The dog as a genetic model for immunoglobulin A (IgA) deficiency: identification of several breeds with low serum IgA concentrations

Immunoglobulin A (IgA) serves as the basis of the secretory immune system by protecting the lining of mucosal sites from pathogens. In both humans and dogs, IgA deficiency (IgAD) is associated with recurrent infections of mucosal sites and immune-mediated diseases. Low concentrations of serum IgA have previously been reported to occur in a number of dog breeds but no generally accepted cut-off value has been established for canine IgAD. The current study represents the largest screening to date of IgA in dogs in terms of both number of dogs (n = 1267) and number of breeds studied (n = 22). Serum IgA concentrations were quantified by using capture ELISA and were found to vary widely between breeds. We also found IgA to be positively correlated with age (p < 0.0001). Apart from the two breeds previously reported as predisposed to low IgA (Shar-Pei and German shepherd), we identified six additional breeds in which ≥10% of all tested dogs had very low (<0.07 g/l) IgA concentrations (Hovawart, Norwegian elkhound, Nova Scotia duck tolling retriever, Bullterrier, Golden retriever and Labrador retriever). In addition, we discovered low IgA concentrations to be significantly associated with canine atopic dermatitis (CAD, p < 0.0001) and pancreatic acinar atrophy (PAA, p = 0.04) in German shepherds.

Stability of 3-bromotyrosine in serum and serum 3-bromotyrosine concentrations in dogs with gastrointestinal diseases

Abstract BACKGROUND: 3-Bromotyrosine (3-BrY) is a stable product of eosinophil peroxidase and may serve as a marker of eosinophil activation. A gas chromatography/mass spectrometry method to measure 3-BrY concentrations in serum from dogs has recently been established and analytically validated. The aims of this study were to determine the stability of 3-BrY in serum, to determine the association between peripheral eosinophil counts and the presence of an eosinophilic infiltrate in the gastrointestinal tract, and to compare serum 3-BrY concentrations in healthy dogs (n = 52) and dogs with eosinophilic gastroenteritis (EGE; n = 27), lymphocytic-plasmacytic enteritis (LPE; n = 25), exocrine pancreatic insufficiency (EPI; n = 26), or pancreatitis (n = 27). RESULTS: Serum 3-BrY concentrations were stable for up to 8, 30, and 180 days at 4°C, -20°C, and -80°C, respectively. There was no significant association between peripheral eosinophil count and the presence of eosinophils in the GI tissues (P = 0.1733). Serum 3-BrY concentrations were significantly higher in dogs with EGE (median [range] = 5.04 [≤0.63-26.26] μmol/L), LPE (median [range] = 3.60 [≤0.63-15.67] μmol/L), and pancreatitis (median [range] = 1.49 [≤0.63-4.46] μmol/L) than in healthy control dogs (median [range] = ≤0.63 [≤0.63-1.79] μmol/L; P < 0.0001), whereas concentrations in dogs with EPI (median [range] = 0.73 [≤0.63-4.59] μmol/L) were not different compared to healthy control dogs. CONCLUSIONS: The present study revealed that 3-BrY concentrations were stable in serum when refrigerated and frozen. No relationship between peripheral eosinophil count and the presence of eosinophils infiltration in the GI tissues was found in this study. In addition, serum 3-BrY concentrations were increased in dogs with EGE, but also in dogs with LPE and pancreatitis. Further studies are needed to determine whether measurement of 3-BrY concentrations in serum may be useful to assess patients with suspected or confirmed EGE or LPE.

Serum and fecal canine α1-proteinase inhibitor concentrations reflect the severity of intestinal crypt abscesses and/or lacteal dilation in dogs.

