Mitrecin A, an endolysin-like bacteriolytic enzyme from a newly isolated soil streptomycete.

UNLABELLED: An open reading frame with homology to known endolysin genes was identified in the genome of Streptomyces sp. strain 212, which is a newly isolated soil bacterium. The heterologously expressed gene product of this endolysin-like gene, called Mitrecin A, demonstrated bacteriolytic activity against several Gram-negative bacteria. The genome of the bacterial strain was sequenced to draft quality using pyrosequencing followed by genome assembly and gene annotation. Within the sequence, a chromosomally located endolysin-like open reading frame was predicted. The gene product, designated Mitrecin A, was heterologously expressed and isolated from contaminating proteins as a fusion protein to a 6-histidine tag. Mitrecin A consists of 127 amino acids arranged in modular domains of activity. It has an estimated molecular weight of 14.3 kDa and retains sequence homology to the M15C peptidase subfamily of zinc metallocarboxypeptidases. The heat-labile purified recombinant protein has an overall positive charge, has optimal catalytic activities at 26°C in solution of pH 9 with 1% saline and has bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. SIGNIFICANCE AND IMPACT OF THE STUDY: The gene of a new protein antimicrobial, Mitrecin A, was discovered in the genome of a soil bacterium. The purified recombinant enzyme, resulting from heterologous over expression of the gene, was found to be tolerant of increased pH conditions and to have bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. Characterization of enzymes such as Mitrecin A from previously uncharacterized bacteria provides potential options for new biocontrol agents in medically and economically important applications like therapeutics, disinfectants, food preservatives, agricultural livestock antimicrobials, and inhibitors of biofilm production.

Renal flares are common in patients with severe proliferative lupus nephritis treated with pulse immunosuppressive therapy: long-term followup of a cohort of 145 patients participating in randomized controlled studies.

OBJECTIVE: Immunosuppressive agents have become the standard of therapy for proliferative lupus nephritis, but some patients may relapse after discontinuing treatment. We reviewed the cases of renal flares in a cohort of patients who participated in 2 randomized controlled clinical trials at the National Institutes of Health and explored the prevalence, outcome, and predictive factors of renal flares. METHODS: Data were obtained on 145 patients treated with pulse cyclophosphamide, pulse methylprednisolone, or the combination of both. Patients had not received immunosuppressive therapy for at least 6 months and had experienced complete or partial response according to defined criteria. Renal flares were classified as either proteinuric or nephritic based on changes in urinary protein and sediment. Most patients who experienced a flare received additional immunosuppressive therapy. RESULTS: Seventy-three patients had a complete response, and 19 had partial response/stabilization. Forty-one of these patients (45%) experienced renal flares (nephritic in 33, proteinuric in 8) after a mean followup of 117 months; 31 of them received additional immunosuppressive therapy. The median time to renal flare was 36 months in the complete responders and 18 months in the partial responders. Eleven of the 41 patients (27%) progressed to end-stage renal disease (ESRD); 9 had nephritic flares (all severe except for 1) and 2 had proteinuric flares (1 in each responder group). Compared with patients who had a complete response, those with a partial response were more likely to experience a flare, to have a severe nephritic flare, or to progress to ESRD. Low C4 at the time of response and African American ethnicity were significant independent risk factors for renal flare, by multivariate Cox proportional hazards analysis. CONCLUSION: Nephritic flares are common in patients with proliferative lupus nephritis, even in those with a complete response to therapy, but they do not necessarily result in loss of renal function if treated with additional immunosuppressive agents. Renal flares are an important feature of the natural history of lupus nephritis and provide an opportunity for additional preventive strategies, as well as measures of efficacy in future therapeutic trials.

Combination therapy with pulse cyclophosphamide plus pulse methylprednisolone improves long-term renal outcome without adding toxicity in patients with lupus nephritis.

