LEKTI domain 6 displays anti-inflammatory action in vitro and in a murine atopic dermatitis model.

BACKGROUND: Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a serine protease inhibitor consisting of multiple domains. A loss of function mutation is described in Netherton patients that show severe symptoms of atopic lesions and itch. OBJECTIVES: LEKTI domain 6 (LD6) has shown strong serine protease-inhibitory action in in vitro assays and thus it was tested in vitro and in vivo for potential anti-inflammatory action in models of atopic skin disease. METHODS: Human skin equivalents were treated with LD6 and an inflammatory reaction was challenged by kallikrein-related endopeptidase 5 (KLK5). Furthermore, LD6 was tested on dorsal root ganglia cells stimulated with KLK5, SLIGRL and histamine by calcium imaging. The effect of topically administered LD6 (0.4-0.8%) in lipoderm was compared to a topical formulation of betamethasone-diproprionate (0.1%) in a therapeutic setting on atopic dermatitis-like lesions in NC/Nga mice sensitized to house dust mite antigen. Endpoints were clinical scoring of the mice as well as determination of scratching behaviour. RESULTS: KLK5 induced an upregulation of CXCL-8, CCL20 and IL-6 in skin equivalents. This upregulation was reduced by pre-incubation with LD6. KLK5 as well as histamine induced calcium influx in a population of neurons. LD6 significantly reduced the calcium response to both stimuli. When administered onto lesional skin of NC/Nga mice, both LD6 and betamethasone-dipropionate significantly reduced the inflammatory reaction. The effect on itch behaviour was less pronounced. CONCLUSION: Topical administration of LD6 might be a new therapeutic option for treatment of lesional atopic skin.

Lysozyme: an endogenous antimicrobial protein with potent activity against extracellular, but not intracellular Mycobacterium tuberculosis.

Endogenous antimicrobial peptides (AMPs) play a key role in the host defense against pathogens. AMPs attack pathogens preferentially at the site of entry to prevent invasive infection. Mycobacterium tuberculosis (Mtb) enters its host via the airways. AMPs released into the airways are therefore likely candidates to contribute to the clearance of Mtb immediately after infection. Since lysozyme is detectable in airway secretions, we evaluated its antimicrobial activity against Mtb. We demonstrate that lysozyme inhibits the growth of extracellular Mtb, including isoniazid-resistant strains. Lysozyme also inhibited the growth of non-tuberculous mycobacteria. Even though lysozyme entered Mtb-infected human macrophages and co-localized with the pathogen we did not observe antimicrobial activity. This observation was unlikely related to the large size of lysozyme (14.74 kDa) because a smaller lysozyme-derived peptide also co-localized with Mtb without affecting the viability. To evaluate whether the activity of lysozyme against extracellular Mtb could be relevant in vivo, we incubated Mtb with fractions of human serum and screened for antimicrobial activity. After several rounds of sub-fractionation, we identified a highly active fraction-component as lysozyme by mass spectrometry. In summary, our results identify lysozyme as an antimycobacterial protein that is detectable as an active compound in human serum. Our results demonstrate that the activity of AMPs against extracellular bacilli does not predict efficacy against intracellular pathogens despite co-localization within the macrophage. Ongoing experiments are designed to unravel peptide modifications that occur in the intracellular space and interfere with the deleterious activity of lysozyme in the extracellular environment.

EPI-X4, a CXCR4 antagonist inhibits tumor growth in pancreatic cancer and lymphoma models.

