β-Conglycinin is one of the key thermostable anti-nutritional factors in soybean, which has strong immunogenicity that usually leads to weaning in some young animals such as piglets and calves and allergic reaction in rats. Neutrophils are involved in the pathogenesis of an allergy. However, the contribution of functional neutrophils to allergy needs to be clarified. The formation of neutrophil extracellular traps is a novel effector mechanism of neutrophils and has been extensively investigated in recent years. To the best of our knowledge, there is no information available on β-conglycinin-induced NETs. In this study, β-conglycinin-induced NET formation in mice was examined via immunofluorescence analysis and fluorescence microplate reader. The mechanism of β-conglycinin-induced NETs was investigated using inhibitors and fluorescent microplate methods. The results showed that β-conglycinin induced the classical characteristics of NETs, which mainly consist of DNA as the backbone and decorated with histones, myeloperoxidase (MPO) and neutrophil elastase (NE). Moreover, β-conglycinin significantly induced the formation of NETs in a dose-dependent way. NET degrading enzyme DNase I markedly reduced β-conglycinin-induced NETs, which suggests that β-conglycinin indeed triggered the release of NETs. Further investigation showed that the quantitation of NETs was markedly decreased by the inhibitors of reactive oxygen species (ROS)-derived-NADPH oxidase, ERK1/2, p38, Rac and PAD4 signaling pathways, indicating the crucial role of these signaling pathways in β-conglycinin-induced NETs. Furthermore, our findings revealed that β-conglycinin induced the formation of NETs, which is dependent on NADPH oxidase-derived ROS, ERK1/2, p38, Rac and PAD4 signaling pathways. This study is the first to demonstrate the underlying mechanisms of β-conglycinin-induced NET formation, and it could be helpful to understand diarrhea caused by β-conglycinin overexposure in young animals and provides the corresponding theoretical basis for clinical applications.

The following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium, rabbit uterine myocytes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated myocytes were maintained in culture in 75-cm2 flasks containing Waymouth's MB 751/1 medium-10% fetal bovine serum along with 10(-8) M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes, both the primary and F1 generation. After prelabeling the myocytes with [3H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates. Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine, an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle.

An in vitro culture system for the proliferation of IgG-forming plasma cells from mouse bone marrow cultures has previously been described. The present study attempts to elucidate the mode of action of thymic RNA in these cultures. Autoradiography after using radiolabeled thymic RNA showed that radioactive material was mainly incorporated into the nuclei of IgG-forming plasma cells. No radiolabeled thymic RNA was incorporated into the cells except immunoblasts. The incorporated thymic RNA was acid insoluble and digested by RNase, but resistant to DNase and pronase. Radioactivity in the nucleotide pool after the cells were cultured with radiolabeled thymic RNA was negligible, indicating that reutilization of degraded RNA did not occur in the nuclei of the plasma cells. Moreover, the incorporation of radiolabeled thymic RNA by the cells was not prevented by excess unlabeled nucleosides. Escherichia coli transfer RNA, L-cell RNA and synthetic polynucleotide poly(A-U) were incorporated but were distributed in a different manner in the cells. A derivative of rifampicin, 2'5'-dimethyl N(4') benzyl-N(4')[desmethyl]rifampicin (AF/ABDMP), a possible inhibitor of RNA-dependent DNA polymerases, suppressed both the incorporation of thymic RNA and the differentiation of immunoblasts. AF/ABDMP suppressed DNA synthesis by bone marrow cultures to the same level as those pretreated with anti-mouse B-cell antibodies and complement. DNA dependent RNA polymerase activity was observed in the supernatant of bone marrow cultures stimulated by normal syngeneic thymic RNA and human gammaglobulin as antigen. These results imply a possible relationship between B-cell differentiation and RNA-dependent DNA polymerases.

A series of novel 4-benzyl-morpholine-2-carboxylic acid N'-[2-(4-benzoyl-phenoxy)-acetyl]-hydrazide derivatives 8a-j has been synthesized from (4-hydroxy-aryl)-aryl methanones through a multi-step reaction sequence and then evaluated for anti-proliferative activity in vitro against various types of neoplastic cells of mouse and human such as DLA, EAC, MCF-7 and A549 cells. From the cytotoxic studies and structural activity relationship of compounds 8a-j, it is clear that methyl group on the B ring of benzophenone is essential for antiproliferative activity and bromo at ortho position (compound 8b) and methyl at para position (compound 8f) on A ring of benzophenone are significant for extensive anti-mitogenic activity. Investigation on clonogenesis and Fluorescence-activated cell sorting suggests that compounds 8b and 8f have the potency to exhibit the prolonged activity with cell cycle arrest on G2/M phase against cancer progression. Further, the compounds 8b and 8f inhibit murine ascites lymphoma through caspase activated DNase mediated apoptosis.

