Effects of dietary antioxidant supplementation on metabolism and inflammatory biomarkers in heat-stressed dairy cows.

Heat-stress-induced inflammation may be ameliorated by antioxidant supplementation due to the purported effects of increased production of reactive oxygen species or oxidative stress on the gastrointestinal tract barrier. Thus, study objectives were to evaluate whether antioxidant supplementation [AGRADO Plus 2.0 (AP); EW Nutrition] affects metabolism and inflammatory biomarkers in heat-stressed lactating dairy cows. Thirty-two mid-lactation multiparous Holstein cows were assigned to 1 of 4 dietary-environmental treatments: (1) thermoneutral (TN) conditions and fed a control diet (TN-CON; n = 8), (2) TN and fed a diet with AP (10 g antioxidant; n = 8), (3) heat stress (HS) and fed a control diet (HS-CON; n = 8), or (4) HS and fed a diet with AP (HS-AP; n = 8). The trial consisted of a 23-d prefeeding phase and 2 experimental periods (P). Respective dietary treatments were top-dressed starting on d 1 of the prefeeding period and continued daily throughout the duration of the experiment. During P1 (4 d), baseline data were collected. During P2 (7 d), HS was artificially induced using an electric heat blanket (Thermotex Therapy Systems Ltd.). During P2, the effects of treatment, day, and treatment-by-day interaction were assessed using PROC MIXED of SAS (SAS Institute Inc.). Heat stress (treatments 3 and 4) increased rectal, vaginal, and skin temperatures (1.2°C, 1.1°C, and 2.0°C, respectively) and respiration rate (33 breaths per minute) relative to TN cows. As expected, HS decreased dry matter intake, milk yield, and energy-corrected milk yield (32%, 28%, and 28% from d 4 to 7, respectively) relative to TN. There were no effects of AP on body temperature indices or production. Milk fat, protein, and lactose concentrations remained unaltered by HS or AP; however, milk urea nitrogen was increased during HS regardless of AP supplementation (26% relative to TN). Circulating glucose remained unchanged by HS, AP, or time. Additionally, HS decreased circulating glucagon (29% from d 3 to 7 relative to TN), but there was no additional effect of AP. There was a tendency for nonesterified fatty acid concentrations to be increased in HS-AP cows throughout P2 (60% relative to TN-CON), whereas it remained similar in all other treatments. Blood urea nitrogen increased for both HS treatments from d 1 to 3 before steadily decreasing from d 5 to 7, with the overall increase being most pronounced in HS-CON cows (27% relative to TN-CON). Further, supplementing AP decreased blood urea nitrogen in HS-AP on d 3 relative to HS-CON (15%). Circulating serum amyloid A tended to be and lipopolysaccharide binding protein was increased by HS, but neither acute-phase protein was affected by AP. Overall, AP supplementation appeared to marginally alter metabolism but did not meaningfully alter inflammation during HS.

Effects of spent coffee grounds on production traits, haematological parameters, and antioxidant activity of blood and milk in dairy goats.

Spent coffee ground (SCG) is a byproduct of coffee beverage preparation and a potential source of carbohydrate, protein, and phenolic compounds for livestock feeding. In this study, the effects of SCG supplementation in the diet of lactating goats on milk production traits and health status were studied. The antioxidant status of blood and milk was also evaluated. Twenty-four Saanen goats were fed a total mixed ration containing commercial concentrate, soybean, and haylage; they were divided into three groups: control diet (CON), SCG50 (50 g/d SCG), and SCG100 (100 g/d SCG). The experiment lasted 6 weeks. Linear and quadratic contrasts were used to evaluate the effects of the byproduct doses. SCG supplementation did not affect milk production, but influenced some milk fatty acids. SCG supplementation increased the contents of C18:1, cis-9, trans-11 C18:2, odd and branched-chain fatty acids, and total conjugated linoleic acid. Most of the haematological and biochemical parameters were within the physiological range for goats. The basophil, eosinophil, and glucose contents were quadratically affected by SCG, whereas platelet count increased linearly with the SCG dose. The SCG supplementation had a positive effect on the blood antioxidant status, as evidenced by an increase in ferric reducing antioxidant power and a decrease in malondialdehyde. The SCG supplementation had no effect on the milk antioxidant status. The results show that SCG (up to 100 g/d) did not negatively affect milk production and health status in goats. However, quadratic effects on some antioxidant and biochemical parameters suggest that further investigations are necessary, especially with regard to the optimisation of the supplement dose.

