OBJECTIVE

Group A beta-hemolytic streptococci (GAS) are known to be capable of evoking sterile arthritis. Reactive arthritis (ReA) has been reported sporadically following primary infection with group C and group G beta-hemolytic streptococci (GCS, GGS). We prospectively studied 4 cases of ReA secondary to throat infection with GCS and GGS.

METHODS

Four patients with arthritis secondary to throat infection were seen. Three patients were Dutch, one was Indonesian; female/male ratio was 1/3; mean age was 30 years (range 18-46). Diagnostic evaluation included culture of throat swab and serological screening.

RESULTS

All patients presented with a nonmigratory asymmetrical arthritis: monoarthritis in one patient, oligoarthritis in 3. Culture of throat swab was positive in all. Antistreptolysin-O (ASO) titer rose significantly in 2 patients, and anti-DNase-B rose in 2 patients. ASO was maximal (mean 1000 U/ml; range 890-1110) and anti-DNase-B was 395 U/ml (range 290-500). Treatment consisted of feneticillin for 5 days; nonsteroidal antiinflammatory drugs were prescribed on demand. All patients recovered fully in 3 to 12 weeks.

CONCLUSIONS

These cases provide evidence of a benign non-group A streptococcal ReA, i.e., secondary to GCS or GGS. The presence of the organism in the throat along with the elevation of antibody to streptococcal products is important for the diagnosis of GCS/GGS associated ReA. A positive throat culture is needed for differentiation from GAS associated poststreptococcal ReA, because prophylactic measures are effective only in GAS associated sequelae, but not in GCS/GGS associated ReA.

LF was found to bind to deoxyribonucleic acid as assessed by immunofluorescence studies on cell nuclei, affinity chromatography of DNA on immobilized LF, and gel chromatography of an LF-DNA reaction mixture. LF immobilized on Sepharose 4-B was reacted with 125I-labeled DNA in both its double-stranded and single-stranded configurations; dsDNA eluted with a 0.69M NaCl buffer, whereas ssDNA eluted with a 0.25M NaCl buffer. Additional evidence for a preferential reactivity with dsDNA was provided by the enzymatic treatment of preformed dsDNA-LF and ssDNA-LF complexes with S1 endonuclease, and DNAse 1--DNase digestion alone liberated free LF. The interaction of LF with DNA partially inhibited the binding of anti-DNA antibodies from patients with SLE, as assayed in a standard Farr assay. Furthermore, DNA-anti-DNA (labeled with 125I-IgG) complexes could be dispersed in vitro by the addition of LF. It is hypothesized that the release of LF by neutrophils chemotactically attracted to DNA-anti-DNA complexes may act as a feedback loop to modulate the inflammatory response in SLE.

Familial Mediterranean fever (FMF) is an autosomal recessive disorder. Although the pathogenesis of the disease is not yet completely understood, enhanced acute-phase responsiveness is considered to be one of the most important mechanisms. The presence of high levels of antistreptolysin O (ASO) antibodies and streptococcus-associated diseases, such as acute poststreptococcal glomerulonephritis (AGN) and acute rheumatic fever (ARF), has been reported in patients with FMF. In order to better understand the effect of FMF on antistreptococcal antibody response, we measured ASO and antideoxyribonuclease B (anti-DNAse B) levels in patients with FMF and compared them with those in healthy controls. The study consisted of two parts. In the first step, antistreptococcal antibody levels were analysed in 44 patients with FMF and 165 healthy children who had no history or clinical evidence of upper respiratory tract infection (URTI) for the last 4 months. In the second step, antistreptococcal antibody levels were measured in 15 patients with FMF and 22 healthy controls in response to documented group A beta-haemolytic streptococcal pharyngitis. In the first part of the study, ASO and anti-DNAse B levels in patients with FMF were found to be significantly higher than those in healthy controls (P<0.001). In the second part, ASO and anti-DNAse B titres were found to be significantly higher in patients with FMF than in controls (P<0.001 and <0.05, respectively) 4 weeks after a positive throat culture. We concluded that patients with FMF have an exaggerated response to streptococcal antigens and might be prone to poststreptococcal non-suppurative complications, such as ARF.