Gastrointestinal (GI) protein loss, due to lymphangiectasia or chronic inflammation, can be challenging to diagnose. This study evaluated the diagnostic accuracy of serum and fecal canine α1-proteinase inhibitor (cα1PI) concentrations to detect crypt abscesses and/or lacteal dilation in dogs. Serum and fecal cα1PI concentrations were measured in 120 dogs undergoing GI tissue biopsies, and were compared between dogs with and without crypt abscesses/lacteal dilation. Sensitivity and specificity were calculated for dichotomous outcomes. Serial serum cα1PI concentrations were also evaluated in 12 healthy corticosteroid-treated dogs. Serum cα1PI and albumin concentrations were significantly lower in dogs with crypt abscesses and/or lacteal dilation than in those without (both P <0.001), and more severe lesions were associated with lower serum cα1PI concentrations, higher 3 days-mean fecal cα1PI concentrations, and lower serum/fecal cα1PI ratios. Serum and fecal cα1PI, and their ratios, distinguished dogs with moderate or severe GI crypt abscesses/lacteal dilation from dogs with only mild or none such lesions with moderate sensitivity (56-92%) and specificity (67-81%). Serum cα1PI concentrations increased during corticosteroid administration. We conclude that serum and fecal α1PI concentrations reflect the severity of intestinal crypt abscesses/lacteal dilation in dogs. Due to its specificity for the GI tract, measurement of fecal cα1PI appears to be superior to serum cα1PI for diagnosing GI protein loss in dogs. In addition, the serum/fecal cα1PI ratio has an improved accuracy in hypoalbuminemic dogs, but serum cα1PI concentrations should be carefully interpreted in corticosteroid-treated dogs.

Simultaneous determination of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone concentrations in rat serum by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry

An assay using high performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry (ESI-MS-MS) was developed for simultaneously determining concentrations of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone, in 50 mul samples of rat serum. Deuterated (d(3)) analogues of each compound were used as internal standards. Samples were treated with acetonitrile to precipitate plasma proteins: acetonitrile was removed from the supernatant by centrifugal evaporation before analysis. Limits of quantitation (ng/ml) and their between-day accuracy and precision (%deviation and %CV) were-morphine, 3.8 (4.3% and 7.6%); morphine-3-glucuronide, 5.0 (4.5% and 2.9%); oxycodone, 4.5 (0.4% and 9.3%); noroxycodone, 5.0 (8.5% and 4.6%). (C) 2004 Elsevier B.V. All rights reserved.

Effect of oat bran on time to exhaustion, glycogen content and serum cytokine profile following exhaustive exercise

The aim of this study was to evaluate the effect of oat bran supplementation on time to exhaustion, glycogen stores and cytokines in rats submitted to training. The animals were divided into 3 groups: sedentary control group (C), an exercise group that received a control chow (EX) and an exercise group that received a chow supplemented with oat bran (EX-O). Exercised groups were submitted to an eight weeks swimming training protocol. In the last training session, the animals performed exercise to exhaustion, (e.g. incapable to continue the exercise). After the euthanasia of the animals, blood, muscle and hepatic tissue were collected. Plasma cytokines and corticosterone were evaluated. Glycogen concentrations was measured in the soleus and gastrocnemius muscles, and liver. Glycogen synthetase-alpha gene expression was evaluated in the soleus muscle. Statistical analysis was performed using a factorial ANOVA. Time to exhaustion of the EX-O group was 20% higher (515 +/- 3 minutes) when compared with EX group (425 +/- 3 minutes) (p = 0.034). For hepatic glycogen, the EX-O group had a 67% higher concentrations when compared with EX (p = 0.022). In the soleus muscle, EX-O group presented a 59.4% higher glycogen concentrations when compared with EX group (p = 0.021). TNF-alpha was decreased, IL-6, IL-10 and corticosterone increased after exercise, and EX-O presented lower levels of IL-6, IL-10 and corticosterone levels in comparison with EX group. It was concluded that the chow rich in oat bran increase muscle and hepatic glycogen concentrations. The higher glycogen storage may improve endurance performance during training and competitions, and a lower post-exercise inflammatory response can accelerate recovery.