Endogenous peptide inhibitor for CXCR4 (EPI-X4) is a CXCR4 antagonist with potential for cancer therapy. It is a processed fragment of serum albumin from the hemofiltrate of dialysis patients. This study reports the efficacy of fifteen EPI-X4 derivatives in pancreatic cancer and lymphoma models. In vitro, the peptides were investigated for antiproliferation (cytotoxicity) by MTT assay. The mRNA expression for CXCR4 and CXCL12 was determined by RT-PCR, chip array and RNA sequencing. Chip array analysis yielded 634 genes associated with CXCR4/CXCL12 signaling. About 21% of these genes correlated with metastasis in the context of cell motility, proliferation, and survival. Expression levels of these genes were altered in pancreatic cancer (36%), lymphoma models (53%) and in patients' data (58%). EPI-X4 derivatives failed to inhibit cell proliferation due to low expression of CXCR4 in vitro, but inhibited tumor growth in the bioassays with significant efficacy. In the pancreatic cancer model, EPI-X4a, f and k inhibited mean tumor growth by > 50% and even caused complete remissions. In the lymphoma model, EPI-X4b, n and p inhibited mean tumor growth by > 70% and caused stable disease. Given the non-toxic and non-immunogenic properties of EPI-X4, these findings underscore its status as a promising therapy of pancreatic cancer and lymphoma and warrant further studies. SIMPLE SUMMARY: This study examined the value of chemokine receptor CXCR4 as an antineoplastic target for the endogenous peptide inhibitor of CXCR4 (EPI-X4), a 12-meric peptide derived from serum albumin. EPI-X4 inhibits CXCR4 interaction with its natural ligand, CXCL12 (SDF1). Therefore, malignancies (including pancreatic cancer and lymphoma) that depend on the CXCR4/CXCL12 pathway for progression can be targeted with EPI-X4. Of 634 genes that were linked to the CXCR4/CXCL12 pathway, 21% were associated with metastasis. In cultured human Suit2-007 pancreatic cancer cells, CXCR4 showed low to undetectable expression, which was why EPI-X4 did not inhibit pancreatic cancer cell proliferation. These findings were different in vivo, where CXCR4 was highly expressed and EPI-X4 inhibited tumor growth in rodents harboring pancreatic cancer or lymphoma. In the pancreatic cancer model, EPI-X4 derivatives a, f and k caused complete remissions, while in lymphomas EPI-X4 derivatives b, n and p caused stable disease.

Retrospective study of tetanus in 18 dogs-Causes, management, complications, and immunological status.

Objective: Tetanus is a severe neurologic disease caused by Clostridium tetani, resulting in spastic paralysis. Canine tetanus is associated with serious complications such as aspiration and a high mortality rate of up to 50%. Materials and methods: Medical records of all dogs diagnosed with tetanus over 8 years (2014-2022) were analyzed for severity grade, treatment protocols, nutritional management, and complications, as well as outcome, vaccination, and antibody production in some dogs. No medical records were excluded. Normality was analyzed by the D'Agostino-Pearson test. Parametric, normally distributed data were presented as mean ± standard deviation. Non-parametric, non-normally distributed data were presented as median (m) and range (minimum-maximum). The association between tetanus grade, progression of diseases, and duration of hospitalization was analyzed using the t-test, Mann-Whitney U test, and Kruskal-Wallis test. A P ≤ 0.05 was considered significant. Results: Eighteen dogs were identified. Most affected dogs were classified into severity grade II (66.7%, 12 of 18). Clinical signs deteriorated in 55.6% of dogs (10 of 18). A source was identified in 88.9% of dogs (16 of 18). Nine dogs required surgical wound revision. A percutaneous endoscopic gastropexy tube was placed in 83.3% of dogs (15 of 18) for nutritional support. Medical treatment included metronidazole, methocarbamol, and combinations of different sedatives adapted to the patient's requirements. Tetanus antitoxin was used in 72.2% of dogs (13 of 18) without reported adverse events. The survival rate was 88.9% (16 of 18). Complications, such as hypertension, aspiration pneumonia, and laryngeal spasm occurred in 12 of 18 dogs. Median hospitalization time (8 days; range 0-16 days) was associated with the maximum tetanus severity grade (p = 0.022). Rapid eye movement behavior disorder was observed in 72.2% of dogs (13 of 18). In 5 dogs, antibodies were measured after recovery, and in 4 of 5 dogs, no antibodies were detectable despite generalized tetanus disease. Vaccination with tetanus toxoid was performed in five dogs following the disease. Conclusion: In the present study, the mortality rate was lower than previously reported. Tetanus is still a life-threatening disease, but the prognosis may be good if adequate management and monitoring can be ensured.