The antibody response to the group A carbohydrate moiety of the streptococcal cell wall is of special interest because of its postulated role in the pathogenesis of rheumatic valvulitis. The immune response to this somatic antigen was measured in 159 children with culture-proved group A streptococcal pharyngitis and was compared with that to two extracellular antigens of the Group A streptococcus: streptolysin O and streptococcal DNase B. The data suggest that the maximum anti-A-carbohydrate rise occurs soon after the onset of streptococcal pharyngitis in a fashion similar to the response to some streptococcal extracellular antigens. However, the anti-A-carbohydrate antibody response appeared to be a less sensitive indicator of streptococcal upper respiratory tract infection.

Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgGs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.

Using a serum from a patient with an autoimmune disease, we have recently described a novel 55 000-dalton antigen (p55) in the nucleus of several animal cells including human ones. This antigen, designated PSL, was not related to the previously defined antigens recognized by sera from patients with systemic rheumatic diseases (Sm, n-RNP, SS-B, Scl-70). We have now found that p55 is associated with chromatin structures as it is released from the nucleus of mink cell fibroblasts by saline + DNase treatments. Analysis by sucrose gradient centrifugation of the nuclear material released in these conditions indicated that p55 co-migrated with core histones. Meanwhile, p55 was absent from the residual nuclear matrices (achromatinic nuclei). Localization of p55 in synchronized cells was performed by indirect immunofluorescence and immunoprecipitation. P55 appeared to accumulate in the nucleus during the S phase. Finally, it was not recognized by an anti-SV40 tumor serum that specifically precipitated the protein p53, which has been recently related to cell proliferation. Thus, PSL an p53, although apparently not antigenically related, appear to be implicated in the same step of the cell cycle.

The inhibitor of caspase-3-activated DNase (ICAD) is a caspase-3 substrate that controls nuclear apoptosis. ICAD has two isoforms: a functional isoform of M(r) 45,000, ICAD-L/DNA fragmentation factor (DFF) 45; and a M(r) 35,000 isoform, ICAD-S/DFF35. ICAD-deficient murine cells display resistance to apoptotic stimuli and absence of typical nuclear changes of apoptosis. Our aim was to: (a) characterize the ICAD expression in several human colonic cancer cell lines compared with human normal colonocytes; and (b) correlate the phenotypic features of apoptosis to the level of ICAD expression. ICAD expression was assessed by immunoblot analysis. Early markers of apoptosis of cultured cells included lactate dehydrogenase retention in dying cells, cytokeratin 18 cleavage, and caspase-3 activation. Nuclear markers of apoptosis were assessed by Hoechst staining of nuclei, electron microscopy, and DNA electrophoresis. Inhibition of caspases was performed using a broad-spectrum caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone. ICAD expression was restricted to the functional ICAD-L/DFF45 isoform in colonic cancer cells as well as in human normal colonocytes. In a clonal derivative of HT29 cells (HT29-Cl.16E cells), ICAD expression was found to be down-regulated during the exponential phase of growth, and the cell death triggered by IFN-gamma, anti-Fas antibody plus Adriamycin was characterized by the expression of early markers of apoptosis, whereas the key nuclear features of apoptosis were absent. In contrast, exposure of confluent cells to this treatment led to a typical apoptotic nuclear fragmentation. Both forms of apoptosis, in exponentially growing and confluent cells, were sensitive to the broad spectrum inhibitor of caspases, z-Val-Ala-Asp-fluoromethyl ketone. Our findings support the concept that the expression of ICAD is essential to the execution of full-blown apoptosis in colonic cancer cells. Altogether, our results point to ICAD as a potential target for restoring a normal apoptotic signal transduction pathway in colonic cancer cells.

OBJECTIVE

Mesenchymal stem cells (MSCs) isolated from healthy dental tissues are being investigated as an alternative source of MSCs for the treatment of damaged tissues and inflammatory diseases. Here we investigated whether MSCs from periapical lesions (PL-MSCs) also possess multi-lineage differentiation capacity and immunomodulatory properties.

METHODS

PL-MSCs, isolated by collagenase/DNAse digestion from surgically extracted PLs, were compared with MSCs from non-inflamed dental pulp (DP-MSCs) and dental follicle (DF-MSCs) for their phenotype and multi-potent differentiation potential. The anti-inflammatory and immunomodulatory effects of PL-MSCs were studied in co-culture with peripheral blood mononuclear cells (PB-MNCs) and PL-inflammatory cells (PL-ICs).

