Clinical efficacy of nutraceutical diet for cats with clinical signs of cutaneus adverse food reaction (CAFR).

Food allergies and food intolerances are clinically difficult to discriminate. Most often, along with cutaneous adverse food reactions or CAFR, they are classified as adverse food reactions, whose causes are numerous, including toxic compounds. Eighteen indoor-housed domestic cats with evident clinical symptoms related to CAFR (drooling, back and neck intense itching, neck eczema, chronic conjunctivitis and stomatitis) involving skin lesions were studied. Cytological evaluations of ear, skin and gingival swabs revealed an increased turnover of keratinocytes while the oxytetracycline ELISA determination showed an unexpected high amount of oxytetracycline in all cats at the first visit. All cats were then randomly assigned to receive a standard (SD group) or a nutraceutical diet (ND group) for 60 days. In the ND group a significant reduction of the mean serum concentration of oxytetracycline, pruritus intensity and skin lesion severity (**p<0.01, ***p<0.001, and ***p<0.001, respectively) was observed after 60 days, and associated with a significant improvement in the clinical picture. Although a direct correlation between oxytetracycline presence within cat sera and CAFR-related symptoms has never been described, this study highlights the benefit of a specific nutraceutical diet supplementation in improving clinical symptoms and skin lesions in cats with CARF.

Toxicological Implications and Inflammatory Response in Human Lymphocytes Challenged with Oxytetracycline.

Antibiotics are widely used in zoo technical and veterinary practices as feed supplementation to ensure wellness of farmed animals and livestock. Several evidences have been suggesting both the toxic role for tetracyclines, particularly for oxytetracycline (OTC). This potential toxicity appears of great relevance for human nutrition and for domestic animals. This study aimed to extend the evaluation of such toxicity. The biologic impact of the drug was assessed by evaluating the proinflammatory effect of OTC and their bone residues on cytokine secretion by in vitro human peripheral blood lymphocytes. Our results showed that both OTC and OTC-bone residues significantly induced the T lymphocyte and non-T cell secretion of interferon (IFN)-γ, as cytokine involved in inflammatory responses in humans as well as in animals. These results may suggest a possible implication for new potential human and animal health risks depending on the entry of tetracyclines in the food-processing chain.

Spray-dried chestnut extract containing Lactobacillus rhamnosus cells as novel ingredient for a probiotic chestnut mousse.

AIMS: Consumers' demand for innovative probiotic products has recently increased. In previous studies, chestnuts were evaluated as substrate for the growth of lactobacilli and chestnut extract was found to enhance acid tolerance of probiotic strains. The main objectives of this study were to evaluate the suitability of chestnut extract as carrier for spray drying of two probiotic Lactobacillus rhamnosus strains and to develop a probiotic food chestnut based. METHODS AND RESULTS: The optimal settings for the spray-drying processes were defined and the loads of undamaged cells in the dried powders were quantified. Spray-dried cultures were incorporated into an anhydrous basis for chestnut mousse developed ad hoc. In this form, viable cells remained stable over 10(8) CFU g(-1) during a 3 months long storage at 15°C. Sensorial analysis did not highlighted significant differences (P < 0·05) in preference between probiotic-supplemented and control mousses. CONCLUSIONS: Results suggest that chestnut mousse, a food product naturally rich in antioxidant compounds, may represent an excellent carrier for probiotics delivering. SIGNIFICANCE AND IMPACT OF THE STUDY: To authors' knowledge, this is the first information on the survival of lactobacilli in an anhydrous basis for dessert.

Anti-hemorrhagic agents in oral and dental practice: an update.

Many oral surgeons in their daily practice have the problem of controlling postoperative bleeding. In surgical, oral and maxillofacial practice, standard anti-hemorrhagic protocols, especially in high risk patients, are obviously required and need to be continuously updated. The purpose of this review is to give a rational insight into the management of bleeding in oral and dental practice through modern drugs and medical devices such as lysine analogues and serine protease inhibitors, desmopressin, fibrin sealants, cyanoacrylates, gelatins, collagen and foams, protein concentrates, recombinant factors, complementary and alternative medicine and other compounds.

Delayed expansion of atrophic mandible (deam): a case report.

