Glutamate dehydrogenase (GDH) isoenzymes were purified from control, and ribonucleoside triphosphate (NTP)-treated peanut seedlings. GDH purification was by preparative-scale, free solution isoelectric focusing, followed by native PAGE, and the cryoelectrophoretic elution of the isoenzymes from the gel. SDS-PAGE of the purified GDH isoenzymes, followed by either silver staining of the gel, or western analysis using anti-GDH antibody, gave identical GDH polypeptide (a, alpha, and b) bands, thus, confirming the purity of the isoenzymes. The substrate specificities in the aminating activity of the GDH isoenzymes, or disaggregated polypeptides were determined by photometry, but the substrate specificities in the RNA synthesis activity were determined in cocktails containing 0.06-0.8 mM of each of UTP, ATP, GTP, and CTP, 0-100.0 mM NH4Cl, 0-50.0 mM alpha-ketoglutaratr (alpha-KG), 0-0.2 mM NADH, 0-10.0 mM CaCl2 5 units of DNase 1, antibiotics, and approximately 5 microg pure GDH isoenzymes or polypeptides at pH 8.0, and overnight at 16 degrees C. The GDH polypeptides were active only in amination reaction, but the GDH isoenzymes were active in both amination and RNA synthesis. Whereas, NADH, NH4Cl and alpha-KG served as the substrates for the amination reaction, and as modulators in the RNA synthetic reaction, ATP, GTP, UTP, and CTP served as substrates for theisoenzymes in RNA synthesis reaction. The product RNA was up to 2 microg microg(-1) GDH, and consisted of RNA species in the size ranges of 26, 16, and 5 S rRNAs. DNAse 1 in the assay cocktail ruled out transcription as the mechanism of the RNA synthesis. Addition of [alpha-32P] NTP led to the production of labeled RNA, thus confirming the specificity of NTPs as substrates, and that the RNA was not pre-existing in the reaction cocktail.

Anti-DNAse B test was performed with microtechnique in 160 subjects including patients with acute streptococcal pharyngitis, rheumatic fever and rheumatic heart disease as well as normal controls. It was shown that the antibody titer varied with age, stage of rheumatic fever and rheumatic heart disease as well as the frequency of benthazine penicillin injections. It was also shown in this study that the school children group had considerably higher geometric mean antibody titer than the adult group. The upper normal limit of anti-DNAse B titer was 240 units in the school children group and 160 units in the adult group. The anti-DNAse B test shows a higher positive detection rate for streptococcal infection than ASO, especially when these two tests are used in combination. In patients with manifestations of acute rheumatic fever without elevation of ASO titer, anti-DNAse B test will be of great diagnostic value.

Regulation of nerve growth factor (NGF) production in neuronal targets is critical for the differentiation and survival of NGF-responsive neurons. A principal cell type that produces NGF in neuronal targets is the fibroblast. Using primary kidney and established L929 cell fibroblast cultures we have studied the regulation of NGF production at the transcriptional level. A reporter gene containing the NGF promoter region including a downstream AP-1 element was efficiently expressed in stably transfected L929 cells. DNase-1 footprinting and gel shift analyses showed that the AP-1 element was bound by L929 cell nuclear factors. Mutation of the AP-1 element prevented nuclear factor binding and severely reduced expression in stably transfected L929 cells. Similarly, a reporter gene lacking the AP-1 element and carried by transgenic mice is not expressed in cultured kidney fibroblasts although the endogenous NGF gene is expressed. In addition to basal transcription, the AP-1 element may also be involved in modulation of NGF production. In support of this role, the phorbol ester TPA was shown to induce NGF mRNA in L929 cells by Northern analysis. The induction was preceded by transient increases in c-fos and jun-B mRNAs. TPA treatment also increased the binding of nuclear proteins immunoreactive with anti-fos and anti-jun antisera to the AP-1 element. A reporter gene containing the AP-1 element was transactivated by c-fos and c-jun in cotransfection experiments. Thus, the NGF promoter region including the AP-1 element is important in basal and modulated NGF gene expression in cells within neuronal targets.

BACKGROUND

Monoarticular arthritis in children is most often suppurative septic arthritis (SA) of bacterial origin. We recently cared for 3 patients with monoarticular arthritis who developed carditis while receiving antibiotics for SA. Distinguishing SA from rheumatic fever (RF) is critical to avoid lifelong cardiac complications associated with RF.

