Changes in the salivary proteome of beagle dogs after weight loss.

Being overweight or obese represents an important health issue in humans and pets. The aim of this study was to investigate changes in the salivary proteome of overweight beagles after induced weight loss to better understand the physiological changes involved in this process. Five overweight/obese neutered males of pure breed beagles were evaluated. During the 3-mo period of weight loss, each animal received a strictly controlled amount of a low fat commercial diet per day. Body condition scores (BCS), body weight (BW), and serum biochemical parameters (total cholesterol, triglycerides, and C-reactive protein) were assessed weekly. Quantitative proteomics analysis by SWATH was used to evaluate the salivary proteome changes induced by weight loss treatment. BCS, BW, serum total cholesterol concentration, and abundances of 23 salivary proteins differed significantly between before and after treatment. Some of the altered protein amounts, namely of peptidyl-prolyl cis-trans isomerase, fructose-bisphosphate aldolase C, and 78-kDa glucose-regulated protein, increased after weight loss. These proteins are related with the immune system, inflammatory status, oxidative stress, and glucose metabolism. The results obtained suggest a potential use of salivary proteins in monitoring physiological changes in dogs subjected to weight loss. Moreover, the type of changes identified reinforces the postulated physiological improvements, which weight loss induces. Further research is needed to determine whether the changes observed in this study are due to weight loss, dietary changes, or a combination of both.

Vitamin E nanoemulsion activity on stored red blood cells.

BACKGROUND: Stored red blood cells (RBCs) undergo numerous changes that have been termed RBC storage lesion, which can be related to oxidative damage. Vitamin E is an important antioxidant, acting on cell lipids. Thus, this study aimed to investigate vitamin E activity on stored RBCs. METHODS: We prepared a vitamin E nanoemulsion that was added to RBC units and stored at 4 °C. Controls, without vitamin E, were kept under the same conditions. Reactive oxygen species (ROS) production was monitored for up to 35 days of storage. RBC elasticity was also evaluated using an optical tweezer system. RESULTS: Vitamin E-treated samples presented a significant decrease in ROS production. Additionally, the elastic constant for vitamin E-treated RBCs did not differ from the control. CONCLUSION: Vitamin E decreased the amount of ROS in stored RBCs. Because vitamin E acts on lipid oxidation, results suggest that protein oxidation should also be considered a key factor for erythrocyte elastic properties. Thus, further studies combining vitamin E with protein antioxidants deserve attention, aiming to better preserve overall stored RBC properties.

Resistance of Nonmelanoma Skin Cancer to Nonsurgical Treatments. Part II: Photodynamic Therapy, Vismodegib, Cetuximab, Intralesional Methotrexate, and Radiotherapy. / Resistencias al tratamiento no quirúrgico en cáncer cutáneo no melanoma. Parte II: terapia fotodinámica, vismodegib, cetuximab, metotrexato intralesional y radioterapia.

A wide range of treatments is now available for nonmelanoma skin cancer, including 5-fluorouracil, ingenol mebutate, imiquimod, diclofenac, photodynamic therapy, methotrexate, cetuximab, vismodegib, and radiotherapy. All are associated with high clinical and histologic response rates. However, some tumors do not respond due to resistance, which may be primary or acquired. Study of the resistance processes is a broad area of research that aims to increase our understanding of the nature of each tumor and the biologic features that make it resistant, as well as to facilitate the design of new therapies directed against these tumors. In this second article, having covered the topical treatments of nonmelanoma skin cancer, we review resistance to other nonsurgical treatments, such as monoclonal antibodies against basal and squamous cell carcinomas, intralesional chemotherapy, photodynamic therapy, and radiotherapy.

Post-translational allosteric activation of the P2X7 receptor through glycosaminoglycan chains of CD44 proteoglycans.