The Importance of Protein-Protein Interactions on the pH-Induced Conformational Changes of Bovine Serum Albumin: A Small-Angle X-Ray Scattering Study

The combined effects of concentration and pH on the conformational states of bovine serum albumin (BSA) are investigated by small-angle x-ray scattering. Serum albumins, at physiological conditions, are found at concentrations of similar to 35-45 mg/mL (42 mg/mL in the case of humans). In this work, BSA at three different concentrations (10, 25, and 50 mg/mL) and pH values (2.0-9.0) have been studied. Data were analyzed by means of the Global Fitting procedure, with the protein form factor calculated from human serum albumin (HSA) crystallographic structure and the interference function described, considering repulsive and attractive interaction potentials within a random phase approximation. Small-angle x-ray scattering data show that BSA maintains its native state from pH 4.0 up to 9.0 at all investigated concentrations. A pH-dependence of the absolute net protein charge is shown and the charge number per BSA is quantified to 10(2), 8(l), 13(2), 20(2), and 26(2) for pH values 4.0, 5.4, 7.0, 8.0, and 9.0, respectively. The attractive potential diminishes as BSA concentration increases. The coexistence of monomers and dimers is observed at 50 mg/mL and pH 5.4, near the BSA isoelectric point. Samples at pH 2.0 show a different behavior, because BSA overall shape changes as a function of concentration. At 10 mg/mL, BSA is partially unfolded and a strong repulsive protein-protein interaction occurs due to the high amount of exposed charge. At 25 and 50 mg/mL, BSA undergoes some refolding, which likely results in a molten-globule state. This work concludes by confirming that the protein concentration plays an important role on the pH-unfolded BSA state, due to a delicate compromise between interaction forces and crowding effects.

Effect of Food Restriction and Intense Physical Training on Estrous Cyclicity and Plasma Leptin Concentrations in Rats

Intense physical training and dietary energy restriction have been associated with consequences such as nutritional amenorrhea. We investigated the effects of intense physical training, food restriction or the combination of both strategies on estrous cyclicity in female rats, and the relationship between leptin ad these effects. Twenty-seven female Wistar rats were distributed into four groups: SF: sedentary, fed ad libitum; SR: sedentary subjected to 50% food restriction (based on the food intake of their fed counterparts); TF: trained (physical training on a motor treadmill with a gradual increase in speed and time), fed ad libitum; TR; trained with 50% food restriction. We analysed estrous cyclicity, plasma leptin and estradiol as well as chemical composition of the carcass, body weight variation. and weight of ovaries and perirenal adipose tissue. Data demonstrate that physical training alone was not responsible for significant modifications in either carcass chemical composition or reproductive function. Food restriction reduced leptin levels in all animals and interrupted the estrous cyclicity in some animals, but only the combination of food restriction and physical training was capable of interrupting the estrous cyclicity in all animals. Leptin was not directly related to estrous cyclicity. From our findings, it may be concluded that there is an additive or synergistic effect of energy intake restriction and energy expenditure by intense physical training on estrous cyclicity. Leptin appears to be one among others factors related to estrous cycle, but it probably acts indirectly.

Association between decreased vitamin levels and MTHFR, MTR and MTRR gene polymorphisms as determinants for elevated total homocysteine concentrations in pregnant women

Objectives: To examine the association between methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C), methionine synthase (MTR) A2756G and methionine synthase reductase (MTRR) A66G gene polymorphisms and total homocysteine (tHcy), methylmalonic acid (MMA) and S-adenosylmethionine/ S-adenosylhomocysteine (SAM/SAH) levels; and to evaluate the potential interactions with folate or cobalamin (Cbl) status. Subjects/ Methods: Two hundred seventy-five healthy women at labor who delivered full-term normal babies. Cbl, folate, tHcy, MMA, SAM and SAH were measured in serum specimens. The genotypes for polymorphisms were determined by PCR-restriction fragment length polymorphism ( RFLP). Results: Serum folate, MTHFR 677T allele and MTR 2756AA genotypes were the predictors of tHcy levels in pregnant women. Serum Cbl and creatinine were the predictors of SAM/SAH ratio and MMA levels, respectively. The gene polymorphisms were not determinants for MMA levels and SAM/SAH ratios. Low levels of serum folate were associated with elevated tHcy in pregnant women, independently of the gene polymorphisms. In pregnant women carrying MTHFR 677T allele, or MTHFR 1298AA or MTRR 66AA genotypes, lower Cbl levels were associated with higher levels of tHcy. Lower SAM/SAH ratio was found in MTHFR 677CC or MTRR A2756AA genotypes carriers when Cbl levels were lower than 142 pmol/l. Conclusions: Serum folate and MTHFR C677T and MTR A2576G gene polymorphisms were the determinants for tHcy levels. The interaction between low levels of serum Cbl and MTHFR (C677T or A1298C) or MTRR A66G gene polymorphisms was associated with increased tHcy.

