs examined either from RA patients or healthy controls.

Some Bence-Jones proteins enter cultured renal tubular cells, reach nuclei and induce cell death.

Eighteen monoclonal Bence-Jones proteins (BJPs) were examined for their effects on cultured LLC-PK1 (porcine kidney proximal tubule) cells as well as for their amidase and DNase activities. Five proteins were found to enter the cell and to gain access to the nucleus without degradation of epitopes. Intranuclear BJPs ultimately induced DNA fragmentation and cell death. BJPs with relatively high amidase activity were cytotoxic. On the other hand, three of four BJPs with DNase activity had a cytocidal effect on cultured cells; the remaining BJP, which had a relatively high DNase activity but a very low amidase activity, failed to enter the cell and was not cytotoxic in vitro. These results suggest that catalytic and cytotoxic activities of some BJPs may make a significant contribution, in a substantial proportion of myeloma patients, to the development and/or deterioration of the disease.

Increased interleukin-4 production by NK T cells in systemic lupus erythematosus.

It has been reported that production of interleukin (IL)-4, a T helper (Th)-2-type cytokine, might play an important role in the pathogenesis of systemic lupus erythematosus (SLE). On the other hand, it is known that NK1.1(+) cells which belong to CD4, CD8 double-negative, or CD4(+) cells are associated with initial IL-4 production and Th2 differentiation in mice although human equivalent cells are unknown. In order to study the profile of IL-4-producing cells in SLE, cytoplasmic IL-4 and various surface antigens on peripheral mononuclear cells were analyzed. Peripheral mononuclear cells were stimulated for 5 h by phorbol ester and ionomycin in the presence of monensin, fixed, and permeabilized with paraformaldehyde and saponin solution. Then cytoplasmic IL-4 and various surface antigens were analyzed by flow cytometry. IL-4-producing cells in SLE were phenotypically the same as those which produce IL-4 normally and frequently bore activated T-cell (CD7, CD25, CD28, CD29) and NK-cell markers (CD56, CD57). Double-negative T cells and CD57(+) T cells were increased in number and were more frequently positive for cytoplasmic IL-4 in SLE compared with normal controls and various infectious diseases. It was suggested that T cells with NK cell markers, CD57(+) T cells, which are known to extrathymically differentiate, might be involved in the pathogenesis of SLE as a counterpart of mouse NK1.1(+) cells.

Amidolytic and peptidolytic activities of immunoglobulin G present in sera from patients with rheumatoid arthritis, Sjogren's syndrome and systemic lupus erythematosus.

Polyclonal immunoglobulin G (IgG) from healthy subjects was found to be capable of hydrolyzing carbobenzoxy-Val-Gly-Arg-p-nitroanilide (a synthetic chromogenic substrate for trypsin) and D-Pro-Phe-Arg-p-nitroanilide (a substrate for plasma kallikrein). Statistically significant elevation of activity against the former substrate was found in patients with rheumatoid arthritis (RA), but not in patients with Sjogren's syndrome (SjS) or systemic lupus erythematosus (SLE). On the other hand, IgG samples from the patients with these three autoimmune diseases showed reduced activity against d-Pro-Phe-Arg methylcoumarinamide, although the differences were not statistically significant. Preliminary studies have shown that two out of three IgG samples from RA patients exhibited the activity of cleaving a pentapeptide, Gln-Arg-Arg-Ala-Ala, whereas virtually no cleavage of the same peptide was observed with IgG from healthy controls or from patients with SjS or SLE.

Prolactin modulates the disease activity of systemic lupus erythematosus accompanied by prolactinoma.

We present a 31-year-old female patient with systemic lupus erythematosus (SLE) which was accompanied by prolactinoma. Her SLE flared when the plasma levels of prolactin (PRL) increased, and subsided when these levels decreased following the administration of bromocriptine, irrespective of the glucocorticoid dosage. For the 29 plasma samples obtained during the clinical course, PRL levels were significantly correlated to the serum anti-DNA antibody titers (r = 0.55, p < 0.05) and inversely to the serum complement activity (r = -0.33, p < 0.05). This result suggests that PRL may play a role in the pathogenesis of SLE in some patients.