RESULTS

PL-MSCs were characterized by typical MSCs phenotype, lower clonogenicity and self-renewal rate, compared to DF-MSCs and DP-MSCs. These cells possess the potential to differentiate into adipocyte-, osteoblast- and chondrocyte-like cells in vitro, which differs from that of DP-MSCs and DF-MSCs. PL-MSCs inhibited phytohemaglutinine-induced proliferation of PB-MNCs and production of IL-2, IFNγ and IL-5 in the co-culture, probably via TGF-β-dependent mechanisms. These cells also suppressed the production of IL-1β, IL-6, and TNF-α by PL-ICs via soluble mediators, whereas the suppression of IL-8 production required a direct cell-to-cell contact.

CONCLUSIONS

The differentiation potential of PL-MSCs and their immunosuppressive/anti-inflammatory properties could be beneficial for the treatment of chronic periodontal diseases.

Staphylococcus aureus is a major causative agent for biofilm-associated infections. Inside biofilms, S. aureus cells are embedded in an extracellular matrix (ECM) composed of polysaccharide-intercellular adhesins (PIA), proteins, and/or extracellular DNA (eDNA). However, the importance of each component and the relationship among them in biofilms of diverse strains are largely unclear. Here, we characterised biofilms formed by 47 S. aureus clinical isolates. In most (42/47) of the strains, biofilm formation was augmented by glucose supplementation. Sodium chloride (NaCl)-triggered biofilm formation was more prevalent in methicillin-sensitive S. aureus (15/24) than in methicillin-resistant strain (1/23). DNase I most effectively inhibited and disrupted massive biofilms, and Proteinase K was also effective. Anti-biofilm effects of Dispersin B, which cleaves PIA, were restricted to PIA-dependent biofilms formed by specific strains and showed significant negative correlations with those of Proteinase K, suggesting independent roles of PIA and proteins in each biofilm. ECM profiling demonstrated that eDNA was present in all strains, although its level differed among strains and culture conditions. These results indicate that eDNA is the most common component in S. aureus biofilms, whereas PIA is important for a small number of isolates. Therefore, eDNA can be a primary target for developing eradication strategies against S. aureus biofilms.

BACKGROUND

Lytic reactivation of EBV has been reported to play an important role in human diseases, including NPC carcinogenesis. Inhibition of EBV reactivation is considered to be of great benefit in the treatment of virus-associated diseases. For this purpose, we screened for inhibitory compounds and found that apigenin, a flavonoid, seemed to have the ability to inhibit EBV reactivation.

METHODS

We performed western blotting, immunofluorescence and luciferase analyses to determine whether apigenin has anti-EBV activity.

RESULTS

Apigenin inhibited expression of the EBV lytic proteins, Zta, Rta, EAD and DNase in epithelial and B cells. It also reduced the number of EBV-reactivating cells detectable by immunofluorescence analysis. In addition, apigenin has been found to reduce dramatically the production of EBV virions. Luciferase reporter analysis was performed to determine the mechanism by which apigenin inhibits EBV reactivation: apigenin suppressed the activity of the immediate-early (IE) gene Zta and Rta promoters, suggesting it can block initiation of the EBV lytic cycle.

CONCLUSIONS

Taken together, apigenin inhibits EBV reactivation by suppressing the promoter activities of two viral IE genes, suggesting apigenin is a potential dietary compound for prevention of EBV reactivation.

Molecular studies have cast light on new facts about the virulence factors of bacteria responsible for implant infections. Recently, the biofilm matrix has been shown to include a variety of important structural components, including DNA, in addition to polysaccharides and proteins. This finding has stimulated interest in substances able to disrupt biofilms by attacking both the poly-N-acetylglucosamine surface polysaccharide (PNAG) and the extracellular DNA (eDNA), namely, dispersin B, a PNAG-degrading enzyme, and DNase I. The therapeutic potential of these enzymes are reviewed in this issue of IJAO, as well as the ability of the excretions/secretions of the medicinal maggot Lucilia sericata to disrupt Staphylococcus epidermidis biofilms. The activity of bacterial proteases causes the release of eDNA, critical for the early development of biofilms. This complex process, including suicidal and fratricidal mechanisms, is also presented in the current issue of IJAO. The different sensitivities of S. aureus and S. epidermidis to enzymatic anti-biofilm agents and to the host's first line of defense, as well as the importance of knowing the cascade of regulatory genes in bacteria so as to interfere with biofilm production using gene therapy or quorum sensing inhibitors are also discussed in this issue. Other innovative approaches consist in disrupting biofilms by exposing them to photodynamic substances and simultaneously to visible light, and in coating biomaterial surfaces with organic molecules to prevent protein adsorption and biofilm formation.