PURPOSE: The present case report presents the clinical results of delayed expansion of mandibles by ultrasonic surgery in case of mono edentulous. MATERIALS AND METHODS: The patients with a residual alveolar ridge thickness between 2,3 and 4,1 mm in the coronal area of posterior mandible was threated. In the first stage, four linear corticotomies were carried out by ultrasonic surgical device; bone expansion was not performed. After 4 weeks, in the second stage, adequate bone expansion, without compromising cortical vascularisation, by utilising a combination of scalpel, thin chisels and threaded osteotomes was achieved and one implant was placement, after filling the gaps with a cortico bovine biomaterial. RESULTS: The postoperative course was uneventful and final width ridge was 6,17±0,26mm. CONCLUSIONS: The present case report showed that mandibular ridge expansion using a delayed split-crest technique by means of ultrasonic surgery and association with biomaterial clinically lead to a good horizontal bone gain with no fractures of the buccal plate and high implant success rate.

Bone regeneration in aesthetic areas using titanium micromesh. Three case reports.

PURPOSE: An adequate bone volume for complete circumferential coverage of the implants is very important for obtaining a long-term success of oral implants. To avoid these problems various membranes and biomaterials were used, but soft tissue pressure could cause a membrane collapse toward the defect. The present work describes a ridge augmentation with titanium mesh shaped by adapting it to a bone defect in aesthetic areas. MATERIALS AND METHODS: Three patients with alveolar crest defects received three implants (Bone System, Milano, Italy) and the defects were filled with bone chips. The defects were covered with a titanium micromesh above which was positioned a resorbable membrane (Biogide, Geistlich, Wohlhusen, Switzerland). RESULTS: At the re-entry procedure the titanium micromesh appeared to be surrounded by a dense connective tissue with no clinical signs of inflammation. Clinically in all patients, no residual bone defects were observed, and a significant increase of the alveolar width or height was found. CONCLUSIONS: In conclusion the clinical results of the present study show that most certainly the space for the bone regeneration is one of the most critical factors in the success of the regenerative techniques.

Effects in dogs with behavioural disorders of a commercial nutraceutical diet on stress and neuroendocrine parameters.

The well-being of dogs can be affected by changes in human lifestyle, eating habits and increased stressors that lead to behavioural disorders including fear, hyperactivity and anxiety, followed by negative affective moods and poor welfare. This randomised, controlled clinical evaluation involved 69 dogs, 38 males and 31 females, of different breeds, with behavioural disorders related to anxiety and chronic stress. They were fed a control diet or a nutraceutical diet (ND group) for 45 days. Neuroendocrine (serotonin, dopamine, ß-endorphins, noradrenaline and cortisol) and stress (derivatives of reactive oxygen metabolites (dROMs) and biological antioxidant potential (BAP)) parameters related to behavioural disorders were evaluated at the beginning and end of the study period. Results showed a significant increase in serotonin, dopamine and ß-endorphins plasma concentrations (*P<0.05, *P<0.05 and **P<0.01, respectively) and a significant decrease in noradrenaline and cortisol plasma concentrations in the ND group (*P<0.05). dROMs significantly decreased in the ND group (*P<0.05) while BAP was not affected. This study demonstrated for the first time that a specific diet significantly and positively affected neuroendocrine parameters and dROMs. These results open significant perspectives concerning the use of diet and nutraceuticals in the treatment of behavioural disorders.

[Gastrointestinal parasites in goat farms from Bergamo province (Lombardy, north Italy)]. / Parassitismo gastrointestinale in allevamenti caprini della provincia di Bergamo (Lombardia).

A survey on gastrointestinal parasites in goat farms in Bergamo province was carried out from May 2005 to January 2006. Thirty-one dairy goat farms and 836 adult female goats were examined. Faecal sampling was carried out before the antihelminthic treatments, and the sampling times were grouped in three seasonal periods (June-August, September-October and November-March). Faecal exams were carried out by a McMaster method and eggs per gram (EPG) were calculated for each gastrointestinal parasite. Differences in prevalence of Strongylida, Trichuris and Nematodirus were found between farms with goats on pasture and those where animals were permanently housed indoor. Strongyloides spp. showed higher values of prevalence in goats housed indoor. With regard to the sampling seasons, Strongylida eggs were more prevalent in summer while Nematodirus in winter in goats at pasture. Strongyloides occurred more frequently in autumn in stabled goats.

