Oral cinacalcet administration decreases serum ionized calcium and parathyroid hormone concentrations in healthy dogs.

Cinacalcet is an oral calcimimetic that has potential to non-invasively treat primary hyperparathyroidism in dogs (Canis lupis familiaris). There is minimal data assessing its efficacy in dogs. This study aimed to determine whether a single dose of cinacalcet decreases serum ionized calcium (iCa), total calcium (tCa), and parathyroid hormone (PTH) concentrations. Twelve dogs received a median dose of 0.49 mg/kg (range 0.30-0.69 mg/kg) cinacalcet per os. Venous blood samples were collected at time 0 (before cinacalcet administration), 3, 8, and 24 h following cinacalcet administration. PTH, iCa, and tCa concentrations were measured at each time point and compared to 0 hour concentrations. A significant (50%) decrease in serum PTH occurred at 3 h with a median PTH of 4.6 pmol/L (range 2.7-10.8) at baseline and 1.65 pmol/L (range 0.5-14.7) at 3 h; p = .005. A significant, but not clinically relevant, decrease in serum iCa from a median baseline of 1.340 mmol/L (range 1.32-1.41) to a 3 h median of 1.325 mmol/L (range 1.26-1.39), p = .043, was also observed. tCa concentrations were not different. This study showed that a single dose of cinacalcet leads to transient decreases in iCa and PTH concentrations in healthy dogs.

FATAL ACUTE HEMOLYSIS FOLLOWING TRIAZOLE THERAPY IN AFRICAN PENGUINS (SPHENISCUS DEMERSUS).

Aspergillosis is a major cause of morbidity and mortality in penguins, with triazole antifungal drugs being commonly used for prophylaxis and treatment. This report describes 15 cases of fatal hemolysis associated with liquid itraconazole and voriconazole formulations administered to African penguins (Spheniscus demersus) from four institutions. All penguins underwent stressful events (e.g. relocation, induced molt) and were administered commercial liquid itraconazole formulations or compounded voriconazole liquid suspension. Observed clinical signs in affected penguins prior to death included hyporexia, weight loss, lethargy, dyspnea, red-tinged droppings, and obtunded mentation. Intra- and extravascular hemolysis and hemoglobinuric nephrosis were the primary pathologic manifestations on postmortem examination. The concentration-dependent hemolytic potentials of itraconazole, voriconazole, and commercial and compounded vehicle suspensions were evaluated in vitro by exposing chicken whole blood as a surrogate for penguin blood. Hemoglobin content in blood plasma was then measured by spectrophotometry. Neither itraconazole nor voriconazole alone induced hemolysis in vitro. The vehicle ingredients sorbitol and hydromellose induced hemolysis, but not at predicted plasma levels in chicken erythrocytes, suggesting neither the azole antifungals nor their major vehicles alone were likely to contribute to hemolysis in vivo in these penguins. Potential mechanisms of toxicosis include generation of an unmeasured reactive metabolite causing hemolysis, preexisting erythrocyte fragility, or species-specific differences in hemolytic thresholds that were not assessed in the chicken erythrocyte model. More research is needed on the potential for toxicosis of azole antifungal drugs and carrier molecules in this and other avian species.

Report from the National Institute of Allergy and Infectious Diseases workshop on drug allergy.

Allergic reactions to drugs are a serious public health concern. In 2013, the Division of Allergy, Immunology, and Transplantation of the National Institute of Allergy and Infectious Diseases sponsored a workshop on drug allergy. International experts in the field of drug allergy with backgrounds in allergy, immunology, infectious diseases, dermatology, clinical pharmacology, and pharmacogenomics discussed the current state of drug allergy research. These experts were joined by representatives from several National Institutes of Health institutes and the US Food and Drug Administration. The participants identified important advances that make new research directions feasible and made suggestions for research priorities and for development of infrastructure to advance our knowledge of the mechanisms, diagnosis, management, and prevention of drug allergy. The workshop summary and recommendations are presented herein.