We identified, by anticomplement immunofluorescence, a nuclear antigen (hepatitis B virus-associated nuclear antigen [HBNA]) in two human hepatoma cell lines containing integrated hepatitis B virus DNA but not in three hepatoma cell lines lacking it. The antigen resembled neoantigens associated with the oncogenesis of certain papovaviruses, adenoviruses, and herpesviruses. Antibody to the antigen (anti-HBNA) was found in 7.3% of hepatitis B surface antigen-positive sera from patients with hepatocellular carcinoma but not in surface antigen-negative sera. The staining of HBNA was characterized by two patterns, reticular nuclear fluorescence and nucleolar fluorescence. The expression of HBNA did not parallel the production of extracellular hepatitis B surface antigen. Treatment of cells with proteinase K, RNase, DNase, or cycloheximide significantly diminished the staining of HBNA.

This paper describes a method for the preparation of co-cultures of rat heart cells and bovine adrenal chromaffin paraneurons. The most suitable condition for heart cell isolation was when a combination of trypsin-DNAse I in Locke's solution was used for digestion. The best co-culture conditions were obtained when 10(6) heart cells were plated on 7- to 8-d-old adrenal chromaffin paraneuron cultures containing 0.5 x 10(6) cells per 35-mm diameter culture dishes. Measurements of DNA (heart cells and chromaffin paraneurons), monitoring of beating frequency (heart cells), and catecholamine (chromaffin paraneurons) levels and release indicated that both cell types remain viable and functional for several weeks. Heart cells started their characteristic contractile activity 24 h earlier when plated either on viable or lysed chromaffin paraneurons, an effect apparently due to faster surface adhesion of heart cells. The beating frequency of heart cells increased after treatment of co-cultures with either noradrenaline or nicotine, with the latter agent acting indirectly through the release of chromaffin paraneuron catecholamines. Propranolol produced a dose-related inhibition of the responses to either noradrenaline or nicotine, thus suggesting that the increase in myocyte's beating activity was mediated through beta-receptors. Anti-myosin and anti-dopamine-beta-hydroxylase immunostaining was used for cell type identification and for the demonstration of body-to-body and process-to-process contacts between adrenal chromaffin paraneurons and heart cells. This co-culture system will serve as a starting point of further studies directed to understand a) the influence of a cell type on the development and on the phenotypic characteristics of a second cell type and b) the interaction of cells derived from different organs and species.

Most Cystic Fibrosis (CF) patients succumb to airway inflammation and pulmonary infections due to Pseudomonas aeruginosa. D-BMAP18, a membrane-permeabilizing antimicrobial peptide composed of D-amino acids, was evaluated as a possible antibacterial aimed to address this issue. The antipseudomonal activity of D-BMAP18 was tested in a pathophysiological context. The peptide displayed activity against CF isolates of Pseudomonas aeruginosa in the presence of CF sputum when combined with sodium chloride and DNase I. In combination with DNase I, D-BMAP18 discouraged the deposition of new biofilm and eradicated preformed biofilms of some P. aeruginosa strains. In addition, D-BMAP18 down regulated the production of TNF-α, IL1-β, and TGF-β in LPS-stimulated or IFN-γ macrophages derived from THP-1 cells indicating an anti-inflammatory activity. The biocompatibility of D-BMAP18 was assessed using four different cell lines, showing that residual cell-specific cytotoxicity at bactericidal concentrations could be abolished by the presence of CF sputum. Overall, this study suggests that D-BMAP18 may be an interesting molecule as a starting point to develop a novel therapeutic agent to simultaneously contrast lung infections and inflammation in CF patients.

Keywords: BMAP-18; P. aeruginosa infection; antimicrobial peptide; cystic fibrosis; inflammation.