METHODS

We compared the 3 cases of RF presenting with monoarticular arthritis with 12 cases of culture-confirmed SA to assess the clinical and laboratory differences between the 2 groups.

RESULTS

Erythrocyte sedimentation rate, C-reactive protein and mean synovial fluid white blood cell counts were elevated in both groups. Mean antistreptolysin O (ASO) and anti-DNase B titers were elevated in patients with RF.

CONCLUSIONS

The clinical and laboratory features of RF and SA demonstrate substantial overlap. Patients with monoarticular arthritis and sterile synovial fluid cultures should have RF included in their differential diagnoses. Patients with an elevated ASO and/or anti-DNase B titer should have a careful cardiac examination looking for clinical evidence of carditis. Echocardiogram should be considered if clinical carditis associated with RF is suspected.

In systemic lupus erythematosus hyperactive helper T-cells drive polyclonal B-cell activation and secretion of pathogenic auto-antibodies. The auto-antibodies form immune complexes with their respective auto-antigens, which in turn deposit in sites such as the kidney and initiate a destructive inflammatory reaction. Lupus nephritis can be managed successfully in the majority of cases; however, the most widely used immunosuppressive therapies, notably corticosteroids and cyclophosphamide are non-specific and are associated with substantial toxicities. Novel treatments for lupus nephritis have to be at least as effective and less toxic than existing therapies. The ultimate aim is to develop treatments that target specific steps in the disease process. Novel therapeutic strategies in the short-term more likely will focus on refining regimens of drugs that are already in use (mycophenolate mofetil, adenosine analogues) and combinations of existing chemotherapeutic agents, as well as attempts to achieve immunological reconstitution using immunoablative chemotherapy with or without haematopoietic stem cell rescue. Several new agents targeting specific steps in the pathogenesis of lupus are in various phases of clinical development. Interrupting the interactions between T-lymphocytes and other cells by blocking co-stimulatory molecules, such as CD40 ligand or CTLA4-Ig, may interfere with the early steps of pathogenesis. Blocking IL-10 may decrease auto-antibody production and help normalise T-cell function. Treating patients with DNase or interfering with the complement cascade by blocking C5, or neutralising pathogenic antibodies by administering specific binding peptides or inducing specific anti-idiotype antibodies may prevent immune complex formation and/or deposition.

OBJECTIVE

To determine antistreptokinase antibody (anti-SK) titres in patients with the acute coronary syndrome from communities with endemic group A streptococcal infection because of the implications for streptokinase (SK) thrombolysis.

METHODS

Anti-SK titres were determined using a standard method in 47 consecutive SK naive patients, presenting to the Mt Isa Hospital emergency department, Australia, with an acute coronary syndrome. Both indigenous and non-indigenous subjects were enrolled. Antistreptolysin O (ASOT) and anti-DNAse B (ADB) titres were also determined.

RESULTS

Indigenous patients were more likely to have anti-SK antibodies (p < 0.001) than the non-indigenous cohort. Anti-SK antibody titres also correlated well with ASOT/ADB titres.

CONCLUSIONS

Anti-SK antibodies are highly prevalent in SK naive indigenous patients presenting with the acute coronary syndrome. Streptokinase should not be used for thrombolysis in populations with endemic group A streptococcal infection.

Although the incidence of rheumatic fever has declined significantly over the last decade, testing for antibodies to streptococcal extracellular enzymes maintains an important role in differentiating this disease from others with similar characteristics. Detection of antibodies to streptolysin O and DNase-B remain the more popular single antibody tests while the streptozyme test, which detects antibodies to five distinct streptococcal extracellular products, has been increasingly used in recent years as a screening test. Several new procedures detecting antibodies to different somatic antigens have been developed, the most promising of which seem to be anti-Group A carbohydrate tests. Because antibodies to the group A carbohydrate remain for several years in patients with persistent rheumatic valvular disease, this test should aid in the differentiation of rheumatic from non-rheumatic heart disease.