Polymorphisms in the carcinogen detoxification genes CYB5A and CYB5R3 and breast cancer risk in African American women.

PURPOSE: Cytochrome b 5 (encoded by CYB5A) and NADH cytochrome b 5 reductase (encoded by CYB5R3) detoxify aromatic and heterocyclic amine mammary carcinogens found in cigarette smoke. We hypothesized that CYB5A and CYB5R3 polymorphisms would be associated with breast cancer risk in women. METHODS: We characterized the prevalence of 18 CYB5A and CYB5R3 variants in genomic DNA from African American (AfrAm) and Caucasian (Cauc) women from the Carolina Breast Cancer Study population (1,946 cases and 1,747 controls) and determined their associations with breast cancer risk, with effect modification by smoking. RESULTS: A CYB5R3 variant, I1M+6T (rs8190370), was significantly more common in breast cancer cases (MAF 0.0238) compared with controls (0.0169, p = 0.039); this was attributable to a higher MAF in AfrAm cases (0.0611) compared with AfrAm controls (0.0441, p = 0.046; adjusted OR 1.41, CI 0.98-2.04; p = 0.062). When smoking was considered, I1M+6T was more strongly associated with breast cancer risk in AfrAm smokers (adjusted OR 2.10, 1.08-4.07; p = 0.028) compared with never smokers (OR = 1.21; 0.77-1.88; p for interaction = 0.176). I1M+6T and three additional CYB5R3 variants, -251T, I8-1676C, and *392C, as well as two CYB5A variants, 13G and I2-992T, were significantly more common in AfrAms compared with Caucs. CONCLUSIONS: CYB5R3 I1M+6C>T should be considered in future molecular epidemiologic studies of breast cancer risk in AfrAms. Further, variants in CYB5A and CYB5R3 should be considered in the evaluation of other tumors in AfrAms that are associated with aromatic and heterocyclic amine exposures, to include prostate, bladder, and colon cancers.

Cutaneous pythiosis in two dogs from Wisconsin, USA.

BACKGROUND: Pythium insidiosum is an oomycete that causes cutaneous lesions or infiltrative gastrointestinal disease in dogs, cats, humans, horses and other mammals, primarily in tropical and subtropical climates. HYPOTHESIS/OBJECTIVES: We report the clinicopathological findings associated with cutaneous pythiosis in two dogs from a Northern temperate climate zone. ANIMALS: A 3-year-old intact male Chesapeake Bay retriever was presented with an ulcerated soft-tissue swelling over the left eye. A 4-year-old spayed female German shepherd dog was presented with a soft-tissue swelling overlying the right hock. Both dogs lived in northern latitudes (between 43 and 45°N) and neither had travelled outside of Wisconsin or Michigan's upper peninsula, USA. METHODS: Histopathological examination and culture of affected tissues on specialized media, serology for anti-P. insidiosum antibodies, P. insidiosum-specific PCR and ribosomal RNA gene sequencing were carried out. RESULTS: Histopathological examination revealed pyogranulomatous and eosinophilic inflammation associated with wide, poorly septate hyphae. CONCLUSIONS AND CLINICAL IMPORTANCE: Even clinicians who practice in temperate climates should consider pythiosis as a differential diagnosis for young to middle-aged adult dogs presented with ulcerated cutaneous nodules or infiltrative gastrointestinal disease.

Use of linezolid to treat MRSP bacteremia and discospondylitis in a dog.

A 1.5 yr old male German shepherd dog was evaluated for recurrent intermittent episodes of fever and lethargy. Clinicopathologic abnormalities were suggestive of a discospondylitis at the seventh and eighth thoracic vertebrae. Blood and urine cultures yielded growth of methicillin-resistant Staphylococcus pseudintermedius (MRSP) that was resistant to all commonly used antibiotics. Extralabel antibiotic susceptibility testing demonstrated susceptibility of both blood and urine isolates to linezolid. The prescribed dose was extrapolated from pharmacokinetic (PK) studies and the isolate's plasma minimum inhibitory concentration (MIC). Linezolid was administered for 23 wk and resulted in successful resolution of bacteremia, bacteriuria, and discospondylitis. When justified, linezolid should be considered to treat methicillin-resistant infections.