Sera of 157 baboons (Papio hamadryas and P. anubis) 21 chimpanzees (Pan troglodytes) five orang utans (Pongo pygmaeus) three mountain gorillas (Gorilla gorilla beringei) and three gibbons (Hylobates lar lar) were examined for the content of antistreptolysin O, antidesoxyribonuclease B, for the presence of rheumatoid factors as well as for the level and type of haptoglobin. The mean antistreptolysin O titer (AST) in baboons was 106 ASU +/- 18 in dextransulfate absorbed sera ("real" AST) and 182 ASU +/- 34 in non-absorbed sera. The mean decrease after absorption was 20% (i.e., 20% inhibitor + 80% antistreptolysin O), a value that is lower than previously found in rhesus monkeys (55%) or in man (40%). Raised values of anti-DNase B were found in two baboons only, and none of the sera of that species displayed presence of rheumatoid factors. In chimpanzees, the mean AST was 68 +/- 29 in absorbed and 87 ASU +/- 57 in non-absorbed sera. Anti-DNase B was raised in three animals, and in two cases the increase was correlated with raised AST. Of 19 chimpanzee sera examined, 13 were found to contain antigammaglobulins ("rheumatoid factors") the titers of which reached 1:64 or more. All primate sera tested so far showed haptoglobin type 1-1 or Hp 1-1-like patterns. The haptoglobin level in chimpanzees and baboons was comparable to that established in man; in rhesus monkey, on the other hand, much lower values (40-62 mg/100 ml) or ahaptoglobinemia were observed. The sera of all monkeys and apes tested so far showed a very low (undiluted, or up to 1:10 titer at most) agglutinating activity against T4-antigen-carrying streptococci. This is in agreement with our observations made previously which indicated that human or animal sera of haptoglobin type 1-1 agglutinated streptococci to a much lower degree than type Hp 2-2 or type Hp 2-1 sera.

Sarcoma arises extremely rarely on foreign bodies in man, but is aggressive and often lethal. A coating for implants which would further reduce the risk in man is desirable. The incidence in mice is much greater, and responds to chemical treatment of the implant surface. Coating with histones increases tumour yield. Accordingly, related substances, foreign DNA, DNase and a mixture of the two, were tested for anticancer activity by application to 25 mm nitrocellulose filters in groups of 30-45 BALB/c mice, in comparison with untreated filters. Other substances reported to influence neoplasia, paprika, beta-carotene, rhodamine and tuftsin; and substances expected to be neutral, oxyprenolol, liquid paraffin, iodine, and adenosine diphosphate were similarly tested against concurrent untreated controls for comparison. Bovine DNA (p = 0.01) and DNA/DNase mixture (p = 0.04) and DNase fomented tumour growth by 55, 45 and 59% respectively. Paprika and beta-carotene did so by 70% (p = 0.05). The other substances were inert. None were candidates for an anti-sarcoma coating.

A fraction extracted from Mycobacterium bovis BCG was found to exhibit strong antitumor activity. This fraction, which was designated MY-1, caused some animal tumors to regress and/or prevent metastasis very effectively. MY-1 after digestion with DNase had almost completely reduced activity, while MY-1 digested with RNase did not. MY-1 also augmented natural killer (NK) cell activity of mouse spleen cells in vitro, and produced factors which showed anti-viral activity and rendered macrophages cytotoxic towards tumor cells. The function of the factor to activate macrophages was destroyed by treatment with anti-interferon (IFN)-gamma antibody, while the anti-viral activity was destroyed by treatment with anti-INF alpha/beta antibody. The oligonucleotides contained in MY-1 distributed in a broad range of molecular size, and peaked at 45 nucleotides. We synthesized 13 kinds of 45-mer nucleotides with sequence present in the known cDNA encoding various BCG proteins. Six out of these oligonucleotides, which contained one or more hexameric palindromic structures, showed strong antitumor activity, while the other without palindrome did not. These active oligonucleotides possessed the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotide to augment NK activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Conjugated linoleic acids (CLA) have attracted more attention as functional lipids due to their potential physiological activities including <em>anti</em>-cancer, <em>anti</em>-inflammatory, <em>anti</em>-cardiovascular disease and <em>anti</em>-dia<em>b</em>etes. Micro<em>b</em>iological synthesis of CLA has <em>b</em>ecome a compelling method due to its high isomer selectivity and convenient separation and purification process. In <i>Lacto<em>b</em>acillus plantarum,</i> the generation of CLA from linoleic acids (LA) requires the com<em>b</em>ination of CLA oleate hydratase (CLA-HY), CLA short-chain dehydrogenase (CLA-DH) and CLA acetoacetate decar<em>b</em>oxylase (CLA-DC) which are separately encoded <em>b</em>y <i>cla-hy</i>, <i>cla-dh</i> and <i>cla-dc</i> However, the regulatory mechanisms of CLA synthesis remain unknown. In this study, we found that a lysR-family transcriptional regulator LTTR directly <em>b</em>ound to the promoter region of <i>cla</i> operon and activated the transcription of <i>cla-dh</i> and <i>cla-dc</i> The <em>b</em>inding motif was also predicted <em>b</em>y <em>b</em>ioinformatics analysis and verified <em>b</em>y EMSA and <em>DNase</em> I footprinting assay. The <i>lttR</i> overexpression strain showed a 5-fold increase in CLA production. Moreover, we uncovered that the transcription of <i>lttR</i> is activated <em>b</em>y LA. These results indicate that LttR senses LA and promotes CLA production <em>b</em>y activating the transcription of <i>cla-dh</i> and <i>cla-dc</i> This study reveals a new regulatory mechanism in CLA <em>b</em>iotransformation and provides a new potential meta<em>b</em>olic engineering strategy to increase the yield of CLA.(<em>b</em>)Importance</<em>b</em>) Our work has identified a novel transcriptional regulator LTTR that regulates the production of CLA <em>b</em>y activating the transcription of <i>cla-dh</i> and <i>cla-dc</i>, essential genes participating in the CLA synthesis in <i>Lacto<em>b</em>acillus plantarum</i> This provides the insight into the regulatory mechanism of CLA synthesis and <em>b</em>roadens our understanding a<em>b</em>out synthesis and regulatory mechanisms of <em>b</em>iosynthesis of CLA.

