LEI/L-DNase II is the key protein of a caspase-independent pathway activated by serine proteases. LEI (Leukocyte elastase inhibitor), L-DNase II precursor, is a member of the clade B serpins (also called serpin b1). In its native conformation it inhibits several intracellular proteases and has an anti-apoptotic activity. Following a metabolic stress and the increase of protease activity in the cell, LEI is cleaved and transformed into L-DNase II (LEI-derived DNase II). This transformation is due to a conformational modification that exposes a nuclear localization signal and an endonuclease active site. In this paper we show that LEI can bind the exportin Crm1, and we identify on LEI a nuclear export signal involved in the control of LEI/L-DNase II nuclearization in healthy cells. Point mutation of this site increases the accumulation of the molecule in the nucleus and triggers cell death.

ClC-5 is a chloride/proton exchanger that plays an obligate role in albumin uptake by the renal proximal tubule. ClC-5 forms an endocytic complex with the albumin receptor megalin/cubilin. We have identified a novel ClC-5 binding partner, cytosolic aspartyl aminopeptidase (DNPEP; EC 3.4.11.21), that catalyzes the release of N-terminal aspartate/glutamate residues. The physiological role of DNPEP remains largely unresolved. Mass spectrometric analysis of proteins binding to the glutathione-S-transferase (GST)-ClC-5 C terminus identified DNPEP as an interacting partner. Coimmunoprecipitation confirmed that DNPEP and ClC-5 also associated in cells. Further experiments using purified GST-ClC-5 and His-DNPEP proteins demonstrated that the two proteins bound directly to each other. In opossum kidney (OK) cells, confocal immunofluorescence studies revealed that DNPEP colocalized with albumin-containing endocytic vesicles. Overexpression of wild-type DNPEP increased cell-surface levels of ClC-5 and albumin uptake. Analysis of DNPEP-immunoprecipitated products from rat kidney lysate identified β-actin and tubulin, suggesting a role for DNPEP in cytoskeletal maintenance. A DNase I inhibition assay showed a significant decrease in the amount of G actin when DNPEP was overexpressed in OK cells, suggesting a role for DNPEP in stabilizing the cytoskeleton. DNPEP was not present in the urine of healthy rats; however, it was readily detected in the urine in rat models of mild and heavy proteinuria (diabetic nephropathy and anti-glomerular basement membrane disease, respectively). Urinary levels of DNPEP were found to correlate with the severity of proteinuria. Therefore, we have identified another key molecular component of the albumin endocytic machinery in the renal proximal tubule and describe a new role for DNPEP in stabilizing the actin cytoskeleton.

The proximal mouse IL-5 promoter was examined using a mouse TH2 clone stimulated through the T cell receptor using anti-CD3 monoclonal antibody. DNase I protection defined four protein binding regions [IL-5RE-A, -69/-45; -B, (-90/-76); -C, (-154/-130); and -D (-176/-157)]. Stimulation-dependent binding, which was seen in the IL-5RE-B, -D regions and the 5' end of tIL-5RE-A, did not require new protein synthesis inhibitor during cell activation. EMSA using probes targeted to the IL-5RE-B, -C, -D regions demonstrated the multimeric nature of the bound proteins. By transfection analysis using a series of truncated IL-5 promoter-luciferase constructs, IL-5RE-C and -D contributed little to constitutive or inducible activity. The CLE0 site in the IL-5RE-A region contributed to full transcriptional activity but was not sufficient to mediate full activity. Full stimulation-dependent activity required the IL-5RE-B region and/or the GATA site (-70/-60).

OBJECTIVE

To evaluate the feasibility of using magnetic iron oxide (Fe(3)O(4))-dextran-anti-β-human chorionic gonadotropin (HCG) nanoparticles as a gene vector for cellular transfections.

METHODS

Fe(3)O(4)-dextran-anti-β-HCG nanoparticles were synthesized by chemical coprecipitation. The configuration, diameter, and iron content of the nanoparticles were detected by transmission electron microscopy (TEM), light scatter, and atomic absorption spectrophotometry. A3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay was used to evaluate the cytotoxicity of Fe(3)O(4)-dextran-anti-β-HCG nanoparticles. Enzyme-linked immunosorbent assay and indirect immunofluorescence were used to evaluate immunoreactivity. The efficiency of absorbing DNA and resisting deoxyribonuclease I (DNase I) digestion when bound to Fe(3)O(4)-dextran-anti-β-HCG nanoparticles was examined by agarose gel electrophoresis. The ability of Fe(3)O(4)-dextran-anti-β-HCG nanoparticles to absorb heparanase antisense oligodeoxynucleotides (AS-ODN) nanoparticles in different cell lines was evaluated by flow cytometry. The tissue distribution of heparanase AS-ODN magnetic nanoparticles in choriocarcinoma tumors transplanted in nude mice was detected by atomic absorption spectrophotometry.