Human lysozyme inhibits the fibrillation of serum amyloid a protein from systemic AA amyloidosis.

BACKGROUND: Systemic AA amyloidosis is a world-wide occurring protein misfolding disease in humans and animals that arises from the formation of amyloid fibrils from serum amyloid A (SAA) protein and their deposition in multiple organs. OBJECTIVE: To identify new agents that prevent fibril formation from SAA protein and to determine their mode of action. MATERIALS AND METHODS: We used a cell model for the formation of amyloid deposits from SAA protein to screen a library of peptides and small proteins, which were purified from human hemofiltrate. To clarify the inhibitory mechanism the obtained inhibitors were characterised in cell-free fibril formation assays and other biochemical methods. RESULTS: We identified lysozyme as an inhibitor of SAA fibril formation. Lysozyme antagonised fibril formation both in the cell model as well as in cell-free fibril formation assays. The protein binds SAA with a dissociation constant of 16.5 ± 0.6 µM, while the binding site on SAA is formed by segments of positively charged amino acids. CONCLUSION: Our data imply that lysozyme acts in a chaperone-like fashion and prevents the aggregation of SAA protein through direct, physical interactions.

Bacterial Contamination of Inhalation Chambers Used for Cats and Dogs with Chronic Airway Diseases.

Inhalation chambers (ICs) are regularly used in veterinary medicine for the inhalative treatment of chronic respiratory diseases in dogs and cats. Since therapy is usually required lifelong and daily, devices are frequently in use. The aim of this study was to identify bacterial contamination of ICs used for cats and dogs in relation to the applied cleaning measures. Swabs from ICs of 66 cats and 19 dogs with chronic airway diseases were obtained using a standardized protocol and subsequently cultured. A questionnaire was completed by the pet owners regarding the history of their pet's illness and applied device cleaning measures. Overall, 64% (54/86) of the ICs were found to be contaminated; the mask was significantly (p < 0.001) more often contaminated than other device parts. Most cultured bacteria were environmental contaminants; however, some harbored pathogenic potential. Cleaning frequency and method did not significantly influence the presence of contamination. Bacterial contamination of ICs, used for cats and dogs, is common but is not significantly influenced by the type or frequency of cleaning. To avoid potential infection by opportunistic bacteria, the instruction of pet owners regarding the maintenance of the ICs is recommended.

[Influence of the Verordnung über Tierärztliche Hausapotheken on antimicrobial use in dogs and cats in Bavaria]. / Einfluss der Verordnung über Tierärztliche Hausapotheken auf den Antibiotikaeinsatz bei Hund und Katze in Bayern.

OBJECTIVE: Aim of the study was to collect data concerning the use of antibiotics (AB) in dogs and cats in veterinary practices and clinics in Bavaria, Germany. It was evaluated, whether changes in the use of AB since the amendment of the Verordnung über Tierärztliche Hausapotheken (TÄHAV, German Regulation on the Veterinary In-house Pharmacy) in March 2018 could be documented. MATERIAL AND METHODS: Using 2 anonymous online surveys in 2017 and 2020, veterinarians treating dogs and cats in Bavaria were questioned about their usage of AB and their assessment of the current antimicrobial resistance situation. The results of both surveys were evaluated statistically and compared with each other. RESULTS: While in 2017 a total of 238 questionaries were evaluated, 160 could be included in 2020. The three most commonly used antibiotics for systemic therapy were Amoxicillin/Clavulanic acid (74.8 % of veterinarians), Enrofloxacin (56.7 %) and Amoxicillin (53.4 %) in 2017; and Amoxicillin/Clavulanic acid (88.8 %), Amoxicillin (67.5 %) and Metronidazole (33.8 %) in 2020, respectively. The participating veterinarians stated that their overall use of 3 rd and 4th generations cephalosporins (from 20.2 % of veterinarians in 2017 to 9.4 % in 2020, p = 0,005) as well as fluoroquinolones (from 80.3 % to 33.1 %, p < 0.001) had significantly declined. In 2020, the choice of AB in veterinarians was affected by legal requirements (83.8 %), tolerability (81.3 %), way of application (76.9 %), acceptance by the patient (70.0 %), and frequency of application (64.4 %), with the last parameter being significantly more important to veterinarians working in a practice (83.8 %, p = 0.004) than to veterinarians in a clinic. CONCLUSION: Veterinarians report a significantly reduced usage of fluoroquinolones and 3 rd and 4th generation cephalosporines in dogs and cats compared to 2017. These changes in prescribing practice could be a consequence of the amendment of the TÄHAV, which dictates a prohibition of rededication as well as an obligation for microbial sensitivity testing for these AB classes. CLINICAL RELEVANCE: Legal restrictions could have a positive influence on the amount and type of antibiotics used and therefore help to prevent antimicrobial resistance.