Here, we present evidence for the positive allosteric modulation of the P2X7 receptor through glycosaminoglycans (GAGs) in CHO (cell line derived from the ovary of the Chinese hamster) cells. The marked potentiation of P2X7 activity through GAGs in the presence of non-saturating agonists concentrations was evident with the endogenous expression of the receptor in CHO cells. The presence of GAGs on the surface of CHO cells greatly increased the sensitivity to adenosine 5'-triphosphate and changed the main P2X7 receptor kinetic parameters EC50, Hill coefficient and E max. GAGs decreased the allosteric inhibition of P2X7 receptor through Mg(2+). GAGs activated P2X7 receptor-mediated cytoplasmic Ca(2+) influx and pore formation. Consequently, wild-type CHO-K1 cells were 2.5-fold more sensitive to cell death induced through P2X7 agonists than mutant CHO-745 cells defective in GAGs biosynthesis. In the present study, we provide the first evidence that the P2X7 receptor interacts with CD44 on the CHO-K1 cell surface. Thus, these data demonstrated that GAGs positively modulate the P2X7 receptor, and sCD44 is a part of a regulatory positive feedback loop linking P2X7 receptor activation for the intracellular response mediated through P2X7 receptor stimulation.

Cryptomphalus aspersa mollusc eggs extract promotes migration and prevents cutaneous ageing in keratinocytes and dermal fibroblasts in vitro.

BACKGROUND: The search of substances that minimize cutaneous ageing has increased in the last few years. Previous studies have described the regenerative properties of the secretion of the mollusc Cryptomphalus aspersa (C. aspersa) when applied topically. OBJECTIVE: We evaluate the in vitro effects of a new product derived from the eggs of C. aspersa, IFC-CAF, on cell proliferation, migration, distribution of cytoskeletal proteins, production of extracellular components as well as its ability to prevent cutaneous ageing because of intrinsic or extrinsic factors (exposure to UVB) by determination of ageing markers. METHODS: We have used the human keratinocyte cell line (HaCaT cells), primary dermal fibroblasts (HDF) and senescent dermal fibroblasts (SHDF). The effects of the compound on cell proliferation and on the cell cycle were determined by the MTT colorimetric assay, estimation of total protein and/or trypan blue test and by flow cytometry, respectively. We also studied cell migration using the wound-healing migration assay, whereas ELISA assays, Western Blot and immunofluorescence microscopy were carried out to test the expression of proteins related to cytoskeleton, extracellular matrix and with ageing. RESULTS: We have found that IFC-CAF does not promote proliferation but induces migration of HaCaT, HDF and SHDF in a time- and dose-dependent manner; a better organization of cytoskeletal proteins (F-actin and vimentin) and promotes the production of extracellular components (fibronectin, collagen 1 and MMPs) and the adhesion to cell-substrate vinculin protein. IFC-CAF also prevents cutaneous ageing. The treatment decreases the expression of the ageing-related markers b-Gal, p53 and p16INK4 in SDDF cells, and improves cell survival after UVB irradiation and nuclear repair in HaCaT cells. CONCLUSION: IFC-CAF has regenerative properties and protects against ageing factors being, therefore, a potential therapeutic agent for treating or preventing skin ageing.

Effect of rapamycin on spleen size in longstanding renal transplant recipients.

