Can montelukast sodium be an alternative treatment in the treatment of interstitial cystitis?

Background: The leukotriene D4 receptors have been detected in human bladder detrusor myocytes, and they can play the role of interstitial cystitis etiology. Aim: Our study aims to explain the role of mast cells histologically and immunohistochemically in the pathogenesis and the effectiveness of montelukast that leukotriene D4 receptor antagonist in the treatment of interstitial cystitis. Subjects and Methods: Twenty-four Wistar albino adult female rats were used. Group 1 (n = 8): control (sham) group, Group 2 (n = 8): interstitial cystitis group, and Group 3 (n = 8): treatment group. Groups 2 and 3 rats were administered 75 mg/kg cyclophosphamide four times every three days intraperitoneally. The rats in the treatment group were started on montelukast sodium as 10 mg/kg, 1 × 1/day per orally after the last administration of cyclophosphamide and were given for 14 days. Mast cells in the bladder tissues were examined histologically, and the presence of IL-6, 8, VEGF, and TNF alpha was examined immunohistochemically. Results: Thin transitional epithelium, loose connective tissue, weak smooth muscle bundles, and signs of chronic inflammation were observed in the interstitial cystitis group. Regenerated transitional epithelium, intact basement membrane, compact lamina propia, thick smooth muscle bundles, and rare inflammatory cells were observed after the treatment with the montelukast. Mast cells were decreased in bladder tissue after treatment. IL-6, IL-8, VEGF, and TNF alpha levels were significantly decreased after treatment. Conclusions: We found that inflammatory mediators were significantly reduced after treatment with montelukast in the interstitial cystitis group. Montelukast can be used as an effective drug in the treatment of interstitial cystitis.

Development of an improved and specific inhibitor of NADPH oxidase 2 to treat traumatic brain injury.

NADPH oxidases (NOX's), and the reactive oxygen species (ROS) they produce, play an important role in host defense, thyroid hormone synthesis, apoptosis, gene regulation, angiogenesis and other processes. However, overproduction of ROS by these enzymes is associated with cardiovascular disease, fibrosis, traumatic brain injury (TBI) and other diseases. Structural similarities between NOX's have complicated development of specific inhibitors. Here, we report development of NCATS-SM7270, a small molecule optimized from GSK2795039, that inhibited NOX2 in primary human and mouse granulocytes. NCATS-SM7270 specifically inhibited NOX2 and had reduced inhibitory activity against xanthine oxidase in vitro. We also studied the role of several NOX isoforms during mild TBI (mTBI) and demonstrated that NOX2 and, to a lesser extent, NOX1 deficient mice are protected from mTBI pathology, whereas injury is exacerbated in NOX4 knockouts. Given the pathogenic role played by NOX2 in mTBI, we treated mice transcranially with NCATS-SM7270 after injury and revealed a dose-dependent reduction in mTBI induced cortical cell death. This inhibitor also partially reversed cortical damage observed in NOX4 deficient mice following mTBI. These data demonstrate that NCATS-SM7270 is an improved and specific inhibitor of NOX2 capable of protecting mice from NOX2-dependent cell death associated with mTBI.

Granulibacter bethesdensis, a Pathogen from Patients with Chronic Granulomatous Disease, Produces a Penta-Acylated Hypostimulatory Glycero-D-talo-oct-2-ulosonic Acid-Lipid A Glycolipid (Ko-Lipid A).