An exclusive or predominant nucleolar location of antinuclear antibodies is rare in the course of lupus disease: less than 1 p. cent of our patients. Thirteen cases of lupus disease with exclusive or predominant antinucleolar antibodies are analyzed: clinically, the only difference from other lupus diseases is the absence of pleurisy (p less than 0.03) and the frequent thrombopenia (p = 0.05). A proliferative glomerular lesion was found 4 times on renal biopsy. No patient presented any indications of overlapping sign with sclerodermia, polymyositis or Gougerot-Sjögren syndrome which are usually associated to a nuclear fluorescence of nucleolar type. 11 out of 13 patients have natural anti-DNA antibodies, including 9 with a very weak titer. Six patients present cytoplasmic anti-organic antibodies. Four patients have antibodies which precipitates on gelose, identifying nuclear or cytoplasmic antibodies: in 1 instance anti-SS-B et SS-A (Ro), in 1 instance anti-ribosomes associated with anti-ADN, in one instance anti-DNA. No serum contained anti-histones antibodies. The study of the sensitivity of nucleolar antigens to digestion by various enzymes (DNAse, RNAse and trypsin) showed that antinucleolar sera could be placed into three groups: 8/10 recognize a ribonucleic antigen, 1/10 a ribonucleoproteic antigen and 1/10 an antigen resisting to various enzymatic digestions. Therefore, in spite of its rarity, an exclusively nucleolar fluorescence should not rule out the diagnosis of lupus disease.

This study investigated the effects of immune complexes (ICs) on tumor necrosis factor α (TNF-α) and B cell-activating factor (BAFF) production from U937 cells and further explored the mechanism.

U937 cells were incubated with necrosis supernatant or systemic lupus erythematosus (SLE) sera alone, or their combination. The expression of TNF-α and <em>B</em>AFF was determined by Real-time polymerase chain reaction and enzyme-linked immunosorbent assay. High mobility group box protein 1(HMG<em>B</em>1) A-box was produced by gene recombination. HMG<em>B</em>1 A-box and anti-receptor for advanced glycation end products (RAGE) antibody were adopted in the blocking experiments. The importance of DNA for cytokine induction was investigated by DNase treatment.

The combination of necrosis supernatant and SLE sera induced the expression of TNF-α and <em>B</em>AFF significantly increased compared to necrosis supernatant or SLE sera alone. Recombinant HMG<em>B</em>1 A-box protein was purified, and TNF-α and <em>B</em>AFF production, which were induced by this combination, was blocked via HMG<em>B</em>1 A-box and anti-RAGE antibody. Moreover, we found that DNA component is important for the immunostimulatory activity of this combination.

ICs containing DNA can promote TNF-α and BAFF production in U937 cells, and this process can be mediated by HMGBBAFF production in SLE is proposed in this study whereby B cell activation, antibody production and ICs stimulated monocytes may create a vicious cycle that leads to B cell hyperactivity, which can be of importance for SLE etiopathogenesis.

Antibodies against the group polysaccharide of group A streptococci were estimated by means of a haemagglutination reaction. In this reaction human erythrocytes of blood group O were sensitized with polysaccharide esterified with myristoylchloride. The optimal conditions of the reactions were determined by varying the ester group content in the antigen and the amount of ester used for sensitization. The specificity of the reaction could be established by reacting sensitized erythrocytes with homologous and heterologous sera and by absorption experiments. Antistreptococcal group A polysaccharide titres (ASPAT) and antibody levels to streptolysine O and DNase-B were compared in a group of 52 children with proved streptococcal infection and in 52 age- and season-matched controls. Antibody levels were significantly higher in the patient group than in the controls. In the ASPAT there was clearly less overlap between patients and controls than in both other reactions. In the patient group the ASO titres were raised above normal in 27 cases (51.9%), anti-DNase-B titres in 18 (34.6%), and ASPAT in 40 (76.9%). Taken together the three reactions gave a positive score in 51 cases (98.1%) in the patient group against 17 cases (32.7%) in the controls. A positive antibody response is usually defined as a rise of two dilution increments between the acute and convalescent sera. According to this definition the ASPAT showed a response in 42%, ASO and/or DNase-B in 42%, and the three reactions taken together in 68% of paired sera from patients. It is believed the ASPAT will prove a welcome addition to the diagnostic outfit when the presence of streptococcal infection in children is considered.