Proton pump inhibitors protect mice from acute systemic inflammation and induce long-term cross-tolerance.

Incidence of sepsis is increasing, representing a tremendous burden for health-care systems. Death in acute sepsis is attributed to hyperinflammatory responses, but the underlying mechanisms are still unclear. We report here that proton pump inhibitors (PPIs), which block gastric acid secretion, selectively inhibited tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) secretion by Toll-like receptor (TLR)-activated human monocytes in vitro, in the absence of toxic effects. Remarkably, the oversecretion of IL-1ß that represents a hallmark of monocytes from patients affected by cryopyrin-associated periodic syndrome is also blocked. Based on these propaedeutic experiments, we tested the effects of high doses of PPIs in vivo in the mouse model of endotoxic shock. Our data show that a single administration of PPI protected mice from death (60% survival versus 5% of untreated mice) and decreased TNF-α and IL-1ß systemic production. PPIs were efficacious even when administered after lipopolysaccharide (LPS) injection. PPI-treated mice that survived developed a long-term cross-tolerance, becoming resistant to LPS- and zymosan-induced sepsis. In vitro, their macrophages displayed impaired TNF-α and IL-1ß to different TLR ligands. PPIs also prevented sodium thioglycollate-induced peritoneal inflammation, indicating their efficacy also in a non-infectious setting independent of TLR stimulation. Lack of toxicity and therapeutic effectiveness make PPIs promising new drugs against sepsis and other severe inflammatory conditions.

Collagen-gelatin-genipin-hydroxyapatite composite scaffolds colonized by human primary osteoblasts are suitable for bone tissue engineering applications: in vitro evidences.

The application of porous hydroxyapatite (HAp)-collagen as a bone tissue engineering scaffold represents a new trend of mimicking the specific bone extracellular matrix (ECM). The use of HAp in reconstructive surgery has shown that it is slowly invaded by host tissue. Therefore, implant compatibility may be augmented by seeding cells before implantation. Human primary osteoblasts were seeded onto innovative collagen-gelatin-genipin (GP)-HAp scaffolds containing respectively 10%, 20%, and 30% HAp. Cellular adhesion, proliferation, alkaline phosphatase (ALP) activity, osteopontin (OPN), and osteocalcin (OC) expressions were evaluated after 3, 7, 15, and 21 days. The three types of scaffolds showed increased cellular proliferation over time in culture (maximum at 21 days) but the highest was recorded in 10% HAp scaffolds. ALP activity was the highest in 10% HAp scaffolds in all the times of evaluation. OC and OPN resulted in higher concentration in 10% HAp scaffolds compared to 20% and 30% HAp (maximum at 21 days). Finally, scanning electron microscopy analysis showed progressive scaffolds adhesion and colonization from the surface to the inside from day 3 to day 21. In vitro attachment, proliferation, and colonization of human primary osteoblasts on collagen-GP-HAp scaffolds with different percentages of HAp (10%, 20%, and 30%) all increased over time in culture, but comparing different percentages of HAp, they seem to increase with decreasing of HAp component. Therefore, the mechanical properties (such as the stiffness due to the HAp%) coupled with a good biomimetic component (collagen) are the parameters to set up in composite scaffolds design for bone tissue engineering.

In vitro exposure of human osteoarthritic chondrocytes to ELF fields and new therapeutic application of musically modulated electromagnetic fields: biological evidence.