BACKGROUND: Based on evidence available in the literature, rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, but not calcineurin inhibitors (CNIs), has been shown to decrease spleen size. Small spleen, in some instances, is associated with hyposplenism, a condition recently reported in patients with longstanding renal transplant. Accordingly, the effect of immunosuppressive drugs on spleen size was evaluated. METHODS: Renal transplant recipients (35 taking mTOR and 68 CNI) were included, in whom a standardized investigation of the kidney allograft and spleen with the use of color Doppler ultrasound was performed and a peripheral smear were reviewed for the presence of Howell-Jolly bodies (HJBs). RESULTS: We enrolled 103 patients (64 men; 66 from a deceased donor). The mean age was 47.7 years (range, 23.0-74.0 y). Mean transplant duration was 1,899 days (range, 181-6,883 d). According to the presence of HJBs, the prevalence of hyposplenism was 47.6% for the entire cohort. The differences between the mTOR and CNI groups regarding sex and the presence of HJBs were not statistically significant (P > .05). Age, creatinine, hemoglobin, leukocytes, platelets, and Doppler parameters in spleen and kidney were similar in both groups (P > .05). mTOR patients had a decreased spleen length size (90.09 ± 13.02 mm vs 111.95 ± 18.66 mm; P < .001), a longer transplant duration (3,576 ± 1,594 d vs 1,036 ± 1,369 d; P < .001) and higher serum cholesterol (227.50 ± 38.75 mg/dL vs 182.67 ± 37.74 mg/dL; P < .001) and triglycerides (194.23 ± 79.88 mg/dL vs 148.70 ± 55.54 mg/dL; P = .003) levels compared with the CNI group. A multivariate analysis showed mTOR inhibitor to be the most important predictor of spleen size. In both the mTOR and CNI groups, the comparison between the subgroups of present and absent HJBs did not show any difference. CONCLUSIONS: The findings of this study suggest that small spleens in transplant recipients may be linked to treatment with an mTOR inhibitor, although this apparently does not compromise splenic function.

Blood clearance of Howell-Jolly bodies in an experimental autogenic splenic implant model.

BACKGROUND: Autogenic splenic implant (ASI) is one of the few alternatives for preservation of splenic tissue when total splenectomy is inevitable. The aim of this study was to determine the morphological and functional regeneration of ASIs, as indicated by the clearance of Howell-Jolly (HJ) bodies, in an experimental model. METHODS: Ninety-nine male Wistar rats were divided into three groups: sham-operated (group 1), total splenectomy alone (group 2), and total splenectomy combined with ASI (group 3). Animals in group 3 were further allocated to nine subgroups of nine rats each, and analysed at different time points (1, 4, 8, 12, 16, 20, 24, 28 and 32 weeks after surgery). Blood smears were prepared at predetermined times for detection of HJ bodies. Morphological regeneration of tissue in the ASI was analysed by histology. RESULTS: At 1 week, the regenerated mass corresponded to about 7 per cent of the tissue implanted, reaching approximately 54 per cent at 24 weeks. The HJ body levels were increased in groups 2 and 3 until 8 weeks after surgery, following which levels in the ASI group became similar to those in the sham-operated group. HJ bodies were difficult to detect when a level of 22.5 per cent of regenerated ASI mass was reached. CONCLUSION: Functional regeneration of ASIs occurred from 8 weeks after surgery. When 22.5 per cent of regenerated ASI mass was reached almost no HJ bodies could be observed in the bloodstream, resembling a spleen in situ. SURGICAL RELEVANCE: Splenectomy has been practised routinely, both in the emergency setting and as a therapeutic elective procedure. There is a correlation between asplenia/hyposplenia and the occurrence of fulminant sepsis, underlining the importance of developing surgical methods for preserving splenic function. Both clinical and experimental studies have shown at least partial morphological and functional regeneration of autogenic splenic implants (ASIs). Experimental studies investigating the immunoprotective effect of ASIs, based mostly on exposure of animals to various bacteria, have demonstrated that ASIs can increase the rate of bacterial clearance and decrease mortality from sepsis. Clinical studies have shown their ability to remove colloidal substances and altered erythrocyte corpuscular inclusions, such as Howell-Jolly, Heinz and Pappenheimer bodies, from the bloodstream. In this experimental study the functional and morphological regeneration of ASIs was studied over time in rats.

Functional hyposplenism in long-standing renal transplant recipients.