Acute kidney injury (AKI) is increasingly emerging as a global emergency. Sepsis, major surgery, and nephrotoxic drugs are the main causes of AKI in hospitalized patients. However, glomerulonephritis accounts for about 10% of AKI episodes in adults, mainly related to rapidly progressive glomerulonephritis resulting from granulomatous polyangiitis (GPA, Wegener granulomatosis), microscopic polyangiitis (MPA), and anti-glomerular basement membrane (GBM) disease. Also, diffuse proliferative lupus nephritis, immunoglobulin A nephropathy, post-streptococcal glomerulonephritis, mixed cryoglobulinemia, mesangiocapillary glomerulonephritis, membranous nephropathy, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and scleroderma can induce acute renal failure. Early diagnosis of AKI due to glomerulonephritis is crucial for prompt, effective management to improve short- and long-term outcomes. Kidney biopsy is the gold standard for the diagnosis of glomerular disease, but it is not frequently performed in critically ill patients because of their clinical conditions. In this setting, a growing number of diagnostic assays can support the working hypothesis, including antineutrophil cytoplasmic antibodies (ANCAs), anti-double-stranded DNA antibodies, anti-GBM antibodies, antistreptolysin O and anti-DNase B antibodies, cryoglobulins, antiphospholipid antibodies, and complement levels. Therapeutic strategies in AKI patients with glomerulonephritis include high-dose corticosteroids, cyclophosphamide, and plasma exchange. This article reviews the wide spectrum of glomerulopathies associated with AKI, describing the immunological mechanisms underlying glomerular diseases and presenting an overview of the therapeutic options.

Keywords: AKI; antibodies; complement; glomerulonephritis; immunosuppression.

OBJECTIVE

We describe a woman with perianal and periumbilical dermatitis secondary to group G Streptococcus, summarize the salient features of this condition, and review other cutaneous conditions that clinically mimic streptococcal dermatitis of the umbilicus.

BACKGROUND

Periumbilical and perianal streptococcal dermatitis are conditions that commonly occur in children and usually result from beta-hemolytic group A Streptococcus. Rarely, non-group A streptococcal and staphylococcal infections have been reported in adults.

METHODS

A 31-year-old woman developed perianal and periumbilical group G streptococcal dermatitis. Symptoms were present for six months and were refractory to clotrimazole 1 percent and betamethasone dipropionate 0.05 percent cream.

RESULTS

The etiology of perianal and periumbilical dermatitis is unclear, but is perhaps explained by virulence of previously asymptomatic colonized bacteria. Perianal streptococcal dermatitis is more common in children. A number of adult infections have been reported, most of which were secondary to group A beta-hemolytic Streptococcus. Men are more often affected than women. Group G Streptococcus is rarely the infective etiology of perianal streptococcal dermatitis. This condition presents as a superficial well demarcated erythematous patch on clinical examination. Diagnosis is ascertained by diagnostic swabs and serological tests: antistreptolysin O (ASO) or anti-DNase titer. Treatments include oral amoxicillin, penicillin, erythromycin, and mupirocin ointment.

CONCLUSIONS

Our patient expands on the clinical presentation typical of streptococcal dermatitis. We describe a rare occurrence of an adult woman infected with non-group A Streptococcus. Several conditions can mimic the presentation of perianal streptococcal dermatitis. Although rare, group G Streptococcus should be considered in the setting of virulent infections usually attributed to group A species. Streptococcal dermatitis can be added to the list of conditions affecting the umbilicus.

Conditioned medium (CM) from 24-hr culture of guinea pig L2C B lymphoblastic leukemia cells contained an inhibitor(s) of mitogen- and antigen-stimulated proliferation of syngeneic (strain 2 guinea pigs), allogeneic (Hartley guinea pigs), and xenogeneic (Balb/c mouse, NZW rabbit) lymphocytes. The proliferation of several lymphoid and nonlymphoid cell lines also was inhibited in the presence of CM. The inhibitor(s) in CM was not toxic to any of the cultures studied. CM inhibited the mitogen-stimulated proliferation of lymphocytes when added to cultures up to 52 hr after addition of mitogen. Normal responsiveness to mitogens could be restored by washing the CM-treated lymphocytes with medium during the first 6 hr of culture. The addition of exogenous IL-2 to lymphocyte cultures did not overcome the CM-mediated suppression of mitogen- or antigen-stimulated proliferation. CM also inhibited the IL-2-dependent proliferation of murine CTLL-2 cells. Preincubation of guinea pig lymphocytes in CM did not inhibit the capacity of these cells to release IL-2 after exposure to mitogen. The antiproliferative activity of CM was stable to heating at low pH (100 degrees C, 10 min, pH 4.0), was resistant to treatment with papain, pronase, DNase, and RNase and did not bind to Con A-Sepharose. Incubation of the L2C cells in indomethacin did not inhibit the release of the inhibitor(s). The inhibitor(s) in CM had an apparent molecular weight of 500-3500 Da as determined by dialysis and ultrafiltration analysis. The inhibitory activity was recovered in the organic phase after extraction with chloroform:methanol and eluted distinct from the thymidine standard after gel filtration on Sephadex-G 25. These data suggest that the inhibitor(s) in CM is a nonspecific, low molecular weight, lipid-like component (not prostaglandin) that exerts its antiproliferative effects subsequent to cell activation. The inhibitor(s) did not appear to suppress other biologic functions associated with activation, such as IL-2 secretion. The inhibitor in CM may be important in promoting tumor survival in vivo by suppressing potential anti-tumor cellular immune responsiveness.