The rat D2 dopamine receptor gene is transcribed from a TATA-less promoter that has an initiator-like sequence and several putative Sp1 binding sites. The main activator of this gene is between nucleotides -75 and -29, and a strong negative modulator is located between bases -217 and -76 (Minowa, T., Minowa, M. T., and Mouradian, M. M. (1992) <em>B</em>iochemistry 31, 8389-8396). In the present investigation, a small deletion series within this negative modulator fused with the reporter gene for chloramphenicol acetyltransferase was used to transfect the D2-expressing cells, N<em>B</em>41A3. Two cis-acting functional DNA sequences were identified: a 41-base pair segment between nucleotides -116 and -76 (D2Neg-<em>B</em>), which decreased transcription from the D2 promoter by about 45%, and a 26-base pair segment between nucleotides -160 and -135 (D2Neg-A), which, in the presence of the downstream negative modulator, reduced transcription down to the level of a promoterless vector. <em>DNase</em> I footprinting, gel mobility shift, and competitive cotransfection experiments suggested that D2Neg-A functions without trans-acting factors, whereas D2Neg-<em>B</em> interacts with nuclear factors at its Sp1 binding sequences. Gel supershift with <em>anti</em>-Sp1 <em>anti</em>body and UV cross-linking experiments revealed that a novel 130-kDa factor as well as Sp1 interact with D2Neg-<em>B</em> in N<em>B</em>41A3 cells. This novel protein recognizing Sp1 binding sequences in the D2 gene negative modulator is also found in nuclear extract from the rat striatum.

OBJECTIVE

To assess the safety of discontinuing prophylaxis with antimicrobial agents in patients judged to be at relatively low risk for recurrence of acute rheumatic fever.

METHODS

Observational cohort study.

METHODS

Public health clinics in the Southeast Health District of Santiago, Chile.

METHODS

Fifty-nine patients (19 men, 40 women) ranging in age at study entry from 15 to 44 years (mean, 24.5 years). Forty-eight had completed their prescribed period of prophylaxis. Eleven refused or were allergic to intramuscular benzathine penicillin G and were non-compliant with oral sulfadiazine.

METHODS

In patients who did not have carditis during their previous attack(s), prophylaxis was discontinued after 5 years or at age 18, whichever was longer. In those with only mild mitral regurgitation or healed carditis, prophylaxis was stopped after 10 years or at age 25. Symptomatic intercurrent streptococcal throat infections were treated with antibiotics.

METHODS

Patients were seen every 3 months during the study (July 1982 to September 1988). For the first 4.25 years, throat cultures as well as sera samples for antistreptolysin O and anti-DNAse B assays were obtained at each visit.

RESULTS

During laboratory surveillance, significant increases in antibody titers were detected in 56 instances (28.1 [95% CI, 21.7 to 36.5] per 100 patient-years), and 29 isolations of group A streptococci occurred (14.5 [CI, 10.1 to 20.8] per 100 patient-years). The patients were followed for a total of 3349 patient-months, during which time two acute rheumatic fever recurrences were observed (0.7 [CI, 0.2 to 2.6] per 100 patient-years). No recurrences occurred during an outbreak of acute rheumatic fever in 52 patients in the study area in 1986.

CONCLUSIONS

These and other data indicate that acute rheumatic fever prophylaxis can safely be discontinued in young adults judged to be at low risk for recurrence and who are maintained under careful prospective surveillance.

OBJECTIVE

Caspase-activated DNase (CAD) is an endonuclease that is activated by active caspase 3 during apoptosis and is responsible for degradation of chromatin into nucleosomal units. These nucleosomal units are then included in apoptotic bodies. The presence of apoptotic bodies is considered important for the generation of autoantigen in autoimmune diseases, such as systemic lupus erythematosus (SLE), that are characterized by the presence of antinuclear antibodies. The present study was carried out to determine the role of CAD in SLE and to investigate the ability of lupus autoantibodies to bind to CAD-deficient or CAD-sufficient apoptotic cells.

