Autoantibodies to recombinant canine proinsulin in canine diabetic patients

Objective

To determine whether dogs with spontaneously-occurring diabetes mellitus demonstrate serological reactivity to proinsulin.

Sample population

Serum samples were collected from 15 newly-diagnosed diabetic, 15 insulin-treated diabetic and 15 non-diabetic control dogs.

Procedures

Canine proinsulin was cloned into a prokaryotic expression vector to generate recombinant poly-histidine-tagged protein in Escherichia coli. A Western blotting assay was developed for detection of proinsulin autoantibodies in canine sera.

Results

Reactivity to canine proinsulin was detected in 3 of 15 control dogs, 8 of 15 newly-diagnosed diabetic dogs and 6 of 15 insulin-treated diabetic patients. Of these reactors, only 1 control dog, 1 newly-diagnosed diabetic dog and 3 insulin-treated diabetic dogs recognised porcine insulin by ELISA, suggesting that the remaining proinsulin reactors might have been recognising proinsulin-specific epitopes.

Conclusions and clinical relevance

This study suggests that proinsulin autoantibodies are present in a proportion of diabetic dogs. Further work is required to refine the assay and clarify the significance of these autoantibodies.     

Quantifying fenbendazole and its metabolites in self-medicating wild red grouse Lagopus lagopus scoticus using an HPLC-MS-MS approach

On red grouse estates in the UK the nematode parasite Trichostrongylus tenuis is often controlled by application of grit medicated with the anthelmintic fenbendazole (FBZ). To date, assessment of the efficacy has been inhibited by the inability to quantify uptake of FBZ by the birds. We have developed a simple and sensitive HPLC-MS-MS method for detecting and quantifying FBZ and its metabolites from a 300 mg sample of red grouse liver. This method could be used to improve the efficacy of medicated grit treatment by allowing the identification of conditions and application methods that optimize the uptake of FBZ. With the necessary modifications, our method will also be applicable to other wildlife species where self-medication is used for parasite control.     

Chromatographic fingerprint analysis and rutin and quercetin compositions in the leaf and whole-plant samples of di- and tetraploid Gynostemma pentaphyllum

Gynostemma pentaphyllum (Thunb.) Makino, also known as jiaogulan, has been shown to have antioxidant, antiproliferative, and anti-inflammatory activities. Flavonoid is considered a major contributor for these beneficial effects. To obtain chemical patterns of flavonoids in G. pentaphyllum of different genotypes (di- versus tetraploids) and different parts (leaf versus whole plant) of plants, the extraction condition was optimized and a fingerprinting approach was established by means of high-performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD-MS). Eight flavonoids were identified, among which rutin and quercetin were quantified. The highest levels of rutin and quercetin were 23.03 and 12.10 mg/g, respectively, observed in the diploid leaf sample 2L3 and 2L2, while the lowest levels of rutin and quercetin were 1.92 and 0.25 mg/g in the tetraploid whole-plant sample 4W3. The chemical patterns were further analyzed by similarity calculation and principal component analysis (PCA). Seven common characteristic peaks were found in all of the tested samples. Flavonoid patterns of tetraploids were significantly different from those of diploids, whereas different parts of plants showed less difference. The flavonoid pattern of the diploid leaf sample was most similar to that of the reference botanical G. pentaphyllum. The combination of chromatographic fingerprint and quantification analysis could be used for quality assessment of G. pentaphyllum and its derived nutraceutical products.     

Censoring of autoreactive B cell development by the pre-B cell receptor

Antibody diversity occurs randomly as B cells recombine their immunoglobulin (Ig) heavy- and light-chain genes during development. This process inevitably generates reactivity against self structures, and several mechanisms prevent the development of autoreactive B cells. We report here a role for the pre-B cell receptor, composed of Ig heavy and surrogate light chains, in the negative selection of cells expressing Ig heavy chains with the potential to generate autoantibodies. Surrogate light-chain-deficient (SLC-/-) mice harbored elevated levels of antinuclear antibodies (ANAs) in their serum and showed evidence of escape of pre-B cells expressing prototypic autoantibody heavy chains from negative selection, leading to mature autoantibody secreting CD21-CD23- B cells in the periphery. Thus, the pre-B cell receptor appears to censor the development of certain autoantibody-secreting cells and may represent an important factor in multifactorial autoimmune diseases.     