A prospective study of acute nephritis in children was conducted at the Universiti Sains Malaysia Hospital, Kubang Kerian between July 1987 and June 1988. One hundred and twenty four children were admitted with acute glomerulonephritis. The aim of the study was to determine the clinical pattern of the nephritis as well as its aetiology. The majority of our patients came from the lower socio-economic group and 54% of the families had incomes below the poverty line. Preceding skin infection was much more common than throat infection. The children showed a high incidence of complications: severe hypertension (43.6%), hypertensive encephalopathy (11.3%), clinical pulmonary oedema (36.3%), severe azotaemia (10.5%), and prolonged gross haematuria (13.7%). By using immunologic indices such as ASOT, anti-DNase B and complement 3, it was concluded that 121 of the 124 patients had post-streptococcal nephritis.

The present study examines the effectiveness of DNA vaccine against Aeromonas hydrophila through oral route using chitosan-tripolyphosphate (Cs-TPP) nanoparticles encapsulation. The virulent gene of outer membrane protein (OMP) and hemolysin (hly) related to pathogenicity of A. hydrophila was used to construct a DNA vaccine using pVAX1, and the construct was named as pVAX-OMP and pVAX-hly DNA vaccines. The pVAX-OMP and pVAX-hly DNA vaccines were encapsulated by Cs-TPP nanoparticles and size measured by field emission scanning electron microscopy (FE-SEM). The encapsulation efficiency of Cs-TPP nanoparticles was found to be 79.6% for pVAX-OMP DNA and 82.3% for pVAX-hly DNA binding with Cs-TPP nanoparticles. The stability and invitro release profile of plasmid DNA was also determined after encapsulation using DNase and chitosanase. DNA vaccines distribution in tissues was investigated in fish fed with the pVAX-OMP, pVAX-hly and pVAX-OMP+pVAX-hly encapsulated in Cs-TPP nanoparticles and confirmed by PCR and multiplex PCR. The results suggest that Cs-TPP nanoparticles encapsulated DNA vaccine delivered into fish by feeding. After oral vaccination of Labeo rohita were challenged with A. hydrophila by intraperitoneal injection. Relatively, gene expression of c- and g-type lysozyme followed by pro- and anti-inflammatory cytokines (Interlukin-10 and Tumor Growth Factor β) was up-regulated in heart and kidney for pVAX-OMP+pVAX-hly vaccinated group. Moreover, fish fed with pVAX-OMP+pVAX-hly encapsulated in Cs-TPP nanoparticles had a significantly higher survival rate (76.2%) against A. hydrophila. This study concludes that pVAX-OMP and pVAX-hly DNA vaccines can be delivered orally using Cs-TPP nanoparticles.

Keywords: Aeromonas hydrophila; Cs-TPP nanoparticles; DNA Vaccine; Haemolysin (hly); Immune gene expression; Outer membrane protein (OMP); pVAX-OMP; pVAX-hly.

