Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNA-binding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal cross-linking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 Å resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß-ß-a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel β-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated.

OBJECTIVE

Narcolepsy-cataplexy has long been thought to have an autoimmune origin. Although susceptibility to narcolepsy, like many autoimmune conditions, is largely genetically determined, environmental factors are involved based on the high discordance rate (approximately 75%) of monozygotic twins. This study evaluated whether Streptococcus pyogenes and Helicobacter pylori infections are triggers for narcolepsy.

METHODS

Retrospective, case-control.

METHODS

Sleep centers of general hospitals.

METHODS

200 patients with narcolepsy/hypocretin deficiency, with a primary focus on recent onset cases and 200 age-matched healthy controls. All patients were DQ<em>B</em>1*0602 positive with low CSF hypocretin-1 or had clear-cut cataplexy.

RESULTS

Participants were tested for markers of immune response to beta hemolytic streptococcus (anti-streptolysin O [ASO]; anti DNAse B [ADB]) and Helicobacter pylori [Anti Hp IgG], two bacterial infections known to trigger autoimmunity. A general inflammatory marker, C-reactive protein (CRP), was also studied. When compared to controls, ASO and ADB titers were highest close to narcolepsy onset, and decreased with disease duration. For example, ASO>> or = 200 IU (ADB>> or = 480 IU) were found in 51% (45%) of 67 patients within 3 years of onset, compared to 19% (17%) of 67 age matched controls (OR = 4.3 [OR = 4.1], P < 0.0005) or 20% (15%) of 69 patients with long-standing disease (OR = 4.0 [OR = 4.8], P < 0.0005]. CRP (mean values) and Anti Hp IgG (% positive) did not differ from controls.

CONCLUSIONS

Streptococcal infections are probably a significant environmental trigger for narcolepsy.

BACKGROUND

Probiotic yeast has become a field of interest to scientists in recent years.

METHODS

Conventional cultural method was employed to isolate and identify yeast and standard methods were used to determine different probiotic attributes, antimicrobial and antioxidant properties.

RESULTS

This study reports potential probiotic properties of a strain of S. cerevisiae IFST 062013 isolated from fruit. The isolate is tolerant to a wide range of temperature and pH, high concentration of bile salt and NaCl, gastric juice, intestinal environment, α-amylase, trypsin and lysozyme. It can produce organic acid and showed resistance against tetracycline, ampicillin, gentamycin, penicillin, polymixin B and nalidixic acid. It can assimilate cholesterol, can produce killer toxin, vitamin BDNase. The isolate showed moderate anti-microbial activity against bacteria and fungi and cell lysate showed better antimicrobial activity than whole cell and culture supernatant. Again, the isolate showed better anti-bacterial activity against gram negative bacteria than gram positive. The isolate showed strong antioxidant activity, reducing power, nitric oxide and hydroxyl radical scavenging activity, significant brine shrimp cytotoxicity and acute toxicity and metal ion chelating activity. The isolate did not induce any detectable change in general health of mice upon oral toxicity testing and found to be safe in mouse model. The isolate improve lymphocyte proliferation and cytokine production in treated mice.

CONCLUSIONS

Such isolate could be potential as probiotic to be used therapeutically.

BACKGROUND

Screening of blood donors for viral nucleic acids has recently been introduced in several countries. With the use of transcription-mediated amplification, a blood donor was detected who had 90,000 copies of hepatitis B virus (HBV) DNA/mL but no hepatitis B surface antigen (HBsAg) or antibody to hepatitis B core antigen (anti-HBc). One month later, anti-HBc and hepatitis B surface antibody (anti-HBs) appeared; HBV DNA disappeared after 2 months. This study asked why HBsAg was undetectable in this rare case of transient occult HBV infection.

METHODS

The HBV DNA in the first sample was cloned and sequenced to identify mutations. The physical nature of the virus was examined by polyethylene glycol precipitation, DNase digestion, density gradient centrifugation, and immunoprecipitation.

RESULTS

Several mutations were found all over the genome, but the HBs antigen loop was unchanged. A stop mutation in the precore region led to loss of hepatitis B e antigen (HBeAg) expression. No HBV DNA–containing immune complexes were present. The plasma did not contain nonencapsidated HBV DNA that could explain the absence of HBsAg. The virus was immune precipitated by antibodies against HBsAg or preS1 antigen. The ratio of HBV to HBsAg subviral particles was estimated to be 1 in less than 20 whereas in overt cases the ratio is 1 in more than 1000.