Comparison of milk culture, direct and nested polymerase chain reaction (PCR) with fecal culture based on samples from dairy herds infected with Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in cattle and other farm ruminants, and is also a suspected pathogen of Crohn’s disease in humans. Development of diagnostic methods for MAP infection has been a challenge over the last few decades. The objective of this study was to investigate the relationship between different methods for detection of MAP in milk and fecal samples. A total of 134 milk samples and 110 feces samples were collected from 146 individual cows in 14 MAP-infected herds in southwestern Ontario. Culture, IS900 polymerase chain reaction (PCR) and nested PCR methods were used for detecting MAP in milk; results were compared with those of fecal culture. A significant relationship was found between milk culture, direct PCR, and nested PCR (P < 0.05). The fecal culture results were not related to any of the 3 assay methods used for the milk samples (P > 0.10). Although fecal culture showed a higher sensitivity than the milk culture method, the difference was not significant (P = 0.2473). The number of MAP colony-forming units (CFU) isolated by culture from fecal samples was, on average, higher than that isolated from milk samples (P = 0.0083). There was no significant correlation between the number of CFU cultured from milk and from feces (Pearson correlation coefficient = 0.1957, N = 63, P = 0.1243). The animals with high numbers of CFU in milk culture may not be detected by fecal culture at all, and vise versa. A significant proportion (29% to 41%) of the positive animals would be missed if only 1 culture method, instead of both milk and feces, were to be used for diagnosis. This suggests that the shedding of MAP in feces and milk is not synchronized. Most of the infected cows were low-level shedders. The proportion of low-level shedders may even be underestimated because MAP is killed during decontamination, thus reducing the chance of detection. Therefore, to identify suspected Johne’s-infected animals using the tests in this study, both milk and feces samples should be collected in duplicate to enhance the diagnostic rate. The high MAP kill rate identified in the culture methods during decontamination may be compensated for by using the nested PCR method, which had a higher sensitivity than the IS900 PCR method used.     

Dirofilaria repens infection in a dog imported to Norway

Dirofilaria repens infection was diagnosed in a dog that had been imported to Norway from Hungary three years previously. The dog was a four-year-old castrated male mixed-breed dog and presented for examination of two masses on the right thoracic wall. Fine needle sampling from the subcutaneous nodules and subsequent cytological examination revealed a high number of microfilariae and a pyogranulomatous inflammation. At re-examination approximately 3 weeks later, both masses had apparently disappeared spontaneously, based on both inspection and palpation. However, examination of peripheral blood by a modified Knott’s test revealed a high number of unsheathed microfilariae with mean length of 360 μm and mean width of 6-7 μm, often with the classic umbrella handle appearance of D. repens. Polymerase chain reaction and sequencing confirmed the D. repens diagnosis. Subcutaneous dirofilariosis caused by D. repens is probably the most common cause of human zoonotic dirofilariosis in Europe, but currently is rarely encountered in northern countries such as Norway. However, travelling, import and relocation of dogs have increased, and thus the geographical range of these parasites is likely to increase from traditionally endemic southern regions. Increasing numbers of autochthonous cases of D. repens infections in dogs have been reported in eastern and central Europe. Although infection with D. repens often induces only mild signs or subclinical infections in dogs, they nevertheless represent a reservoir for zoonotic transmission and thus a public health concern, and, in addition, due to the long prepatent period and the high frequency of subclinical infections or infections with unspecific clinical signs, could easily be missed. Lack of experience and expectation of these parasites may mean that infection is underdiagnosed in veterinary clinics in northern countries. Also, predicted climate changes suggest that conditions in some countries where this infection is currently not endemic are likely to become more suitable for development in the intermediate host, and thus the establishment of the infection in new areas.     

Contamination of Lettuce Irrigated with Sewage Effluent

Faecal contamination on lettuce growing in the open as indicated by the presence of Escherichia coli I was demonstrated for up to 21 days after irrigation with sewage effluent, although the level of contamination usually decreased after 3–7 days. Recovery of other coliform bacteria gave parallel results ; counts of these organisms and of other bacteria also indicated the persistence of more general pollution.     