Osteoarthritis (OA) is the most frequently occurring rheumatic disease, caused by metabolic changes in chondrocytes, the cells that maintain cartilage. Treatment with electromagnetic fields (MF) produces benefits in patients affected by this pathology. Isolated human osteoarthritic (OA) chondrocytes were cultured in vitro under standard conditions or stimulated with IL-1beta or IGF-1, to mimic the imbalance between chondroformation and chondroresorption processes observed in OA cartilage in vivo. The cells were exposed for a specific time to extremely low frequency (ELF; 100-Hz) electromagnetic fields and to the Therapeutic Application of Musically Modulated Electromagnetic Fields (TAMMEF), which are characterized by variable frequencies, intensities, and waveforms. Using flow cytometry, we tested the effects of the different types of exposure on chondrocyte metabolism. The exposure of the cells to both systems enhances cell proliferation, does not generate reactive oxygen species, does not cause glutathione depletion or changes in mitochondrial transmembrane potential and does not induce apoptosis. This study presents scientific support to the fact that MF could influence OA chondrocytes from different points of view (viability, ROS production and apoptosis). We can conclude that both ELF and TAMMEF systems could be recommended for OA therapy and represent a valid non-pharmacological approach to the treatment of this pathology.

Clinical presentation and pathogenesis of cold-induced autoinflammatory disease in a family with recurrence of an NLRP12 mutation.

OBJECTIVE: NLRP12 mutations have been described in patients affected with peculiar autoinflammatory symptoms. This study was undertaken to characterize NLRP12 mutations in patients with autoinflammatory syndromes, particularly a novel missense mutation, p.D294E, affecting a protein sequence crucial for ATP binding, which was identified in a Caucasian family with familial cold-induced autoinflammatory syndrome in some family members. METHODS: Fifty patients were tested for NLRP12 mutations. A Caucasian family with the p.D294E missense mutation of NLRP12 in some family members was clinically characterized. In vitro analysis of the effects of the mutation on NF-κB activity was performed in HEK 293 cells after cotransfection of the cells with a luciferase NF-κB-responsive element and mutant or wild-type (WT) NLRP12 expression plasmids. NF-κB activity was also evaluated 24 hours after stimulation with tumor necrosis factor α in monocytes from individual family members carrying the mutation. Furthermore, secretion of interleukin-1ß (IL-1ß), production of reactive oxygen species (ROS), and activation of antioxidant systems in patient and healthy donor monocytes, under resting conditions and after stimulation with pathogen-associated molecular patterns (PAMPs), were also assessed. RESULTS: In the family assessed, the p.D294E mutation segregated in association with a particular sensitivity to cold exposure (especially arthralgias and myalgia), but not always with an inflammatory phenotype (e.g., urticarial rash or fever). In vitro, the mutant protein maintained the same inhibitory activity as that shown by WT NLRP12. Consistently, NLRP12-mutated monocytes showed neither increased levels of p65-induced NF-κB activity nor higher secretion of IL-1ß. However, the kinetics of PAMP-induced IL-1ß secretion were significantly accelerated, and high production of ROS and up-regulation of antioxidant systems were demonstrated. CONCLUSION: Even with a variable range of associated manifestations, the extreme sensitivity to cold represents the main clinical hallmark in an individual carrying the p.D294E mutation of the NLRP12 gene. Although regulation of NF-κB activity is not affected in patients, redox alterations and accelerated secretion of IL-1ß are associated with this mild autoinflammatory phenotype.

Incidence and risk factors associated with cardiac arrhythmias during rehabilitation after coronary artery bypass surgery.

OBJECTIVE: To evaluate the clinical relevance of cardiac arrhythmias during rehabilitation after coronary artery bypass graft (CABG) surgery. DESIGN: Survey of consecutive patients admitted to an inpatient cardiac rehabilitation center. SETTING: A rehabilitation center acting as a reference for cardiology clinics lacking this facility. The program was performed in the inpatient setting. PATIENTS: Two hundred sixty patients undergoing elective CABG surgery for angina. INTERVENTIONS: Four-week cardiac rehabilitation program. A 24-hour electrocardiographic examination was performed during the first 2 days and, subsequently, on a daily basis. MAIN OUTCOME MEASURES: Incidence and type of cardiac arrhythmias during rehabilitation; identification of factors associated with an increased risk of cardiac arrhythmias. RESULTS: Cardiac arrhythmias were recorded in 33.5% of the patients and were more frequent in patients with hypertension (47%), diabetes (42.4%), and hyperlipidemia (36.5%), compared with patients free from these diseases (15.5%) (p < .05). Arrhythmias were also recorded in patients older than 70 years (49%), compared with those younger (29.2%) (p < .01), and in patients who discontinued amiodarone (64.3%) compared with those who did not interrupt the drug (16%) (p < .005). Arrhythmias precluded rehabilitation in 5% of the patients and caused death in one patient. CONCLUSION: Surveillance for the development of cardiac arrhythmias during cardiac rehabilitation after CABG surgery may allow the early detection and treatment of arrhythmias in a substantial number of patients.