CONCLUSIONS

The acute resolving occult HBV infection was caused by an HBeAg-negative variant, which otherwise was almost normal. The negative HBsAg result was probably due to an unusually low production of surplus HBsAg. The absence of the viral immunomodulator HBeAg and the early appearance of anti-HBs suggested a rapid noncytolytic HBsAg-specific T-cell response leading to low expression of HBsAg.

As a part of studies of streptococcal infections of the upper respiratory tract, sera from 142 individuals (mean age 11.9 years) with signs and symptoms on tonsillitis or pharyngitis and their family contacts were studied to quantitatively evaluate the sensitivity and specificity of the Streptozyme test. We also compared this agglutination test for streptococcal extracellular antibodies with the antistreptolysin O and antideoxyribonuclease B tests. Using different lots of Streptozyme reagent we found evidence suggesting variation in the strength of the reagent from lot to lot. Evaluation of the group specificity of the Streptozyme test suggests that it may not be specific for antibodies to extracellular products of group A streptococci, since a response to this test was seen in individuals with only non-group A strains isolated from their upper respiratory tracts. In patients with group A streptococci isolated from the upper respiratory tract, the quantitative sensitivity of the antibody response as measured by the Streptozyme test is comparable to, but no greater than, the ASO or anti-DNase B tests. Our analysis of upper limits of normal for the Streptozyme test in the study population indicates that previous recommendations for values for elevated titers are too low, especially for predominantly pediatric populations, so that previous reports of patients demonstrating an antibody response to Streptozyme-measured antibodies may include a significant number of patients with "normal" levels. This study indicates the need for both further documentation of standardization of the reagents used in this agglutination test and additional quantitative studies of the response measured by Streptozyme.

We found and previously reported a new mammalian DNA polymerase inhibitor from a sea alga, Gigartina tenella, (Ohta K., et al., Chem. Pharm. Bull., 46, 684-686, 1998). It was a new sulfolipid compound that belonged in the class of sulfoquinovosyldiacylglycerol. The biochemical properties have been investigated here. The compound, temporarily designated KM043, potently inhibited the activities of mammalian DNA polymerase alpha(pol. alpha) and DNA polymerase beta(pol. beta) and terminal deoxynucleotidyl transferase (TdT), and moderately, human immunodeficiency virus reverse transcriptase (HIV-RT). KM043 dose-dependently inhibited their activities, and each of their IC50 values was 0.25 microM for pol. alpha, 0.38 microM for TdT, 3.6 microM for pol. beta, or 11.2 microM for HIV-RT, and almost complete inhibition of each was achieved at 1.0 to 2.0 microM for pol. alpha and TdT, 7.5 microM for pol. beta and about 30 microM for HIV-RT. However, the compound did not influence the activities of prokaryotic DNA polymerases such as E. coli DNA polymerase I, and DNA metabolic enzymes like DNase 1. Inhibition of pol. alpha or beta by KM043 was non-competitive with both the DNA template and the substrate deoxythymidine 5'-triphosphate (dTTP). KM043 was weakly cytotoxic to cultured HeLa-S3 cells, and the IC50 value was 80 microM. KM043 could synergistically enhance the cytocidal effect of an anti-cancer chemotherapy agent, bleomycin. In the presence of 50 microM KM043, the effect ratio of (bleomycin plus KM043)/(bleomysin only) decreased from 0.76 to 0.22.

Lupus glomerulonephritis (LGN) is one of the most threatening manifestations of systemic lupus erythematosus (SLE) and a major predictor of poor prognosis. The mechanisms leading to kidney inflammation are not completely clear; however, autoantibodies seem to play a pivotal role. Apoptosis dysregulation in SLE is likely to trigger generation of autoantibodies, the released nucleosomes being the driving autoantigen for further epitope amplification and selection of DNA or nucleosome-specific B cells. Growing evidence supports a multistep path to LGN involving initial autoantibody binding to chromatin fragments in the mesangial matrix, where they can induce mesangial inflammation leading to a shut-down of the renal DNase gene, generation and deposition of secondary necrotic chromatin on the glomerular basement membrane favouring antibody binding, complement activation and development of membrano-proliferative glomerular lesions. Anti-DNA IgG antibodies display the major pathogenetic potential in LGN initiation; however, other isotypes (IgA or IgE) as well as autoantibodies targeting other molecules (e.g. anti-C1q, anti-C reactive protein) can perpetuate renal injury. Conversely, protective autoantibodies are also likely in SLE which can contain renal damage targeting either DNA (i.e. IgM anti-DNA) or other molecules (e.g. pentraxin 3). Thus, lupus nephritogenic-antinephritogenic antibodies orchestrate the balance between harm and defence of renal tissue.