Granulibacter bethesdensis can infect patients with chronic granulomatous disease, an immunodeficiency caused by reduced phagocyte NADPH oxidase function. Intact G. bethesdensis (Gb) is hypostimulatory compared to Escherichia coli, i.e., cytokine production in human blood requires 10-100 times more G. bethesdensis CFU/mL than E. coli. To better understand the pathogenicity of G. bethesdensis, we isolated its lipopolysaccharide (GbLPS) and characterized its lipid A. Unlike with typical Enterobacteriaceae, the release of presumptive Gb lipid A from its LPS required a strong acid. NMR and mass spectrometry demonstrated that the carbohydrate portion of the isolated glycolipid consists of α-Manp-(1→4)-ß-GlcpN3N-(1→6)-α-GlcpN-(1⇿1)-α-GlcpA tetra-saccharide substituted with five acyl chains: the amide-linked N-3' 14:0(3-OH), N-2' 16:0(3-O16:0), and N-2 18:0(3-OH) and the ester-linked O-3 14:0(3-OH) and 16:0. The identification of glycero-d-talo-oct-2-ulosonic acid (Ko) as the first constituent of the core region of the LPS that is covalently attached to GlcpN3N of the lipid backbone may account for the acid resistance of GbLPS. In addition, the presence of Ko and only five acyl chains may explain the >10-fold lower proinflammatory potency of GbKo-lipidA compared to E. coli lipid A, as measured by cytokine induction in human blood. These unusual structural properties of the G.bethesdensis Ko-lipid A glycolipid likely contribute to immune evasion during pathogenesis and resistance to antimicrobial peptides.

Low Plasma Gelsolin Concentrations in Chronic Granulomatous Disease.

Plasma gelsolin (pGSN) is the secreted isoform of an intracellular actin remodeling protein found in high concentrations in human plasma. Clinical studies demonstrate reduced pGSN concentrations in several disease states, including severe trauma, burns, and sepsis. Markedly decreased pGSN concentrations in these conditions precede and predict adverse clinical outcomes. In this study, we measured pGSN in patients with chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent infections and dysregulated inflammation. pGSN was quantified using a sandwich ELISA in plasma from healthy volunteers, clinically stable CGD patients, and X-linked CGD carriers and in sera from 12 CGD patients undergoing bone marrow transplantation. pGSN was also quantified in healthy volunteers challenged with intravenous endotoxin. pGSN concentrations were lower in CGD patients without active infection or systemic inflammation compared with healthy control subjects. In CGD patients undergoing bone marrow transplantation, pGSN concentrations increased significantly following successful transplant. X-linked carriers of CGD had normal pGSN. Despite reduction of pGSN in CGD patients, we did not detect significant changes in pGSN over 24 h following challenge of healthy volunteers with intravenous endotoxin (4 ng/kg) that elicited a febrile response. We describe, for the first time, significantly lower pGSN in clinically stable patients with CGD compared with age- and sex-matched healthy volunteers. Low pGSN levels in CGD patients significantly increased following bone marrow transplantation. X-linked carriers of CGD had normal pGSN. In healthy volunteers challenged with intravenous endotoxin, pGSN is not an acute phase reactant.

An immune-based biomarker signature is associated with mortality in COVID-19 patients.

Immune and inflammatory responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contribute to disease severity of coronavirus disease 2019 (COVID-19). However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity and assessed type I IFN-, type II IFN-, and NF-κB-dependent whole-blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and nonhematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients' first samples revealed 12 biomarkers (CCL2, IL-15, soluble ST2 [sST2], NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and 2 additional biomarkers (lactoferrin, CXCL9) that were substantially associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.

Immunopathogenesis of canine chronic ulcerative stomatitis.

Canine Chronic Ulcerative Stomatitis is a spontaneously occurring inflammatory disease of the oral mucosa. An immune-mediated pathogenesis is suspected though not yet proven. We have recently reported on the clinical and histologic features, and identification of select leukocyte cell populations within the lesion. A clinical and histologic similarity to oral lichen planus of people was proposed. In the present study, these initial observations are extended by examining lesions from 24 dogs with clinical evidence of chronic ulcerative stomatitis. Because dogs with chronic ulcerative stomatitis often have concurrent periodontal disease, we wondered if dental plaque/biofilm may be a common instigator of inflammation in both lesions. We hypothesized that dogs with chronic ulcerative stomatitis would exhibit a spectrum of pathologic changes and phenotype of infiltrating leukocytes that would inform lesion pathogenesis and that these changes would differ from inflammatory phenotypes in periodontitis. Previously we identified chronic ulcerative stomatitis lesions to be rich in FoxP3+ and IL17+ cells. As such, we suspect that these leukocytes play an important role in lesion pathogenesis. The current study confirms the presence of moderate to large numbers of FoxP3+ T cells and IL17+ cells in all ulcerative stomatitis lesions using confocal immunofluorescence. Interestingly, the majority of IL17+ cells were determined to be non-T cells and IL17+ cell frequencies were negatively correlated with severity on the clinical scoring system. Three histologic subtypes of ulcerative stomatitis were determined; lichenoid, deep stomatitis and granulomatous. Periodontitis lesions, like stomatitis lesions, were B cell and plasma cell rich, but otherwise differed from the stomatitis lesions. Direct immunofluorescence results did not support an autoantibody-mediated autoimmune disease process. This investigation contributes to the body of literature regarding leukocyte involvement in canine idiopathic inflammatory disease pathogenesis.