RESULTS

TEM demonstrated that the shape of nanoparticles is irregular. Light scatter revealed nanoparticles with a mean diameter of 75.5 nm and an iron content of 37.5 μg/mL. No cytotoxicity was observed when the concentration of Fe(3)O(4)-dextran-anti-β-HCG nanoparticles was <37.5 μg/mL. Fe(3)O(4)-dextran nanoparticles have a satisfactory potential to combine with β-HCG antibody. Agarose gel electrophoresis analysis of binding experiments showed that after treatment with sodium periodate, Fe(3)O(4)-dextran-anti-β-HCG nanoparticles have a satisfactory potential to absorb DNA, and the protection experiment showed that nanoparticles can effectively protect DNA from DNase I digestion. Aldehyde Fe(3)O(4)-dextran-anti-β-HCG nanoparticles can transfect reporter genes, and the transfection efficiency of these nanoparticles is greater than that of liposomes (P < 0.05). Fe(3)O(4)-dextran-anti-β-HCG nanoparticles can concentrate in choriocarcinoma cells and in transplanted choriocarcinoma tumors.

CONCLUSIONS

The results confirm that Fe(3)O(4)-dextran-anti-β-HCG nanoparticles have potential as a secure, effective, and choriocarcinoma-specific targeting gene vector.

We have previously reported that human placental cytotrophoblasts (C-cells) contain nuclear 3,5,3'-triiodo-L-thyronine (T3) receptors. Using a C-cell culture system, the present study was undertaken to clarify some of the effects of T3 and EGF on trophoblastic cells. C-cells were purified from human term placenta by treatment with trypsin-DNAse and percoll gradient centrifugation aggregated, then fused, differentiating into multinuclear syncytiotrophoblasts (S-cells) with incubation times up to 96 h in vitro. As the incubation time increased, the number of immunocytochemically reactive cells with antibodies to hCG-alpha, hCG-beta and hPL increased. Anti-EGF antibody reacted only with the initial C-cells, while anti-EGF receptor antibody reacted only with fused S-cells. Maximum secretion of hCG and hCG-alpha by the cultured cells was evident only when the cells were cultured in T3 (10(-8)M) or EGF (10 ng/ml) containing medium. When the initial cells were exposed to 10(-8) M T3 from 0 to 48 h of incubation, the secretion in 48-96 h was significantly accelerated. However, exposure from 48 to 96 h had no effect on peptide excretion. Although an exposure of these cells to 10 ng/ml EGF during 48-96 h of incubation stimulated the secretion of hCG and hCG-alpha, 0-48 h exposure did not produce any positive effect regardless of incubation time. These results indicated that the main target cell of T3 is the C-cell, while that of EGF is the S-cell. Furthermore, it is suggested that the interaction between T3 and its receptor facilitated functional cell differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Putrescine, spermidine, spermine and two unknowns designated as A and B were detected in first seedling leaves of barley (Hordeum vulgare L. var. Wolfe). The levels of these polyamines in first seedling leaves from 4-day-old barley plants grown in darkness or in light were comparable and did not change significantly after exposure of dark grown plants to light for 24 h. No significant consistent changes in the amounts of above polyamines, except perhaps decline in spermidine, were noted during senescence of intact or excised first seedling leaves of barley and this spermidine decline was suppressed during retardation of senescence of excised leaves by 10 mg/l kinetin in the dark. In addition, putrescine, spermidine, spermine, cadaverine and diaminopropane (0.2 mM, 1 mM, 10 mM) had no effect on senescence of excised barley leaves in the dark and both spermine and spermidine induced bleaching of the leaves in the light. Both spermine and spermidine (approx. 10 mM) inhibited RNase and DNase activities but stimulated phosphodiesterase activity (assayed with bis-p-nitrophenyl phosphate as substrate) in crude soluble extracts from barley leaves. Purified snake venom phosphodiesterase activity assayed with RNA as substrate was, however, stimulated by 300-400% by 7-14 mM spermine or spermidine indicating similar possibilities for barley phosphodiesterase. These results together with the presence of multiple species of these enzymes and a decline in net soluble RNase and DNase activities during senescence in barley leaves reported previously, make it unlikely that inhibition of RNase activity in vitro by polyamines could be correlated with their effect on senescence. Putrescine, spermidine and spermine were detected in normal and crown gall tumor tissue cultures of tobacco (Nicotiana tabacum var Wisconsin 38) and in tobacco mosaic virus (TMV)-infected freshly excised pith tissue from tobacco which represented non-proliferating tissue. The level of all three polyamines was several-fold higher in cultured tissues compared to the non-dividing freshly excised pith tissue and the tumor cultures had several-fold higher spermidine and putrescine respectively compared to normal tissue cultures. These results indicate high levels of polyamines in growing tissues but no consistent pivotal changes in polyamines during senescence. The results also do not support polyamines being natural anti-senescent compounds in plants or that their anti-senescent compounds effect could result from inhibition of RNase activity.