Hepatitis B virus (HBV) infection is highly specific to primates. To cross the species barrier, we injected naked plasmid DNA containing a 1.5-fold-overlength genome of HBV into immunocompetent mice via the tail vein with acute circulatory overload. The injection resulted in production of viral transcripts specifically in the liver and expression of hepatitis B core protein in approximately 3% of the hepatocytes. HBV was secreted into the blood, evidenced by the presence of DNase I-resistant HBV sequence in fractionated serum at a density of 1.21g/ml. The HBV DNA positivity in the serum persisted for 1 week. Most mice became positive for hepatitis B surface antigen for 2 weeks and later seropositive for anti-hepatitis B surface antibody. This simple and efficient HBV replication system in mice could be useful for investigating whether viral mutations as well as host genetic background may affect viral replication and induction of immune response.

The cellular distribution of the fission yeast mitotic cyclin B, p63cdc13, was investigated by a combination of indirect immunofluorescence light microscopy, immunogold electron microscopy, and nuclear isolation and fractionation. Immunofluorescence microscopy of wild-type cells and the cold-sensitive mutant dis2.11 with a monospecific anti-p63cdc13 antiserum was consistent with the association of a major subpopulation of fission yeast M-phase protein kinase with the nucleolus. Immunogold electron microscopy of freeze-substituted wild-type cells identified two nuclear populations of p63cdc13, one associated with the nucleolus, the other with the chromatin domain. To investigate the cell cycle regulation of nuclear labeling, the mutant cdc25.22 was synchronized through mitosis by temperature arrest and release. Immunogold labeling of cells arrested at G2M revealed gold particles present abundantly over the nucleolus and less densely over the chromatin region of the nucleus. Small vesicles around the nucleus were also labeled by anti-p63cdc13, but few gold particles were detected over the cytoplasm. Labeling of all cell compartments declined to zero through mitosis. Cell fractionation confirmed that p63cdc13 was substantially enriched in both isolated nuclei and in a fraction containing small vesicles and organelles. p63cdc13 was not extracted from nuclei by treatment with RNase A, Nonidet P40 (NP-40), Triton X-100, and 0.1 M NaCl, although partial solubilization was observed with DNase I and 1 M NaCl. A known nucleolar protein NOP1, partitioned in a similar manner to p63cdc13, as did p34cdc2, the other subunit of the M-phase protein kinase. We conclude that a major subpopulation of the fission yeast mitotic cyclin B is targeted to structural elements of the nucleus and nucleolus.

Inositol lipid-derived second messengers have long been known to have an important regulatory role in cell physiology. Phosphatidylinositol 3-kinase (PI3K) synthesizes the second messenger 3,4,5'-phosphatidylinositol trisphosphate (Ptdlns 3,4,5P3) which controls a multitude of cell functions. Down-stream of PI3K/PtdIns 3,4,5P3 is the serine/threonine protein kinase Akt (protein kinase B, PKB). Since the PI3K/ PtdIns 3,4,5P3 /Akt pathway stimulates cell proliferation and suppresses apoptosis, it has been implicated in carcinogenesis. The lipid phosphatase PTEN is a negative regulator of this signaling network. Until recently, it was thought that this signal transduction cascade would promote its anti-apoptotic effects when activated in the cytoplasm. Several lines of evidence gathered over the past 20 years, have highlighted the existence of an autonomous nuclear inositol lipid cycle, strongly suggesting that lipids are important components of signaling pathways operating at the nuclear level. PI3K, PtdIns(3,4,5)P3, Akt, and PTEN have been identified within the nucleus and recent findings suggest that they are involved in cell survival also by operating in this organelle, through a block of caspase-activated DNase and inhibition of chromatin condensation. Here, we shall summarize the most updated and intriguing findings about nuclear PI3K/ PtdIns(3,4,5)P3/Akt/PTEN in relationship with carcinogenesis and suppression of apoptosis.

