Hemophilia B-Leyden is characterized by the gradual amelioration of bleeding after the onset of puberty. All Leyden phenotype mutations found to date lie within the Leyden-specific region, which spans roughly nt-40 to +20 in the 5' end of the human factor IX gene. With HepG2 cell nuclear extracts, the Leyden-specific region and its immediate neighboring region of the normal factor IX gene showed five DNase I footprints: FP-I (nt +4 to +19), FP-II (nt -16 to -3), FP-III (nt -27 to -19), FP-IV (nt -67 to -49), and FP-V (nt -99 to -77). Protein binding affinities of short oligonucleotides containing sequences of FP-I, FP-II, or FP-III were substantially reduced in the presence of Leyden phenotype mutations in these areas, correlating well with the negative effects of these mutations on factor IX gene expression. A Leyden phenotype mutation at nt -20 (T to A) caused a loss of both footprints FP-III and FP-II but generated a new footprint, FP-III' (nt -34 to -23), partially overlapping with FP-III, indicating mutation-dependent competitive protein binding at these sites. Although the FP-III' area contains an androgen responsive element-like sequence, the nuclear protein that binds at FP-III' is not androgen receptor. The protein was not recognized by anti-androgen receptor antibody and, furthermore, was present not only in liver but also in both androgen receptor-positive and androgen receptor-negative cells in electrophoretic mobility shift assays. The nuclear concentration of this protein increased significantly upon treatment of the HepG2 cells with testosterone. Its binding affinity to an oligonucleotide (-32sub) containing the FP-III' sequence was greatly reduced in the presence of exogenous androgen receptor, suggesting a possible interaction of this protein with androgen receptor. The affinities of both this protein and a protein which binds to FP-III (presumably HNF-4) to -32sub with a mutation at nt -26 were grossly lowered. These findings suggest that the amelioration of hemophilia B-Leyden with a mutation at nt -20 after puberty involves binding of a specific non-androgen receptor nuclear protein at FP-III' and it is able to substitute for the function of a protein bound at FP-III in the normal gene optimally through its elevated interaction with androgen receptor upon a surge of testosterone.(ABSTRACT TRUNCATED AT 400 WORDS)

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.

Nuclear matrix prepared from mouse leukemia L5178Y cells contained not only the two common actin isomers, beta and gamma actins, but also two additional acidic species of actin (pI 5.1 and 5.3). An anti-actin antibody recognized these acidic species as well as beta and gamma actins on a nitrocellulose filter following western blotting of two-dimensional electrophoresis. These acidic species were co-purified with beta and gamma actins using DNase I-Sepharose affinity chromatography on the nuclear matrix. Limited digestion of the acidic actin with protease V8 or trypsin gave very similar peptide fragments as did digestion of beta and gamma actins. These acidic actins were found to be distributed in the nuclear fraction, but were scarcely detectable in the cytoplasmic fraction. One of the acidic actins (pI 5.3) was found in all subnuclear fractions (DNase extract, high-salt extract and nuclear matrix), while the other species, the most acidic actin (pI 5.1), was localized predominantly in the nuclear matrix.

OBJECTIVE

Autoantibodies to cell membrane associated DNA are described in systemic lupus erythematosus (SLE). The specificity of these antibodies differ from antibodies to nuclear DNA.

METHODS

Using indirect immunofluorescence, a specific IgG was detected giving a characteristic pattern of continuous peripheral membrane fluorescence on cultured B-lymphocytes.

RESULTS

This pattern was observed in 53 of 80 serum samples of SLE patients but absent in the serum samples of the control populations: 15 rheumatoid arthritis, 38 ankylosing spondylarthritis, 17 non-inflammatory osteopenic patients, and 224 blood donors. In 34 Sjögren syndrome's patients one only showed a positive test. The cmDNA specificity of these antibodies was confirmed by pattern extinction with DNAse but not RNase or protease pre-treatment of the cells. IgG to cmDNA, separated by absorption/elution from purified cmDNA immobilised on DEAE-nitrocellulose reproduced the immunofluorescence pattern pictures. Extensive serum depletion of anti-double strand or single strand DNA antibodies by absorption to cellulose bound ds- or ss-DNA affected marginally the pericellular fluorescence revealing some minor cross reactivity with nuclear DNA. Moreover, in SLE patients without detectable antibody to ds-DNA, pericellular fluorescence could be visible.