METHODS

The Sle1, Sle123, and 3H9 mouse models of SLE, in which autoimmunity is genetically predetermined, were used. To determine the role of chromatin fragmentation in SLE, CAD deficiency was introduced in these mouse models.

RESULTS

Deficiency of CAD resulted in increased anti-double-stranded DNA antibody titers in lupus-prone mice. Surprisingly, the absence of CAD exacerbated only genetically predetermined autoimmune responses. To further determine whether nuclear modifications are needed in order to maintain tolerance to nuclear autoantigens, we used the 3H9 mouse, an anti-DNA heavy chain knockin; in this model, the autoreactive B cells are tolerized by anergy. In accordance with findings in the CAD-mutant Sle1 and Sle123 mice, CAD-deficient 3H9 mice spontaneously generated anti-DNA antibodies. Finally, we showed that autoantibodies with specificities toward histone-DNA complexes bind more to CAD-deficient apoptotic cells than to CAD-sufficient apoptotic cells.

CONCLUSIONS

We propose that in mice that are genetically predisposed to lupus development, nuclear apoptotic modifications are needed to maintain tolerance. In the absence of these modifications, apoptotic chromatin is abnormally exposed, facilitating the autoimmune response.

OBJECTIVE

The purpose of this work was to assess the capacity of tumor-infiltrating leukocytes (TIL) from human non-small-cell lung carcinoma (NSCLC) specimens to synthesize type-1 and type-2 cytokines.

METHODS

TIL were isolated from tumors following digestion with collagenase/DNase and further enriched by ficoll-hypaque gradient centrifugation. Membrane phenotypes and intracellular cytokine protein expression of TIL were assessed by flow cytometry.

RESULTS

The majority of TIL expressed the CD3 antigen with a CD4:CD8 ratio of approximately 2:1. Other leukocytes such as macrophages (CD14), B lymphocytes (CD20), and natural killer (NK) cells (CD56) were also found to infiltrate the tumors, but in significantly lower numbers. Owing to the limited recovery of non-CD3(+) leukocytes, our analysis of cytokine biosynthesis has focused on T lymphocytes. In the absence of activation, a small percentage of CD3(+) TIL synthesized cytokines ( <4%). Following activation with anti-CD3+interleukin-2 (IL-2), CD3(+) TIL synthesized predominantly a type-1 cytokine profile; however, the type-2 cytokines, IL-6 and IL-10, were also detected in a small percentage of infiltrating cells. Following activation with phorbol 12-myristate 13-acetate + ionomycin, CD3(+) TIL also expressed more type-1 than type-2 cytokines and in significantly greater numbers of cells. The CD3(+)CD8(+) component of the TIL synthesized only type-1 cytokines, whereas the CD3(+)CD4(+) component synthesized both type-1 and type-2 cytokines.

CONCLUSIONS

These results show that the majority of the TIL isolated from NSCLC specimens are T lymphocytes with the capacity to synthesize type-1 cytokines.

An ELISA method for determining the streptococcal anti--DNase B titers in human sera is presented. The details of this technique, a method for converting A493 readings into titers, and a standardization procedure are discussed. Anti--DNase B titers of 20 sera determined by ELISA were compared with titers determined by the microtechnique. A correlation coefficient of 0.96 between the two methods was obtained. The reproducibility of the ELISA method was established by comparing titers obtained from two separate determinations on the 20 sera. Twenty-four pairs of acute and convalescent sera were assayed for anti--DNase B titers by both ELISA and microtechnique to compare the ability of the two techniques to identify significant titer rises. The ELISA was as specific and possibly more sensitive than the microtechnique in identifying significant anti--DNase B titer rises. This ELISA method is simple and rapid and has the potential for automation.