BACKGROUND: A nephrologist with expertise in ultrasonography noticed that patients with longstanding renal grafts had smaller spleens than subjects undergoing initial post-transplantation imaging. This putative finding prompted us to pursue a further investigation into splenic function based on Doppler ultrasound and hematologic parameters. METHODS: We enrolled 47 patients with functioning long-standing kidney grafts, measuring longitudinal diameter of the spleen, hilar and intrasplenic peak systolic velocities (PSV), and hilar and intrasplenic resistivity indices of the splenic artery as well as mean arterial blood pressure (MAP). Giemsa-stained peripheral blood smears were examined for the presence of Howell-Jolly bodies (HJBs) using light microscopy. The patients were then divided into HJB present (HJ(+)) or absent (HJ(-)) groups for further comparison. RESULTS: The overall mean age of 21 females and 26 males was 47.8 ± 12.0 years, and the mean time after transplantation was 2750 ± 1818 days (range, 208-6446). HJBs were detected in 23/47 patients (48.9%). The intrasplenic artery PSV was significantly lower and MAP higher in the HJ(+) group (P < .05). There was no difference in spleen size between the groups. DISCUSSION: HJBs in peripheral blood red cells, an indicator of hyposplenism, was associated with reduced intrasplenic artery PSV, suggesting dysfunction, which may play a role in the known vulnerability of renal transplant recipients to infections.

Relationship between digestive enzymes and food habit of Lutzomyia longipalpis (Diptera: Psychodidae) larvae: Characterization of carbohydrases and digestion of microorganisms.

The sandfly Lutzomyia longipalpis (Lutz and Neiva, 1912) is the main vector of American Visceral Leishmaniasis. In spite of its medical importance and several studies concerning adult digestive physiology, biochemistry and molecular biology, very few studies have been carried out to elucidate the digestion in sandfly larvae. Even the breeding sites and food sources of these animals in the field are largely uncharacterized. In this paper, we describe and characterize several carbohydrases from the gut of L. longipalpis larvae, and show that they are probably not acquired from food. The enzyme profile of this insect is consistent with the digestion of fungal and bacterial cells, which were proved to be ingested by larvae under laboratory conditions. In this respect, sandfly larvae might have a detritivore habit in nature, being able to exploit microorganisms usually encountered in the detritus as a food source.

Patients' satisfaction and functional assessment of existing complete dentures: correlation with objective masticatory function.

The aim of this study was to evaluate the correlation between patients' and dentist's assessment of dentures and to correlate these variables with objective measures of masticatory function. A sample of 28 edentulous individuals was selected, all wearing both complete dentures for at least 6 months and with no signs or symptoms of temporomandibular joint disorders. They rated their level of satisfaction with their dentures from 0 to 100 by means of a visual analogue scale, and dentures were scored by a dentist from 0 to 9 considering functional aspects. Tooth wear was evaluated on the posterior teeth of dentures considering the lack of occlusal anatomy. Masticatory performance and swallowing threshold tests were performed with an artificial test food (Optocal), and the median particle size was determined by the sieving method. The results showed that the mean satisfaction value of volunteers with their dentures was 49·1 and the median score of dentist's evaluation was 6. The Spearman correlation coefficient revealed no significant correlation between patients' and dentist's assessment of dentures (P>0·05). The median particle size for masticatory performance and swallowing thresholds was 5·5 ± 1·0 and 4·9 ± 1·2 mm, respectively. Data of both masticatory tests showed no significant correlation with patients' satisfaction or with dentist's evaluation of dentures. There was no difference of food comminution between subjects with and without excessive posterior tooth wear. It can be concluded that dentist's and patients' assessment of dentures were not correlated, and no correlation was observed between these variables and masticatory function.

Clinical scoring system for paediatric tuberculosis in HIV-infected and non-infected children in Rio de Janeiro.

The scoring system for diagnosing tuberculosis (TB) used in Brazil has already been validated in human immunodeficiency virus (HIV) negative tuberculous children. Here we studied HIV-positive children. The individuals were divided into four groups: 1) tuberculous infection; 2) other pulmonary diseases; 3a) pulmonary TB and 3b)pulmonary TB and HIV. Group 3a had a significantly higher score than Groups 1 and 2, and Group 1 had a higher score than Group 2. Scores for Group 3b were lower than for Group 3a, although they were still >30 points, which is enough to establish a TB diagnosis (P = 0.0005). The system was useful for the diagnosis of pulmonary TB in HIV-negative and -positive children.