Echinococcus multilocularis in north Italy.

Alveolar echinococcosis is a zoonotic infection caused by the metacestode of the tapeworm Echinococcus multilocularis. Fox populations living in the Alpine regions of Italy had been considered free from this parasite until 2002, when two infected foxes were detected in Bolzano province (Trentino Alto Adige region) near Austrian border. A modified nested PCR analysis was used to detect E. multilocularis DNA in faecal samples belonging to red fox populations from five Italian regions. A total of 522 faecal samples were analysed from foxes shot in Valle d'Aosta (N = 65), Liguria (N = 44), Lombardy (N = 105), Veneto (N = 67), and Trentino Alto Adige (N = 241) regions. Among these, 24 samples, all from the Trentino Alto Adige region, were found positive. Moreoever, 1406 faecal samples of red foxes were analyzed by CA-ELISAs commercial test kit. This paper provides an update of the epidemiological knowledge of this parasite in north Italy.

Echinococcus multilocularis in red foxes (Vulpes vulpes) of the Italian Alpine region: is there a focus of autochthonous transmission?

Alveolar echinococcosis, caused by the metacestode of Echinococcus multilocularis, is a zoonosis with a wider distribution area than described in the past. Fox populations living in the Alpine regions of Italy had been considered free from this parasite until 2002, when two infected foxes (Vulpes vulpes) were detected in the Bolzano province (Trentino Alto Adige region) near the Austrian border. The aim of this work was to evaluate the prevalence of infection in red fox populations from five Italian regions. A modified nested PCR analysis was used to detect E. multilocularis DNA in faecal samples. Amplicons were confirmed by sequencing. Of 500 faecal samples from foxes shot in Valle d'Aosta (n=57), Liguria (n=44), Lombardy (n=102), Veneto (n=56), and Trentino Alto Adige (n=241) regions, 24 animals, all from the Trentino Alto Adige region, were found positive. Twenty-two positive animals originated from the Bolzano province and two positive animals from the Trento province. Several localities of the Bolzano province, in which positive foxes were detected, are the same as those where alveolar echinococcosis had been described in humans in the second half of the 19th century, suggesting an old endemicity for the investigated area, which is adjacent to endemic areas of Austria. Therefore, the question arises if we are observing an increase and expansion of foci, or if the new records are due to the more sensitive and specific methods used to detect the worm DNA.

Skeletal involvement in female acromegalic subjects: the effects of growth hormone excess in amenorrheal and menstruating patients.

Bone involvement is a common clinical feature in acromegalic patients, though previous studies gave divergent results possibly because of the different gonadal status of the patients studied. To study the influence of estrogen milieu in these patients, we evaluated 23 acromegalic patients with active disease, subdivided into two groups: menstruating and amenorrheal patients, comparable for duration and activity of disease. Forty-two matched women served as controls. Skeletal involvement was studied by measuring: (a) the main biomarkers of bone turnover: serum alkaline phosphatase total activity (AP), bone GLA protein (BGP), serum carboxy-terminal propeptide of type I collagen (PICP), serum type I cross-linked N-telopeptide (ICTP), and urinary pyridinoline and deoxypyridinoline corrected for creatinine (Pyr/Cr, D-Pyr/Cr) and urinary calcium/creatinine ratio (Ca/Cr); (b) bone mineral density (BMD), as measured by quantitative computed tomography both at lumbar spine and distal radius, and by dual X-ray absorptiometry both at lumbar spine and at three femoral sites (Ward's triangle, femoral neck, and great trochanter). AP, BGP, ICTP, Pyr/Cr, D-Pyr/Cr were significantly higher in patients than in controls, independent of the menstrual pattern. Higher PICP levels were found in the whole group and in menstruating acromegalics when compared with control women; no difference was found in amenorrheal patients, who in turn showed higher urinary Ca/Cr values. When patients were considered all together, BMD at spine, femoral neck, and trochanter was higher than in controls. In contrast, when the gonadal status was taking into account and, menstruating and amenorrheal subjects were considered separately, BMD at spine, but not in other sites, was significantly higher in menstruating patients than in controls. In contrast, no difference of BMD values at any site was observed between amenorrheal patients and controls. The mean BMD Z scores allowed us to detect an unequal involvement of different skeletal sites. Our results show that bone turnover is increased in acromegalic women and suggest that GH anabolic effect on bone is more evident in the presence of estrogens and that different skeletal sites may be affected differently by hormone excess.