Introduction: The neurobiology of Gilles de la Tourette syndrome (GTS) is known to involve cortico-striatal loops possibly under genetic control. Less is known about possible environmental triggers of GTS. Specifically, immune-related events following possible environmental inducers have been evoked, but important controversies still exist.

Objectives: In this systematic review and meta-analysis, we then aimed to look for evidence in favor of such possibilities.

Method: We performed a systematic review and meta analysis of all immunological data in PubMed.

Results: We found large discrepancies concerning immune dysfunctions in GTS and meta-analyzing cytokines data did not allow us to conclude to an involvement of specific cytokines in GTS neurobiology. When looking specifically to the PANDAS/PANS entity, we found some important evidence of a possible infectious involvement but in limited number of studies. Our meta-analysis found an increased level of anti-streptolysin O antibodies in GTS patients, but level of anti-DNase B antibodies was not increased.

Conclusions: Too many questions still exist to allow us to definitively reach the conclusion that there is an infectious and immunological etiology in GTS. Much work is still needed to elucidate the possible role of immunology in GTS neurobiology and to favor immunological treatment rather than classical treatment.

Keywords: Autoimmunity; Cytokines; Immunology; PANS/PANDAS; Tic disorders.

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.

Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 is the causative agent of leptospirosis in animals and humans. This organism carries a functional cas1 gene classified under CRISPR-Cas I-B. In this study, using various nuclease assays and bioinformatics analysis, we report that the recombinant Cas1 (LinCas1) possesses metal-ion dependent DNase activity, which is inhibited upon substitution or chelation of metal-ion and/or interaction with recombinant Cas2 (LinCas2) of L. interrogans. Model of LinCas1 structure shows a shorter N-terminal domain unlike other Cas1 orthologs reported to date. The C-terminal domain of LinCas1 contains conserved divalent-metal binding residues (Glu108, His176, and Glu191) and the mutation of these residues leads to abolition in DNase activity. Immunoassay using anti-LinCas2 demonstrates that LinCas1 interacts with LinCas2 and attains a saturation point. Moreover, the nuclease activity of the LinCas1-Cas2 mixture on ds-DNA displayed a reduction in activity compared to the pure core LinCas proteins under in vitro condition. The DNase activity for LinCas1 is consistent with a role for this protein in the recognition/cleavage of foreign DNA and integration of foreign DNA as spacer into the CRISPR array.

Keywords: CRISPR-Cas; Cas protein; DNase; Leptospira; Nucleases.

(<em>b</em>)<em>B</em>ackground:</<em>b</em>) Previous studies have esta<em>b</em>lished that streptococcal <em>anti</em><em>b</em>ody titer is correlated with a diagnosis of acute rheumatic fever (ARF). However, results vary in the usefulness of GAS <em>anti</em><em>b</em>odies, particularly <em>anti</em>-streptolysin-O (ASO) and <em>anti</em>-<em>DNase</em> <em>B</em>, in confirming a recent GAS infection. Therefore, we sought to provide, from pu<em>b</em>lished studies, an evidence-<em>b</em>ased synthesis of the correlation of streptococcal serology to esta<em>b</em>lish the usefulness of immunological data in aiding the diagnosis of ARF. These findings are <em>anti</em>cipated to have implications where echocardiography is not freely availa<em>b</em>le, especially where ARF is rampant. (<em>b</em>)Methods:</<em>b</em>) We conducted a comprehensive search across a num<em>b</em>er of data<em>b</em>ases. Applying a priori criteria, we selected articles reporting on studies, regardless of study design, that evaluate the levels of <em>anti</em><em>b</em>odies against GAS-specific <em>anti</em>gens in ARF su<em>b</em>jects against control values or a pu<em>b</em>lished standard. Data were extracted onto data extraction forms, captured electronically, and analyzed using Stata software. Risk of <em>b</em>ias was assessed in included studies using the Newcastle-Ottawa Scale (NOS). (<em>b</em>)Results and Conclusion:</<em>b</em>) The search strategy yielded 534 studies, from which 24 met the inclusion criteria, reporting on evaluation of titers for SLO (<i>n</i> = 10), <em>DNase</em> <em>B</em> (<i>n</i> = 9), <em>anti</em>-streptokinase (ASK) (<i>n</i> = 3) amongst others. Elevation in titers was determined <em>b</em>y comparison with controls and upper limit of normal (ULN) <em>anti</em><em>b</em>ody values as determined in healthy individuals. Meta-analysis of case-controlled studies revealed moderate odds ratio (OR) correlations <em>b</em>etween ARF diagnosis and elevated titers for SLO (OR = 10.57; 95% CI, 3.36-33.29; 10 studies) and <em>DNAse</em> <em>B</em> (OR = 6.97; 95% CI, 2.99-16.27; 7 studies). While providing support for incorporating SLO and <em>DNase</em> <em>B</em> in the diagnosis of ARF, we present the following reflections: an elevation in SLO and <em>DNase</em> <em>B</em> levels are not consistently associated with an ARF diagnosis; increasing the num<em>b</em>er of GAS proteins in the test is warranted to improve sensitivity; paired (acute and convalescent) samples could provide a more accurate indication of a rising titer. Use of community-<em>b</em>ased controls as a standard is not a relia<em>b</em>le marker <em>b</em>y which to gauge recent GAS infection.