Neutrophil extracellular traps (NETs) are the main source of autoantigens in systemic lupus erythematosus (SLE). The aim of this study was to evaluate the clinical importance of NETs-associated markers in SLE. We compared NETs-associated markers in SLE patients (n = 111) with healthy controls (n = 50). Moreover, in 35 patients with drug-naïve SLE (n = 35), we investigated correlation between NETs-associated markers [DNase I concentration, myeloperoxidase (MPO) activity, anti-MPO antibodies, cell-free DNA (cfDNA), NETolytic activity] with serological parameters [anti-dsDNA antibodies, C3, C4 and B-cell activating factor (BAFF) levels] and disease activity measured by modified SLE Disease Activity Index (M-SLEDAI-2K). In comparison with healthy controls, SLE patients had higher cfDNA, MPO activity, anti-MPO antibodies (p < 0.001), BAFF and DNase I concentration (p < 0.01). Contrary, NETolytic activity was lower in SLE patients (p < 0.05), despite higher concentration of DNase I. MPO activity and cfDNA levels showed correlation with DNase I concentration (p < 0.001, p < 0.01, respectively). BAFF levels correlated with cfDNA, DNase I concentration and MPO activity (p < 0.05). Anti-dsDNA antibodies showed correlation with MPO activity (p < 0.01), cfDNA and BAFF levels (p < 0.001). Anti-dsDNA and C3 levels were independent predictors of M-SLEDAI-2K in multivariate analysis (p < 0.01). We demonstrated that sera of SLE patients have decreased NETolytic activity, leading to increased levels of various NETs-associated markers, which correlate with anti-dsDNA antibodies in drug-naïve SLE. We showed that BAFF participates in a complex relationship between NETosis and anti-dsDNA antibodies production. These findings have important implications for a better understanding of SLE pathogenesis and development of therapy that inhibits NETs persistence and disease progression.

The present study was undertaken to determine the upper 95% confidence limits (CL) for titres of antibodies to streptolysin O (ASO) and streptococcal DNase B (ADNB) using 741 sera from a general population. The ASO titres in sera from children increased abruptly with increasing age, from a mean of 21 (CL 67) in infants of 3 years or less to 211 (CL 462) in children 7 to 8 years old. In children 9 to 12 years of age titres exhibited a plateau with mean values of 168-258 (CL 436-611). In individuals over 20 years the mean ASO titre diminished with increasing age, individuals of 70 years or more showing a mean titre of 83 (CL 169). A similar variation was seen for ADNB. Infants 3 years of age or younger exhibited no detectable ADNB whereas individuals 70 years or more had a mean titre of 11 (CL 91). A maximal mean ADNB titre of 184 (CL 706) was detected in 9-year-old-children. It was concluded that age-related reference values for anti-streptococcal antibodies increase the clinical relevance of the tests.

<p><div>(<em>b</em>)BACKGROUND</<em>b</em>)</div>Air pollution, including particulates and gazes such as ozone (O<su<em>b</em>)3</su<em>b</em>)), is detrimental for patient's health and has repeatedly <em>b</em>een correlated to increased mor<em>b</em>idity and mortality in industrialised countries. Although studies have descri<em>b</em>ed a link <em>b</em>etween am<em>b</em>ient particulate matter and increased lung cancer mor<em>b</em>idity, no direct relation has yet <em>b</em>een esta<em>b</em>lished <em>b</em>etween O<su<em>b</em>)3</su<em>b</em>) exposure and metastatic dissemination to lungs.</p><p><div>(<em>b</em>)OBJECTIVES</<em>b</em>)</div>To outline the mechanisms through which pulmonary O<su<em>b</em>)3</su<em>b</em>) exposure modulates metastasis kinetics in an experimental mouse model of O<su<em>b</em>)3</su<em>b</em>) exposure.</p><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>Metastatic responses to pulmonary O<su<em>b</em>)3</su<em>b</em>) exposure were assessed using a relia<em>b</em>le experimental mouse model of concomitant pulmonary O<su<em>b</em>)3</su<em>b</em>) exposure and tumour cell injection. Roles of neutrophils in O<su<em>b</em>)3</su<em>b</em>)-induced lung metastasis were highlighted using <em>b</em>locking <em>anti</em>-Ly6G <em>anti</em><em>b</em>odies; moreover, the implication of neutrophil extracellular traps (NETs) in metastatic processes was evaluated using <i>MRP8cre-Pad4lox/lox</i> mice or <em>b</em>y treating mice with <em>DNase</em> I.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Pulmonary O<su<em>b</em>)3</su<em>b</em>) exposure strongly facilitates the esta<em>b</em>lishment of lung metastasis <em>b</em>y (1) Inducing a pulmonary injury and neutrophilic inflammation, (2) Influencing very early steps of metastasis, (3) Priming neutrophils' phenotype to release NETs that favour tumour cell colonisation in lungs. The a<em>b</em>ility of O<su<em>b</em>)3</su<em>b</em>)-primed neutrophils to enhance lung colonisation <em>b</em>y tumour cells was proven after their adoptive transfer in Bal<em>b</em>/c mice unexposed to O<su<em>b</em>)3</su<em>b</em>).</p><p><div>(<em>b</em>)CONCLUSIONS</<em>b</em>)</div>Pulmonary neutrophils induced <em>b</em>y O<su<em>b</em>)3</su<em>b</em>) promote metastatic dissemination to lungs <em>b</em>y producing NETs. These findings open new perspectives to improve treatment and prevention strategies in patients affected <em>b</em>y metastatic diseases.</p>