Subgroups of Graves' patients identified on the basis of the biochemical activities of their immunoglobulins.

Graves' patient immunoglobulins (IgG) are known to activate adenylyl cyclase. Recently, we have shown that they also stimulate phospholipase A2 (PLA2). Here we analyze the relationship of these biochemical activities of Graves' IgG to thyroid growth in vitro ([3H]thymidine incorporation) and in vivo (patient goiter size) as well as to clinical indicators of severity of the disease, such as ophthalmopathy, T3 levels, T3/T4 molar ratio, and TSH binding-inhibiting IgG activity. A cluster analysis of the biochemical parameters referring to the whole population (158 subjects) led to the identification of 4 subgroups of Graves' patients based on the different capabilities of IgG to stimulate adenylyl cyclase, PLA2, and [3H]thymidine incorporation. Importantly, a trend of increasing severity of the disease from group 1 to group 4 could be identified. In particular, patients in group 4 (characterized by elevated stimulation of adenylyl cyclase, PLA2, and [3H]thymidine incorporation) had the largest goiter, highest serum concentration of T3, highest T3/T4 molar ratio, and highest prevalence of ophthalmopathy. These results indicate that Graves' IgG induce thyroid growth by stimulating both adenylyl cyclase and PLA2, and suggest a method for the subclassification of Graves' patients that identifies four groups with different degrees of severity of the disease. Moreover, this classification might lead to the targeted use of a novel therapeutic approach based on the inhibition of PLA2 and arachidonic acid metabolism.

Immunoglobulins from Graves' patients stimulate phospholipase-A2 in FRTL5 thyroid cells.

The well documented ability of immunoglobulins G (IgGs) from Graves' patients to stimulate cAMP production is believed to be involved in the pathophysiology of this disease. It is still under discussion whether other intracellular messengers known to regulate thyroid function might play a similar role. This study shows that phospholipase-A2, a signal pathway unrelated to cAMP, is activated by Graves' IgGs. The IgGs from 67 patients with active Graves' disease, 8 patients with Graves' disease in remission, 5 patients with idiopathic myxedema, 2 patients with Hashimoto's thyroiditis, 57 patients with nonautoimmune thyroid disease, and 65 normal subjects were tested for their ability to stimulate phospholipase-A2 activity, as measured by arachidonic acid release from FRTL5 thyroid cells. The IgGs from patients with active Graves' disease caused a significant increase in arachidonic acid release compared to those from normal subjects, patients with nonautoimmune thyroid diseases, and patients with Graves' disease in remission (P less than 0.0001). The IgGs from active Graves' patients were also able to increase cAMP accumulation in FRTL5 cells. This effect did not correlate with the ability of the same IgGs to induce arachidonic acid release, suggesting that Graves' IgGs stimulate these two pathways by separate mechanisms. Moreover, a subgroup of IgGs that stimulated phospholipase-A2 did not increase the cAMP levels in FRTL5 cells. Our data suggest a novel mechanism of action of Graves' IgGs, the activation of phospholipase-A2, well distinguishable from the known effect on cAMP accumulation. The assay we describe could be helpful in improving the diagnosis and therapy of Graves' disease and in distinguishing it from nonautoimmune thyroid diseases. It also supplies the basis for a prospective subclassification of the Graves' patients, which might become useful to clarify the pathophysiology of this disease.

Cyclooxygenase-dependent thyroid cell proliferation induced by immunoglobulins from patients with Graves' disease.

IgG associated with Graves' disease bind to the TSH receptor and alter thyroid growth and function, mainly through the stimulation of adenylyl cyclase. In addition, Graves' IgG are able to interact with the phospholipase C (PLC)/Ca2+ and phospholipase A2 (PLA2)/arachidonic acid (AA) cascades. The activation of this latter pathway leads to thyroid cell growth in vitro. The elucidation of additional mechanisms of action of Graves' IgG has made possible the identification of four subgroups of patients, characterized by IgG with different biochemical activities (extent of cAMP and AA release stimulation in in vitro assays). On the basis of these results, a novel therapeutic approach could be proposed based on the inhibition of PLA2 and AA metabolism. To test this hypothesis, the ability of IgG from 56 Graves' patients to stimulate [3H]thymidine incorporation in FRTL5 thyroid cells in the presence and absence of the cyclooxygenase inhibitor indomethacin (2.5 x 10(-6) mol/L) was measured. A significant reduction in [3H]thymidine incorporation was found (33% inhibition; P < 0.0001) upon pretreatment with indomethacin, suggesting that in vitro thyroid cell growth is regulated by cyclooxygenase metabolites. This strengthens the argument for involvement of the PLA2/AA cascade in the pathophysiology of Graves' disease and the proposal for novel selective pharmacological treatments of these patients.