Poly(dA).poly(dT), but not B-form DNA, is specifically recognized by experimentally induced anti-kinetoplast or anti-poly(dA).poly(dT) immunoglobulins. Antibody binding is completely competed by poly(dA).poly(dT) and poly(dA).poly(dU) but not by other single- or double-stranded DNA sequences in a right-handed B-form. Antibody interaction with poly(dA).poly(dT) depends on immunoglobulin concentration, incubation time and temperature, and is sensitive to elevated ionic strengths. Similar conformations, for example, (dA)4-6 X (dT)4-6, in the kinetoplast DNA of the parasite Leishmania tarentolae are also immunogenic and induce specific anti-poly(dA).poly(dT) antibodies. These antibody probes specifically recognize nuclear and kinetoplast DNA in fixed flagellated kinetoplastid cells as evidenced by immunofluorescence microscopy. Anti-poly(dA).poly(dT) immunofluorescence is DNase-sensitive and competed by poly(dA).poly(dT), but not other classical double-stranded B-DNAs. Thus, these unique cellular B'-DNA helices are immunogenic and structurally similar to synthetic poly(dA).poly(dT) helices in solution.

BACKGROUND

Cationic streptococcal proteinase (erythrotoxin B) and its precursor, zymogen, are putative nephritogenic antigens. The present study was designed to test whether serum titers to these antigens were good markers of streptococcal infection associated with glomerulonephritis.

METHODS

We studied 153 patients (male/female = 104/49, age range, 2 to 23 years old) with acute poststreptococcal glomerulonephritis (APSGN) from three countries (Venezuela, Chile and Argentina). The site of the initial infection was the skin in 84 patients, the throat in 55 patients and was unknown in 14 patients. In addition, we studied 23 patients (1 to 24 years old) with streptococcal infection not associated with glomerulonephritis (14 patients with impetigo and 9 patients with pharyngitis). As control group, 93 healthy individuals (54 males, 2 to 19 years old) were studied. Anti-zymogen and anti-proteinase titers were determined in a single laboratory by ELISA, and the intra- and interassay coefficients of variation were 5.3% and 8.5%, respectively. ASO titers and anti-DNAse B titers were also done.

RESULTS

Anti-zymogen titers of 1:800 to 1:3200 had likelihood ratios (sensitivity/1-specificity) for detection of streptococcal infection in APSGN patients ranging from 2.00 to 44.2 in Argentina, Chile and Venezuela. Anti-zymogen titers decreased one to two months after APSGN and they were 1 to 3 log2 dilutions higher that anti-proteinase titers. Receiver operating characteristic (ROC) curves showed that anti-zymogen titers were consistently superior to anti-streptolysin O and anti-DNAse B titers as markers for streptococcal infection in APSGN.

CONCLUSIONS

These results suggest that increased anti-zymogen antibody titers are the best available marker for streptococcal infection associated with acute glomerulonephritis.

1. To study proteins transported with actin in axons, we pulse-labeled motoneurons in the chicken sciatic nerve with [35S]methionine and, 1-20 days later, isolated actin and its binding proteins by affinity chromatography of Triton soluble nerve extracts on DNase I-Sepharose. The DNase I-purified proteins were electrophoresed on two-dimensional gels and the specific activity of the radioactively labeled protein spots was estimated by fluorography. 2. In addition to actin, which binds specifically to DNase I, a small number of other proteins were labeled, including established actin monomer binding proteins and a protein of 36 kDa and pI 8.5. On the basis of its molecular mass, pI, amino acid composition, and immunostaining, the unrecognized protein was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 3. The high-affinity binding of GAPDH to actin was confirmed by incubation of Triton-soluble nerve extracts with either mouse anti-GAPDH (or antiactin) and indirect immunomagnetic separation with Dynabeads covalently linked to sheep anti-mouse antibody. Analysis by one-dimensional gel electrophoresis and immunoblotting showed that actin and GAPDH were the main proteins isolated by these methods. 4. Analysis of labeled nerves at 12 and 20 days after pulse labeling showed that GAPDH and actin were transported at the same rate, i.e., 3-5 mm/day, which corresponds to slow component b of axonal transport. These proteins were not associated with rapidly transported proteins that accumulated proximal to a ligation 7 cm from the spinal cord 9 hr after injection of radioactivity. 5. Our results indicate that GAPDH and actin are transported as a complex in axons and raise the possibility that GAPDH could act as a chaperone for monomeric actin, translocating it to intraaxonal sites for exchange with or assembly into actin filaments. Alternatively, actin could be involved in translocating and anchoring GAPDH to specialized sites in axons and nerve terminals that require a source of ATP by glycolysis.