Bacterial urinary tract infection and subclinical bacteriuria in dogs receiving antineoplastic chemotherapy.

BACKGROUND: Immunosuppressive treatment with glucocorticoids and cyclosporine increases the risk for positive urine cultures (PUCs) in dogs. OBJECTIVE: To investigate the prevalence and incidence of PUC in dogs diagnosed with cancer and treated with antineoplastic chemotherapy while distinguishing between subclinical bacteriuria (SB) and urinary tract infection (UTI). ANIMALS: Forty-six client-owned dogs with nonurogenital cancer treated with antineoplastic chemotherapy. METHODS: Prospective observational longitudinal clinical study. Dogs in which a urine culture was performed before the start of and at least once during antineoplastic chemotherapy were included. A McNemar's test was used to investigate if the prevalence of PUC increased during antineoplastic chemotherapy. Positive urine cultures were categorized into SB and UTI and multiple PUCs from the same dog and category were grouped together as 1 episode of PUC. RESULTS: Urine culture was positive in 21/185 urine samples in 8/46 dogs. Antineoplastic chemotherapy did not influence the prevalence of PUC (P = 1.00), which was 11% (5/46 dogs; 95% confidence interval: 5-23%) before the start of and 13% (6/46 dogs; 95% confidence interval: 6-26%) during antineoplastic chemotherapy. Eight dogs had 10 episodes of PUC; 7/10 episodes were classified as SB, and in 3/10 episodes UTI (chronic prostatitis, prostatic abscess, and emphysematous cystitis) was diagnosed. Escherichia coli was the most common pathogen, isolated in 9/10 episodes. CONCLUSIONS AND CLINICAL IMPORTANCE: We did not find evidence that antineoplastic chemotherapy is a major predisposing factor for the development of PUC. Most dogs with PUC had SB.

A Fibrinogen Alpha Fragment Mitigates Chemotherapy-Induced MLL Rearrangements.

Rearrangements in the Mixed Lineage Leukemia breakpoint cluster region (MLLbcr) are frequently involved in therapy-induced leukemia, a severe side effect of anti-cancer therapies. Previous work unraveled Endonuclease G as the critical nuclease causing initial breakage in the MLLbcr in response to different types of chemotherapeutic treatment. To identify peptides protecting against therapy-induced leukemia, we screened a hemofiltrate-derived peptide library by use of an enhanced green fluorescent protein (EGFP)-based chromosomal reporter of MLLbcr rearrangements. Chromatographic purification of one active fraction and subsequent mass spectrometry allowed to isolate a C-terminal 27-mer of fibrinogen α encompassing amino acids 603 to 629. The chemically synthesized peptide, termed Fα27, inhibited MLLbcr rearrangements in immortalized hematopoietic cells following treatment with the cytostatics etoposide or doxorubicin. We also provide evidence for protection of primary human hematopoietic stem and progenitor cells from therapy-induced MLLbcr breakage. Of note, fibrinogen has been described to activate toll-like receptor 4 (TLR4). Dissecting the Fα27 mode-of action revealed association of the peptide with TLR4 in an antagonistic fashion affecting downstream NFκB signaling and pro-inflammatory cytokine production. In conclusion, we identified a hemofiltrate-derived peptide inhibitor of the genome destabilizing events causing secondary leukemia in patients undergoing chemotherapy.