BACKGROUND: Controlled trials in lupus nephritis have demonstrated that cyclophosphamide therapy is superior to corticosteroid therapy alone. The long-term effectiveness and side-effect profiles of pulse immunosuppressive regimens warrant further study. OBJECTIVE: To define the long-term risk and benefit of monthly treatment with boluses of methylprednisolone, cyclophosphamide, or both. DESIGN: Extended follow-up (median, 11 years) of a randomized, controlled trial. SETTING: U.S. government research hospital. PATIENTS: 82 patients with proliferative lupus nephritis. MEASUREMENTS: Rates of treatment failure (defined as need for supplemental immunosuppressive therapy or doubling of serum creatinine concentration, or death) and adverse events. RESULTS: In an intention-to-treat survival analysis, the likelihood of treatment failure was significantly lower in the cyclophosphamide (P = 0.04) and combination therapy (P = 0.002) groups than in the methylprednisolone group. Combination therapy and cyclophosphamide therapy alone did not differ statistically in terms of effectiveness or adverse events. Of patients who completed the protocol (n = 65), the proportion of patients who had doubling of serum creatinine concentration was significantly lower in the combination group than in the cyclophosphamide group (relative risk, 0.095 [95% CI, 0.01 to 0.842]). CONCLUSION: With extended follow-up, pulse cyclophosphamide continued to show superior efficacy over pulse methylprednisolone alone for treatment of lupus nephritis. The combination of pulse cyclophosphamide and methylprednisolone appears to provide additional benefit over pulse cyclophosphamide alone and does not confer additional risk for adverse events.

Do DNA vaccines induce autoimmune disease?

This report examines whether plasmid DNA vaccines induce the production of anti-DNA or anti-muscle cell autoantibodies. A three-fold increase in the number of B cells secreting immunoglobulin G (IgG) anti-DNA autoantibodies was detected in BALB/c mice immunized and boosted with any of three DNA plasmids (p < 0.004). This correlated with a transient increase in serum anti-DNA autoantibody titers but was not associated with the development of glomerulonephritis or autoimmune disease. None of the DNA vaccines examined stimulated the production of anti-muscle cell autoantibodies or the development of myositis. The effect of DNA vaccines on the development of nascent autoimmunity in lupus-prone (NZB x NZW)F1 mice was also examined. Repeated vaccination did not alter the onset or course of disease in these animals. These findings suggest that DNA vaccines neither initiate nor accelerate the development of systemic autoimmunity.

Testing mode and surface treatment effects on dentin bonding.

The goal of this project was to evaluate the effect of the following variables on shear dentin-bonding test results: mode of testing (cyclic fatigue versus static loading), surface treatments (32% phosphoric acid, 10% phosphoric acid, and no treatment [unetched]), and type of shear test (traditional planar versus push-out). All teeth were stored in distilled water and tested in a shear mode at a loading rate of 2 mm/ min. The specimens were loaded in static or cycled for 1000 cycles using a staircase approach or until fracture, whichever occurred first. On samples with etched dentin surfaces, the push-out test did not demonstrate a significant difference in measured bond strength when compared with results from the planar test, although sample preparation was more labor-intensive. The bond strength resulting from cyclic fatigue of the etched specimens was approximately 51% of the static loading value. Ten percent phosphoric acid was as effective as 32% phosphoric acid for dentin bonding. Finite-element analysis indicated that the traditional planar shear test produces flexure of the specimen and high tensile stress magnitudes within the resin bonding layer. The push-out test produces elevated compressive stresses localized in the composite along the circumference of the punch. Shear stresses in the resin bonding layer are similar for both testing methods at the same loading element contact force.

Methylprednisolone and cyclophosphamide, alone or in combination, in patients with lupus nephritis. A randomized, controlled trial.