CONCLUSIONS

This novel rapid immunofluorescence method may serve as an identification test of SLE patients. Given its positive (97.1%) and negative (92.9%) predictive value, sensitivity (66%) and specificity (99.5%), it improves on other diagnostic tests such as the detection of antibodies to Sm.

The chromatin structure of a diploid precursor B-cell line (REH), in vitro-stimulated normal B-lymphocytes, and reactive and malignant lymph node B-lymphocytes was studied by staining formaldehyde-fixed, permeabilized cells with the DNA-specific fluorophore 7-aminoactinomycin D (7-AMD) and measuring single-cell fluorescence by flow cytometry. Resting peripheral blood B- and T-lymphocytes (G0 cells) bound low amounts of 7-AMD (7-AMD- phenotype), while G1 REH cells and purified B-cells stimulated with anti-mu + B-cell growth factor bound nearly twice as much 7-AMD (7-AMD+ phenotype). 7-AMD binding increased up to threefold and the differences in binding between G0 and G1 cells were nearly abolished when nuclei were isolated prior to fixation or when fixed whole cells were treated with DNase 1. 7-AMD binding increased in parallel with autofluorescence and approximately linearly with time during the G0-G1 transition of in vitro stimulated B-cells, as was determined by simultaneous measurements of 7-AMD fluorescence and autofluorescence or fluorescence of fluorescein isothiocyanate-labeled antibodies to the early activation antigen 4F2 and to the transferrin receptor. In cell suspensions from lymph node biopsies, the 7-AMD+ phenotype was a property of tumor cells in patients with high grade non-Hodgkin's lymphoma (H-NHL, Kiel classification, 5/5); cells with this phenotype were only found in one of nine low grade non-Hodgkin's lymphoma samples (L-NHL, 1/9). The other (8/9) L-NHL samples and the reactive lymph node contained only 7-AMD- cells. All tumors were diploid. The correlation observed between 7-AMD binding and DNase 1 susceptibility of DNA in chromatin (P less than 0.001) suggests that 7-AMD binding is a marker of general transcriptional activity. Surprisingly, the percentage of tumor cells in S phase did not correlate significantly with 7-AMD stainability (P = 0.07), while the light scattering (cell size) of G0/G1 cells was highly correlated to 7-AMD binding (P less than 0.001).

Serological tests are commonly employed to aid the diagnosis of Streptococcus pyogenes infections, particularly when non-suppurative sequelae are suspected. Conventional laboratory practice is to measure antibody levels to various combinations of the extracellular group A Streptococcus (GAS) antigens streptolysin O (SLO), DNase B, streptokinase and hyaluronidase. Antibody to the extracellular cysteine proteinase streptococcal pyrogenic exotoxin B (SPE B) and its precursor zymogen is also produced in response to GAS infections. An indirect hemagglutination test for antibody to zymogen/SPE B was established and evaluated in serum samples from 168 patients with proven (n = 27) or suspected GAS (n = 141) infections, which were also screened for antibodies using the 4 conventional tests. For comparison, sera from 56 patients infected with a variety of other pathogens, as well as sera from 16 patients infected with either S. agalactiae or S. pneumoniae and 34 sera from healthy subjects, were tested. Statistical analysis confirmed that antibody to zymogen/SPE B is a serological marker that can discriminate GAS infections. It can be ranked with the anti-SLO titer, currently the most widely used test, as a marker of an antecedent GAS infection.

Antigen binding to the B-cell receptor (BCR) of pre-mature B lymphocytes induces their apoptotic cell death, but binding to the BCR of mature B lymphocytes triggers activation and proliferation. Binding to pre-mature B lymphocytes is thought not only to function as a mechanism to exclude B-cell clones that possess the ability to react with self-antigen, but also to act as a defense mechanism in auto-immune diseases. Cross-linking of BCR of pre-mature B-cell lines, including the chicken DT40 cell line, with anti-immunoglobulin IgG induces apoptotic cell death. Treatment with phorbol 12-myristate 13-acetate/ionomycin, which mimics BCR stimulation, is used to study intracellular signal transduction of B lymphocytes. Here, by analyzing the E2A-deficient DT40 cell line, E2A(-/-), we show that E2A deficiency prevents certain levels of apoptotic cell death mediated by BCR signaling. In addition, E2A deficiency-linked BCR signaling controls the mimicked pre-mature B-cell apoptosis by PMA/ionomycin through elevated survivin plus inhibitor of apoptosis 2 levels, and reduced caspase-3 and caspase-8 activities, resulting in increased amounts of ICAD (inhibitor of caspase-activated DNase), compared with those in the presence of E2A, followed by reduction of DNA fragmentation. These findings will contribute to the resolution of molecular mechanisms of negative selection of B cells and also auto-immune diseases.