Environmental radon, fracking wells, and lymphoma in dogs.

BACKGROUND: Multicentric lymphoma (ML) in dogs resembles non-Hodgkin lymphoma (NHL) in humans. Human NHL is associated with multiple environmental exposures, including to radon and volatile organic compounds (VOCs). HYPOTHESIS/OBJECTIVES: The objective of this study was to determine whether ML in dogs was associated with environmental radon or proximity to horizontal oil and drilling (fracking), a source of VOC pollution. METHODS: We identified dogs from the Golden Retriever Lifetime Study that developed ML (n = 52) along with matched controls (n = 104). Dog home addresses were categorized by Environmental Protection Agency radon zone and average residential radon by county, as well as by distance from fracking and associated wastewater wells. RESULTS: We found no significant differences in county level radon measurements. Individual household radon measurements were not available. There was no difference in residential proximity to active fracking wells between dogs with ML and unaffected dogs. While dogs with ML lived closer to wastewater wells (123 vs 206 km; P = .01), there was no difference in the percentage of cases vs controls that lived in close proximity (20 km) to a fracking well (11.5% for cases, 6.7% for controls; OR 1.81, 95% CI 0.55 to 5.22; P = .36), or a wastewater well (6.7% for cases, 4.4% for controls; P > .99). CONCLUSIONS AND CLINICAL IMPORTANCE: These data suggest that more proximate sources of chemical exposures need to be assessed in dogs with ML, including measurements of individual household radon and household VOC concentrations.

Urinary and household chemical exposures in pet dogs with urothelial cell carcinoma.

Urothelial cell carcinoma (UCC) has been linked to environmental chemical exposures in people, but these risk factors are not well understood in dogs with UCC. We hypothesised that household chemical exposures contribute to the risk of UCC in pet dogs. This prospective cross-sectional case-control study included 37 dogs with UCC and 37 unaffected breed-, sex-, and age-matched controls. Dog owners completed an environmental questionnaire and household samples were collected and analysed for arsenic (in tap water and room dust) and acrolein (in room air). Urine samples from UCC dogs, control dogs, and consenting owners were analysed for inorganic arsenic species, the acrolein metabolite 3-HPMA, and the phenoxy herbicide 2,4-D. Public data on chlorination byproducts (total trihalomethanes) in municipal drinking water were also compared between case and control households. Dogs with UCC were more likely to swim in a pool (15.2%) compared with control dogs (0%) (OR 1.69, 95% CI = 1.69-∞; p = .02). Dogs with UCC also had more than 4-fold higher reported municipal water concentrations of chlorination byproducts (median 28.0 ppb) compared with controls (median 6.9 ppb; p < .0001). Dust arsenic concentrations were unexpectedly lower in case households (median 0.277 ng/cm2) compared with control households (median 0.401 ng/cm2; p = .0002). Other outcomes were not significantly different between groups. These data suggest that dog owners, especially those of breeds known to be at higher risk for UCC, consider limiting access to swimming pools and installing water filtration units that remove total trihalomethanes.

Challenges in exploring the cytochrome P450 system as a source of variation in canine drug pharmacokinetics.

The cytochrome P450 (CYP) superfamily constitutes a collection of enzymes responsible for the metabolism of a wide array of endo- and xenobiotic compounds. Much of the knowledge on substrate specificity and genetic identification of the various CYP isoforms is derived from research in rodents and humans and only limited information has been captured in the dog. Currently, there exist many gaps in our knowledge of canine CYP diversity as a result of the paucity of studies focusing on canine CYPs, canine CYP polymorphisms, and the therapeutic consequences of these genetic variants. Challenges engendered by this lack of information is further amplified by inter- and intraspecies differences in the specificity and affinity of substrates and inhibitors, prohibiting a simple extrapolation of probe substances used in human CYP research. This creates a need to develop and validate canine-specific CYP probes. Failure to understand this potential metabolic and pharmacogenomic diversity can also influence the interpretation of data generated in dogs to support human drug development. It is with these objectives in mind that we provide an overview of what is currently known about canine CYPs with the hope that it will encourage further exploration into this important area of research.