Influence of acute and chronic smoking on leukocyte aggregation.

The aim of the present study was to evaluate whether alterations of leukocyte aggregation are present before the development of smoking-associated diseases. Leukocyte aggregation, studied by means of the leukergy test, was significantly higher in 24 healthy smokers as compared to 18 healthy non-smokers (mean + SE) 4.4 +/- 2.2 vs 1.5 +/- 1.3; P < 0.02. Intermediate values were found in ex-smokers. A significant increase was observed in smokers who underwent the acute smoking of three cigarettes in 20 minutes (mean +/- SE) 3.5 +/- 1.9 vs 7.5 +/- 2.6; P < 0.02. These data indicate that changes of leukocyte aggregation induced by smoking are present in healthy subjects before the development of clinically evident diseases associated with smoking.

Endocrine response to HIV infection. Involvement of thyroid gland.

Endocrine dysfunctions were associated at different stages of infection, including those early in the course of HIV infection. In fact adrenal insufficiency, hyporeninemic hypoaldosteronism, hyponatremia, reduced gonadotropins levels, gonadal abnormalities and changes in hormone-binding proteins were reported in HIV infection. Also a thyroid involvement with autoimmune phenomena was observed in HIV infection by several studies giving different explanations of altered thyroid conditions. These findings suggest an effect of HIV on endocrine system. Recognition of endocrine manifestations in these patients may contribute to better characterize different stages of the infection and improve the management of HIV patients.

Isoelectric focusing and immunoblotting analysis of thyroglobulin from different thyroid diseases.

Thyroglobulins (Tgs) have been obtained from macro-microfollicular goiter, toxic adenomas, papillary carcinomas and metastatic lympho-node and chromatographed by gel filtration. The 19S Tgs so obtained have been characterized by isoelectric focusing (IEF) and immunoblotting. IEF showed microheterogeneity pattern of Tgs from normal tissue, macro-microfollicular goiter and toxic adenomas to be within pH range from 4.2 to 4.5, while that from papillary carcinomas and metastatic lympho-node showed a wider microheterogeneity. Passive blotting of focused Tgs and immunoreaction with rabbit anti-Tg peroxidase conjugated antibody have been carried out and a positive reaction for all examined samples have been evidenced. Also two bands, focused at more cathodic pH value (pH 4.7), observed in IEF patterns of Tgs from carcinomas and metastatic lympho-node, gave a positive reaction with anti-Tg conjugated antibody. These differences observed in IEF and immunoblotting patterns of Tgs from papillary carcinomas and metastatic lympho-node are discussed in this report.

[Congenital hypothyroidism: organization and coordination of neonatal screening in Italy]. / Ipotiroidismo congenito: organizzazione e coordinamento dello screening neonatale in Italia.

Congenital hypothyroidism (IC) is the most frequent endocrine disease of the infancy and it is caused by primary deficiency of thyroid hormones. The damages derived by protracted hormone deficiency are diffused to all organs and systems and particularly severe for the development of central nervous system. Mental retardation can be prevented by early diagnosis and therapy. Early diagnosis is assured by neonatal thyroid screening performed on all newborns in the first days of life. The progress report on the screening situation in Italy as well as the national coordination realized in this field are presented.

Monoclonal autoantibody to thyroglobulin as a possible vector in immunodiagnosis and immunotherapy of differentiated thyroid cancer.