Methylprednisolone administration alters adenine nucleotide hydrolysis in rat blood serum.

The effect of methylprednisolone on the hydrolysis of adenine nucleotides by rat blood serum enzymes was studied. Adult male Wistar rats were submitted to three different treatments with synthetic steroid methylprednisolone: one dose of 50 mg/kg, i.p. (acute); or oral doses of 6 mg/kg dissolved in drinking water for 15 (sub-chronic) or 30 (chronic) days. Decreased ADP hydrolysis was observed after acute and sub-chronic treatments. Furthermore, ATP, ADP and AMP hydrolysis decreased after chronic treatment. These alterations may constitute one of the mechanisms that mediate the development of some of the side effects associated with corticosteroid use.

Ecto-nucleotidase activities in spinal cord of rats changes as function of age.

An increase in ADP hydrolysis was observed in spinal cord synaptosomal fractions of 2-month-old Wistar male rats, when compared to other ages (1, 4 and 6 months of age), while no change in ATPase activity was observed. Conversely, in female rats, whilst no change in ADPase activity was observed in the spinal cord synaptosomal fraction, ATPase activity diminished with age, in 1-6-month-old animals. 5'-Nucleotidase activity was higher in the 4-month-old male and female rats in relation to 1 and 2-month-old animals. In the female rats, this activity continued to increase at least until 6 months of age. In conclusion, adenine nucleotides hydrolysis in synaptosomes from rat spinal cord is influenced by age and by gender. Since both ATP and adenosine may act as neuromodulators in the spinal cord, influencing several processes such as nociception, the regulation of ATP-metabolizing enzymes in spinal cord is probably important for the normal function of this tissue at different ages.

Kinetics of eosinophil and IgE-mast cell changes following infection with Angiostrongylus costaricensis in Wistar rats.

Human abdominal angiostrongyliasis is a severe eosinophilic disease caused by Angiostrongylus costaricensis. Previous studies have demonstrated that wild rodents are critically involved as definitive hosts to this nematode in nature. In this study, we have evaluated the susceptibility of Wistar rats (Rattus norvegicus) to A. costaricensis infection. Kinetics of parasitological and pathological changes, including the number of adult worms recovered from mesenteric arteries, and of IgE, mast cell and eosinophil levels in several compartments have been assessed. The oral inoculation of third-stage larvae (L3) into adult Wistar rats led to a marked accumulation of worms in the branches of the mesenteric arteries 25 and 50 days post-inoculation. Intense bone marrow eosinophilia ranging from 7 to 50 days was accompanied by marked accumulation of eosinophils in the blood, peritoneal and bronchoalveolar spaces. Eosinophilic periarteritis, oedema and granuloma in the intestinal and lung tissues were also histologically evident. Total serum IgE and specific anti-parasite IgE peaked at 25 days post-infection, as measured by ELISA and by the passive cutaneous anaphylaxis test, respectively. At that time point, there was a drastic reduction in the number of intact mast cells in the peritoneal effluent. These findings indicate that Wistar rats are permissive to A. costaricensis infection. IgE-mast cell activation and massive tissue eosinophil infiltration are marked features in the process and are likely to play a crucial role in the immune-response evoked by this parasite.

The effect of stress upon hydrolysis adenine nucleotides in blood serum of rats.

Alterations of enzyme activities involved in adenine nucleotide hydrolysis have been reported in spinal cord and blood serum after repeated restraint stress. On the other hand, no effect was observed in the spinal cord of rats after acute stress. In the present study, we investigated the effect of acute stress on the hydrolysis of adenine nucleotides in rat blood serum. Adult male Wistar rats were submitted to 1-h restraint stress and were sacrificed at 0, 6, 24 and 48 h. Increased ATP and ADP hydrolysis were observed in the blood serum of stressed rats 24 h after stress (58% and 54%, respectively, when compared to controls). On the other hand, the AMP hydrolysis was increased after 6 h (68% when compared to controls) and at 24 h (94% when compared to controls) after stress. The results suggest that altered activity of soluble enzymes in serum may be a biochemical marker for stress situations.