Pseudolaric acid B is a major compound found in the bark of Pseudolarix kaempferi Gordon. In our study, pseudolaric acid B inhibited growth of human melanoma cells, A375-S2 in a time- and dose-dependent manner. A375-S2 cells treated with pseudolaric acid B showed typical characteristics of apoptosis including morphologic changes, DNA fragmentation, sub-diploid peak in flow cytometry, cleavage of poly-ADP ribose polymerase (PARP) and degradation of inhibitor of caspase-activated DNase (ICAD). P53 protein expression was upregulated while cells were arrested at the G2/M phase of the cell cycle. There was a decrease in the expression of anti-apoptotic Bcl-2 and Bcl-xL proteins, whereas pro-apoptotic Bax was increased. The two classical caspase substrates, PARP and ICAD, were both decreased in a time-dependent manner, indicating the activation of downstream caspases.

Inefficient and abnormal clearance of apoptotic cells (efferocytosis) contributes to systemic autoimmune disease in humans and mice, and inefficient chromosomal DNA degradation by DNAse II leads to systemic polyarthritis and a cytokine storm. By contrast, efficient clearance allows immune homeostasis, generally leads to a non-inflammatory state for both macrophages and dendritic cells (DCs), and contributes to maintenance of peripheral tolerance. As many as 3 × 108 cells undergo apoptosis every hour in our bodies, and one of the primary "eat me" signals expressed by apoptotic cells is phosphatidylserine (PtdSer). Apoptotic cells themselves are major contributors to the "anti-inflammatory" nature of the engulfment process, some by secreting thrombospondin-1 (TSP-1) or adenosine monophosphate and possibly other immune modulating "calm-down" signals that interact with macrophages and DCs. Apoptotic cells also produce "find me" and "tolerate me" signals to attract and immune modulate macrophages and DCs that express specific receptors for some of these signals. Neither macrophages nor DCs are uniform, and each cell type may variably express membrane proteins that function as receptors for PtdSer or for opsonins like complement or opsonins that bind to PtdSer, such as protein S and growth arrest-specific 6. Macrophages and DCs also express scavenger receptors, CD36, and integrins that function via bridging molecules such as TSP-1 or milk fat globule-EGF factor 8 protein and that differentially engage in various multi-ligand interactions between apoptotic cells and phagocytes. In this review, we describe the anti-inflammatory and pro-homeostatic nature of apoptotic cell interaction with the immune system. We do not review some forms of immunogenic cell death. We summarize the known apoptotic cell signaling events in macrophages and DCs that are related to toll-like receptors, nuclear factor kappa B, inflammasome, the lipid-activated nuclear receptors, Tyro3, Axl, and Mertk receptors, as well as induction of signal transducer and activator of transcription 1 and suppressor of cytokine signaling that lead to immune system silencing and DC tolerance. These properties of apoptotic cells are the mechanisms that enable their successful use as therapeutic modalities in mice and humans in various autoimmune diseases, organ transplantation, graft-versus-host disease, and sepsis.

To define the cis-acting elements that regulate LPS-stimulated IL-1 beta gene transcription, we analyzed the murine IL-1 beta gene by digestion with DNase I. At least two hypersensitive sites were located between 2200 and 2600 bp upstream of the transcription start site in mononuclear phagocytes, but not in an IL-1 nonproducing immature T cell line. Specific DNA sequences required for LPS induction of IL-1 beta gene expression were identified within the DNase I hypersensitive (DH) region using transfection of reporter constructs that contained portions of the IL-1 beta 5'-flanking region. Two specific DNA sequences were targets for nuclear factor binding as assessed with use of electrophoretic mobility shift analysis (EMSA). One site contained a consensus sequence for NFIL-6 binding. Base substitutions within this NFIL-6 site resulted in virtual elimination of LPS-induced IL-1 beta gene transcription. Introduction of multimers of the NFIL-6-like sequence immediately 5' to homologous or heterologous promoters conferred LPS-induced transcription, indicating that this NFIL-6-like consensus site was a transcriptional activator. Anti-C/EBP beta (NFIL-6) and anti-C/EBP delta (NFIL-6 beta) Abs identified both of these proteins in complexes formed between the NFIL-6-like element and mononuclear cell nuclear extracts. C/EBP delta (NFIL-6 beta) was not detected in complexes utilizing extracts from the IL-1 nonproducing T cell line. These data are consistent with the requirement for C/EBP beta (NFIL-6) and C/EBP delta (NFIL-6 beta) in the activation of murine IL-1 beta gene expression by endotoxin.