Corynebacterium rouxii, a recently described member of the C. diphtheriae group isolated from three dogs with ulcerative skin lesions.

Corynebacterium (C.) diphtheriae is one of the two etiological pathogens for human diphtheria with significant morbidity and mortality. Recently, members of its biovar Belfanti have been described as two novel species, C. belfantii and C. rouxii. The most important virulence factor and also the premise to cause diphtheria is the isolate's capacity to encode and express the diphtheria toxin (DT). In contrast to C. ulcerans, which represents a potentially zoonotic pathogen, C. diphtheriae (incl. the novel deduced species) has almost exclusively been found to comprise a human pathogen. We here report three rare cases of C. rouxii isolation from dogs suffering from disseminated poly-bacterial exsudative to purulent dermatitis and a traumatic labial defect, respectively. The isolates were identified as C. diphtheriae based on commercial biochemistry and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) analysis. However, recently described specific spectral peaks were highly similar to spectra of C. rouxii, which was confirmed by whole genome sequencing. Further investigations of the dog isolates for the presence of DT by tox gene qPCR revealed negative results. The findings from this study point out that skin infections in companion animals can be colonized by uncommon and so believed human specific pathogens, thereby resembling the clinical signs of cutaneous diphtheria.

Natural cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling.

GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4+ T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.

Evaluation of different cleaning methods for feline inhalation chambers after bacterial contamination.

OBJECTIVES: Inhalation chambers are commonly used for the delivery of aerosol drugs to cats with respiratory disease. The aim of the study was to identify successful cleaning methods for inhalation devices after standardised bacterial contamination. METHODS: Spacer devices of two different manufacturers were used: RC Chamber (Cegla Medizintechnik) and Aerokat (Trudell Medical International). The bacterial contamination was performed using Pseudomonas aeruginosa. Previously marked areas of the chamber were contaminated with 50 µl of bacterial solution, containing between 2.2 ×105 and 2.1 ×108 colony-forming units/ml each. After cleaning the devices as recommended by each manufacturer (RC Chamber: special microwave cleaning bag [n = 5] or boiling water with liquid dish detergent for 15 mins [n = 5]; Aerokat: rinsing in a solution of lukewarm water and liquid dish detergent for 15 mins), chambers were air-dried for 24 h and samples for bacterial culture were taken from three defined areas. Sample material was applied on Müller-Hinton agar plates and subsequently incubated for 24 h at 37°C. RESULTS: Bacterial contamination was not detected in any of the examined inhalation devices using the recommended cleaning methods. CONCLUSIONS AND RELEVANCE: If inhalation chambers are cleaned following the manufacturers' recommendations, successful bacterial decontamination can be expected.

A Placenta Derived C-Terminal Fragment of ß-Hemoglobin With Combined Antibacterial and Antiviral Activity.

The placenta acts as physical and immunological barrier against the transmission of viruses and bacteria from mother to fetus. However, the specific mechanisms by which the placenta protects the developing fetus from viral and bacterial pathogens are poorly understood. To identify placental peptides and small proteins protecting from viral and bacterial infections, we generated a peptide library from 10 kg placenta by chromatographic means. Screening the resulting 250 fractions against Herpes-Simplex-Virus 2 (HSV-2), which is rarely transmitted through the placenta, in a cell-based system identified two adjacent fractions with significant antiviral activity. Further rounds of chromatographic purification and anti-HSV-2 testing allowed to purify the bioactive peptide. Mass spectrometry revealed the presence of a 36-mer derived from the C-terminal region of the hemoglobin ß subunit. The purified and corresponding chemically synthesized peptide, termed HBB(112-147), inhibited HSV-2 infection in a dose-dependent manner, with a mean IC50 in the median µg/ml range. Full-length hemoglobin tetramer had no antiviral activity. HBB(112-147) did not impair infectivity by direct targeting of the virions but prevented HSV-2 infection at the cell entry level. The peptide was inactive against Human Immunodeficiency Virus Type 1, Rubella and Zika virus infection, suggesting a specific anti-HSV-2 mechanism. Notably, HBB(112-147) has previously been identified as broad-spectrum antibacterial agent. It is abundant in placenta, reaching concentrations between 280 and 740 µg/ml, that are well sufficient to inhibit HSV-2 and prototype Gram-positive and -negative bacteria. We here additionally show, that HBB(112-147) also acts potently against Pseudomonas aeruginosa strains (including a multi-drug resistant strain) in a dose dependent manner, while full-length hemoglobin is inactive. Interestingly, the antibacterial activity of HBB(112-147) was increased under acidic conditions, a hallmark of infection and inflammatory conditions. Indeed, we found that HBB(112-147) is released from the hemoglobin precursor by Cathepsin D and Napsin A, acidic proteases highly expressed in placental and other tissues. We propose that upon viral or bacterial infection, the abundant hemoglobin precursor is proteolytically processed to release HBB(112-147), a broadly active antimicrobial innate immune defense peptide.