To determine the regulatory mechanism of the expression of the mouse platelet-derived growth factor (PDGF) beta-receptor gene, a 1.9-kb 5' flanking genomic fragment was cloned and analyzed. Site-directed mutagenesis of a CCAAT motif, located 60 bp upstream of the transcriptional-start site, completely abolished the promoter activity [Ballagi, A. E., Ishisaki, A., Nelin, J.-O. & Funa, K. (1995) Biochem. Biophys. Res. Commun. 210, 165-1751. The sequence around the intact CCAAT motif was protected by in vitro DNase-I-footprinting analysis. Electrophoresis-mobility-shift assays with anti-[nuclear factor Y(NF-Y)]Ig revealed binding of the NF-Y complex to the CCAAT box. Furthermore, the double-stranded oligonucleotides corresponding to the sequence around the CCAAT motif were conjugated with DNA-affinity magnetic beads. The binding proteins were affinity purified and identified as the NF-Y transcription factor by western blotting. Our results indicate that NF-Y controls the basal transcription activity of the mouse PDGF beta-receptor gene.

Immunofluorescent methods using a monoclonal antibody against chick DNA polymerase alpha and a rabbit antibody against chick DNA polymerase beta demonstrated that both DNA polymerases alpha and beta are present mainly in nuclei of cultured chick embryo cells. Fluorescence produced by anti-DNA polymerase alpha was more intense in the small granules than in other parts of the nucleus but, fluorescence produced by anti-DNA polymerase beta was distributed evenly in the nucleus. Cells first were treated with Nonidet P-40, followed by treatment with 50 micrograms/ml pancreatic DNase and 2 M NaCl in order to prepare the nuclear matrix. Fluorescence produced by anti-DNA polymerase alpha was still detectable in the granules after these treatments, but most of the fluorescence produced by anti-DNA polymerase beta disappeared. Our results indicate that a part of DNA polymerase alpha is tightly bound to a special structure present in the nuclear matrix which presumably is the DNA replication machinery.

In previous studies a subset of complement-component-C3 (C3) epitopes, C3(D), expressed in denatured and surface-bound C3 and C3 fragments, has been described. These epitopes were detected by antibodies raised against denatured C3. In the present study we used a cDNA expression strategy to localize epitopes recognized by monoclonal and polyclonal anti-C3(D) antibodies. First, DNAse I digestion of C3 cDNA was used to generate 200-300 bp fragments. These cDNA fragments were expressed as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector. The fusion proteins were tested by Western-blot analysis for reactivity with monoclonal and polyclonal anti-C3 antibodies, and the location of the epitopes were determined by sequencing the cDNA fragments. Affinity-purified polyclonal anti-C3(D) antibodies specific for denatured C3 reacted strongly with the C3 fusion fragments corresponding to segments of the 40 kDa subunit of C3c (residues 1477-1510) and the C3d fragment (residues 1117-1155 and 1234-1294) of C3. Adsorption of the polyclonal antibodies with a mixture of EAC3b and EAC3bi (degradation fragments of C3 bound to sheep erythrocytes) abolished binding to fusion proteins spanning the C3d region, but not the 40 kDa fragment of C3c. No effect was seen with the corresponding soluble C3 fragments. The monoclonal anti-C3(D) antibodies (mAbs) 7D326.1 and 7D331.1, specific for EAC3b and EAC3bi, bound to a fusion protein corresponding to amino acid residues 1312-1404, whereas mAb 7D9.2, specific for EAC3d, reacted with a fusion protein spanning amino acid residues 1082-1118. mAbs 4SD11.1 and 4SD18.1, which did not bind to any physiological C3 fragment, detected a fusion protein covering residues 1477-1510. In summary, the segments of C3 represented by amino acid residues 1082-1118, 1117-1155, 1234-1294 and 1312-1404 accommodate C3(D) epitopes that are expressed by erythrocyte-bound C3 fragments, but not by the corresponding fluid-phase fragment, whereas the segments spanning residues 973-1026 and 1477-1510 contain C3(D) epitopes that are exposed exclusively in denatured C3 and therefore hidden in physiological fragments of the protein.