Potential mechanism for hyperhomocysteinemia in Greyhound dogs.

BACKGROUND: Greyhounds have been reported to have hyperhomocysteinemia (HHC), but the underlying mechanisms and clinical implications are unclear. HYPOTHESIS: Our primary aim was to assess serum concentrations of homocysteine (HCy) and related analytes in Greyhounds and to identify a likely metabolic pathway for HHC. A secondary aim was to determine whether HHC is associated with evidence of oxidative stress. ANIMALS: Healthy pet Greyhounds (n = 31) and non-sighthound control dogs (n = 15). METHODS: Analysis of serum HCy, cobalamin, folate, and methionine, and plasma cysteine, glutathione, and total 8-isoprostane concentrations. RESULTS: Homocysteine concentrations were higher in Greyhounds (median, 25.0 µmol/L) compared to controls (13.9 µmol/L; P < .0001). Cobalamin concentrations were lower in Greyhounds (median, 416 ng/L) compared to controls (644 ng/L; P = .004) and were inversely correlated with HCy (r = -0.40, P = .004). Serum concentrations of folate, which is regenerated when HCy is converted to methionine, also were inversely correlated with HCy (r = -0.47, P = .002). Serum methionine concentrations were more than 4-fold lower in Greyhounds (median, 3.2 µmol/L) compared to controls (median, 15.0 µmol/L), but this difference was not significant (P = .3). Plasma cysteine, glutathione, and 8-isoprostane concentrations did not differ significantly between groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Our findings suggest a primary defect in conversion of HCy to methionine in Greyhounds, with related impaired folate generation. Ineffective cycling by methionine synthase could lead to secondary cobalamin depletion. Notably, low serum folate and cobalamin concentrations can be observed in Greyhounds without signs of intestinal disease.

Evaluation of polymorphisms in the sulfonamide detoxification genes NAT2, CYB5A, and CYB5R3 in patients with sulfonamide hypersensitivity.

OBJECTIVE: To determine whether polymorphisms in the sulfonamide detoxification genes, CYB5A (encoding cytochrome b(5)), CYB5R3 (encoding cytochrome b(5) reductase), or NAT2 (encoding N-acetyltransferase 2) were over-represented in patients with delayed sulfonamide drug hypersensitivity, compared with control patients who tolerated a therapeutic course of trimethoprim-sulfamethoxazole without adverse event. METHODS: DNA from 99 nonimmunocompromised patients with sulfonamide hypersensitivity who were identified from the Personalized Medicine Research Project at the Marshfield Clinic, and from 99 age-matched, race-matched, and sex-matched drug-tolerant controls, were genotyped for four CYB5A and five CYB5R3 polymorphisms, and for all coding NAT2 SNPs. RESULTS: CYB5A and CYB5R3 SNPs were found at low allele frequencies (<3-4%), which did not differ between hypersensitive and tolerant patients. NAT2 allele and haplotype frequencies, as well as inferred NAT2 phenotypes, also did not differ between groups (60 vs. 59% slow acetylators). Finally, no difference in NAT2 status was found in a subset of patients with more severe hypersensitivity signs (drug reaction with eosinophilia and systemic symptoms) compared with tolerant patients. CONCLUSION: We found no evidence of a substantial involvement of these nine CYB5A or CYB5R3 polymorphisms in sulfonamide hypersensitivity risk, although minor effects cannot be completely ruled out. Despite careful medical record review and full resequencing of the NAT2 coding region, we found no association of NAT2 coding alleles with sulfonamide hypersensitivity (predominantly cutaneous eruptions) in this adult Caucasian population.

Incidence of hepatopathies in dogs administered zonisamide orally: A retrospective study of 384 cases.