Effect of chronic and acute stress on ectonucleotidase activities in spinal cord.

We have previously observed that, while acute stress induces analgesia, chronic stress causes a hyperalgesic response in male rats. No effect was observed in females. There is increasing evidence that both ATP and adenosine can modulate pain. Extracellular ATP and ADP are hydrolyzed by an apyrase in synaptosomes from the peripheral and central nervous systems. In the present study, we investigated the effect of chronic and acute stress on ATPase-ADPase and 5'-nucleotidase activities in spinal cord of male and female rats. Adult male and female Wistar rats were submitted to 1 h restraint stress/day for 1 day (acute) or 40 days (chronic) and were sacrificed 24 h later. ATPase-ADPase activities were assayed in the synaptosomal fraction obtained from the spinal cord of control and stressed animals. ADP hydrolysis was decreased 25% in chronically stressed males, while no change was observed on ATPase activity. There was an increase in the 5'-nucleotidase activity in the same group. No effect on ADPase, ATPase or on 5'-nucleotidase activity was observed in females with chronic stress, or after acute stress neither in males or females. Chronic stress reduced ADP hydrolysis and increased 5'-nucleotidase activity in the spinal cord in male rats.

Cytokine accumulation in osteitis fibrosa of renal osteodystrophy

Bone marrow fibrosis occurs in association with a number of pathological states. Despite the extensive fibrosis that sometimes characterizes renal osteodystrophy, little is known about the factors that contribute to marrow accumulation of fibrous tissue. Because circulating cytokines are elevated in uremia, possibly in response to elevated parathyroid hormone levels, we have examined bone biopsies from 21 patients with end-stage renal disease and secondary hyperparathyroidism. Bone sections were stained with antibodies to human interleukin-1alpha (IL-1alpha), IL-6, IL-11, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-ß (TGF-ß) using an undecalcified plastic embedding method. Intense staining for IL-1alpha, IL-6, TNF-alpha and TGF-ß was evident within the fibrotic tissue of the bone marrow while minimal IL-11 was detected. The extent of cytokine deposition corresponded to the severity of fibrosis, suggesting their possible involvement in the local regulation of the fibrotic response. Because immunoreactive TGF-ß and IL-6 were also detected in osteoblasts and osteocytes, we conclude that selective cytokine accumulation may have a role in modulating bone and marrow cell function in parathyroid-mediated uremic bone disease

Cytokine accumulation in osteitis fibrosa of renal osteodystrophy.

Bone marrow fibrosis occurs in association with a number of pathological states. Despite the extensive fibrosis that sometimes characterizes renal osteodystrophy, little is known about the factors that contribute to marrow accumulation of fibrous tissue. Because circulating cytokines are elevated in uremia, possibly in response to elevated parathyroid hormone levels, we have examined bone biopsies from 21 patients with end-stage renal disease and secondary hyperparathyroidism. Bone sections were stained with antibodies to human interleukin-1alpha (IL-1alpha), IL-6, IL-11, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) using an undecalcified plastic embedding method. Intense staining for IL-1alpha, IL-6, TNF-alpha and TGF-beta was evident within the fibrotic tissue of the bone marrow while minimal IL-11 was detected. The extent of cytokine deposition corresponded to the severity of fibrosis, suggesting their possible involvement in the local regulation of the fibrotic response. Because immunoreactive TGF-beta and IL-6 were also detected in osteoblasts and osteocytes, we conclude that selective cytokine accumulation may have a role in modulating bone and marrow cell function in parathyroid-mediated uremic bone disease.