Physiological concentrations of thyrotropin increase cytosolic calcium levels in primary cultures of human thyroid cells.

The activity of TSH, the main regulator of growth and differentiation in the thyroid, has been mainly related to the activation of the adenylyl cyclase cascade. TSH also activates phospholipase-C and -A2; these effects, however, have been reported to require concentrations of the hormone up to 1000-fold higher than those effective on adenylyl cyclase, suggesting that the main physiological mechanism involved in the action of TSH is the activation of this enzyme. Using primary cultures of human thyroids, we here show that physiological concentrations of TSH (0.01-10 mU/L) are also able to increase intracellular Ca2+ levels. Cells were loaded with the fluorescent Ca2+ probe fura-2 and analyzed by single cell Ca2+ recording. The basal Ca2+ level was 105 +/- 30 nmol/L, and physiological concentrations of TSH increased it by 2- to 7-fold. The Ca2+ increase was transient and lasted up to 10 min. It is also shown that the TSH-dependent Ca2+ increase involves both the activation of phospholipase-C and the entry of extracellular Ca2+. TSH (100-10000 mU/L) increased cAMP levels by up to 20-fold in parallel experiments performed on the same cell preparations. These data demonstrate that physiological concentrations of TSH are able to increase cytosolic Ca2+ levels, indicating that this second messenger might directly mediate the action of this hormone in the thyroid.

Graves' immunoglobulins activate phospholipase A2 by recognizing specific epitopes on thyrotropin receptor.

Thyroid-stimulating IgG from Graves' patients bind to the TSH receptor and activate both adenylyl cyclase (AC) and phospholipase A2 (PLA2) in FRTL5 thyroid cells. Both activities have been associated with increased thyroid cell growth and function; evidence exists that subpopulations of Graves' IgG can stimulate either AC or PLA2 cascades and that the activation of both is associated with the largest goiters in patients. Studies using chimeras of the human TSHR receptor (hTSHR) and the LH-CG receptor show that most patients with Graves' disease have cAMP-stimulating IgG that require epitopes on the N-terminal portion of the TSHR extracellular domain; epitopes associated with PLA2 activation are not clear. To address this question we used stably transfected Chinese hamster ovary (CHO) cells containing the wild-type hTSHR and the hTSHR chimera with residues 8-165 (Mc1+2) substituted by equivalent residues of the LH-CG receptor. PLA2 activity, measured as arachidonic acid (AA) release, was determined in 32 patients with Graves' disease. We show that 72% of Graves' patients have IgG able to stimulate PLA2 in CHO cells transfected with the TSHR and that AA release induced by Graves' IgG was significantly reduced (P = 0.022) in the CHO-Mc1+2-transfected cells (193 +/- 88% vs. 131 +/- 67%, respectively). Unlike IgG, the effect of TSH was not modified in the CHO-Mc1+2-transfected cells. When we compared the AC- and PLA2-stimulating activities of these 32 IgG in wild-type TSHR transfectants, we found that 63% of Graves' patients have antibodies able to stimulate both PLA2 and AC, whereas some patients' IgG were active only in AC or PLA2 assays. Of the patients with IgG having activity in both assays in wild-type TSHR transfectants, 50% of the IgG lost their stimulatory activities in both AA release and cAMP assays in Mc1+2 cells. Of the remainder, some IgG maintained their activity in one (AA release) or the other (cAMP) assay when measured in Mc1+2 chimeras. Thus, our data show that the N-terminal portion of extracellular domain of the TSHR is required for PLA2 as well as AC activation by IgG from patients with Graves' disease. These data also demonstrate that patients with Graves' disease have heterogeneous autoantibodies that selectively activate AC and PLA2 pathways and suggest that patients with autoantibodies active in both assays have more severe disease, with higher thyroid hormone levels and larger goiters.