BACKGROUND: Acute hepatopathy secondary to administration of zonisamide has been reported in 2 dogs, but overall incidence of hepatopathy is unknown. OBJECTIVE: To characterize the incidence of hepatopathy in dogs administered zonisamide PO. ANIMALS: Three hundred eighty-four dogs administered zonisamide PO. METHODS: Multicenter retrospective study. Medical records were searched for dogs prescribed zonisamide PO and which had follow-up for at least 3 months (acute exposure) and >3 months (chronic exposure). Reported clinical signs, physical examination findings, and serum biochemical panels were reviewed for possible hepatotoxicosis. Serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activity and albumin concentration were documented for all available cases. RESULTS: Acute clinical hepatopathy was found in 2 of 384 treated dogs (0.52%, 95% confidence interval [CI], 0.06-1.9) after 13-16 days of zonisamide treatment. One additional dog had elevated serum ALT activity with no clinical signs. Of these 3 dogs, 2 recovered after administration of zonisamide was stopped, and 1 was euthanized because of liver failure. Of the 117 cases chronically administered zonisamide, 10 had an increase in ALP, 6 had an increase in ALT, and 1 had hypoalbuminemia. No clinical signs of liver disease were noted in dogs chronically treated with zonisamide (median, 20 months; range, 5-94 months). CONCLUSIONS AND CLINICAL IMPORTANCE: Acute, potentially life-threatening hepatopathy associated with oral administration of zonisamide to dogs is estimated to occur in less than 1% of dogs and was observed in the first 3 weeks of treatment. Subclinical abnormalities in ALT and ALP activity were noted in <10% of dogs during chronic administration of zonisamide, with no clinical signs of liver disease noted.

Risk of bladder cancer and lymphoma in dogs is associated with pollution indices by county of residence.

Human urothelial cell carcinoma (UCC) and non-Hodgkin lymphoma are considered environmental cancers in people, but less is known about environment risk for UCC and lymphoma in dogs. The objective of this study was to determine whether dogs with these cancers, compared to unaffected control dogs, live in counties with higher tap water contaminants or higher levels of air pollution as measured by the Environmental Protection Agency (EPA) and by National Air Toxics Assessment chemical exposure risk estimates. Dogs with available home addresses from two previously published case-control populations were included: 66 dogs with UCC and 70 unaffected controls; and 56 boxer dogs with lymphoma and 84 unaffected boxer controls. Tap water total trihalomethanes, which are water disinfection by-products, were more than threefold higher in UCC case counties of residence compared to controls (p < .0001), and a higher proportion of dogs with UCC lived in counties exceeding EPA ozone limits (41.8%) compared to controls (13.6% p = .0008). More boxers with lymphoma lived in counties exceeding EPA ozone limits (52.1%) compared to controls (29.0%; p = .018), with higher exposure risk estimates for airborne 1,3-butadiene and formaldehyde (p = .004-.005). These data support the hypothesis that tap water contaminants and airborne environmental pollutants contribute to the risk of both urothelial carcinoma and lymphoma in dogs. If these findings reflect causal relationships, then it is possible that tap water filtration units and more effective air pollution controls could decrease the overall incidence of these cancers in dogs.

Genotoxicity from metronidazole detected in vitro, but not in vivo, in healthy dogs in a randomized clinical trial.