Decreased Th1-like and increased Th2-like cells in systemic lupus erythematosus.

The proportion of the lymphocytes which produce the cytokines corresponding to murine T helper- (Th1) or Th2 cells was studied using flow cytometry in systemic lupus erythematosus (SLE). When the peripheral mononuclear cells were stimulated with phorbol myristate acetate and ionomycin in the presence of monensin, which blocks the secretion of cytokines, the positive rates for the cytoplasmic IL-2 and IFN-gamma were lower and those for the cytoplasmic IL-4 and IL-10 were higher in SLE than in normal subjects. When the cells were cultured with monensin alone, the positive rates for these 4 cytokines were slightly increased in SLE. These data suggest that the mononuclear cells are already activated in vivo and a deviation of the proportion of the Th cells to the Th2-like ones might be associated with the polyclonal B cell activation seen in SLE.

DNA-hydrolyzing activity of Bence Jones proteins.

Of 18 monoclonal Bence Jones proteins purified from urine of patients with multiple myeloma, 4 were found to have a DNA-nicking activity. In contrast, all Bence Jones proteins tested showed detectable amidase activity against carbobenzoxy-L-valyl-glycyl-L-arginine p-nitroanilide. No correlation between the two activities was found. Four patients excreting Bence Jones protein with the DNA-nicking activity showed somewhat severe symptoms, suggesting that this activity may be related to the progressive deterioration of clinical status.

Crossroads of the effects of cyclophosphamide pulse therapy for lupus nephritis--experience of 11 cases.

In this study time for initial assessment of monthly intravenous cyclophosphamide (CP) pulse therapy is discussed for a better outcome with less complications. Eleven patients with lupus nephritis (LN) resistant to conventional therapy (serum creatinine level < or = 2.7 mg/dl) were given 500 mg/m2 of CP 7-9 times with an interval of one month. Urinary protein (Up) decreased in all patients after 3 courses of CP pulse therapy and kept similar levels thereafter. In one group of patients (n = 7), Up decreased to < 2 g/day after 3 courses, while in the other group (n = 4), it did not decrease to < 4 g/day. Creatinine clearance increased by 0-100% in the former group, while it decreased by 5-20% in the latter group after 6-9 courses. Renal function of the patients with insufficient response after 3 courses tended to show no further improvement or worsened thereafter, although Up decreased during CP pulse therapy. A relatively small dose of CP (500 mg/m2) pulse therapy was useful in most LN patients regardless of the renal histology and it was thought important to assess its effects after 3 courses for a prediction of the clinical course. Modification of the protocol at that time might be necessary in regard to dose or interval of CP administration especially for patients with insufficient outcome.

Combination of immunoadsorption therapy and high-dose methylprednisolone in patients with lupus nephritis; possible indications in patients with early stage.

OBJECTIVE: Therapeutic protocol and indication of immunoadsorption therapy (IA) for lupus nephritis (LN) have not been established, although it has been reported to be effective in resistant cases. Here, we performed IA and double filtration plasmapheresis (DFPP) in combination with high-dose methyprednisolone in patients with LN, and studied possible indications of IA. METHODS: IA and DFPP were performed in 9 patients each with LN. They were immediately followed by intravenous infusion of 500 mg of methylprednisolone for prevention of rebound phenomenon. After these treatments 1-2 times a week, a total of 4-6 times, clinical findings were observed for 6 months. RESULTS: The effects on clinical findings such as erythema, fever and arthralgia, serum complement activity, mean urinary protein and reduction of dose of adrenocorticosteroids were comparable in both treatments. Serum titers of ADNA decreased by IA more than DFPP (16% in IA, 38% in DFPP in 3 months), while serum immunoglobulins decreased by IA less than DFPP. Responses in urinary protein after IA tended to be better in patients with high titer of serum ADNA and without nephrotic syndrome, and not associated with disease activity of SLE. CONCLUSION: Removal of ADNA was more selective in IA than in DFPP, and the effects of IA were comparable with those of DFPP. Since patients with low titers of serum ADNA and nephrotic syndrome showed poor responses to IA, it might be worth trying rather in patients with early phase of lupus nephritis.