Signaling pathways involved in thyroid hyperfunction and growth in Graves' disease.

The elucidation of the multiple signaling cascades coupled to the TSH receptor has offered new approaches in the understanding of the pathogenesis of Graves' disease. Here we review findings showing that immunoglobulins from Graves' patients are heterogeneous, bind to different epitopes and, similarly to TSH, activate different signaling pathways, including adenylyl cyclase, phospholipase C and phospholipase A2. Evidence that the multiplicity of signals correlates with the different manifestations of the disease is also summarized. We believe that the dissection of the molecular mechanisms involved in the pathogenesis of Graves' disease offers the basis for developing novel therapeutical approaches to this disease.

A thyroid hormone receptor beta gene polymorphism associated with Graves' disease.

Autoimmune thyroid diseases (AITDs) are clustered in families, but the nature of this clustering is still poorly understood. One possible approach to the identification of genetic factors interacting with the AITDs is the study of the association between polymorphic markers and AITDs themselves. In the present study we have shown an association between an allele of a HindIII restriction fragment length polymorphism (EA beta H) intragenic to c-erbA beta, which codes for the thyroid hormone beta receptor, and Graves' disease. This polymorphism can be detected by PCR followed by digestion with the restriction enzyme HindIII. The allelic frequencies were analysed in a panel of DNAs extracted from a population of individuals affected by thyroid disease and originating from southern Italy. A control group (n = 120) from the same area was also analysed. The distribution of EA beta H alleles was significantly different (P < 0.001) in Graves' disease (n = 94) but not in autoimmune thyroiditis (n = 60), as compared with controls. Also the distribution of the EA beta H genotypes was significantly different in Graves' patients (P = 0.003), as compared with controls, the homozygous state EA beta H+/EA beta H+ being more frequent in Graves' patients than in all the other groups. We did not find any association between EA beta H genotypes and clinical parameters in Graves' patients, including eye signs, thyroid volume and level of TSH-binding inhibiting immunoglobulins. Our data support the idea that Graves' disease is a genetically distinct group within the AITDs.

Thyroperoxidase microsatellite polymorphism in thyroid diseases.

Autoimmune thyroid diseases (AITD) cluster in families, although the nature of this phenomenon is still poorly understood. One possible approach to the identification of genetic factors contributing to the pathogenesis of AITD is the study of association between polymorphic markers and AITD themselves. In the present study we have analyzed the allelic distribution of sRA-1, a TPO tetranucleotide repeat, among patients with AITD, in comparison with patients with nonautoimmune thyroid diseases and the general population. The polymorphic marker was analyzed by PCR followed by electrophoresis on polyacrylamide denaturant gel. Our data show that no association exists between AITD and any of sRA-1 alleles, despite the important role that TPO plays as a thyroid autoantigen.

Screening of thyrotropin receptor mutations by fine-needle aspiration biopsy in autonomous functioning thyroid nodules in multinodular goiters.

Multinodular goiter (MNG) is characterized by nodules of different size and function. Areas of increased function may emerge, appearing as single, or more frequently, multiple autonomously functioning thyroid nodules (AFTN). The molecular mechanism for the autonomous growth and function of these nodules has been related to mutations in the thyrotropin receptor (TSHR) that constitutively activate the adenylyl cyclase. We searched for mutations in a limited area of the TSHR gene, covering the major mutational hotspot, in 38 AFTNs found in 37 patients with MNGs. We used reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction enzyme analysis of fine-needle aspiration biopsy (FNAB) samples to rapidly identify 4 of the more frequently occurring TSHR mutations: D619G, F631C, T632I and D633E. Mutations were identified in 5 nodules (1 D619G mutation and 4 T632I mutations). Subsequently, the entire transmembrane portion of the TSHR gene was sequenced in a random sample of 12 AFTN samples that were free of mutations by RT-PCR and restriction enzyme analysis. By direct sequencing we identified a new mutation, F666L, in the seventh transmembrane domain in a sample from 1 nodule. Analysis of FMA samples of AFTN is an effective approach to identify TSHR gene mutations because individual mutations may be associated with different growth and function in vitro, our approach might, allow correlation of a given mutation with the clinical behavior in vivo.