OBJECTIVE: To determine whether metronidazole (MTZ), at recommended therapeutic dosages in dogs, induces peripheral blood cell (PMBC) genotoxicity, using the γ-H2AX assay as a sensitive measure of DNA breaks. The secondary aim was to assess dose-dependent genotoxicity in vitro in dog and cat PBMCs exposed to increasing MTZ concentrations. ANIMALS: 12 healthy employee- and student-owned dogs and blood samples from 2 other healthy untreated dogs and 2 healthy untreated cats. PROCEDURES: Screened dogs were randomized to receive lower-dose MTZ (7.5 mg/kg, PO, q 12 h) or higher-dose MTZ (20 mg/kg, PO, q 12 h) for 7 days. Blood was drawn at baseline, after the 1 week of treatment, and after a 1-week washout, for DNA damage assessment and serum MTZ concentration measurements. For in vitro studies, PBMCs from untreated healthy dogs and cats were exposed to 0 to 500 µg/mL MTZ. RESULTS: No dogs showed a significant increase in DNA damage at these MTZ dosages for 1 week. The highest serum MTZ concentration observed 1 hour after dosing was 36 µg/mL. In vitro, MTZ led to a significant increase in DNA damage at 100 µg/mL in both canine and feline PBMCs. CLINICAL RELEVANCE: Although MTZ was not significantly genotoxic in vivo in the healthy dogs in this study, MTZ was significantly genotoxic to canine PBMCs in vitro at 3-fold higher concentrations than those documented in vivo. The safety of MTZ in clinically ill dogs, which may have impaired MTZ clearance or DNA repair, should be assessed next.

Environmental exposures and lymphoma risk: a nested case-control study using the Golden Retriever Lifetime Study cohort.

Lymphoma is the second most common cancer affecting Golden Retrievers and is hypothesized to arise through a complex interaction of genetic and environmental factors. The aim of this nested case-control study was to investigate the association between potential environmental pollutant sources and lymphoma risk among Golden Retrievers participating in the Golden Retriever Lifetime Study. Forty-nine Golden Retrievers with non-cutaneous lymphoma and 98 Golden Retrievers without a history of cancer matched by age, sex and neuter status were selected from the Golden Retriever Lifetime Study cohort. Geographic proximity between each dog's primary residence and nine potential sources of environmental pollution was determined. In addition, the average annual ozone and airborne fine particulate matter levels for each dog's county of residence and owner-reported secondhand smoke exposure were evaluated. Environmental pollution sources of interest included chemical plants, municipal dumps, manufacturing plants, incineration plants, railroad embankment tracks, landfills, coal plants, high-voltage transmission lines, and nuclear power plants. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for each exposure of interest. Subgroup analyses were conducted to evaluate whether associations differed among 1) dogs with multicentric lymphoma, 2) dogs with B-cell lymphoma, and 3) dogs with T-cell lymphoma. No variables reached statistical significance when evaluating all cases together. However, cumulative exposure burden (household proximity to 3 or more pollution sources) approached significance within the multicentric lymphoma subgroup (OR = 2.60, 95%CI 0.99-6.86, p-value = 0.053). Patterns emerged among B- and T-cell subgroups, but none reached statistical significance. Ongoing research is warranted to discern if different environmental mechanisms may be driving B- and T-cell lymphoma immunophenotypes, consistent with previously reported regional differences in subtype prevalence.

Lymphoma is a common cancer affecting dogs, particularly Golden Retrievers. By identifying risk factors for lymphoma, work can be done to reduce harmful exposures or increase monitoring among dogs at a higher risk of disease. Using a subset of dogs from the Golden Retriever Lifetime Study, we sought to investigate whether dogs with lymphoma were more likely to live near certain environmental pollutant sources than dogs without lymphoma.Forty-nine Golden Retrievers with non-cutaneous lymphoma and 98 Golden Retrievers without a history of cancer were selected from the Golden Retriever Lifetime Study Cohort. We evaluated how close each dog lived to nine environmental pollutant sources: chemical plants, municipal dumps, manufacturing plants, incineration plants, railroad embankment tracks, landfills, coal plants, high-voltage transmission lines, and nuclear power plants. Additionally, we evaluated individual exposure to secondhand smoke, and average annual ozone and particulate matter exposure (as surrogate measures for air pollution) for each dog's county of residence.None of the exposures examined were associated with an increased lymphoma risk in this population. More research is needed, including direct biomonitoring, to determine whether specific environmental exposures are associated with lymphoma in the Golden Retriever breed.

Evaluation of sulfonamide detoxification pathways in haematologic malignancy patients prior to intermittent trimethoprim-sulfamethoxazole prophylaxis.