Post-streptococcal autoimmunity affects millions worldwide, targeting multiple organs including the heart, brain, and kidneys. To explore the post-streptococcal autoimmunity spectrum, we used western blot analyses, to screen 310 sera from healthy subjects with (33%) and without (67%) markers of recent streptococcal infections [anti-Streptolysin O (ASLO) or anti-DNAse B (ADB)]. A 58 KDa protein, reacting strongly with post-streptococcal sera, was identified as Protein Disulfide Isomerase (PDI), an abundant protein with pleiotropic metabolic, immunologic, and thrombotic effects. Anti-PDI autoantibodies, purified from human sera, targeted similar epitopes in Streptolysin O (SLO, P51-61) and PDI (P328-338). The correlation between post-streptococcal status and anti-human PDI auto-immunity was further confirmed in a total of 2987 samples (13.6% in 530 ASLO positive versus 5.6% in 2457 ASLO negative samples, p<0.0001). Finally, anti-PDI auto-antibodies inhibited PDI-mediated insulin degradation in vitro (n = 90, p<0.001), and correlated with higher serum insulin (14.1 iu/ml vs. 12.2 iu/ml, n = 1215, p = 0.039) and insulin resistance (Homeostatic Model Assessment (HOMA) 4.1 vs. 3.1, n = 1215, p = 0.004), in a population-based cohort. These results identify PDI as a major target of post-streptococcal autoimmunity, and establish a new link between infection, autoimmunity, and metabolic disturbances.

Silibinin, derived from the milk thistle plant, Silybum marianum, has been traditionally used as an antihepatotoxic agent for the treatment of liver disease. Our preliminary study demonstrated that silibinin has protected rat cardiac myocytes against beta-adrenergic agonist isoproterenol-induced injury through resuming mitochondrial function and regulating the expression of SIRT1 and Bcl-2 family members. In this study, we investigate whether silibinin has anti-apoptotic effect on isoproterenol-treated rat cardiac myocytes. DNA damage, detected by the TUNEL and DNA fragmentation assay, was diminished after treatment of silibinin. Results of nitrite and Western blot assays showed that the amount of NO and the expression of iNOS were decreased after treatment with silibinin, while the expression of procaspase-3 and digestion of caspase-3 substrates, the inhibitor of caspase-activated DNase (ICAD) and poly-(ADP-ribose) polymerase (PARP), were increased simultaneously. The DNA damage was reversed by down-regulation of p53 phosphorylation after treatment with silibinin. Result of flowcytometric analysis showed that the cell cycle was not affected, and the expression of cell cycle regulatory protein p21 also had no change. Consequently, silibinin protected cardiac myocytes against isoproterenol-induced DNA damage through caspase pathway and the expression of p53, but independent on regulation of cell cycle.

We have shown that young autoimmune and normal strain mice possess autoantigen-sensitive cells potentially capable of producing anti-sDNA autoantibody in the absence of normal regulatory mechanisms in vitro. In certain strains such as B/W mice, these regulatory mechanisms presumably break down with increasing age, and autoimmunity develops. These regulatory mechanisms might consist of sDNA, T cells, or some combination of these since both of these agents suppressed the anti-sDNA PFC response in vitro. The sDNA may have inhibited PFC development by a receptor blockade mechanism since i) spleen cells pulsed with sDNA for short periods and then washed were suppressed after 5 days of culture; ii) treatment of these blocked cells with trypsin and DNase I restored the anti-sDNA response; iii) the PFC remaining in partially blocked cultures were of lower avidity than PFC in unblocked cultures; and iv) the target of sDNA may be a B cell. Thymocytes and splenic T cells suppressed the anti-sDNA response but not the anti-SRBC response in vitro in a dose-dependent manner. The suppressive capacity of thymus cells did not decline with age in B/W mice. In addition, thymus cells activated by competing foreign antigens could also suppress the anti-sDNA response. The relationship between these modes of regulating autoreactivity remains to be investigated.