Characteristics of the clinical findings in patients with idiopathic thrombocytopenic purpura who are positive for anti-phospholipid antibodies.

It has been reported that anti-phospholipid antibodies are detected in some patients with idiopathic thrombocytopenic purpura (ITP). To study the significance of determination of anti-phospholipid antibodies in patients with ITP, clinical and laboratory findings were compared between patients whose sera were positive for these antibodies (Group A) and non-positive patients (Group B). Anti-cardiolipin antibody (aCL) was determined by enzyme-linked immunosorbent assay and lupus anticoagulant (LA) was determined by activated partial thromboplastin time (aPTT) and thromboplastin time inhibition test. Seven out of 27 cases of ITP belonged to Group A and 3 of the 7 were confirmed to have anti-phospholipid antibody syndrome (APS). There was a tendency for habitual abortion, and thrombosis, megakaryocytes in the bone marrow and platelet-associated IgG (PAIgG)-positive cells were more frequent in Group A than in Group B. However, it was difficult to discriminate APS from ITP alone, when there were no symptoms or signs of APS. Therefore, measurement of anti-phospholipid antibodies in ITP was thought to be useful for the differential diagnosis of APS and subsequently for the prevention of thrombosis.

High-affinity anti-DNA antibody parallels clinical course of immunoadsorption therapy for systemic lupus erythematosus.

Anti-DNA antibody, especially high-affinity anti-DNA antibody (ADNA), is thought to have an important role in the pathogenesis of lupus nephritis. In this study, ADNA which binds to double-stranded DNA under a high concentration of sodium chloride was measured in patients who had received immunoadsorption (IA) therapy with a dextran-sulfate column. Titers of high-affinity ADNA in the cases with renal dysfunction tended to be higher than in those without renal dysfunction. The change in the titer of high-affinity ADNA paralleled the clinical course. These findings suggest that measurement of high-affinity ADNA is useful for follow-up of the clinical course of patients who have undergone IA therapy.

Enhancement of Clostridium difficile toxin production in biotin-limited conditions.

The effect of biotin on toxin production by Clostridium difficile was examined in a defined medium. When toxin production by strain KZ 1647, which was isolated from a healthy adult, was examined in relation to its biotin requirement, it was found that with decreasing concentrations of biotin, bacterial growth was decreased, but production of both toxins A and B were remarkably increased, particularly with 0.05 nM biotin. The time course of production of both toxins in biotin-limited conditions was similar to that in biotin-enriched conditions. The biotin effect on toxin production was also observed in 15 other strains, suggesting that the effect occurs frequently amongst toxigenic C. difficile strains. The biotin effect is discussed in relation to the pathogenesis of C. difficile colitis.

A defined growth medium for Clostridium difficile.

Minimal requirements of amino acids and vitamins were determined in chemically defined medium for five strains of Clostridium difficile. Cysteine, isoleucine, leucine, proline, tryptophan and valine were essential amino acids for growth of C. difficile. Arginine, glycine, histidine, methionine and threonine enhanced growth. Biotin, pantothenate and pyridoxine were essential vitamins. A defined medium containing the minimal requirements of amino acids and vitamins produced a rapid and heavy growth which was comparable to that in modified brain heart infusion, a complex medium. Adenine was able to substitute for glycine and threonine, suggesting that the two amino acids may be utilized as precursors of purine nucleotides. The defined medium developed here will assist physiological and biochemical studies on C. difficile.