Absence of oxysterol-like side effects in human monocytic cells treated with phytosterols and oxyphytosterols

Oxysterols, found in some commonly consumed foods, can induce a wide range of cytotoxic effects, which have been extensively studied. On the other hand, the side effects of phytosterols and oxyphytosterols are less well-known. Over the past few years, different types of foods have been enriched with phytosterols on the basis of the properties of these compounds that reduce circulating cholesterol levels in certain experimental conditions. It is therefore important to gain better knowledge of the risks and benefits of this type of diet. In this study, conducted in human monocytic U937 cells, the ability of phytosterols (sitosterol, campesterol) and oxyphytosterols (7β-hydroxysitosterol, 7-ketositosterol) to induce cell death, polar lipid accumulation, and pro-inflammatory cytokine (MCP-1; IL-8) secretion was determined and compared to that of oxysterols (7-ketocholesterol, 7β-hydroxycholesterol). Phytosterols and oxyphytosterols had no significant effects on the parameters studied; only 7β-hydroxysitosterol slightly increased cell death, whereas at the concentration used (20 μg/mL), strong cytotoxic effects were observed with the oxysterols. With sitosterol, campesterol, and 7-ketositosterol, IL-8 secretion was decreased, and with campesterol the intracellular polar lipid level was reduced. The data show that phytosterols and oxyphytosterols have no oxysterol-like side effects, and they rather argue in favor of phytosterols' beneficial effects.     

Neuroprotective Effects of Longan ( Dimocarpus longan Lour.) Flower Water Extract on MPP(+)-Induced Neurotoxicity in Rat Brain

In this study, the neuroprotective effect of Dimocarpus longan Lour. flower water extract (LFWE) was investigated. First, an in vitro study showed that LFWE concentration-dependently inhibited lipid peroxidation of brain homogenates incubated at 37 °C. The antioxidative activity of LFWE was more potent than that of glutathione or Trolox. Furthermore, an ex vivo study found that the basal lipid peroxidation (0 °C) and lipid peroxidation incubated at 37 °C were lower in the brain homogenates of LFWE-treated (500 mg/day) rats, indicating that the brain of LFWE-treated rats was more resistant to oxidative stress. Moreover, a Parkinsonian animal model was employed to demonstrate that oral administration of LFWE (125-500 mg/kg/day) dose-dependently attenuated 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity in the nigrostriatal dopaminergic system of rat brain. In conclusion, this study suggests that LFWE is antioxidative, anti-inflammatory, and anti-apoptotic. Furthermore, oral administration of LFWE appears to be useful in preventing and/or treating central nervous system neurodegenerative diseases, including Parkinsonism.     

Tissue distribution and cytochrome p450 inhibition of sesaminol and its tetrahydrofuranoid metabolites

Sesame lignans such as sesamin, sesaminol, and sesamolin are major constituents of sesame oil, and all have a methylenedioxyphenyl group and multiple functions in vivo. It was previously reported that sesaminol, a tetrahydrofurofuran type lignin, was metabolized to mammalian lignans. The present study examined the tissue distribution of sesaminol in Sprague-Dawley (SD) rats. Changes in the concentration of sesaminol and its metabolites (sesaminol glucuronide/sulfate, hydroxymethylsesaminol-tetrahydrofuran, enterolactone, and enterodiol) were determined in tissues within a 24 h period after tube feeding (po 220 mg/kg) to SD rats. The concentrations of enterodiol and enterolactone were significantly higher than those of sesaminol and its tetrahydrofuranoid metabolites in the organs (liver, heart, brain, and kidney). This study demonstrates that sesaminol has potent inhibition of cytochrome P450 (CYPs), compared to tetrahydrofuranoid metabolites. The IC(50) values of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 for sesaminol were determined as 3.57, 3.93, 0.69, 1.33, and 0.86 μM, respectively. In addition, hydroxymethylsesaminol-tetrahydrofuran and enterodiol were weak inhibitors of CYP2C9 and CYP1A2, respectively.