We investigated a possible association of leukosialin (CD43), the major surface sialoglycoprotein of leukocytes, with neutrophil cytoskeleton. We first analysed the solubility of CD43 in Triton X-100 and observed that CD43 of resting neutrophils was mostly soluble. The small proportion of CD43 molecules, which 'spontaneously' precipitated in Triton, appeared associated with F-actin, as demonstrated by the fact that this insolubility did not occur when cells were incubated with cytochalasin B or when F-actin was depolymerized with DNase I in the Triton precipitate. Cell stimulation with anti-CD43 mAb (MEM59) enhanced this CD43-cytoskeleton association. By immunofluorescence as well as by electron microscopy, we observed a redistribution of CD43 on the neutrophil membrane, initially in patches followed by caps, during anti-CD43 cross-linking at 37 degrees C. This capping did not occur at 4 degrees C and was inhibited by cytochalasin B and by a myosin disrupting drug butanedione monoxime, thus providing evidence that the actomyosin contracile sytem is involved in the capping and further suggesting an association of CD43 with the cytoskeleton. Some of the capped cells exhibited a front-tail polarization with CD43 caps located in the uropod at the rear of the cell. Surprisingly, colchicine and the chemotactic factor fNLPNTL which induce neutrophil polarization associated with cell motility, also resulted in a clustering of CD43 in the uropod, independently of a cross-linking of the molecule by mAbs. An intracellular redistribution of F-actin, mainly at the leading front and of myosin in the tail, was observed during CD43 clustering induced by colchicine and in cells polarized by anti-CD43 mAbs cross-linking. We conclude that neutrophil CD43 interacts with the cytoskeleton, either directly or indirectly, to redistribute in the cell uropod under antibodies stimulation or during cell polarization by colchicine, thus highly suggesting that CD43 may be involved in cell polarization.

Class-switched antibodies to double-stranded DNA (dsDNA) are prevalent and pathogenic in systemic lupus erythematosus (SLE), yet mechanisms of their development remain poorly understood. Humans and mice lacking secreted DNase DNASE1L3 develop rapid anti-dsDNA antibody responses and SLE-like disease. We report that anti-DNA responses in Dnase1l3^-/- mice require CD40L-mediated T cell help, but proceed independently of germinal center formation via short-lived antibody-forming cells (AFCs) localized to extrafollicular regions. Type I interferon (IFN-I) signaling and IFN-I-producing plasmacytoid dendritic cells (pDCs) facilitate the differentiation of DNA-reactive AFCs in vivo and in vitro and are required for downstream manifestations of autoimmunity. Moreover, the endosomal DNA sensor TLR9 promotes anti-dsDNA responses and SLE-like disease in Dnase1l3^-/- mice redundantly with another nucleic acid-sensing receptor, TLR7. These results establish extrafollicular B cell differentiation into short-lived AFCs as a key mechanism of anti-DNA autoreactivity and reveal a major contribution of pDCs, endosomal Toll-like receptors (TLRs), and IFN-I to this pathway.

Keywords: DNASE1L3; TLR7; TLR9; anti-DNA antibodies; extrafollicular B cell response; plasmacytoid dendritic cells; systemic lupus erythematosus; type I interferon.

Systemic lupus erythematosus is an autoimmune disease characterized by autoantibodies and systemic inflammation that results in part from dendritic cell activation by nucleic acid containing immune complexes. There are many mouse models of lupus, some spontaneous and some induced. We have been interested in an induced model in which estrogen is the trigger for development of a lupus-like serology. The R4A transgenic mouse expresses a transgene-encoded H chain of an anti-DNA Ab. This mouse maintains normal B cell tolerance with deletion of high-affinity DNA-reactive B cells and maturation to immunocompetence of B cells making nonglomerulotropic, low-affinity DNA-reactive Abs. When this mouse is given estradiol, normal tolerance mechanisms are altered; high-affinity DNA-reactive B cells mature to a marginal zone phenotype, and the mice are induced to make high titers of anti-DNA Abs. We now show that estradiol administration also leads to systemic inflammation with increased B cell-activating factor and IFN levels and induction of an IFN signature. DNA must be accessible to B cells for both the production of high-affinity anti-DNA Abs and the generation of the proinflammatory milieu. When DNase is delivered to the mice at the same time as estradiol, there is no evidence for an abrogation of tolerance, no increased B cell-activating factor and IFN, and no IFN signature. Thus, the presence of autoantigen is required for positive selection of autoreactive B cells and for the subsequent positive feedback loop that occurs secondary to dendritic cell activation by DNA-containing immune complexes.