Mouse sarcoma 180 cells have a polypeptide that has the same molecular weight as actin but it is more acidic than alpha-actin. Its tryptic peptide pattern on reversed-phase HPLC was very similar to that of beta + gamma-actin, an actin sample prepared by affinity chromatography on DNase I-Sepharose contained the acidic polypeptide, and monoclonal anti-actin antibody reacted with it; therefore, the polypeptide is considered an actin isoform. The mRNA for this variant actin was identified by analyzing the polypeptides translated in vitro, which indicated that the variant actin is not a post-translationally modified form of any known actin. The variant actin was not stained by polyclonal anti-gizzard actin antibody which reacts with gamma-cytoplasmic, alpha-smooth and gamma-smooth muscle actins, nor by polyclonal anti-skeletal muscle actin antibody which reacts with skeletal, cardiac and alpha-smooth muscle actins. These results suggest that this variant actin is related to beta-cytoplasmic actin or, is a novel species whose N-terminal amino acid sequence is not Glu-Glu-Glu.

Bacterial adherence to extracellular matrix proteins (ECMp) plays important roles during host-pathogen interaction, however its genetic regulation remains poorly understood. yloA of the model bacterium Bacillus subtilis shows high homology to genes encoding fibronectin-binding proteins of Gram-positive pathogens. Here, we characterized the regulatory network of YloA-dependent adhesive properties of the probiotic B. subtilis natto (Bsn). YloA-proficient, but not YloA-deficient, Bsn specifically bound to ECMp in a concentration-dependent manner and were proficient in biofilm formation. yloA expression showed a continuous increase in activity during the growth phase and decreased during the stationary phase. The transcription factors AbrB and DegU downregulated yloA expression during the logarithmic and stationary growth phases respectively. Analysis of the yloA promoter region revealed the presence of AT-rich direct and inverted repeats previously reported to function as DegU-recognized binding sites. In spo0A cells, yloA expression was completely turned off because of upregulation of AbrB throughout growth. Accordingly, DNase I footprinting analysis confirmed that AbrB bound to the promoter region of yloA. Interestingly, Bsn bound fibronectin with higher affinity, lower Kd, than several bacterial pathogens and competitively excluded them from binding to immobilized-fibronectin, a finding that might be important for the anti-infective properties of B. subtilis and its relatives.

Among the four known Streptococcal nucleases comprising of DNase A, B, C and D; DNase B is the most common, and determination of the levels of antibody to DNase B (ADB) is often used to confirm a clinical diagnosis of Streptococcus pyogenes/group A Streptococcal (GAS) infection. The commonly used assays for antibodies that neutralize DNase B or streptolysin O activity use partially purified antigens that often fail to detect antibody changes subsequent to culture documented infections. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed employing his-tagged recombinant DNase B as plate antigen for comparison to the commonly used DNA methyl green micromethod (DMGM). DNAs from various Streptococcal species were screened for presence of dnaseB gene by PCR. Measurements of ADB in sera collected from subjects belonging to different ages, and ethnic groups were used to compare the two methods. dnaseB was not detected by PCR in DNA samples isolated from different strains of group B (GBS), C (GCS) and G (GGS) Streptococci. The ADB based ELISA proved to be highly sensitive and more responsive to changes in antibody concentration than DMGM. Use of recombinant DNase B eliminates the variability associated with the enzyme, partially purified from Streptococcal culture supernatants from various commercial sources and may provide a more reliable source of antigen to a wider group of laboratories concerned with GAS diagnosis.