BACKGROUND: Uncertainty exists about the efficacy and toxicity of bolus therapy with methylprednisolone or of the combination of methylprednisolone and cyclophosphamide in the treatment of lupus nephritis. OBJECTIVE: To determine 1) whether intensive bolus therapy with methylprednisolone is an adequate substitute for bolus therapy with cyclophosphamide and 2) whether the combination of methylprednisolone and cyclophosphamide is superior to bolus therapy with methylprednisolone or cyclophosphamide alone. DESIGN: Randomized, controlled trial with at least 5 years of follow-up. SETTING: Government referral-based research hospital. PATIENTS: 82 patients with lupus nephritis who had 10 or more erythrocytes per high-power field, cellular casts, proteinuria (> 1 g of protein per day), and a renal biopsy specimen that showed proliferative nephritis. INTERVENTIONS: Bolus therapy with methylprednisolone (1 g/m2 body surface area), given monthly for at least 1 year; bolus therapy with cyclophosphamide (0.5 to 1.0 g/m2 body surface area), given monthly for 6 months and then quarterly; or bolus therapy with both methylprednisolone and cyclophosphamide. MEASUREMENTS: 1) Renal remission (defined as < 10 dysmorphic erythrocytes per high-power field, the absence of cellular casts, and excretion of < 1 g of protein per day without doubling of the serum creatinine level), 2) prevention of doubling of the serum creatinine level, and 3) prevention of renal failure requiring dialysis. RESULTS: Renal remission occurred in 17 of 20 patients in the combination therapy group (85%), 13 of 21 patients in the cyclophosphamide group (62%), and 7 of 24 patients in the methylprednisolone group (29%) (P < 0.001). Twenty-eight patients (43%) did not achieve renal remission. By life-table analysis, the likelihood of remission during the study period was greater in the combination therapy group than in the methylprednisolone group (P = 0.028). Combination therapy and cyclophosphamide therapy were not statistically different. Adverse events were amenorrhea (seen in 41% of the cyclophosphamide group, 43% of the combination therapy group, and 7.4% of the methylprednisolone group), cervical dysplasia (seen in 11% of the cyclophosphamide group. 7.1% of the combination therapy group, and 0% of the methylprednisolone group), avascular necrosis (seen in 11% of the cyclophosphamide group, 18% of the combination therapy group, and 22% of the methylprednisolone group), herpes zoster (seen in 15% of the cyclophosphamide group, 21% of the combination therapy group, and 3.7% of the methylprednisolone group) and at least one infection (seen in 26% of the cyclophosphamide group. 32% of the combination therapy group, and 7.4% of the methylprednisolone group). CONCLUSIONS: Monthly bolus therapy with methylprednisolone was less effective than monthly bolus therapy with cyclophosphamide. A trend toward greater efficacy with combination therapy was seen.

Platelet association with gingival tissue inflammation.

Platelets (PL) may be involved in the inflammatory process through the release of a variety of factors which could contribute to gingival tissue injury. Thus, conditions which result in the localized discharge of PL constitutents could lead to amplification of the inflammatory process at these sites. The purpose of this study was to determine if there was evidence of PL activation in gingival crevicular fluid and whether the degree of gingival inflammation, as measured by the gingival index (GI), was associated with the degree of platelet activation. This was monitored by assaying for beta-thromboglobulin (beta-TG), a platelet specific protein released from alpha granules of PL when activated. One uL samples of the fluids were obtained from human subjects from gingival sites with various GI scores. Fluid samples were also obtained at probe-induced bleeding gingival crevicular sites. beta-TG levels in the various fluids obtained from the crevice were determined by radioimmunoassay (RIA). The RIA data indicated that detectable beta-TG levels were observed in all samples, the means ranging from 5.5 ng/ml to 45.2 ng/ml. Additionally, a positive association between the GI scores of 0 and 1 and the beta-TG levels where observed. For GI scores of 2 and above the beta-TG concentrations appeared to approach a maximum value. These findings provide evidence for PL activation and suggest a relationship with gingival inflammation.

Complexity of the cytokine and antibody response elicited by immunizing mice with Plasmodium yoelii circumsporozoite protein plasmid DNA.

The number, type, and location of cytokine- and Ab-secreting cells activated in mice immunized and boosted with plasmid DNA encoding the circumsporozoite protein of the malarial parasite Plasmodium yoelii (PyCSP) were monitored. The initial humoral response was localized to the draining lymph nodes and was characterized by production of IgG1 anti-PyCSP Abs and the Th2 cytokine IL-4. In contrast, the secondary response was dominated by IFN-gamma production (a Th1 cytokine) and the secretion of IgG2a anti-PyCSP Abs in the spleen. PyCSP DNA and mRNA were detected only in the quadriceps muscles (sites of plasmid injection), yet these sites lacked either cytokine- or Ab-secreting cells. These findings indicate that circulating lymphocytes encounter plasmid-encoded Ag in the muscle bed, initiate a humoral response in the draining lymph nodes, and then seed distal lymphoid organs. Profound differences were observed between the primary and secondary immune responses induced by plasmid immunization, which may influence vaccine efficacy.