A group of prokaryotic actin-related proteins (PARP) with an Mr of 43000 was detected in Actinobacillus pleuropneumoniae. These proteins were enriched by a depolymerization/polymerization cycle, under similar conditions to those used to polymerize muscle actin, and purified by affinity chromatography on a DNase I-Sepharose column. Three isoforms of A. pleuropneumoniae PARP (Ap-PARP) with pI values of 5.8, 6.15 and 6.2 were detected. Ap-PARP were recognized by four different anti-actin antibodies (one anti-muscle and three anti-cytoplasmic isoforms). Ap-PARP were also recognized by antibodies against Anabaena variabilis PARP (Av-PARP) and against actin-binding proteins such as alpha-actinin and spectrin, and also by a monoclonal antibody against heat-shock cognate protein 70 (Hsc70). Specific binding of phalloidin to Ap-PARP was detected both in permeabilized cells and in vitro. Purified Ap-PARP can polymerize under similar conditions to those required for skeletal muscle actin polymerization and the filaments formed appear to be decorated with myosin subfragment-1(S1) as observed by transmission electron microscopy. The amino acid composition of Ap-PARP revealed more similarities to muscle gamma-actin and the cytoplasmic beta-actin isoform than to eukaryotic actin-related proteins.

(<em>b</em>)O<em>b</em>jective:</<em>b</em>) Pediatric autoimmune neuropsychiatric disorder associated with <i>Streptococcus pyogenes</i> infection (PANDAS) and pediatric acute-onset neuropsychiatric syndrome (PANS) are emerging immune-mediated encephalopathies characterized <em>b</em>y sudden onset of seemingly inexplica<em>b</em>le complex neuropsychiatric symptoms, including o<em>b</em>sessions, compulsions, and heterogeneous tics, which occur in children. Main goal of this study was to report our experience in a large cohort of Italian children affected <em>b</em>y either PANDAS or PANS and treated long term with an <em>anti</em><em>b</em>iotic regimen similar to that used for acute rheumatic fever. (<em>b</em>)Patients and Methods:</<em>b</em>) The clinical charts of a cohort of 371 consecutive Italian children, 345 with PANDAS (93.0%) and 26 with PANS (7.0%), were retrospectively evaluated. <em>Anti</em>streptococcal, <em>anti</em>nuclear <em>anti</em><em>b</em>odies, and serologic evaluation for a group of common auto<em>anti</em><em>b</em>odies and micro<em>b</em>ial agents were also assessed. A strict differential diagnosis with other autoimmune diseases displaying neuropsychiatric manifestations was performed. (<em>b</em>)Results:</<em>b</em>) <em>Anti</em>streptolysin O and <em>anti</em>-<em>DNase</em> <em>B</em> <em>anti</em><em>b</em>ody titers were tested and were positive in all PANDAS su<em>b</em>jects, <em>b</em>ut negative in PANS. <em>Anti</em>-<i>Mycoplasma pneumoniae</i> <em>anti</em><em>b</em>odies and <em>anti</em>-Epstein-<em>B</em>arr virus Nuclear <em>Anti</em>gen <em>anti</em><em>b</em>odies were found positive in 11 (42.3%) and 5 (19.2%) patients with PANS, respectively. Among PANDAS cases, a clear streptococcal infection was clinically evident at the onset of neurological symptoms in only 74 patients (21.4%), whereas the relationship with <i>Streptococcus pyogenes</i> was confirmed <em>b</em>y serologic tests in the other 271 (78.6%). All patients fulfilling the diagnostic criteria for PANDAS (<i>n</i> = 345) received amoxicillin/clavulanic acid for 10-21 days at diagnosis, while those who were diagnosed with PANS (<i>n</i> = 26) received treatment according to the causative agent. Thereafter, all PANDAS/PANS patients received prophylaxis with <em>b</em>enzathine <em>b</em>enzylpenicillin for an overall period of at least 5 years to prevent su<em>b</em>sequent potential streptococcal infections. To date, 75.0% of PANDAS patients (<i>n</i> = 258) have shown an improvement of neurologic symptoms, mainly o<em>b</em>served within 3-5 months of treatment for PANDAS cases, while 88.4% of PANS patients (<i>n</i> = 23) have improved after 6-12 months. Infection-related relapses of neurologic manifestations were o<em>b</em>served in <em>b</em>oth PANDAS and PANS patients (<i>n</i> = 167 out of 371; 45% of the total cohort) in the long term. (<em>b</em>)Conclusions:</<em>b</em>) Our study has confirmed the usefulness of the preliminary diagnostic criteria for PANDAS and PANS, revealing also the importance of early diagnosis to reduce the risk of evolution toward disa<em>b</em>ling chronic neurologic sequelae. Long-term <em>anti</em><em>b</em>iotic prophylaxis has resulted in a su<em>b</em>st<em>anti</em>al <em>b</em>enefit to reduce neurological symptoms for the majority of PANDAS and PANS patients over a 7-year period.