Multifocal discrete osteolysis in a horse with silicate associated osteoporosis.

Silicate associated osteoporosis (SAO) was diagnosed post mortem in an adult horse with the shortest documented exposure to cytotoxic silicates of 2 years. The horse was evaluated for a 6-months history of progressive back tenderness and acute onset of lameness. The horse had a marked (4/5) [American Association of Equine Practitioners scale] left forelimb lameness, moderate (2/5) hindlimb ataxia and weakness, and cervical pain upon palpation. Physical examination did not reveal clinical skeletal deformities or respiratory compromise. Radiographs revealed widespread, discrete, sharply delineated, osteolytic lesions in the skull, vertebral column, ribs, scapulae and middle phalanx (P2) of the left forelimb and a diffuse bronchointerstitial lung pattern. The presumptive clinical diagnosis was widespread, metastatic osteolytic neoplasia. Due to the poor quality of life and grave prognosis, the horse was humanely euthanised. Post mortem examination revealed pulmonary silicosis in the lungs and hilar lymph nodes and osteolytic lesions with numerous, large osteoclasts and disorganised bone remodeling both consistent with SAO. SAO should be included as a differential diagnosis for horses with widespread, multifocal, discrete osteolysis and history of exposure to endemic regions with possible cytotoxic silicate inhalation. Exposure time of 2 years is potentially sufficient to develop SAO.

TNF overproduction impairs epithelial staphylococcal response in hyper IgE syndrome.

Autosomal dominant hyper IgE syndrome (AD-HIES), or Job's syndrome, is a primary immune deficiency caused by dominant-negative mutations in STAT3. Recurrent Staphylococcus aureus skin abscesses are a defining feature of this syndrome. A widely held hypothesis that defects in peripheral Th17 differentiation confer this susceptibility has never been directly evaluated. To assess the cutaneous immune response in AD-HIES, we induced suction blisters in healthy volunteers (HVs) and patients with AD-HIES and then challenged the wound with lethally irradiated bacteria. We show that cutaneous production of IL-17A and IL-17F was normal in patients with AD-HIES. Overproduction of TNF-α differentiated the responses in AD-HIES from HVs. This was associated with reduced IL-10 family signaling in blister-infiltrating cells and defective epithelial cell function. Mouse models of AD-HIES recapitulated these aberrant epithelial responses to S. aureus and involved defective epithelial-to-mesenchymal transition (EMT) rather than a failure of bacterial killing. Defective responses in mouse models of AD-HIES and primary keratinocyte cultures from patients with AD-HIES could be reversed by TNF-α blockade and by drugs with reported modulatory effects on EMT. Our results identify these as potential therapeutic approaches in patients with AD-HIES suffering S. aureus infections.

Early Intracellular Trafficking of Granulibacter bethesdensis in Human Macrophages.