Effect of amoxicillin-clavulanic acid on clinical scores, intestinal microbiome, and amoxicillin-resistant Escherichia coli in dogs with uncomplicated acute diarrhea.

BACKGROUND: Despite limited evidence of efficacy, antibiotic treatment is still frequently prescribed in dogs with uncomplicated acute diarrhea (AD). OBJECTIVE: To assess whether amoxicillin-clavulanic acid has a clinical benefit, an effect on the fecal microbiome, and the proportion of amoxicillin-resistant Escherichia coli in dogs with AD. ANIMALS: Sixteen dogs with AD of <3 days duration. METHODS: Prospective, placebo-controlled, double-blinded study. Clinical scores were compared between client-owned dogs randomly assigned to an antibiotic (AG) or a placebo (PG) group. The intestinal microbiome was analyzed using quantitative PCR assays. Amoxicillin-resistant fecal E. coli were assessed semiquantitatively with microbiological methods. RESULTS: There was no difference in clinical recovery between treated dogs or controls (CADS index day 10: AG group median: 2 (range: 1-3; CI [1.4; 2.6]); PG group median: 1.6 (range: 1-3; CI [1.1; 2.4]); P > .99). All dogs gained normal clinical scores (CADS index ≤3) after 1 to 6 days (median 2 days) after presentation. There was no significant difference in the fecal dysbiosis index (during treatment: AG mean -2.6 (SD 3.0; CI [-5.1; 0.0]); PG mean -0.8 (SD 4.0; CI [-4.2; 2.5]; P > .99) or its bacterial taxa. The proportion of resistant fecal E. coli increased (to median: 100%; range: 35%-100%) during treatment with amoxicillin-clavulanic acid and was still increased (median: 10%; range 2%-67%) 3 weeks after treatment, both of which were significantly higher proportions than in the placebo group for both time points (during treatment AG median 100% versus PG median 0.2% (P < .001); after treatment AG median 10% versus PG median 0.0% (P = .002)). CONCLUSIONS AND CLINICAL IMPORTANCE: Our study suggests that treatment with amoxicillin-clavulanic acid confers no clinical benefit to dogs with AD, but predisposes the development of amoxicillin-resistant E. coli, which persist for as long as 3 weeks after treatment. These findings support international guideline recommendations that dogs with diarrhea should not be treated with antimicrobials unless there are signs of sepsis.

Unbiased Identification of Angiogenin as an Endogenous Antimicrobial Protein With Activity Against Virulent Mycobacterium tuberculosis.