The open reading frames of the phosphoprotein pp58 (BMRFI) and the deoxyribonuclease (BGLF5) of the Epstein-Barr-virus (EBV) strain M-ABA were cloned in the baculovirus expression vectors pAc373 and pAc360 and expressed in the Spodoptera frugiperda (SF158) insect cells. The recombinant phosphoprotein pp58 expressed in SF158 cells was recognized by the anti-pp58 rabbit anti-sera which were generated by immunizing rabbits with a TrpE-BMRFI fusion protein expressed in E. coli. DNA-cellulose chromatography showed that the recombinant pp58 exhibited DNA-binding activities. Immunofluorescence, immunoblot and ELISA analysis indicated that sera from patients with nasopharyngeal carcinoma (NPC) contained antibodies against pp58. The recombinant EBV DNase expressed in SF158 cells was recognized by the anti-EBV DNase rabbit anti-sera which were generated by immunizing rabbits with a TrpE-C-terminal part of BGLF5 fusion protein expressed in E. coli. The anti-EBV DNase rabbit anti-sera recognized also a protein of about 52 kDa in the EBV-harboring human B-cell lines Raji, Jijoye, BBA and BL74 induced by TPA and n-butyrate. The recombinant EBV DNase exhibited exonuclease and endonuclease activities, a requirement for magnesium, and a high pH optimum (8.0). Its enzyme activities could be inhibited by sera from NPC patients and anti-EBV DNase rabbit anti-sera. Comparable studies of Raji EBV-DNase and recombinant EBV-DNase implied that recombinant EBV-DNase could also be used in the enzyme activity assay for the detection of NPC. In contrast to the enzyme inhibition test, immunofluorescence and immunoblot analysis demonstrated that the recombinant EBV DNase exhibited only a weak immunological reaction with NPC sera.

OBJECTIVE

To evaluate the clinical characteristics, complications and outcome of post-infectious glomerulonephritis (PIGN).

METHODS

This prospective observational study was conducted from January 2013 through July 2014 at a tertiary care hospital in south India. Post-streptococcal glomerulonephritis (PSGN) was diagnosed in the presence of: a) Hematuria and proteinuria b) Clinico-serological evidence of recent streptococcal infection [recent pyodermas or pharyngitis; positive antistreptolysin-O (ASO) titres, anti-DNAse B titres or throat swab positivity for Group A streptococcus], and c) Low serum C3 levels, with normalization on 8 wk follow up. PIGN included PSGN and other infectious etiologies. AKI was classified as per Acute Kidney Injury Network (AKIN) criteria. Clinical features, biochemical and serological investigations in the study subjects were recorded.