Differences in both matrix metalloproteinase 9 concentration and zymographic profile between plasma and serum with clot activators are due to the presence of amorphous silica or silicate salts in blood collection devices

Matrix metalloproteinases (MMPs) are promising diagnostic tools, and blood sampling/handling alters MMP concentrations between plasma and serum and between serum with and without clot activators. To explain the higher MMP-9 expression in serum collected with clot accelerators relative to serum with no additives and to plasma, we analyzed the effects of increasing amounts of silica and silicates (components of clot activators) in,citrate plasma, serum, and huffy coats collected in both plastic and glass tubes from 50 healthy donors, and we analyzed the effects of silica and silicate on cultured leukemia cells. The levels of MMP-2 did not show significant changes between glass and plastic tubes, between serum and plasma, between serum with and without clot accelerators, or between silica and silicate treatments. No modification of MMP-9 expression was obtained by the addition of silica or silicate to previously separated plasma and serum. Increasing the amounts of nonsoluble silica and soluble silicate added to citrate and empty tubes prior to blood collection resulted in increasing levels of MMP-9 relative to citrate plasma and serum. Silica and silicate added to buffy coats and leukemia cells significantly induced MMP-9 release/secretion, demonstrating that both silica and silicate induce the release of pro- and complexed MMP-9 forms. We recommend limiting the misuse of serum and avoiding the interfering effects of clot activators. (c) 2007 Elsevier Inc. All rights reserved.

Characterization of serum antibodies to Porphyromonas gingivalis in individuals with and without periodontitis

Although Porphyromonas gingivalis is a defined pathogen in periodontal disease, many subjects control the infection without experiencing loss of attachment. Differences in host susceptibility to the disease may be reflected in the pattern of humoral antibodies against specific P. gingivalis antigens. The aim of this study was to determine the presence of antibodies against immunodominant P. gingivalis antigens as well as the isotype and subclass of anti-P. gingivalis antibodies against outer membrane antigens in four groups of patients: P. gingivalis-positive, 1) with and 2) without periodontitis, and P. gingivalis-negative, 3) with and 4) without periodontitis. Antigens of molecular weight 92, 63, and 32 kDa and lipopolysaccharide were found to be immunodominant. Group 1 subjects showed a significantly higher response to the 92 and 63 kDa antigens compared with other groups. The response to lipopolysaccharide was significantly higher in group 1, and lower in group 4 than in groups 2, 3. Immunoglobulin G(1) (IgG(1)), IgG(2) and IgM antibodies against P. gingivalis outer membrane were present in all subjects, while only some subjects were seropositive for IgG(3), IgG(4) and IgA. There were no differences in concentrations for IgG(1), IgG(3) and IgM. The IgG(2) concentration in group 4 was significantly higher than in groups 1 and 2, while the IgG(4) concentration in group 4 was significantly lower than in other groups. The frequency of seropositivity for IgG(4) and IgA was lowest in group 4, while IgG; seropositivity was almost exclusively seen in healthy patients iii groups 2, 4. These findings suggest that the presence of IgG(3) may reflect non-susceptibility to the disease, while lack of IgG(4) may be indicative of periodontal health and lack of infection.