Tuberculosis is a highly prevalent infectious disease with more than 1.5 million fatalities each year. Antibiotic treatment is available, but intolerable side effects and an increasing rate of drug-resistant strains of Mycobacterium tuberculosis (Mtb) may hamper successful outcomes. Antimicrobial peptides (AMPs) offer an alternative strategy for treatment of infectious diseases in which conventional antibiotic treatment fails. Human serum is a rich resource for endogenous AMPs. Therefore, we screened a library generated from hemofiltrate for activity against Mtb. Taking this unbiased approach, we identified Angiogenin as the single compound in an active fraction. The antimicrobial activity of endogenous Angiogenin against extracellular Mtb could be reproduced by synthetic Angiogenin. Using computational analysis, we identified the hypothetical active site and optimized the lytic activity by amino acid exchanges. The resulting peptide-Angie1-limited the growth of extra- and intracellular Mtb and the fast-growing pathogens Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Toward our long-term goal of evaluating Angie1 for therapeutic efficacy in vivo, we demonstrate that the peptide can be efficiently delivered into human macrophages via liposomes and is not toxic for zebrafish embryos. Taken together, we define Angiogenin as a novel endogenous AMP and derive the small, bioactive fragment Angie1, which is ready to be tested for therapeutic activity in animal models of tuberculosis and infections with fast-growing bacterial pathogens.

Canine Brucellosis: Insights Into the Epidemiologic Situation in Europe.

Brucella canis is one of many responsible pathogens of discospondylitis in dogs and infections require specific management. Little is known about the epidemiologic situation in Europe. The purpose of the study was to get insights into the occurrence of brucellosis in dogs in Europe. The database of a European veterinary laboratory was screened for Brucella positive samples. Additionally, medical records of a veterinary hospital in Germany were screened for diagnosis of discospondylitis and brucellosis. The laboratory received samples from 20 European countries for Brucella testing in dogs: 3.7% of submitted samples were Brucella spp. PCR-positive (61/1,657), and Brucella canis antibodies were identified in 5.4% of submitted samples (150/2,764). Brucella spp. PCR-positive samples originated from Spain (11.1% of submitted samples), Poland (6.7% of submitted samples) and rarely from Italy and France. Samples with Brucella canis antibodies originated from 13 European countries (Sweden, Belgium, Austria, Switzerland, Italy, Finland, Germany, Denmark, Hungary, Norway, Poland, France, Netherlands). Young dogs (0-24 months) had a 5.4-fold increased risk of PCR positive samples. The supplementary medical records search identified four young female dogs (7-30 months) with Brucella canis discospondylitis in Germany. The four dogs had been imported to Germany from Eastern European countries (Moldavia, Romania, Macedonia). In conclusion, infection with Brucella canis needs to be considered in dogs in Europe and diagnostics for Brucella canis infection appear indicated in young dogs with discospondylitis.

Determination of minimum inhibitory concentrations for silver sulfadiazine and other topical antimicrobial agents against strains of Pseudomonas aeruginosa isolated from canine otitis externa.

BACKGROUND: Otitis externa is a common presenting complaint in practice. Ear infections by Pseudomonas aeruginosa are particularly problematic due to the organism's high level of resistance and ability to damage the tympanum. Treatment should be based on susceptibility testing although minimum inhibitory concentrations (MICs) are not available for all treatment options. Silver sulfadiazine has been used in cases of recurrent P. aeruginosa otitis, although a MIC for silver sulfadiazine as a single agent has not been established. OBJECTIVES: To describe susceptibility patterns of P. aeruginosa isolated from canine otitis externa and determine the MIC for silver sulfadiazine and other topical antimicrobials. ANIMALS: Thirty-six P. aeruginosa isolates were collected from client-owned dogs, suffering from otitis externa. METHODS AND MATERIALS: Susceptibility patterns were established using disc diffusion susceptibility testing against 17 antimicrobial agents. For determination of the MIC, selected strains were tested against increasing concentrations of marbofloxacin, enrofloxacin, gentamicin, polymyxin B and silver sulfadiazine using broth microdilution. RESULTS: For nine of 17 antimicrobial agents, complete resistance was seen in all isolates tested via disk diffusion susceptibility testing. Approximately 94% and 96% of isolates were susceptible to gentamicin and imipenem, respectively. These findings were consistent with broth dilution, where all strains were susceptible to gentamicin. Resistance was higher against polymyxin B and the fluoroquinolones. Silver sulfadiazine was effective in vitro with a MIC ranging from 1 to 64 µg/mL. CONCLUSIONS AND CLINICAL SIGNIFICANCE: As the MIC of silver sulfadiazine was lower than the concentration in a 1% preparation, such a product potentially represents a treatment option for dogs with P. aeruginosa otitis.