RESULTS

Among 83 children with acute nephritic syndrome (ANS) recruited, 72 (86.7 %) had PIGN. PSGN was the most common etiology [65(90.3 %)] among the PIGN cases. Pyodermas, upper respiratory infections and varicella preceded hematuria in 58 (80.6 %), 4 (5.6 %) and 2 (2.8 %) cases respectively. Pneumonia, mumps and liver abscess caused PIGN in 7 (9.7 %) cases. Complications included AKI in 15 (20.8 %), hypertensive emergency in 14 (19.4 %), cardiac failure in 8 (11.1 %), encephalopathy in 3 (4.2 %) cases and retinopathy in 1 (1.4 %) case. Among the AKI patients, 3(20 %) were in AKI stage 3, while 1 child required hemodialysis. Twenty three cases (31.9 %) had evidence of residual renal injury at discharge. Renal biopsy showed diffuse proliferative glomerulonephritis in 4 and crescentic glomerulonephritis in one case of PIGN. At 6 mo follow up, one patient continued to have microalbuminuria.

CONCLUSIONS

PIGN (including PSGN) remains a significant contributor to morbidity in children with ANS. The study is notable for high incidence of hypertensive emergency and AKI, that often required intensive care management.

P-815 mastocytoma cells from DBA/2 mice and a 3-methylcholanthrene-induced fibrosarcoma from C57BL/6 mice produced in culture at least two soluble anti-inflammatory factors that inhibited macrophage accumulation in vivo when the factors were injected sc into syngeneic recipients. One factor was a low-molecular-weight peptide (less than 1,000), as judged by ultrafiltration, failure of extraction by lipid solvents, nonsusceptibility to DNase or RNase, partial inactivation by trypsin, and complete inactivation by carboxypeptidase B. The second anti-inflammatory factor had a molecular weight between 30,000 and 100,000 and was also not extractable with lipid solvents. Production of anti-inflammatory factors by P-815 mastocytoma cells was inhibited by cycloheximide and cell irradiation but not by colchicine pretreatment of the cells, suggesting a relationship to protein synthesis rather than cell growth. Soluble anti-inflammatory factors depressed granulocyte as well as macrophage responses. Anti-inflammatory factors were not found in supernatants from cultures of splenocytes, peritoneal exudate cells, or murine lung fibroblasts.

Bacterial biofilms are one of the major issues in the treatment of chronic infections such as chronic wounds, where biofilms are typically polymicrobial. The synergy between species can occur during most polymicrobial infections, where antimicrobial resistance enhances as a result. Furthermore, self-produced extracellular polymeric substance (EPS) in biofilms results in a high tolerance to antibiotics that complicates wound healing. Since most antibiotics fail to remove biofilms in chronic infections, new therapeutic modalities may be required. Disruption of EPS is one of the effective approaches for biofilm eradication. Therefore, degradation of EPS using enzymes may result in improved chronic wounds healing. In the current study, we investigated the efficacy of trypsin, β-glucosidase, and DNase I enzymes on the degradation of dual-species biofilms of Pseudomonas aeruginosa and Staphylococcus aureus in a wound-like medium. These species are the two most common bacteria associated with biofilm formation in chronic wounds. Moreover, the reduction of minimum biofilm eradication concentration (MBEC) of meropenem and amikacin was evaluated when combined with enzymes. The minimum effective concentrations of trypsin, β-glucosidase, and DNase I enzymes to degrade biofilms were 1 μg/ml, 8 U/ml, and 150 U/ml, respectively. Combination of 0.15 μg/ml trypsin and 50 U/ml DNase I had a significant effect on S. aureus-P. aeruginosa biofilms which resulted in the dispersal and dissolution of all biofilms. In the presence of the enzymatic mixture, MBECs of antibiotics showed a significant decrease (p < 0.05), at least 2.5 fold. We found that trypsin/DNase I mixture can be used as an anti-biofilm agent against dual-species biofilms of S. aureus-P. aeruginosa.

Type III CRISPR-Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3' protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.