Granulibacter bethesdensis is a Gram-negative bacterium that infects patients with chronic granulomatous disease (CGD), a primary immunodeficiency marked by a defect in NOX2, the phagocyte NADPH oxidase. Previous studies have shown that NOX2 is essential for killing of G. bethesdensis by neutrophils and monocytes and that the bacteriostatic activity of monocyte-derived macrophages (MDM) requires NOX2 and gamma interferon (IFN-γ) pretreatment. To determine whether G. bethesdensis evades phagolysosomal killing, a host defense pathway intact in both normal and CGD MDM, or whether it occupies a distinct intracellular niche in CGD MDM, we assessed the trafficking patterns of this organism. We observed colocalization of G. bethesdensis with an early endosome antigen 1 (EEA1)-positive compartment, followed by colocalization with lysosome-associated membrane protein 1 (LAMP1)-positive and LysoTracker-positive late phagosomes; these characteristics were similar in both normal and CGD MDM. Despite localization to acidified late phagosomes, viable G. bethesdensis cells were recovered from viable MDM in numbers greater than in the initial input up to 6 days after infection. G. bethesdensis remains, and in some cases appears to divide, within a membrane-bound compartment for the entire 6-day time course. These findings indicate that this organism resists both oxygen-dependent and oxygen-independent phagolysosomal antimicrobial systems of human macrophages.

CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease.

Gene repair of CD34+ hematopoietic stem and progenitor cells (HSPCs) may avoid problems associated with gene therapy, such as vector-related mutagenesis and dysregulated transgene expression. We used CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated 9) to repair a mutation in the CYBB gene of CD34+ HSPCs from patients with the immunodeficiency disorder X-linked chronic granulomatous disease (X-CGD). Sequence-confirmed repair of >20% of HSPCs from X-CGD patients restored the function of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and superoxide radical production in myeloid cells differentiated from these progenitor cells in vitro. Transplant of gene-repaired X-CGD HSPCs into NOD (nonobese diabetic) SCID (severe combined immunodeficient) γc-/- mice resulted in efficient engraftment and production of functional mature human myeloid and lymphoid cells for up to 5 months. Whole-exome sequencing detected no indels outside of the CYBB gene after gene correction. CRISPR-mediated gene editing of HSPCs may be applicable to other CGD mutations and other monogenic disorders of the hematopoietic system.

Clinical and Histopathologic Characterization of Canine Chronic Ulcerative Stomatitis.

Canine chronic ulcerative stomatitis, also known as chronic ulcerative paradental stomatitis, is a painful condition of the oral cavity. The purpose of this study was to determine if there are commonalities in clinical and radiographic features among patients, whether the histopathologic evaluation might inform the pathogenesis, and whether the condition appears similar to human oral mucosal diseases. To do this, we prospectively collected clinical, radiographic, and histopathologic data from 20 dogs diagnosed with the disease. Clinical data were based on a clinical disease activity index, oral and periodontal examination parameters, and full-mouth dental radiographs. The histopathological and immunohistochemical data were based on oral mucosal samples obtained from erosive or ulcerated areas. Our findings revealed that canine chronic stomatitis is clinically characterized by painful oral mucosal ulcers of varying size, pattern, appearance, and distribution, most often associated with teeth with early periodontitis. Histologic examination revealed a subepithelial lichenoid band (interface mucositis) where B cells, T cells, and Forkhead-box protein 3 (FoxP3)- and interleukin-17-expressing cells were present. These cells might play a role in the underlying immune response and an immune-mediated pathogenesis is suspected. The clinical and histopathologic features of this chronic inflammatory mucosal disease in dogs resemble those of oral lichen planus in humans.

Cytoskeletal abnormalities and neutrophil dysfunction in WDR1 deficiency.

Cell motility, division, and structural integrity depend on dynamic remodeling of the cellular cytoskeleton, which is regulated in part by actin polymerization and depolymerization. In 3 families, we identified 4 children with recurrent infections and varying clinical manifestations including mild neutropenia, impaired wound healing, severe stomatitis with oral stenosis, and death. All patients studied had similar distinctive neutrophil herniation of the nuclear lobes and agranular regions within the cytosol. Chemotaxis and chemokinesis were markedly impaired, but staphylococcal killing was normal, and neutrophil oxidative burst was increased both basally and on stimulation. Neutrophil spreading on glass and cell polarization were also impaired. Neutrophil F-actin was elevated fourfold, suggesting an abnormality in F-actin regulation. Two-dimensional differential in-gel electrophoresis identified abnormal actin-interacting protein 1 (Aip1), encoded by WDR1, in patient samples. Biallelic mutations in WDR1 affecting distinct antiparallel ß-strands of Aip1 were identified in all patients. It has been previously reported that Aip1 regulates cofilin-mediated actin depolymerization, which is required for normal neutrophil function. Heterozygous mutations in clinically normal relatives confirmed that WDR1 deficiency is autosomal recessive. Allogeneic stem cell transplantation corrected the immunologic defect in 1 patient. Mutations in WDR1 affect neutrophil morphology, motility, and function, causing a novel primary immunodeficiency.