Group A streptococcus (GAS) is the cause of a wide range of acute suppurative and, following a latent period, non-suppurative diseases such as rheumatic fever and poststreptococcal glomerulonephritis. Diagnosis of the latter group requires evidence of preceding GAS infection. The bacteria produce a range of extracellular antigens, including streptolysin O, which induce an antibody response in the host. A rise in antistreptolysin O titre (ASOT) is indicative of preceding GAS infection. In clinical practice, often only a single ASOT measurement is available and its timing in relation to a possible GAS infection is unknown. Interpretation of the result in this context is liable to misdiagnosis. In order to optimise diagnosis of preceding GAS infection, at least two sequential ASOT measurements, together with simultaneous assay for anti-DNase B, a second antistreptococcal antibody, is recommended.

An outbreak of post streptococcal acute glomerulonephritis (PSAGN) was recorded in the South East Health Service (SEHS) of Santiago, Chile, between 1984 and 1987. Some aspects related to its epidemiology and natural history are discussed. This outbreak was preceded by streptococcal skin infection (SSI) in 60% of cases vs. 36% during the previous period. At the same time it was observed an increased isolation rate of group A beta hemolytic streptococci (GABHS) of skin origin, and a high prevalence of anti-DNase B antibodies (ADB) in the general population, what reveals an important skin-GABHS reservoir in the community. In Chile, skin infections with GABHS are frequently secondary to scabies. The trend of scabies in the last years has been similar to that of PSAGN. The need for control and treatment of scabies is emphasized to interrupt the epidemiological chain. The protection of household contacts with Benzathin Penicillin is recommended because lower secondary attack rate has been observed in prophylaxis versus non protected groups.

Molecular heterogeneity of the extracellular deoxyribonuclease (DNase) in group A streptococci was demonstrated in 42 clinical isolates. Although polyacrylamide gel electrophoretic patterns of the extracellular DNase of all the isolates were heterogeneous, they could be divided into five main patterns with respect to the presence or absence of three DNase components including DNase B. By comparing the electrophoretic patterns of DNase in all the isolates with their T-types, we found that the patterns were quite characteristic for their T-types, especially in the prevalent T-types 12 and 1, and that the isolates of T-types 12 and 1 produced DNase B as their major extracellular DNase. Relative DNase B activity in the total extracellular DNase activity of group A, B, and G isolates was determined by the rapid method of neutralization with anti-DNase B antibody. The results showed neutralization of DNase activity in all the isolates of group A streptococci, largely corresponding to their T-types, but not of the isolates of groups B and G. These results indicate that the electrophoretic patterns of the extracellular DNase of group A streptococci are closely correlated with their T-types, suggesting the physicochemical taxonomic value of these properties.

Streptococcus equi subspecies zooepidemicus infection is rare in humans, but a well-known cause of pyogenic disease in cows and horses. S. zooepidemicus uncommonly causes post-strep glomerulonephritis (PSGN) in humans via epidemic outbreaks. We present a sporadic case of post S. zooepidemicus glomerulonephritis in a child most probably contracted from a horse. The 14-year-old girl presented with the typical signs of PSGN, with S. equi zooepidemicus isolated from a blood culture, together with a low C3 and raised anti-DNAse B. This is the first known report of a sporadic case of PSGN in a child caused by this organism.