Self-reactive B cells generated through V(D)J recombination in the bone marrow or through accrual of random mutations in secondary lymphoid tissues are mostly purged or edited to prevent autoimmunity. Yet, 10-20% of all mature naïve B cells in healthy individuals have self-reactive B cell receptors (BCRs). In patients with serologically active systemic lupus erythematosus (SLE) the percentage increases up to 50%, with significant self-DNA reactivity that correlates with disease severity. Endogenous or self-DNA has emerged as a potent antigen in several autoimmune disorders, particularly in SLE. However, the mechanism(s) regulating or preventing anti-DNA antibody production remain elusive. It is likely that in healthy subjects, DNA-reactive B cells avoid activation due to the unavailability of endogenous DNA, which is efficiently degraded through efferocytosis and various DNA-processing proteins. Genetic defects, physiological, and/or pathological conditions can override these protective checkpoints, leading to autoimmunity. Plausibly, increased availability of immunogenic self-DNA may be the key initiating event in the loss of tolerance of otherwise quiescent DNA-reactive B cells. Indeed, mutations impairing apoptotic cell clearance pathways and nucleic acid metabolism-associated genes like DNases, RNases, and their sensors are known to cause autoimmune disorders including SLE. Here we review the literature supporting the idea that increased availability of DNA as an immunogen or adjuvant, or both, may cause the production of pathogenic anti-DNA antibodies and subsequent manifestations of clinical disease such as SLE. We discuss the main cellular players involved in anti-DNA responses; the physical forms and sources of immunogenic DNA in autoimmunity; the DNA-protein complexes that render DNA immunogenic; the regulation of DNA availability by intracellular and extracellular DNases and the autoimmune pathologies associated with their dysfunction; the cytosolic and endosomal sensors of immunogenic DNA; and the cytokines such as interferons that drive auto-inflammatory and autoimmune pathways leading to clinical disease. We propose that prevention of DNA availability by aiding extracellular DNase activity could be a viable therapeutic modality in controlling SLE.

BACKGROUND

Group A Beta-Hemolytic Streptococcus (GABHS) can induce PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection). GABHS is the most important and common bacterial cause of acute pharyngitis in Iranian children. We studied the role of GABHS (anti-streptococcal antibodies) in suspected cases of PANDAS in a cross sectional studies.

METHODS

Across sectional study was done in 2 pediatric psychiatric/and neurologic clinics in Tehran (Rasul Akram and Aliasghar Hospital) during 2008-2010. We studied serum anti-streptococcal antibodies (anti streptolysin O, anti Deoxyribonuclease B, and anti-streptokinase (ABcam-ELISA, USA) in 76 cases with psychiatric manifestation (OCD, ADHD) in compare with 39 healthy controls. These antibodies were studied in 53 cases with movement disorders (Tic/Tourette syndrome) in compare with 76 healthy controls. Sensitivity, specificity and positive predictive value of tests were calculated.

RESULTS

In movement disorders ASOT, Anti-DNase and Anti streptokinase was significantly higher than controls (P<0.0001, P=0.000, P<0.00001) ASOT (cut off level> 200 IU/ml) had 75% sensitivity; 84% specificity and 80% PPV; Anti-streptokinase (cut off level>> 332 IU/ml) had 34% sensitivity; 85% specificity, and 72% PPV; Anti-DNase (cut off level>> 140 IU/ml) had 70% sensitivity; 99% specificity and PPV 90% for differentiating the group. ASOT, Anti-DNase and Anti streptokinase titer was significantly higher than controls (P<0.0001, P=0.000, P<0.0001). ASOT had 90% sensitivity; 82% specificity, PPV 92%; Anti streptokinase: 82% sensitivity; 82% specificity, PPV 95%; Anti DNase: 92% sensitivity; 82% specificity, PPV 92% for differentiation the cases from normal controls.

CONCLUSIONS

These findings support that a post infectious immune mechanism to GABHS may play a role in the pathogenesis of PANDAS in our children. A combination of throat culture, rapid antigen detection test, and serologic testing for GABHS is required to achieve maximum sensitivity and specificity for diagnosis. We prefer to use antibiotic prophylaxis in PANDAS cases for preventing recurrent streptococcal infections. Ongoing research is needed for identifying optimum diagnostic, prevention and therapeutic approach especially, aggressive treatment (intravenous immunoglobulin, plasmaphresis).

The actin associated with membrane-enriched extracts of leukocytes can be quantitated by DNAse 1 inhibition. Using this assay, we previously demonstrated that the actin level in monocytes was significantly higher than that in polymorphonuclear, T and B cells respectively. However, the extracellular location of the actin fraction detected by DNAse 1 inhibition (monomeric "G") remained unclear. This study using the DNAse 1/anti DNAse 1 immunoglobulin fluorescein conjugated system demonstrated that G-actin is present primarily in the cortical cell cytoplasm of leukocytes, in confirmation of our previous biochemical findings. Since the solubilized G-actin activities of membrane-rich lymphoid cell fractions, measured by DNAse 1 inhibition, are a reflection of the migratory potential, this immunofluorescent system may permit identification of the leukocytic cell subpopulations that have a potential for active circulation.