The formation of neutrophil extracellular traps (NETs) is a strategy utilized <em>b</em>y neutrophils for capturing infective agents. Extracellular traps consist in a physical net made of DNA and intracellular proteins externalized from neutrophils, where <em>b</em>acteria and viruses are entrapped and killed <em>b</em>y proteolysis. A complex series of events contri<em>b</em>utes to achieving NET formation: signaling from infectious triggers comes first, followed <em>b</em>y decondensation of chromatin and extrusion of the nucleosome components (DNA, histones) from the nucleus and, after cell mem<em>b</em>rane <em>b</em>reakdown, outside the cell. NETs are composed of either DNA or nucleosome proteins and hundreds of cytoplasm proteins, a part of which undergo post-translational modification during the steps leading to NETs. There is a thin <em>b</em>alance <em>b</em>etween the production and the removal of circulating NETs from <em>b</em>lood where digestion of DNA <em>b</em>y circulating <em>DNases</em> 1 and IL3 has a critical role. A delay in NET removal may have consequences for autoimmunity. Recent studies have shown that circulating NET levels are increased in systemic lupus erythematosus (SLE) for a functional <em>b</em>lock of NET removal mediated <em>b</em>y <em>anti</em>-<em>DNase</em> <em>anti</em><em>b</em>odies or, in rare cases, <em>b</em>y <em>DNase</em> IL3 mutations. In SLE, the persistence in circulation of NETs signifies elevated concentrations of either free DNA/nucleosome components and oxidized proteins that, in some cases, are recognized as non-self and presented to <em>B</em>-cells <em>b</em>y Toll-like receptor 9 (TLR9). In this way, it is activated as an immunologic response, leading to the formation of IgG2 auto-<em>anti</em><em>b</em>ody. Monitoring serum NET levels represents a potential new way to herald the development of renal lesions and has clinical implications. Modulating the <em>b</em>alance <em>b</em>etween NET formation and removal is one of the o<em>b</em>jectives of <em>b</em>asic research that are aimed to design new drugs for SLE. (<em>b</em>)Clinical Trial Registration Num<em>b</em>er:</<em>b</em>) The Zeus study was registered at https://clinicaltrials.gov (study num<em>b</em>er: <a href="http://clinicaltrials.gov/show/NCT02403115" title="See in ClinicalTrials.gov">NCT02403115</a>).

Keywords: Lupus nephritis; anti-C1q antibodies; anti-alpha enolase; anti-histone; biomarker; systemic lupu erythematosus.

<p><div>(<em>b</em>)O<em>b</em>jective</<em>b</em>)</div>Extracellular DNA (eDNA) has <em>b</em>een shown to <em>b</em>e important for <em>b</em>iofilm sta<em>b</em>ility of the endodontic pathogen <i>Enterococcus faecalis</i>. In this study, we hypothesized that treatment with <em>DNase</em> prevents adhesion and disperses young <i>E. faecalis</i> <em>b</em>iofilms in 96-well plates and root canals of extracted teeth.</p><p><div>(<em>b</em>)Methods</<em>b</em>)</div><i>E. faecalis</i> eDNA in 96-well plates was visualized with TOTO-1®. The effect of <em>DNase</em> treatment was assessed in 96-well plates and in extracted single-rooted premolars (n=37) using a two-phase crossover design. <i>E. faecalis</i> was treated with <em>DNase</em> (50 Kunitz/mL) or heat-inactivated <em>DNase</em> for 1 h during adhesion or after 24 h of <em>b</em>iofilm formation. In 96-well plates, adhering cells were qu<em>anti</em>fied using confocal microscopy and digital image analysis. In root canals, the num<em>b</em>er of adhering cells was determined in dentine samples <em>b</em>ased on colony forming unit counts. Data from the 96-well plate were analyzed using one-tailed t-tests, and data from extracted teeth were analyzed using mixed-effect Poisson regressions.</p><p><div>(<em>b</em>)Results</<em>b</em>)</div>eDNA was present in wells colonized <em>b</em>y <i>E. faecalis</i> after 1 h of adhesion and 24 h of <em>b</em>iofilm formation; it was removed <em>b</em>y <em>DNase</em> treatment, as evidenced <em>b</em>y TOTO®-1 staining. <em>DNase</em> treatment reduced the area covered <em>b</em>y cells in 96-well plates after 1 h (P<0.05), <em>b</em>ut not after 24 h (P=0.96). No significant differences in the num<em>b</em>er of adhering cells were o<em>b</em>served in extracted teeth after 1 (P=0.14) and 24 h (P=0.98).</p><p><div>(<em>b</em>)Conclusion</<em>b</em>)</div><em>DNase</em> treatment does not disperse endodontic <i>E. faecalis</i> <em>b</em>iofilms. The sole use of <em>DNase</em> as an <em>anti</em>-<em>b</em>iofilm agent in root canal treatments is not recommenda<em>b</em>le.</p>