The DNase of Epstein-Barr virus (EBV) is a 470-amino-acid protein which possesses both endonuclease and exonuclease activities and accepts both double-stranded DNA and single-stranded DNA as substrates. It has been reported that this protein may be found in the nucleus and/or cytoplasm of infected cells. In this study, using cell fractionation and immunoblotting to determine the distribution of EBV DNase in Akata cells stimulated with anti-human immunoglobulin G antibody (anti-IgG), the DNase was found to be located predominantly in the nucleus. To map the signals in DNase which mediate its nuclear localization, we monitored the nuclear transport of fusion proteins consisting of various fragments of EBV DNase linked to a cytoplasmic protein, beta-galactosidase (beta-Gal). The results demonstrated that two regions of the DNase with nuclear localization signal (NLS) activity, designated NLS-A (amino acids 239-266) and NLS-B (amino acids 291-306), were able independently to localize the beta-Gal to the nuclei of HEp-2 and HeLa cells. Five basic residues (R or K) were found in each NLS and distributed differently in primary structure. The basic domains and flanking residues of NLS-A and NLS-B are 250YKRPCKRSFIRFI262 and 294LKDVRKRKLGPGH306, respectively. Further examination of these sequences revealed that NLS-A contains bulky aromatic amino acids (Y and F) which may diminish its capacity to act as a strong NLS and lacks the typical proline and glycine helix-breakers. However, NLS-B contains typical proline and glycine helix-breakers and the histidine residue at amino acid 306 is required for NLS activity. In addition, two hydrophobic regions within the DNase were found to inhibit the function of NLS-A but not NLS-B, suggesting that these two domains are different types of NLSs and differ in their sensitivity to hydrophobic regions in the context of protein structure.

The pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) are basically characterized by obsessive-compulsive symptoms and/or tics triggered by group-A beta-hemolytic Streptococcus infections. Poor data are available about the clear definition of PANDAS's autoimmune origin. The aim of our study was to evaluate the prevalence of autoimmune phenomena, including thyroid function abnormalities, specific celiac disease antibodies, and positivity of organ- or nonorgan-specific autoantibodies in a large cohort of Caucasian children and adolescents with PANDAS. Seventy-seven consecutive patients (59 males, 18 females; mean age 6.3±2.5 years, range 2.0-14.5 years) strictly fulfilling the clinical criteria for PANDAS diagnosis were recruited. In all subjects we evaluated serum concentrations of free-T3, free-T4, thyrotropin, and the following auto-antibodies: anti-thyroperoxidase, anti-thyroglobulin, anti-thyrotropin receptor, anti-gliadin, anti-endomysium, anti-tissue transglutaminase, anti-nuclear, anti-smooth muscle, anti-extractable nuclear antigens, anti-phospholipid, plus lupus-like anticoagulant. The results were compared with those obtained from 197 age- and sex-matched healthy controls (130 males, 67 females; mean age 6.8±2.9 years, range 2.3-14.8 years). The frequencies of subclinical (3.8% vs 3.6%) and overt hypothyroidism (1.2% vs 0%), autoimmune thyroiditis (2.46% vs 1.14%), celiac disease (1.2% vs 0.05%), and positivity of organ- and nonorgan-specific autoantibodies (5.1% vs 4.8%) were not statistically significant between patients with PANDAS and controls. Evaluating the overall disease duration, we did not observe any significant difference between patients with (3.4±2.15 years) and without (3.4±2.89 years) autoimmune abnormalities. However, PANDAS patients with autoimmune diseases or positivity for any organ- and nonorgan-specific antibodies showed significantly higher anti-streptolysin O and anti-DNAse B titers, as well as a history of more frequent throat infections than controls (p<0.0001). Abnormalities of thyroid function and thyroid autoimmune diseases, as well as the association with celiac disease or organ- and nonorgan-specific autoimmunity seem not more frequent in children and adolescents with PANDAS than in healthy controls. A potential relationship between autoimmunity and PANDAS should be assessed further in larger studies. Children and adolescents with PANDAS should not be actually screened for thyroid function, celiac disease and/or autoimmune diseases.