Differentiated thyroid carcinoma synthetize and secrete thyroglobulin. During its biosynthesis this antigen becomes expressed in the microvilli-bearing surface of carcinoma cells. Attempts have been carried out to target, with specific antithyroglobulin antibodies, the membrane bound absorption thyroglobulin in cancer cells for in vivo diagnosis and therapy. In the serum of patients with autoimmune thyroid diseases a high concentration of antithyroglobulin antibodies is frequently found (1-3 mg/ml). Their purification by immunoabsorption and dissociation is hampered by a low recovery and partial denaturation. It has been recently reported that about 1% of sera from Hashimoto's thyroiditis bear in their electrophoretogram a "myeloma-like protein". In the present report we describe in the serum of a Hashimoto patient a myeloma-like IgG which is an antithyroglobulin autoantibody with restricted functional and structural properties. The serum concentration of this myeloma-like IgG was found to be 40 mg/ml with a capacity of 6.5 nM of human thyroglobulin/mg IgG. The light chain composition was determined to be mostly of the lambda type. The clonal analysis of this myeloma IgG carried out by isoelectrofocusing, immunoblotting and autoradiography resulted in the recognition of several distinct clones, two of which were prominent at pH 8.7 and 7.8. By this technique and in view of the high serum concentration of this myeloma-like IgG, single clones of antithyroglobulin autoantibody can be easily obtained in high yields and without denaturation from human serum. This reagent could offer an ideal immunovector to target membrane-bound thyroglobulin of cancer cells.

Influence of environmental iodine deficiency on neonatal thyroid screening results.

During a multicentric pilot screening program for congenital hypothyroidism a comparison between the results obtained from Urbino, an area characterized by low iodine supply and endemic goiter, and Rome, a non-endemic area, has been made. The evaluation of neonatal urinary iodine excretion in the two areas showed significantly lower iodine urinary excretion levels in Urbino than in Rome. A shift of TSH at screening toward higher values as well as a higher percentage of recall in Urbino area than in Rome was observed. This finding, which well correlates with a low environmental iodine supply, emphasizes the importance of screening for congenital hypothyroidism as a suitable index of the presence and action of goitrogenic factors in the environment.

Longitudinal assessment of children with congenital hypothyroidism detected by neonatal screening.

Clinical and laboratory data from 42 children (31 females and 11 males) with primary congenital hypothyroidism (CH) diagnosed by neonatal screening over a six-year period are reported. The mean age at onset of thyroid hormone therapy was 33 days. The adequacy of replacement therapy was assessed by repeated TT4, FT4, T3 and TSH serum determinations. The high serum TT4 concentrations frequently observed were not accompanied by clinical evidence of hyperthyroidism. rT3 levels determined in 28 CH children with TT4 greater than 15 micrograms/dl were clearly higher than in the controls. The mean weight, length and head circumference remained always between the 50th and 75th centile. The radiological assessment of the knee, mainly the distal femoral surface, has been considered as an important clinical value in the initial diagnosis and in the evaluation of both severity and duration of disease. The psychomotor development was assessed using Brunet-Lezine's test until age 36 months, Stanford-Binet at 4 and 5 and WISC at 6 years of age. The mean global developmental quotients (GDQ) were always between 85 and 97 at 6 to 72 months of age, only eight children were below 85. A significant correlation was found between GDQ at 6 months and the bone age. The neurological examination showed an impairment of posture, coordination and subtle deficits in motor and perceptual abilities in a small percentage of children.

Pilot study for congenital hypothyroidism, preliminary report.

Preliminary results of a pilot study on congenital hypothyroidism performed on 8,025 newborns in Italy over a 10-month period are reported. The determination of both T4 and TSH on dried blood, spotted on filter paper, as screening procedure at day 3-5 of life revealed three cases of primary hypothyroidism and one case of hypo-thyroxin-binding-globulinemia (hypo-TBG-emia). The diagnosis of primary hypothyroidism was established on the basis of low T4 and high TSH levels in two of the three cases; in the third, with elevated TSH and normal T4 levels at screening, thyroid scintiscan showed an ectopic gland located at the base of the tongue. In the case of hypo-TBG-emia, low T4 and TBG levels were associated with normal TSH values. Some cases of transient hypothyroidism were observed in premature infants.