The hepatoma Morris 5123 tumor growth is accompanied by changes in actin content and polymerization (Malicka-Błaszkiewicz et al. (1995) Mat. Med. Pol., 27, 115-118; Nowak et al. (1995) J. Exp. Cancer Res. 14, 37-40). Presently actin isoforms from cytosol and cytoskeleton fractions were separated by SDS/PAGE and identified with antibodies directed against different actin isoforms. Actin isolated from the cytosol by affinity chromatography on DNase I bound to agarose shows the presence of only one protein spot on 2D gel electrophoresis corresponding to the mobility of the rabbit a skeletal muscle actin (Mr 43,000) and isoelectric point equal to 5.3. It interacts only with monoclonal anti beta actin isoform antibodies, posing the question of differential affinity of actin isoforms to DNase I.

In a double-blind controlled study we compared the effectiveness of cephalexin b.i.d. versus q.i.d. in the treatment of group A streptococcal pharyngitis in 65 children. Clinical improvement was noted in 64 patients (98%) and bacteriologic cure in 60 (92%). Despite good compliance, three bacteriologic failures were noted in the q.i.d., and two in the b.i.d. treatment groups. Two of these five were carriers. Significant antibody responses were observed in 61% of the patients by at least one of three tests (ASO, anti-DNase B, Streptozyme). We also investigated the extended microbiology of streptococcal pharyngitis by looking for the presence of viruses, chlamydia and beta-lactamase producing organisms in the pharynx. Respiratory viruses were isolated concomitantly with Streptococcus pyogenes in six patients. Beta-lactamase producing bacteria were present in the pharynx of 98% of the patients at the initiation of treatment and comprised 1-98% of the total bacterial flora. The beta-lactamase producing flora did not significantly change with cephalexin therapy.

Neutrophils release extracellular traps (NETs) after interaction with microorganisms and physiological or synthetic products. NETs consist of decondensed chromatin complexed with proteins, some of them with microbicidal properties. Because NETs can modulate the functioning of HIV-1 target cells, we aimed to verify whether they modify HIV-1 replication in macrophages. We found that exposure of HIV-1-infected macrophages to NETs resulted in significant inhibition of viral replication. The NET anti-HIV-1 action was independent of other soluble factors released by the activated neutrophils, but otherwise dependent on the molecular integrity of NETs, since NET-treatment with protease or DNase abolished this effect. NETs induced macrophage production of the anti-HIV-1 β-chemokines Rantes and MIP-1β, and reduced the levels of integrated HIV-1 DNA in the macrophage genome, which may explain the decreased virus production by infected macrophages. Moreover, the residual virions released by NET-treated HIV-1-infected macrophages lost infectivity. In addition, elevated levels of DNA-elastase complexes were detected in the plasma from HIV-1-infected individuals, and neutrophils from these patients released NETs, which also inhibited HIV-1 replication in in vitro infected macrophages. Our results reveal that NETs may function as an innate immunity mechanism able to restrain HIV-1 production in macrophages.

CH12 is a murine B-cell lymphoma whose surface immunoglobulin (sIg) and concanavalin A (Con A) receptors patch and cap readily. Actin may be involved in CH12 patching and capping, since fodrin and F-actin collect under the cap, and cytochalasin D inhibits sIg capping. We have examined the state of the actin cytoskeleton during patching and capping. A wide range of concentrations of rabbit anti-mouse antibody (RAM) and Con A were used to patch or cap CH12 cells. G-actin was quantitated by DNase I inhibition, F-actin was quantitated by fluorescence-activated cell sorter analysis of fluorescent phalloidin staining, and actin nucleation sites were measured by pyrene actin polymerization. None of these methods detected any significant changes in actin when compared to control cells or untreated cells, leading us to conclude that increased actin polymerization is not necessary for capping to occur. The significance of these data to the membrane flow and cytoskeletal models of capping is discussed.