Three patterns of activity were evident when the differential activation of the DNA polymerase associated with serum Dane particles by nonionic detergent and salt was investigated. The patterns were obtained by plotting the increase in enzyme activity mediated by the detergent Nonidet P-40 (NP-40) in increasing concentrations of KCl compared to the activity observed in the absence of detergent. The pattern of differential activity of hepatitis B (HB) DNA polymerase in detergent and salt was altered by subjecting the HBAg preparations to shearing forces. Hepatitis B DNA polymerase activity was stable even in NP-40 concentrations as high as 10%. In addition to hepatitis B DNA polymerase, DNA polymerase activated by calf thymus DNA was found in pellets containing Dane particles. The latter DNA polymerase activity was also activated by NP-40 and was not decreased by DNAse; this DNA polymerase coprecipitated with hepatitis B antigen (HBAg) upon addition of anti-HBs. However, the DNA polymerase activated by calf thymus DNA was inhibited by 0.4 M KCl. Electron microscopic observations of serum Dane particles in 0.4 M KCl showed no alterations of morphology of these particles when compared to particles in low-salt buffer. The data indicated that KCl activated HB DNA polymerase by a different mechanism from that of shear or NP-40, which removed the surface antigen coat from the Dane particles.

Splenic lymphoid cells of normal and preautoimmune mice escape normal regulatory mechanisms in vitro and spontaneously synthesize anti-ssDNA antibody in the absence of any exogenous antigen. In addition, both the cultured cells and their cell-free supernatants were capable of inducing specific anti-ssDNA responses in vivo upon their injection into syngeneic recipients. The response was host-derived, required the participation of B and T cells, and was not due to polyclonal activation. The immunogen responsible for this response appeared to be membrane-associated, was found predominantly on cultured T cells, and was sensitive to DNase and proteolysis.

OBJECTIVE

Anti-streptolysin O (ASO) and anti-DNase B (ADB) titers are used for the diagnosis of poststreptococcal complications. Ranges of normal values of ASO and ADB titers vary, depending on age, population and different time intervals. Although many studies have been performed for determination of the ASO titer in our country, only a few studies have been conducted for specification the upper limit of normal for (ULN) ADB. In our study we aimed to determine the upper limit of normal of ADB antibody titers in children aged 5-15.

METHODS

One hundred and twenty one children aged from 5-15 who were admitted to our outpatient clinic of Haydarpaşa Numune Training and Research Hospital with noninfectious reasons between November 2013 and March 2014 were included in the study. Patients who met the following criteria were included in the study; absence of streptococcal infection in the last three months in physical examination and/or no growth of group A, C, and G of beta-haemolytic streptococci in throat culture, normal ranges of ASO and C reactive protein (CRP) levels. All serum samples were analyzed collectively by nephelometric method. The upper limit of normal value for anti-DNase B has been defined by separating the upper 20% from the lower 80% of all measurements.

RESULTS

Anti-DNase B antibody levels were ranged between 50-576 IU/ml and its upper limit was 219.2 IU/ml. When analyzed according to age groups, anti-DNase B antibody levels in the group aged between 5-10, ranged between 50-576 IU/ml and its upper limit was 212.2 IU/ml, anti-DNase B antibody levels in the group aged 10-15, ranged between 50-408 IU/ml and its upper limit was 231.2 IU/ml (p=0.008).

CONCLUSIONS

Based on our results, upper normal values ADB antibody showed variations with age in our results. Therefore national reference values should be detected by more comprehensive studies.

High-affinity antibodies to double-stranded DNA are a hallmark of systemic lupus erythematosus (SLE) and are thought to contribute to disease flares and tissue inflammation such as nephritis. Notwithstanding their clinical importance, major questions remain about the development and regulation of these pathogenic anti-DNA responses. These include the mechanisms that prevent anti-DNA responses in healthy subjects, despite the constant generation of self-DNA and the abundance of DNA-reactive B cells; the nature and physical form of antigenic DNA in SLE; the regulation of DNA availability as an antigen; and potential therapeutic strategies targeting the pathogenic DNA in SLE. This review summarizes current progress in these directions, focusing on the role of secreted DNases in the regulation of antigenic extracellular DNA.