Characterization of in vivo mutated T cell clones from patients with systemic lupus erythematosus.

Patients with systemic lupus erythematosus (SLE) have increased percentages of activated T cells and increased numbers of cells with mutations in their hypoxanthineguanine phosphoribosyltransferase (hprt) gene, as judged by growth in the presence of 6-thioguanine. To study the relevance of these mutant T cells to disease pathogenesis, we have assessed the phenotype and functional capabilities of such cells from 21 patients with SLE who never had received cytotoxic drugs. The frequency of T cells with mutations in hprt in the blood of these patients ranged from normal to 25 times normal (mean +/- SEM [21.1 +/- 6.1] x 10(-6) versus [4.8 +/- 0.8] x 10(-6), in 15 age-matched normal individuals, P < 0.001) and correlated significantly with disease duration. CD4+ and CD8+ phenotypes were comparable among mutated and nonmutated clones from both patients and normals. Although the frequency of CD3+CD4-CD8- cells was low, it was increased among SLE-derived T cells (mutated and wild-type) compared with clones derived from normals (5% for SLE vs 1% for normals). A substantial percentage of all clones were able to help autologous B cells to produce anti-ssDNA, 11 of 68 (16%) selected clones and 3 of 28 (11%) nonselected clones. Help for autoantibody production was confined to CD4+ SLE-derived T cell clones. It could be blocked using an anti-HLA-DR mAb, suggesting that classical cognate help was operative. This represents the first estimate of the frequency of T cells able to drive autoantibody production in SLE.

Increased utilization of polyreactive B cells during periods of generalized immune activation.

This work examines the hypothesis that B cells secreting polyreactive antibodies (antibodies capable of binding to more than one self or foreign antigen) are preferentially utilized during periods of generalized immune stimulation. Four conditions characterized by such stimulation were examined: chronic virus infection, mitogen treatment, autoimmune disease and neonatal repertoire development. In normal adult mice, polyreactive IgM secreting lymphocytes constituted 8-9% of the actively expressed repertoire. Under conditions of generalized immune activation, this frequency increased to 13-19% (p. < .01). Polyreactive IgG secreting B cells, which were present at frequencies of < 0.5% in normal adult mice, were found at freqeuncies of 6-10% in mice with autoimmune disease, chronic virus infection or following mitogen treatment (p. < .001). We postulate that polyreactive lymphocytes are preferentially activated when the immune system is confronted with stimuli inadequately controlled by antigen-specific responses.

Systemic lupus erythematosus: theories of pathogenesis and approach to therapy.

Human systemic lupus is a heterogeneous disorder characterized by multisystem inflammatory disease and the production of a variety of autoantibodies. For many years, in our ignorance, we had the freedom to imagine numerous abnormalities which might give rise to lupus: defects of the immune system, viruses, major histocompatibility driven predispositions, complement or complement receptor defects, biochemical abnormalities, impaired DNA repair, sex hormone imbalances, and many others. Now, the basis for lupus in lpr/lpr mice has been uncovered: a mutation in fas. The normal Fas cell surface molecule is important in programmed cell death, apoptosis. The lpr-associated defect in Fas interferes with normal apoptosis, allowing persistence of self-reactive lymphocytes. This finding is especially exciting to those of us who have stressed lupus-associated defects in tolerance. It also illustrates the apparent antigen-nonspecific nature of an etiologic abnormality leading to lupus. The multigenic NZB disorder is less well dissected. Recent work points to defects in an unusual bone marrow progenitor population. Patients with systemic lupus erythematosus may have different and multiple pathogenic factors, including those listed above. We recently have found that some patients with lupus have persistence of T cells with random mutations, consistent with prior abnormal activation and, perhaps, impaired apoptosis. Future therapies of patients must consider both the heterogeneity and the multifactorial pathogenesis of the syndrome we call "lupus."