Inflammatory bowel disease (IBD) that presents in children <6 years of age is known as very early-onset IBD (VEO-IBD). Extraintestinal manifestations in IBD, such as erythema nodosum (EN), pyoderma gangrenosum (PG), and, less likely, leukocytoclastic vasculitis (LV), are more commonly present in Crohn's disease. Association between LV and ulcerative colitis (UC) is not commonly seen. We report a case of a 6-year-old female with a VEO-IBD UC phenotype presenting with multiple episodes of leukocytoclastic vasculitis, each preceded by streptococcal pharyngitis. Prior to the diagnosis of VEO-IBD, a skin biopsy was obtained and had shown leukocytoclastic vasculitis with a negative IgA stain. Initial laboratory results were remarkable for leukocytosis and increased anti-strep O and anti-DNase B titers. Gastrointestinal panel PCR demonstrated Clostridium difficile toxin A/B. Treatment for LV consisted of methylprednisolone IV 20 mg for four days with a weaning schedule of prednisolone for two weeks and naproxen 250 mg BID for three days. Clostridium difficile was treated with metronidazole 250 mg TID for ten days. She remained stable for three years until she presented with continuous bloody stools, newly onset chest pain, and shortness of breath. Computed tomography angiogram (CTA) was normal. Stool calprotectin was elevated at 658 mcg/gm. Abdominal magnetic resonance enterography (MRE), esophagogastroduodenoscopy, and colonoscopy confirmed a VEO-IBD ulcerative colitis phenotype. She was started on infliximab 10 mg/kg every four weeks after infliximab titers, and antibodies were obtained. Currently, the patient remains on clinical and biochemical remission, with no recent LV episodes or recurrence of streptococcal pharyngitis. Our patient is unique as no case report has been published with multiple episodes of leukocytoclastic vasculitis in association with a VEO-IBD UC phenotype.

Balsamin, a type I ribosome-inactivating protein (RIP), has been shown to inhibit HIV-1 replication at the translation step. Our recent studies have shown that balsamin also possess anti-tumor, antibacterial and DNase-like activity, however, the amount of natural balsamin in Momordica balsamina seeds is limited and preclinical studies require large quantities of pure, bioactive balsamin. Therefore, in this study, we cloned the balsamin gene, expressed it in E.coli BL21 (DE3) strain and purified it by nickel affinity chromatography. Functional analysis indicated that balsamin exhibits both RNA N-glycosidase activity, releasing the Endo-fragment from rabbit reticulocyte rRNA, and DNase-like activity, converting the supercoiled form of a plasmid into the linear form in a concentration-dependent manner. Analysis of secondary structure revealed that recombinant balsamin mainly consisted of α-helical and random coiled with minimal turns and β-sheets. Recombinant balsamin was found to be stable in the temperature range of 20-60 °C and pH range of 6-9. Antimicrobial assays showed that the minimum inhibitory concentrations of recombinant balsamin for various pathogens ranged between 1.56 and 12.5 μg/ml. Heterologous expression and purification of balsamin carries great importance as it provides an alternative approach for large-scale preparation of biologically active recombinant balsamin, which is difficult from its natural source.

Background: Curcumin has been demonstrated to exert anti-oxidant, anti-fibrotic, anti-inflammatory, and anti-cancer activities. This study was conducted to observe the effect and inner mechanism of curcumin in rats with radiation-induced liver damage (RILD).

Methods: Thirty SD rats were classified into Control, Radiation group and Curcumin (Cur) + Radiation group (n = 10 in each group). The changes in body weight of the rats were observed on the 3rd, 7th and 14th days after the treatment with curcumin. On the 14th day post treatment, the heart blood of the rats was drawn for measurement of liver function indices including total protein (TP), alanine aminotransfetase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) as well as aspartate aminotransfetase (AST). Subsequently, the rats were euthanized and liver tissues were taken to observe liver morphological changes using hematoxylin-eosin (HE), and to analyze apoptosis condition using transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assays. Meanwhile, the oxidative stress level in liver tissue homogenate was determined by biochemical analysis. The expression of nuclear factor kappa B (NF-κB) pathway-associated and apoptosis-associated proteins was detected using Western blot analysis, and the expression levels of inflammatory factors were measured by Enzyme-linked immunosorbent assay (ELISA).