Keywords: ARF diagnosis; GAS antigens; anti-DNase B; anti-streptolysin-O; systematic review.

The discovery of chimeric <em>anti</em>-melanoma agents is reported. These molecules are potent growth suppressors of melanoma cells <i>in vitro</i> with growth inhi<em>b</em>ition of 50% (GI<su<em>b</em>)50</su<em>b</em>)) values as low as 1.32 µM. Compounds were more toxic to melanoma cells <i>in vitro</i> than commonly used <em>anti</em>-melanoma agent dacar<em>b</em>azine as measured <em>b</em>y TUNEL assay. They induced <em>b</em>oth caspase-independent apoptosis evident <em>b</em>y colocalization of TUNEL with endonuclease G (EndoG) and caspase-mediated apoptosis measured <em>b</em>y colocalization of TUNEL with caspase-activated <em>DNase</em> (CAD). In addition, compounds (<em>b</em>)3</<em>b</em>) and (<em>b</em>)5</<em>b</em>) strongly induced oxidative injury to melanoma cells as measured <em>b</em>y TUNEL colocalization with heme oxygenase-1 (HO1). Dacar<em>b</em>azine induced only caspase-independent apoptosis, which may explain why it is less cytotoxic to melanoma cells than compounds (<em>b</em>)3</<em>b</em>), (<em>b</em>)4</<em>b</em>) and (<em>b</em>)5</<em>b</em>).

Keywords: TUNEL assay; androstenone; apoptosis; flow cytometry; melanoma; thiazole.

(<em>b</em>)Purpose</<em>b</em>): Unilateral Acute Idiopathic Maculopathy (UAIM) is a rare inflammatory macular condition affecting young adults with poorly understood etiology. This case report addresses this <em>b</em>y suggesting potential etiology.(<em>b</em>)Methods</<em>b</em>): A retrospective review of patient records was carried out to o<em>b</em>tain data for case report.(<em>b</em>)Results</<em>b</em>): A 29-year-old male developed sudden visual loss in the left eye 1 day following the development of a sore throat. Optical Coherence Tomography (OCT) and fundal exam demonstrated su<em>b</em>retinal/intraretinal fluid and disruption of the outer retina in keeping with UAIM. Serological testing revealed a raised <em>Anti</em>-Streptolysin O Titer and <em>anti</em>-<em>DNAse</em>-<em>B</em> <em>anti</em><em>b</em>ody.(<em>b</em>)Conclusions</<em>b</em>): To date, UAIM has <em>b</em>een known to <em>b</em>e associated with a flu-like illness and a connection <em>b</em>etween coxsackievirus has <em>b</em>een suggested <em>b</em>ut it has not <em>b</em>een linked to streptococcal pharyngitis. We suggest a potential link <em>b</em>etween UAIM and streptococcal infection and that UAIM may <em>b</em>e on the spectrum of post-streptococcal autoimmune disease.

Keywords: Group A streptococcus; UAIM; streptococcal pharyngitis; unilateral acute idiopathic maculopathy; uveitis.