AIMS: Patients with haematologic malignancies have a reportedly high incidence of sulfamethoxazole (SMX) hypersensitivity. The objective of this study was to determine whether deficiencies in sulfonamide detoxification pathways, to include glutathione (GSH) and ascorbate (AA), and cytochrome b(5) (b5) and cytochrome b(5) reductase (b5R), were prevalent in these patients. A secondary pilot objective was to determine whether the incidence of drug hypersensitivity following intermittent trimethoprim-SMX (TMP-SMX) prophylaxis approached that reported for high dose daily regimens. METHODS: Forty adult patients with haematologic malignancies (HM) and 35 healthy adults were studied; an additional 13 HM patients taking ascorbate supplements (HM-AA) were also evaluated. Twenty-two of 40 HM patients were prescribed and were compliant with TMP-SMX 960 mg three to four times weekly. RESULTS: There were no significant differences between HM and healthy groups in plasma AA (median 37.2 µm vs. 33.9 µm) or red blood cell GSH (1.9 mmvs. 1.8 mm). However, plasma AA was correlated significantly with leucocyte b5/b5R reduction (r= 0.39, P= 0.002). Deficient b5/b5R activities were not found in HM patients. In fact, patients with chronic lymphocytic leukaemia or myeloma had significantly higher median activities (80.7 µmol mg(-1) min(-1)) than controls (18.9 µmol mg(-1) min(-1), P= 0.008). After 3-4 weeks of treatment, no patients developed SMX-specific T cells and only one patient developed rash. CONCLUSIONS: Deficiencies of blood antioxidants and b5/b5R reduction were not found in this population with haematologic malignancies, and the development of skin rash and drug-specific T cells appeared to be uncommon with intermittent TMP-SMX prophylaxis.

Plasma and urinary F2-isoprostane markers of oxidative stress are increased in cats with early (stage 1) chronic kidney disease.

OBJECTIVES: Oxidative stress contributes to chronic kidney disease (CKD) progression in humans and rodent models; F2-isoprostanes (F2-IsoPs) are established biomarkers of oxidative stress. Our primary aim was to evaluate plasma F2-IsoPs in cats with International Renal Interest Society stage 1 and 2 CKD, compared with healthy cats, and to determine whether plasma and urinary F2-IsoPs are equivalent biomarkers. The secondary aim was to assess whether consumption of a renal diet enriched in omega-3 fatty acids led to improvements in plasma and urinary F2-IsoPs. METHODS: Plasma and urinary F2-IsoPs were measured in 24 cats with stage 1 or 2 CKD, and 12 unaffected controls aged ⩾6 years. Twelve CKD cats were re-evaluated after feeding a commercial renal diet for at least 4 weeks. RESULTS: Median plasma F2-IsoPs were significantly higher in stage 1 CKD (96.2 pg/ml), early stage 2 CKD (83.2 pg/ml) and late stage 2 CKD (80.8 pg/ml) compared with healthy cats (22.8 pg/ml; P = 0.03-0.002). Median urinary F2-IsoPs were significantly higher in cats with stage 1 CKD (231.2 pg/mg) compared with healthy cats (152.5 pg/mg) or cats with late stage 2 CKD (124.8 pg/mg; P = 0.01). Plasma F2-IsoPs remained increased, while urinary F2-IsoPs fell with transition from stage 1 to stage 2 CKD. Feeding a commercial renal diet led to significant decreases in plasma F2-IsoPs in the small group of cats with stage 1 CKD (25-75% decrease) compared with cats with stage 2 CKD (20% decrease to 53% increase; P = 0.01). CONCLUSIONS AND RELEVANCE: Oxidative stress is prominent in cats with stage 1 CKD. Plasma and urinary F2-IsoPs are not interchangeable biomarkers in cats with stage 2 CKD. Placebo-controlled studies are indicated to evaluate dietary or pharmacologic doses of omega-3 fatty acids on redox stress and progression of renal dysfunction in cats with stage 1 CKD.

Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction.