The efficacy and safety of a 10-day course of ceftibuten oral suspension (9 mg/kg once daily) were compared with those of penicillin V (25 mg/kg/day in 3 divided doses) in children 3 to 18 years old treated for symptomatic pharyngitis and scarlet fever caused by group A beta-hemolytic streptococci (Streptococcus pyogenes). The study was prospective, randomized, multicenter and investigator-blinded; patients were randomized in a 2:1 ratio (ceftibuten:penicillin V). Overall clinical success (cure/improvement) at the primary end point of treatment (5 to 7 days posttherapy) was achieved in 97% (285 of 294) of ceftibuten-treated patients vs. 89% (117 of 132) of penicillin V-treated patients (P < 0.01). Elimination of infecting streptococci 5 to 7 days posttherapy was achieved in 91% (267 of 294) of ceftibuten-treated patients vs 80% (105 of 132) of penicillin V-treated patients (P < 0.01). A significant rise in anti-streptolysin O or anti-DNase B was observed in approximately 30% of patients in both treatment groups. No patient developed rheumatic fever or nephritis. Treatment-related adverse events were similar between the two groups; mild vomiting (2%) was most frequently reported. These data suggest that once daily ceftibuten is as safe as and more effective than three times daily penicillin V for the treatment of group A beta-hemolytic streptococcal pharyngitis.

After androgen ablation by castration, the epithelial cells of the rat ventral prostate are eliminated by apoptosis. The number of cells showing apoptotic chromatin degradation increases with time up to day 3 after castration as verified by in situ end labeling of fragmented DNA. Apoptotic chromatin degradation is catalyzed by a Ca2+, Mg2+-dependent endonuclease. Recently, evidence has been presented that suggests deoxyribonuclease I (DNase I) is identical or very closely related to the apoptotic endonuclease (Peitsch, M.C., B. Polzar, H. Stephan, T. Crompton, H.R. MacDonald, H.G. Mannherz, and J. Tschopp. 1993. EMBO [Eur. Mol. Biol. Organ.] J. 12:371-377). Therefore, the expression of DNase I in the ventral prostate of the rat was analyzed before and after androgen ablation at the level of protein, enzymatic activity, and gene transcripts using immunohistochemical and biochemical techniques. DNase I immunoreactivity was detected only in a few single epithelial cells before androgen ablation. After castration, a time-dependent increase in DNase I immunoreactivity was observed within the epithelial cells. It first appeared after about 12 h in the apical region of a large number of epithelial cells. Up to day 3 after castration, the intracellular DNase I antigenicity continuously increased, and the cell nuclei gradually became DNase I positive. At day 5, almost all nuclei of the epithelium were stained by anti-DNase I. DNase I immunoreactivity was particularly concentrated in cells showing morphological signs of apoptosis, like nuclear fragmentation, and in many cases was found to persist in apoptotic bodies. DNase I gene transcripts were detected in control animals using dot and Northern blotting as well as RNase protection assay. After androgen ablation, the amount of DNase I gene transcripts in total extractable RNA was found unchanged or only slightly decreased up to day 5. Their exclusive localization within the epithelial cells was verified by in situ hybridization. Before castration, the DNase I gene transcripts were homogeneously distributed in all epithelial cells. At day 3, DNase I-specific mRNA was found to be highly concentrated in cells of apoptotic morphology. Using the zymogram technique, a single endonucleolytic activity of about 32 kD was detected in tissue homogenates before castration. After androgen ablation, the endonucleolytic activity increased about four- to sevenfold up to day 3. At day 5, however, it had dropped to its original level. At day 1, three new endonucleolytic variants of higher molecular mass were expressed. At day 3, the predominant endonucleolytic activity exhibited an apparent molecular mass of 32 kD. Enzymatic analysis of the endonucleases present in prostate homogenates before and after castration demonstrated properties identical to DNase I. They were inhibited by chelators of divalent cations, Zn2+ ions and monomeric actin. Immunodepletion was achieved by immobilized antibodies specific for rat parotid DNase I. A polyclonal antibody raised against denatured DNase I was shown by Western blotting to stain a 32-kD band after enrichment of the endonuclease from day 0 and 3 homogenates by preparative gel electrophoresis. The data thus indicate that androgen ablation leads to translational upregulation of an endonucleolytic activity with properties identical to DNase I in rat ventral prostate, followed by its intracellular retention and final nuclear translocation in those epithelial cells that are destined to apoptotic elimination.