Thoracic Surgery in Chronic Granulomatous Disease: a 25-Year Single-Institution Experience.

INTRODUCTION: Chronic granulomatous disease (CGD) is a genetic disorder in which phagocyte dysfunction leads to recurrent infection. Persistent pulmonary infections sometimes require thoracic surgical intervention. We reviewed our 25-year experience to identify outcomes and prognostic factors associated with thoracic surgery in these patients. METHODS: A retrospective single-institution review of all patients with CGD from 1990 through 2015 was performed. Univariate analysis identified prognostic variables to include in a Cox model. Overall survival was estimated by the Kaplan-Meier method. RESULTS: We identified 258 patients who had 2221 admissions (both scheduled and emergent). During the period examined, 51 thoracic operations were performed in 13.6 % (35/258) of patients and 2.3 % (35/2221) of overall admissions. Patients undergoing surgery did not have statistically significant differences in disease genotype compared to those that did not require surgery. Pathogens were identified from 67 % (34/51) of specimens. Complications occurred in 27 % (14/51), including 10 % (5/51) with wound and 12 % (6/51) with pulmonary infections. Mortality at 30 and 90 days was 0 and 6 % (3/51), respectively. Overall survival probabilities were 75 and 62 % at 5- and 10-year follow-up (median potential follow-up: 16.5 years), respectively. Undergoing thoracic surgery was associated with an increased hazard ratio for death of 3.71 (p < 0.0001). Both chest wall resection and EBL > 500 mL were negative prognostic factors (p < 0.05). CONCLUSIONS: A minority of CGD patients required thoracic surgery for infections refractory to antibiotic or antifungal therapy. Patients who had these operations had significant morbidity and relatively poor long-term survival, particularly in the cases of chest wall resection or significant blood loss.

Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency (SCID-X1) is a profound deficiency of T, B, and natural killer (NK) cell immunity caused by mutations inIL2RGencoding the common chain (γc) of several interleukin receptors. Gamma-retroviral (γRV) gene therapy of SCID-X1 infants without conditioning restores T cell immunity without B or NK cell correction, but similar treatment fails in older SCID-X1 children. We used a lentiviral gene therapy approach to treat five SCID-X1 patients with persistent immune dysfunction despite haploidentical hematopoietic stem cell (HSC) transplant in infancy. Follow-up data from two older patients demonstrate that lentiviral vector γc transduced autologous HSC gene therapy after nonmyeloablative busulfan conditioning achieves selective expansion of gene-marked T, NK, and B cells, which is associated with sustained restoration of humoral responses to immunization and clinical improvement at 2 to 3 years after treatment. Similar gene marking levels have been achieved in three younger patients, albeit with only 6 to 9 months of follow-up. Lentiviral gene therapy with reduced-intensity conditioning appears safe and can restore humoral immune function to posthaploidentical transplant older patients with SCID-X1.

Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease.

Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus(+) HSPCs with 6-16% human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 resulted in ∼15% gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs, 4-11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.

Th17 Pathway As a Target for Multipotent Stromal Cell Therapy in Dogs: Implications for Translational Research.