OBJECTIVES: NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA. METHODS: Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semiquantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. RESULTS: Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06-1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (P<0.0001, r=0.42; P=0.01, r=0.23, respectively), and expression of both proteins correlated with one another (P<0.0001; r=0.74). A novel cSNP in CYB5A (S5A) was associated with very low activity and protein expression. Two novel CYB5R3 SNPs, R59H and R297H, displayed atypical SMX-HA reduction kinetics and decreased SMX-HA reduction efficiency. CONCLUSION: These studies indicate that although novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low-frequency cSNPs seem to only minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.

Combined ascorbate and glutathione deficiency leads to decreased cytochrome b5 expression and impaired reduction of sulfamethoxazole hydroxylamine.

Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated with drug hypersensitivity reactions, particularly in patients with AIDS. A reactive oxidative metabolite, sulfamethoxazole-nitroso (SMX-NO), forms drug-tissue adducts that elicit a T-cell response. Antioxidants such as ascorbic acid (AA) and glutathione (GSH) reduce SMX-NO to the less reactive hydroxylamine metabolite (SMX-HA), which is further reduced to the non-immunogenic parent compound by cytochrome b (5) (b5) and its reductase (b5R). We hypothesized that deficiencies in AA and GSH would enhance drug-tissue adduct formation and immunogenicity toward SMX-NO and that these antioxidant deficiencies might also impair the activity of the b5/b5R pathway. We tested these hypotheses in guinea pigs fed either a normal or AA-restricted diet, followed by buthionine sulfoximine treatment (250 mg/kg SC daily, or vehicle); and SMX-NO (1 mg/kg IP 4 days per week, or vehicle), for 2 weeks. Guinea pigs did not show any biochemical or histopathologic evidence of SMX-NO-related toxicity. Combined AA and GSH deficiency in this model did not significantly increase tissue-drug adduct formation, or splenocyte proliferation in response to SMX-NO. However, combined antioxidant deficiency was associated with decreased mRNA and protein expression of cytochrome b (5), as well as significant decreases in SMX-HA reduction in SMX-NO-treated pigs. These results suggest that SMX-HA detoxification may be down-regulated in combined AA and GSH deficiency. This mechanism could contribute to the higher risk of SMX hypersensitivity in patients with AIDS with antioxidant depletion.

Clinical presentation of 26 anaplasma phagocytophilum-seropositive dogs residing in an endemic area.

Anaplasma (A.) phagocytophilum, the etiological agent of canine granulocytic anaplasmosis, is capable of inciting moderate to severe clinical disease in a variety of mammals and is endemic in the upper midwest. The purpose of this study was fourfold: to describe the range of clinical signs in dogs seropositive to A. phagocytophilum; to examine the prevalence of immune-mediated hemolytic anemia (IMHA) in this population; to evaluate whether specific clinical signs were associated with coexposure to Borrelia (B.) burgdorferi in actively infected dogs; and to determine whether clinical response to doxycycline was complete in treated dogs. Medical records of dogs seropositive for A. phagocytophilum were reviewed retrospectively. Peripheral blood smears were also reviewed retrospectively for granulocytic Anaplasma morulae. Lethargy (81%), inappetence (58%), and lameness (50%) were the most common clinical signs, followed by fever (46%). Thrombocytopenia was the most common laboratory abnormality, and IMHA was diagnosed in three dogs. Dogs that were thrombocytopenic and had antibodies to both A. phagocytophilum and B. burgdorferi had a median platelet count of 51,000/µL (range 20,000 to 171,000/µL), which was significantly lower than the count in dogs with antibodies only to A. phagocytophilum (P=0.04). Some dogs had an apparent relapse of clinical signs after an appropriate course of doxycycline. Testing for A. phagocytophilum by polymerase chain reaction, serum antibody assays, and/or blood smear evaluation should be considered in dogs with IMHA, cough, or epistaxis and that reside in A. phagocytophilum-endemic areas. If moderate to severe thrombocytopenia is present, testing for concurrent B. burgdorferi infection may be warranted.