Randomized double-blind clinical studies of ularitide and other vasoactive substances in acute decompensated heart failure: a systematic review and meta-analysis.

AIMS: Acute decompensated heart failure (ADHF) has a poor prognosis and limited treatment options. No direct comparisons between ularitide-a synthetic natriuretic peptide being evaluated in ADHF-and other vasoactive substances are available. The aim of this meta-analysis was to determine haemodynamic effect sizes from randomized double-blind trials in ADHF. METHODS AND RESULTS: Eligible studies enrolled patients with ADHF requiring hospitalization and haemodynamic monitoring. Patients received 24-48 h of infusion with a vasoactive substance or comparator. Primary outcome measure was pulmonary artery wedge pressure (PAWP). Treatment effects were quantified as changes from baseline using mean differences between study drug and comparator. Results were analysed using random-effects (primary analysis) and fixed-effects meta-analyses. Twelve randomized, double-blind studies were identified with data after 3, 6, and 24 h of treatment (n = 622, 644, and 644, respectively). At 6 h, significant PAWP benefits for ularitide over placebo were seen (Hedges' g effect size, -0.979; P < 0.0001). On meta-analysis, treatment difference between ularitide and pooled other agents was statistically significant (-0.501; P = 0.0303). Effect sizes were numerically higher with ularitide than other treatments at 3 and 24 h. After 6 h, a significant difference in effect size between ularitide and all other treatments was observed for right atrial pressure (Hedges' g, -0.797 for ularitide and -0.304 for other treatments; P = 0.0274). CONCLUSIONS: After 6 h, ularitide demonstrated high effect sizes for PAWP and right atrial pressure. Improvements in these parameters were greater with ularitide vs. pooled data for other vasoactive drugs.

Kallikrein-related peptidases are activators of the CC chemokine CCL14.

Chemokine CCL14 is inactive in its proform. Here, we show that inflammation- and cancer-associated kallikrein-related peptidases KLK5 and KLK8 remove the N-terminal eight amino acids from the proform thereby converting CCL14 to its active state. Activity of the chemokine is demonstrated by migration of myeloid cells expressing relevant receptors.

Intestinal lesions in dogs with acute hemorrhagic diarrhea syndrome associated with netF-positive Clostridium perfringens type A.

Acute hemorrhagic diarrhea syndrome (AHDS), formerly named canine hemorrhagic gastroenteritis, is one of the most common causes of acute hemorrhagic diarrhea in dogs, and is characterized by acute onset of diarrhea, vomiting, and hemoconcentration. To date, histologic examinations have been limited to postmortem specimens of only a few dogs with AHDS. Thus, the aim of our study was to describe in detail the distribution, character, and grade of microscopic lesions, and to investigate the etiology of AHDS. Our study comprised 10 dogs with AHDS and 9 control dogs of various breeds, age, and sex. Endoscopic biopsies of the gastrointestinal tract were taken and examined histologically (H&E, Giemsa), immunohistochemically ( Clostridium spp., parvovirus), and bacteriologically. The main findings were acute necrotizing and neutrophilic enterocolitis (9 of 10) with histologic detection of clostridia-like, gram-positive bacteria on the necrotic mucosal surface (9 of 10). Clostridium perfringens isolated from the duodenum was identified as type A (5 of 5) by multiplex PCR (5 of 5). In addition, each of the 5 genotyped isolates encoded the pore-forming toxin netF. Clostridium spp. (not C. perfringens) were cultured from duodenal biopsies in 2 of 9 control dogs. These findings suggest that the pore-forming netF toxin is responsible for the necrotizing lesions in the intestines of a significant proportion of dogs with AHDS. Given that the stomach was not involved in the process, the term "acute hemorrhagic diarrhea syndrome" seems more appropriate than the frequently used term "hemorrhagic gastroenteritis."