Detrimental Th17 driven inflammatory and autoimmune disease such as Crohn's disease, graft versus host disease and multiple sclerosis remain a significant cause of morbidity and mortality worldwide. Multipotent stromal/stem cell (MSC) inhibit Th17 polarization and activation in vitro and in rodent models. As such, MSC based therapeutic approaches are being investigated as novel therapeutic approaches to treat Th17 driven diseases in humans. The significance of naturally occurring diseases in dogs is increasingly recognized as a realistic platform to conduct pre-clinical testing of novel therapeutics. Full characterization of Th17 cells in dogs has not been completed. We have developed and validated a flow-cytometric method to detect Th17 cells in canine blood. We further demonstrate that Th17 and other IL17 producing cells are present in tissues of dogs with naturally occurring chronic inflammatory diseases. Finally, we have determined the kinetics of a canine specific Th17 polarization in vitro and demonstrate that canine MSC inhibit Th17 polarization in vitro, in a PGE2 independent mechanism. Our findings provide fundamental research tools and suggest that naturally occurring diseases in dogs, such as inflammatory bowel disease, may be harnessed to translate novel MSC based therapeutic strategies that target the Th17 pathway.

Isolation and Functional Analysis of Human Neutrophils.

This unit describes the isolation of human polymorphonuclear neutrophils (PMN) from blood using dextran sedimentation and Percoll or Ficoll-Paque density gradients. Assays of neutrophil functions including respiratory burst activation, phagocytosis, and microbial killing are also described.

Simultaneous Host-Pathogen Transcriptome Analysis during Granulibacter bethesdensis Infection of Neutrophils from Healthy Subjects and Patients with Chronic Granulomatous Disease.

Polymorphonuclear leukocytes (PMN) from patients with chronic granulomatous disease (CGD) fail to produce microbicidal concentrations of reactive oxygen species (ROS) due to mutations in NOX2. Patients with CGD suffer from severe, life-threatening infections and inflammatory complications. Granulibacter bethesdensis is an emerging Gram-negative pathogen in CGD that resists killing by PMN of CGD patients (CGD PMN) and inhibits PMN apoptosis through unknown mechanisms. Microarray analysis was used to study mRNA expression in PMN from healthy subjects (normal PMN) and CGD PMN during incubation with G. bethesdensis and, simultaneously, in G. bethesdensis with normal and CGD PMN. We detected upregulation of antiapoptotic genes (e.g., XIAP and GADD45B) and downregulation of proapoptotic genes (e.g., CASP8 and APAF1) in infected PMN. Transcript and protein levels of inflammation- and immunity-related genes were also altered. Upon interaction with PMN, G. bethesdensis altered the expression of ROS resistance genes in the presence of normal but not CGD PMN. Levels of bacterial stress response genes, including the ClpB gene, increased during phagocytosis by both normal and CGD PMN demonstrating responses to oxygen-independent PMN antimicrobial systems. Antisense knockdown demonstrated that ClpB is dispensable for extracellular growth but is essential for bacterial resistance to both normal and CGD PMN. Metabolic adaptation of Granulibacter growth in PMN included the upregulation of pyruvate dehydrogenase. Pharmacological inhibition of pyruvate dehydrogenase by triphenylbismuthdichloride was lethal to Granulibacter. This study expands knowledge of microbial pathogenesis of Granulibacter in cells from permissive (CGD) and nonpermissive (normal) hosts and identifies potentially druggable microbial factors, such as pyruvate dehydrogenase and ClpB, to help combat this antibiotic-resistant pathogen.

An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease.

There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70-80% of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34(+) cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all five genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.

Serial haemostatic monitoring of dogs with multicentric lymphoma.

Lymphoma is the most common haematopoietic malignancy in dogs and it has been associated with hypercoagulability and subsequent thromboembolism. The objectives of this study were to serially characterize the haemostatic status of dogs with multicentric lymphoma. Thromboelastography, thrombin-antithrombin complex concentration and routine haematology and coagulation panels were measured. Twenty-seven dogs were included in the study and 15 completed the study in remission. At presentation, 81% (22/27) of dogs with multicentric lymphoma had altered haemostatic profiles consistent with hypercoagulability. Laboratory evidence of hypercoagulability did not resolve during treatment or for up to 1 month following attainment of clinical remission. Accelerated rate of clot formation at the time of chemotherapeutic protocol completion was associated with decreased survival time. We concluded that dogs with multicentric lymphoma were frequently hypercoagulable from presentation through 4 weeks after the completion of chemotherapy. Increased angle and shortened K in dogs that have successfully completed their chemotherapeutic protocol may be associated with shorter survival times.