(<em>b</em>)O<em>b</em>jective:</<em>b</em>) The clinical characteristics of patients with PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection) and PANS (pediatric acute-onset neuropsychiatric syndrome) and the efficacy of <em>anti</em><em>b</em>iotic therapy with psychotherapy and <em>anti</em>psychotics were investigated to improve neurological symptoms as well as o<em>b</em>sessive compulsive disorder (OCD). (<em>b</em>)Methods:</<em>b</em>) We retrospectively analyzed 62 patients with a clinical diagnosis of PANDAS/PANS enrolled from May 14, 2013 to Septem<em>b</em>er 15, 2020 in the Neurology Childhood Division, Department of Pediatrics at Sapienza, Rome. Clinical manifestations, neurological and psychiatric, la<em>b</em>oratory investigations, and familiar history were collected to evaluate the differences <em>b</em>etween the two groups. The effects of various therapeutic approaches were examined. Descriptive and comparative statistical analyses were performed. (<em>b</em>)Results:</<em>b</em>) The mean age at onset of PANDAS/PANS symptoms was 6.2 ± 1.2 years. The most common diagnosis was PANDAS, followed <em>b</em>y PANS. Neurological and psychiatric symptoms were mostly evident in <em>b</em>oth groups (>70% of the population), with no significant difference <em>b</em>etween them (<i>P</i> = 0.52 and <i>P</i> = 0.15, respectively). Irrita<em>b</em>ility, aggressivity, and food restriction were more prevalent in children with PANS than in those with PANDAS (<i>P</i> = 0.024 and <i>P</i> = 0.0023, respectively). The levels of <em>anti</em>-streptolysin O and <em>anti</em>-<em>DNAse</em> <em>B</em> 10-fold higher in PANDAS than those in PANS (<i>P</i> < 0.0001). Anti<em>b</em>iotics or psychotherapy were administered in most cases (90.3 and 53.2%, respectively), followed <em>b</em>y <em>anti</em>psychotic treatments (24.2%). In the multivariate analysis, among the therapies used, psychotherapy significantly resulted in the most efficacious relief of OCD, reducing stress in patients and their parents (<i>P</i> = 0.042). (<em>b</em>)Conclusion:</<em>b</em>) Our findings confirm a clear clinical difference <em>b</em>etween the two groups, PANDAS and PANS, using different approaches. In fact, irrita<em>b</em>ility, aggressivity, and food restriction were significantly more frequent in children with PANS and the levels of <em>anti</em>-streptolysin O and <em>anti</em>-<em>DNAse</em> <em>B</em> were higher in PANDAS. Another relevant finding is the efficacy of psychotherapy, especially for o<em>b</em>sessive-compulsive disorder, and of <em>anti</em><em>b</em>iotic prophylaxis in managing acute neurological symptoms.

Keywords: obsessive compulsive disorder; pediatric acute-onset neuropsychiatric syndrome (PANS); pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS); pediatrics; streptococcus beta hemolytic; tics.

Objectives: Rates of acute rheumatic fever, a sequelae of group A Streptococcal (GAS) infection, remain unacceptably high in Indigenous Māori and Pacific children in New Zealand. This prospective study aimed to describe GAS antibody titres in healthy children (5-14 years) by ethnicity, and to determine how paired titres vary with GAS culture positive and negative pharyngitis, and GAS skin infections.

Methods: Analysis included 887 children (32% Māori, 36% Pacific, 33% European/Other) from Auckland, New Zealand. Cases comprise 772 children who had a sore throat or skin infection, which resulted in a swab taken for culture. Healthy controls were asymptomatic (N=154) and matched by age, ethnicity and region. All participants had a serum sample, with a second sample collected from cases only. Sera were analysed for anti-streptolysin (ASO) and anti-DNase B (ADB) antibodies.

Results: Healthy Māori and Pacific children had higher GAS antibody titres than healthy European/Other children. Children with GAS-positive sore throat had the highest mean ASO titres and children with GAS-positive skin infection had the highest mean ADB titres. When a two-fold increase or an upper limit of normal cut-off (ASO 450 IU/ml, ADB 400 U/ml) was applied to titres from children with GAS-positive sore throat, 62.1% were classified as having serologically confirmed GAS pharyngitis and 37.9% had GAS detected without serological response.

Conclusions: Elevated ASO titres were associated with GAS pharyngitis and elevated ADB titres were associated with GAS skin infections in New Zealand children. Higher ASO/ADB titres in healthy Māori and Pacific children could indicate a greater prior exposure to GAS infections.

Keywords: Group A Streptococcus; anti-DNase-B; anti-streptolysin O; children; pharyngitis; serology; skin infection.