Studies of marrow progenitor abnormalities in lupus-prone mice. II. Further studies of NZB Thy 1(neg)Lin(neg) bone marrow cells.

Bone marrow cells from NZB mice were fractionated and enriched in cells lacking surface markers characteristic of mature lineages, termed Thy 1neg Lineage(neg) cells. These cells represent approximately 1% of all marrow cells and constitute a much greater fraction of the bone marrow than do Thy 1lo Lineage(neg) cells. The NZB Thy 1neg Lineage(neg) cells were able to protect nonautoimmune, histocompatible DBA/2 recipients from lethal doses of irradiation, suggesting that this subpopulation contained progenitor cells. Consistent with this observation, fractioned Lip 6+ Thy 1neg Lineage(neg) cells, representing early B lineage cells, were less effective than Lip 6neg Thy 1neg Lineage(neg) cells in radioprotection. NZB marrow contains a great many more CFU-S than does marrow from nonautoimmune strains. DBA/2 mice transplanted with Thy 1neg Lineage(neg) cells from NZB marrow had substantial numbers of CFU-S, much greater than controls. This CFU-S potential was found primarily in the Lip 6neg Thy 1neg Lineage(neg) fractionated marrow, suggesting that that population contained early progenitor cells that had not yet differentiated into B lineage cells. Both radioprotection and increased CFU-S were transmitted serially by bone marrow from DBA/2 recipients of Thy 1neg Lineage(neg) NZB marrow to secondary and tertiary (irradiated) DBA/2 recipients. Also serially transplanted were precursors of antibody forming cells. These findings suggest that NZB Thy 1neg Lineage(neg) marrow cells play a critical role in the development of the abnormal phenotype of NZB mice. However, because this probably is not a homogeneous population, additional work will be necessary to define the surface and molecular properties of the cell or cells within the NZB Thy 1neg Lineage(neg) marrow population which serve as progenitors of the cells which mediate NZB disease.

Modulation of the expression of murine lupus by gonadotropin-releasing hormone analogs.

Recent studies have suggested that hypothalamic and pituitary hormones may directly influence the immune system. One such hormone with immunomodulatory properties is GnRH. We hypothesized that GnRH and/or the gonadotropins might alter the severity of autoimmune disease through mechanisms distinct from their effects on gonadal hormones. This possibility was tested in a murine model of lupus. We assessed disease severity over time in intact and castrated, male and female, lupus-prone (SWR x NZB) F1 hybrid mice during treatment with GnRH agonist, GnRH antagonist, or vehicle. Compared to vehicle administration, GnRH antagonist administration significantly decreased total serum immunoglobulin G and anti-DNA antibodies in castrated male and female mice and significantly improved survival. In contrast, GnRH agonist administration exerted reciprocal effects in castrated mice, leading to early increases in serum anti-DNA and total immunoglobulin G levels. We conclude that GnRH and/or the gonadotropins can modify the expression of murine lupus independently of their regulation of gonadal steroid secretion.

Role of endogenous retroviruses in autoimmune diseases.

Retroviruses have been implicated in the pathogenesis of murine and human lupus; however, many positive findings have been followed by alternative explanations. Initial findings implicating xenotropic retroviruses were subsequently invalidated. The first solid demonstration that endogenous retroviruses mediate disease was the study of SL/Ni mice. Here budding ecotropic retroviral particles from arterial smooth muscle cells caused an antibody response to the particles with subsequent complement deposition. Our laboratory has focused on derangements in endogenous MCF retroviral expression. We found that lupus-prone NZB, BXSB and MRL strains have a marked increase in expression of Mpmv RNA in their thymuses while bone marrow expression did not differ from normal strains. Sequence analysis demonstrated mutations in the NZB endogenous retroviruses which could alter expression. A phosphorothioate antisense oligonucleotide to the initiation sequence of Mpmv caused lymphocyte activation in vivo in normal mice, providing further evidence for in vivo effects of Mpmv and potential for pathological abnormalities in lupus-prone strains.