Lupus nephritis can be managed successfully in the majority of cases; most therapies, however, are associated with significant side-effects. Several new agents aiming at specific stages in the pathogenesis of lupus are in different phases of clinical trials. The central role of lymphocytes makes them targets of various therapeutic approaches. Lymphocyte depletion can be achieved by high-dose chemotherapy with or without bone marrow transplantation. Nucleoside analogs selectively deplete mononuclear cells; antibodies against T or B cell surface antigens target specific subsets of lymphocytes. Synchronized plasmapheresis has been used in an attempt to delete pathogenic lymphocyte clones activated by plasmapheresis. Treating patients with DNase or neutralizing pathogenic antibodies by administering specific binding peptides or inducing specific anti-idiotype antibodies may prevent immune complex formation and/or deposition. Blocking the complement cascade or some of the inflammatory mediators like thromboxane A2 may be efficacious even if immune complex deposition could not be prevented. Inducing antigen-specific tolerance or interfering with important interactions between T-lymphocytes and other cells by blocking CD40 ligand or decreasing the level of interleukin-10 are some of the other approaches currently under clinical investigation.

We studied a 14 year-old boy with partial DiGeorge syndrome (DGS), status post complete repair of Tetralogy of Fallot, who developed antiphospholipid syndrome (APS) and type III mixed cryoglobulinemia. He presented with recurrent fever and dyspnea upon exertion secondary to right pulmonary embolus on chest computed tomography (CT). Coagulation studies revealed homozygous methylene tetrahydrofolate reductase 677TT mutations, elevated cardiolipin IgM antibodies, and elevated beta(2)-glycoprotein I IgM antibodies. Infectious work-up revealed only positive anti-streptolysin O (ASO) and anti-DNAse B titers. Autoimmune studies showed strongly positive anti-platelet IgM, elevated rheumatoid factor (RF), and positive cryocrit. Renal biopsy for evaluation of proteinuria and hematuria showed diffuse proliferative glomerulonephritis (DPGN) with membranoproliferative features consistent with cryoglobulinemia. Immunofixation showed polyclonal bands. Our patient was treated successfully with antibiotics, prednisone, and mycophenolate mofetil (MMF). This is the first report of a patient with partial DGS presenting with APS and type III mixed cryoglobulinemia possibly due to Streptococcal infection.

Narcolepsy-cataplexy is an uncommon sleep disorder which may present in childhood. We report a case of an 8-year-old presenting with narcolepsy-cataplexy following a streptococcal infection. Autoimmune etiology for narcolepsy has been suggested. In our patient increased anti-streptolysin O and anti-DNAse B titers were noted. As suggested by recent cases, the streptococcal infection was likely a trigger for narcolepsy onset in this genetically predisposed child. The patient was initially diagnosed as having Sydenham chorea due to motor movements. However, these transient movements may be due to the narcolepsy onset. Narcolepsy in childhood may present with atypical symptoms; it might be difficult to obtain accurate history and can be misdiagnosed as in the reported case. A high index of clinical suspicion is needed to diagnose these patients.