ObjectiveTo investigate the association between Group-A streptococcal (GAS) infections and tic incidence among unaffected children with a family history of chronic tic disorders (CTD).MethodsIn a prospective cohort study, children with no history for tics aged 3 to 10 years with a first-degree relative with CTD were recruited from the European Multicentre Tics in Children Study (EMTICS) across 16 European centres. Presence of GAS infection was assessed using throat swabs, serum Anti-streptolysin O titres (ASOT) and Anti-DNAse B (ADB) titres blinded to clinical status. GAS exposure was defined using four different definitions based on these parameters. Cox regression analyses with time-varying GAS exposure were conducted to examine the association of onset of tics and GAS exposure during follow-up. Sensitivity analyses were conducted using Cox regression and logistic regression analyses.ResultsA total of 260 children were recruited whilst one subject was found to have tic onsets before study entry and therefore was excluded. 61 children (23.6%) developed tics over an average follow-up period of 1 (SD 0.7) year. There was a strong association of sex and onset of tics, with girls having an approximately 60% lower risk of developing tics compared to boys (HR: 0.4, 95% CI 0.2-0.7). However, there was no statistical evidence to suggest an association of any of the four GAS exposure definitions with tic onset (GAS exposure definition 1: HR=0.310, 95% CI: 0.037-2.590; definition 2: HR=0.561, 95% CI: 0.219-1.436; definition 3: HR=0.853, 95% CI: 0.466-1.561; definition 4: HR=0.725, 95% CI: 0.384-1.370).ConclusionThese results do not suggest an association of GAS exposure and development of tics.Classification of EvidenceThis study provides Class I evidence that Group-A streptococcal exposure does not associate with the development of tics in children with first-degree relatives with chronic tic disorder.

(<em>b</em>)Background:</<em>b</em>) Periodontitis, a chronic inflammatory oral infection is the outcome of distur<em>b</em>ances in the homeostasis of the oral <em>b</em>iofilm micro<em>b</em>iota. A num<em>b</em>er of studies have found the occurrence of <i>Prevotella</i> species in elevated levels in periodontitis compared to healthy su<em>b</em>jects. Even though different aspects of <i>Prevotella</i> as part of oral <em>b</em>iofilm have <em>b</em>een studied, <i>in vitro</i> <em>b</em>iofilms formed <em>b</em>y these species have not <em>b</em>een characterized systematically. The o<em>b</em>jective of this study was to characterize <em>b</em>iofilms formed <em>b</em>y several <i>Prevotella</i> species and further to assess <em>b</em>iofilm inhi<em>b</em>ition and detachment of preformed <em>b</em>iofilms. (<em>b</em>)Methods:</<em>b</em>) Biofilms were grown in 24-well plates containing <em>b</em>rucella <em>b</em>roth in anaero<em>b</em>ic conditions for 3 days, and were quantified using crystal violet staining. Images of SYTO 9 Green fluorescent stained <em>b</em>iofilms were captured using confocal microscopy. Biofilm inhi<em>b</em>ition and detachment <em>b</em>y proteinase and <em>DNase</em> I was tested. The <em>b</em>iochemical characterization included quantification of proteins and DNA in the <em>b</em>iofilms and <em>b</em>iofilm-supernatants. (<em>b</em>)Results:</<em>b</em>) <i>Prevotella loescheii, Prevotella oralis</i> and <i>Prevotella nigrescens</i> showed highest <em>b</em>iofilm formation. <i>P. nigrescens</i> formed significantly higher amounts of <em>b</em>iofilms than <i>P. loescheii</i> (<i>P</i> = 0.005) and <i>P. oralis</i> (<i>P</i> = 0.0013). Inhi<em>b</em>ition of <em>b</em>iofilm formation was significant only in the case of <i>P. oralis</i> when treated with proteinase (<i>P</i> = 0.037), whereas with <em>DNase</em> I treatment, the inhi<em>b</em>ition was not significant (<i>P</i> = 0.531). Overall, proteinase was more effective in <em>b</em>iofilm detachment than <em>DNase</em> I. Protein and DNA content were higher in <em>b</em>iofilm than the supernatant with the highest amounts found in <i>P. nigrescens</i> <em>b</em>iofilm and supernatants. <i>P. oralis</i> <em>b</em>iofilms appeared to secrete large amounts of proteins extracellularly into the <em>b</em>iofilm-supernatants. (<em>b</em>)Conclusion:</<em>b</em>) Significant differences among <i>Prevotella</i> species to form <em>b</em>iofilms may imply their varia<em>b</em>le a<em>b</em>ilities to get integrated into oral <em>b</em>iofilm communities. Of the species that were a<em>b</em>le to grow as <em>b</em>iofilms, <em>DNase</em> I and proteinase inhi<em>b</em>ited the <em>b</em>iofilm growth or were a<em>b</em>le to cause <em>b</em>iofilm detachment.

Keywords: Prevotella; anaerobic; anti-biofilm; biofilm; oral infections.