The immunologic responses to streptolysin O and streptococcal deoxyribonuclease B were evaluated in children with group A streptococci recovered from the upper respiratory tract to re-examine the hypothesis that a limited capacity to respond to group A streptococcal infection may explain the rare occurrence of acute rheumatic fever in very young children. ASO and anti-DNase B titers were determined on serial bleedings from a total of 301 individuals (52 less than or equal to 3 years; 249 older than 3 years). Very young children with group A streptococcal upper respiratory tract infections had an immunologic response to SO greater than the response in older children as reflected by the magnitude of the antibody rise, and comparable to the ASO response in older children as measured by the percentage showing a significant titer rise. Similar analyses of the anti-DNase B responses showed the response in young children to be comparable to those of the older group. Clinical manifestations of group A streptococcal upper respiratory tract infection in very young children differ from those observed in older children and have not changed significantly in the past several decades. These data suggest that the infrequent occurrence of acute rheumatic fever in very young children is not due to a difference in antibody response to streptolysin O or streptococcal DNase B.

The release of neutrophil extracellular traps ( (<em>b</em>)NETs</<em>b</em>) ) <em>b</em>y hyperactive neutrophils is recognized to play an important role in the throm<em>b</em>oinflammatory milieu inherent to severe presentations of COVID-19. At the same time, a variety of functional auto<em>anti</em><em>b</em>odies have <em>b</em>een o<em>b</em>served in individuals with severe COVID-19 where they likely contri<em>b</em>ute to immunopathology. Here, we aimed to determine the extent to which auto<em>anti</em><em>b</em>odies might target NETs in COVID-19 and, if detected, to elucidate their potential functions and clinical associations. We measured glo<em>b</em>al <em>anti</em>-NET activity in 171 individuals hospitalized with COVID-19 alongside 48 healthy controls. We found high <em>anti</em>-NET activity in the IgG and IgM fractions of approximately 40% and 50% of patients, respectively. There was a strong correlation <em>b</em>etween <em>anti</em>-NET IgG and <em>anti</em>-NET IgM, with high <em>anti</em>-NET <em>anti</em><em>b</em>ody levels in general associating with circulating markers of NETs such as myeloperoxidase-DNA complexes and calprotectin. Clinically, <em>anti</em>-NET <em>anti</em><em>b</em>odies tracked with impaired oxygenation efficiency and elevated levels of circulating D-dimer. Furthermore, patients who required mechanical ventilation had higher levels of <em>anti</em>-NET <em>anti</em><em>b</em>odies than those who did not require oxygen supplementation. Mechanistically, <em>anti</em>-NET <em>anti</em><em>b</em>odies of the IgG isotype impaired the a<em>b</em>ility of <em>DNases</em> in healthy serum to degrade NETs. In summary, these data reveal high levels of <em>anti</em>-NET <em>anti</em><em>b</em>odies in individuals hospitalized with COVID-19, where they likely impair NET clearance and there<em>b</em>y potentiate SARS-CoV-2 mediated throm<em>b</em>oinflammation.

The R antigen, a trypsin-resistant protein observed in group A, C, F, G, and L streptococci, has also been found in group B streptococci (GBS). Although four species of the R antigen have been described for GBS, the R4 protein is the most prevalent in GBS isolates recovered from humans. This study examined the prevalence of antibodies against the R4 antigen by Western blot (immunoblot) (WB) in sera from 40 mothers colonized with GBS serotype II and III and from 26 noncolonized mothers; 92.5% of the colonized mothers had anti-R4 antibodies, compared with 54% of the noncolonized mothers (P < 0.001). Findings of antibodies in neonatal cord sera (n = 14) were concordant with maternal results by WB analysis for 71% of mother-infant pairs colonized with serotype II and for 57% of pairs colonized with serotype III. Of mothers known to be colonized with type II/R4 or III/R4, 100% (n = 12) had antibody against R4 by WB. This study also evaluated the prevalence of antibody to the GBS R4 antigen in 48 sera from individuals with high and low group A streptococcal anti-DNase B titers. Of those individuals with an anti-DNase B titer of>> 640, 64% had a positive WB for anti-R4 antibody, compared with 30% of individuals with low anti-DNase B titers (P < 0.05). The R4 antigen of GBS had immunologic identity to the R4 antigen of group A streptococci. Overall, the findings suggested that antibodies to the streptococcal R4 antigen were commonly present in GBS-colonized mothers and that transplacental passage of these antibodies occurred. The presence of antibody to R4 in non-GBS-colonized individuals may be due to immunologic responses to past exposure to the R antigen present in GBS or other streptococcal groups.

Background: Narcolepsy type 1 (NT1) is considered to be an autoimmune disease, and streptococcal infection may be an environmental trigger. However, previous studies from Asian narcolepsy patients did not reveal elevated anti-streptolysin O [ASO]. The aim is to investigate whether large sample Chinese patients with NT1 have an increase in antistreptococcal antibody titers.