Results: The reduced body weight was observed in rats of the Radiation group compared to the Control and Cur + Radiation groups on day 14. In the Radiation group, hepatic cell edema and inflammatory cell infiltration could be visible under the light microscope, and the hepatocytes presented with vacuolar degeneration. In the Cur + Radiation group, the hepatocytes swelled under the microscope, but the pathological changes were alleviated in comparison with the Radiation group. RILD rats with curcumin treatment presented with decreased ALT, AST, ALP, LDH, and maleicdialdehyde (MDA) levels, and elevated TP, superoxide dismutase (SOD), caspase activated DNase (CAD) and glutathione (GSH) levels. Apoptosis and inflammation in rats with RILD were up-regulated, and the NF-κB pathway was activated, but they were reversed after continuously intragastric administration of curcumin for 14 days.

Conclusion: Our study highlights that curcumin treatment reduces the liver damage caused by radiation through the inhibition of the NF-κB pathway.

Keywords: Apoptosis; Curcumin; Inflammation; NF-κB pathway; Oxidative stress; Radiation-induced liver damage.

Bollo is a traditional Goan fermented food in which coarse wheat/wheat and finger millet is leavened with toddy. We here isolated 42 yeast strains from Bollo batter. Initial screening of the isolates with probiotic properties yielded four yeast isolates (DABRP1, DABRP2, DABRP5 and DABRP12). These isolates exhibited tolerance to high bile salt concentration and acidic pH and resistance to various antibiotics, which indicated their probiotic nature. All these yeast isolates were identified as Saccharomyces cerevisiae through D1D2-LSU-rDNA sequencing. These yeast isolates also showed higher percent hydrophobicity towards chloroform followed by n-hexadecane and o-xylene indicating their mucosal surface-adhesive property. To evaluate the safety of the isolates for them to be called as generally recognized as safe, the pathogenic behavior of the isolates determined through the in vitro hemolysis assay and evaluation of DNase and gelatinase activities. None of the isolates exhibited hemolysis or produced DNase or gelatinase and thus were considered potentially safe. In terms of beneficial effects, the most potent isolate S. cerevisiae DABRP5 showed antibacterial activity against the test pathogens. It also showed excellent antioxidant activity with DPPH free radical scavenging activity of 68.85 ± 0.69%, anti-inflammatory activity with 60.39 ± 0.34% inhibition of protein denaturation, and antidiabetic activity with 71.75 ± 0.45% inhibition of α-amylase activity. The isolate produced α-amylase, lipase, and β-galactosidase. The probiotic potential of the isolate S. cerevisiae DABRP5 was similar to that of the reference strain (Saccharomyces boulardii CNCM I-745) used in this study. The results thus indicate that yeast isolates from Bollo batter have probiotic potential.

Keywords: Bollo batter; Fermented foods; Goa; Probiotics; Saccharomyces cerevisiae; Therapeutic properties.

Numerous reagents are used in the collection processing and storage of hematopoietic progenitor cells for transplantation. To decrease potential variations in the final component for transplantation, these reagents should be uniform in safety, potency, and efficacy. Pharmaceutical-grade reagents are ideal but often are not available. Recently, recombinant human deoxyribonuclease (DNase) was approved for the treatment of patients suffering from the pulmonary complications of cystic fibrosis. We tested this pharmaceutical for toxicity to hematopoietic progenitor cells. These cells were exposed to a range of incubation concentrations for both the recombinant enzyme and bovine DNase previously used in this laboratory. No loss of nucleated cells or hematopoietic progenitors was observed after short-term incubation (1 h) or with direct addition to the culture medium. No incremental toxicity was observed in using recombinant enzyme with murine anti-B cell antibodies and rabbit complement in an immunologic purge technique. A variable effect on cell recovery after thawing of cryopreserved bone marrow cells was observed for both enzyme sources. These data suggest that the pharmaceutical-grade, recombinant human DNase may substitute for previously used reagent-grade protein from animal sources.