The o<em>b</em>ligatory intracellular pathogen <i>Ehrlichia chaffeensis</i> lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of <i>Ehrlichia</i> surface invasin, <u>e</u>ntry-<u>t</u>riggering <u>p</u>rotein of <i><u>E</u>hrlichia</i> (EtpE; EtpE-C) directly <em>b</em>inds mammalian <em>DNase</em> X, a glycosylphosphatidylinositol-anchored cell surface receptor and that <em>b</em>inding is required to induce <em>b</em>acterial entry and simultaneously to <em>b</em>lock the generation of reactive oxygen species (ROS) <em>b</em>y host monocytes and macrophages. However, how the EtpE-C-<em>DNase</em> X complex mediates the ROS <em>b</em>lockade was unknown. A mammalian transmem<em>b</em>rane glycoprotein CD147 (<em>b</em>asigin) <em>b</em>inds to the EtpE-<em>DNase</em> X complex and is required for <i>Ehrlichia</i> entry and infection of host cells. Here, we found that <em>b</em>one marrow-derived macrophages (BMDM) from myeloid cell lineage-selective CD147-null mice had significantly reduced <i>Ehrlichia</i>-induced or EtpE-C-induced <em>b</em>lockade of ROS generation in response to phor<em>b</em>ol myristate acetate. In BMDM from CD147-null mice, nucleofection with CD147 partially restored the <i>Ehrlichia</i>-mediated inhi<em>b</em>ition of ROS generation. Indeed, CD147-null mice as well as their BMDM were resistant to <i>Ehrlichia</i> infection. Moreover, in human monocytes, <em>anti</em>-CD147 partially a<em>b</em>rogated EtpE-C-induced <em>b</em>lockade of ROS generation. Both <i>Ehrlichia</i> and EtpE-C could <em>b</em>lock activation of the small GTPase Rac1 (which in turn activates phagocyte NADPH oxidase) and suppress activation of Vav1, a hematopoietic-specific Rho/Rac guanine nucleotide exchange factor <em>b</em>y phor<em>b</em>ol myristate acetate. Vav1 suppression <em>b</em>y <i>Ehrlichia</i> was CD147 dependent. <i>E. chaffeensis</i> is the first example of pathogens that <em>b</em>lock Rac1 activation to colonize macrophages. Furthermore, <i>Ehrlichia</i> uses EtpE to hijack the unique host <em>DNase</em> X-CD147-Vav1 signaling to <em>b</em>lock Rac1 activation.(<em>b</em>)IMPORTANCE</<em>b</em>)<i>Ehrlichia chaffeensis</i> is an o<em>b</em>ligatory intracellular <em>b</em>acterium with the capa<em>b</em>ility of causing an emerging infectious disease called human monocytic ehrlichiosis. <i>E. chaffeensis</i> preferentially infects monocytes and macrophages, professional phagocytes, equipped with an arsenal of <em>anti</em>micro<em>b</em>ial mechanisms, including rapid reactive oxygen species (ROS) generation upon encountering <em>b</em>acteria. As <i>Ehrlichia</i> isolated from host cells are readily killed upon exposure to ROS, <i>Ehrlichia</i> must have evolved a unique mechanism to safely enter phagocytes. We discovered that <em>b</em>inding of the <i>Ehrlichia</i> surface invasin to the host cell surface receptor not only triggers <i>Ehrlichia</i> entry <em>b</em>ut also <em>b</em>locks ROS generation <em>b</em>y the host cells <em>b</em>y mo<em>b</em>ilizing a novel intracellular signaling pathway. Knowledge of the mechanisms <em>b</em>y which ROS production is inhi<em>b</em>ited may lead to the development of therapeutics for ehrlichiosis as well as other ROS-related pathologies.

S1 proteins (A, B, C and D) are a group of nuclear proteins, isolated by lowering pH to 4.9 of the reaction supernatant of hepatocyte nuclei that had been mildly digested with DNase I. Protein B, apparently ubiquitous in vertebrate cells, was prepared from rat liver and used to immunize a rabbit. The raised antiserum specifically reacted with S1 proteins; it reacted not only with protein B, but also with C and D. Immunoblotting demonstrated that these proteins occurred exclusively in the nucleus, being absent in the cytosol, microsome and mitochondrial fractions. Indirect immunofluorescence of liver tissue sections confirmed their nuclear localization, and further showed that the antibody selectively stained extranucleolar regions of the cell nucleus. These findings suggest that the anti-S1 antibody is specific to S1 proteins and may be useful for their structural and functional studies.

Clinical isolates of group A, B, and G streptococci were compared in regard to their extracellular deoxyribonuclease (DNase) activities by the modified assay system for DNase using DNA-methyl green complex as a substrate. DNase activity in a culture supernatant of each strain was measured as a percent decrease in the substrate-specific absorbance at 640 nm. After 1 hr incubation, DNase activities in all the strains of group A streptococci were found to be markedly higher than those of any strain in the other groups, although the DNase activities in groups B and G were considerably heterogeneous. These findings were also seen when commercially available DNA-dye complex reagent for the determination of anti-DNase B antibody titer was used as a substrate in the assay system. Our results suggest that this newly established assay system could be applicable for the rapid measurement of DNase activity to distinguish group A streptococci from the other groups.

Specific anamnestic stimulation of spleen cells from mice immunized 7 days earlier with horse erythrocytes (HRBC) generated the release of a soluble factor that was capable of suppressing the initiation of the in vitro primary gammaM immune response to sheep red blood cells (SRBC), as well as to the immunogen that elicited its formation. Moreover, the suppressive macromolecule (mol. wt yields to 34,000), derived from antigen-activated, HRBC-primed T lymphocytes (but not B cells), inhibited the secondary gammaM and gammaG anti-SRBC plaque-forming cell responses of SRBC-primed spleen cells. The active material was resistant to treatment with DNase and RNase, but was inactivated by protease (10 microgram/ml, 30 min) or exposure to mild heat (56 degrees, 30 min). The antibody initiation suppressor factor (AISF) was concentrated and partially purified by gel filtration, followed by poly-acrylamide gel electrophoresis.