Methods: A total of 214 narcolepsy patients and 360 healthy controls were recruited. All patients were DQBanti DNAse B [ADB]. These patients were divided into five groups according to disease duration, including 29 patients less than 3 months; 25 from 3 months to 1 year; 40 from 1 to 3 years; 61 from 3 to 10 years and 59 patients over 10 years. Comparison was also made between children and adults with age matched controls, respectively.

Results: There were no significant differences between patients and healthy controls in regard to both ASO ≥200 IU (19.2% vs. 16.9%, p = 0.50) and ADB≥480IU (9.8% vs. 10.3%, p = 0.86). For children narcolepsy patients, ASO positive rates (19.8% vs. 18%, p = 0.68) and ADB positive rates (10.4% vs. 12%, p = 0.72) had no differences compared to age matched controls. No difference was observed in adult narcolepsy patients either, with ASO positive rates (18.5% vs. 13.8%, p = 0.39) and ADB positive rates (9.3% vs. 5.3%, p = 0.42) compared to age matched controls, respectively. ASO and ADB positive rates had no significant differences among different disease duration groups (p = 0.55 and 0.9, respectively).

Conclusion: Streptococcus infection reflected by increase of ASO and ADB levels was not found in Chinese patients with type 1 narcolepsy, additional triggers for narcolepsy need to be addressed in this population.

Keywords: Cataplexy; Narcolepsy; Streptococcus.

OBJECTIVE

To better understand the potential effect of ultraviolet light on the photosensitivity of patients with lupus erythematosus (LE), to elucidate the mechanisms of SSA/Ro antibody formation after UV exposure, and to investigate the role of this autoantibody in the pathogenesis of skin lesions.

METHODS

Primary human keratinocytes were cultured in Medium-154. After ultraviolet-B light (UVB) irradiation, the keratinocytes were treated with affinity-purified anti-SSA/Ro sera and stained with FITC-labeled goat-anti-human IgG and propidium iodide (PI), followed by enzyme digestion with RNase, RNase-free DNase or RNase plus DNase. As target cells, the irradiated keratinocytes were incubated with affinity-purified anti-SSA/Ro sera, with or without fresh human sera as complement. The supernatants of irradiated keratinocytes were analyzed with ELISA method for SSA/Ro antigens.

RESULTS

UVB irradiation induced apoptotic blebs on the cell surface. The blebs were composed of ribonucleoproteins and contained SSA/Ro antigens. SSA/Ro antigens expressed on UVB irradiated keratinocytes bound to affinity-purified anti-SSA/Ro sera, leading to complement-dependent cytotoxicity. However, no SSA/Ro antigens were detected in the supernatants.

CONCLUSIONS

SSA/Ro, a ribonucleoprotein antigen expressed on UVB irradiated keratinocytes, may be recognized and presented to immune cells by a direct cell-cell contact other than be eliminated into the circulation.

Some of vanadyl complexes have shown potential to inhibit RNase activity by acting as transition state analogue, while at the same time not inhibiting DNase. To gain an insight into the interaction of protein with vanadate (VO3-) and vanadyl (VO2+) ions, the present study was designed to examine the binding of ribonuclase A (RNase A) with NaVO3 and VOSO4 in aqueous solution at physiological pH with metal ion concentrations of 0.001 mM to 1 mM, and protein concentration of 2% w/v. Absorption spectra and Fourier transform infrared (FTIR) spectroscopy with self-deconvolution and second derivative resolution enhancement were used to determine the cation binding mode, association constant and the protein secondary structure in the presence of vanadate and vanadyl ions in aqueous solution. Spectroscopic results show that an indirect metal ion interaction occurs with the polypeptide C = O, C-N (via H2O) with overall binding constants of K(VO3-) = 3.93x10(2) M(-1) and K(VO2+) = 4.20x10(3) M(-1). At high metal ion concentrations, major protein secondary structural changes occur from that of the alpha-helix 29% (free enzyme) to 23-24%; beta-sheet (pleated and anti) 50% (free enzyme) to 64-66% and turn 21% (free enzyme) to 10-12% in the metal-RNase complexes. The observed structural changes indicate a partial protein unfolding in the presence of high metal ion concentration.