Differential proteomics profile of microcapillary networks in response to sound pattern-driven local cell density enhancement.

Spatial cell organization and biofabrication of microcapillary networks in vitro has a great potential in tissue engineering and regenerative medicine. This study explores the impact of local cell density enhancement achieved through an innovative sound-based patterning on microcapillary networks formation and their proteomic profile. Human umbilical vein endothelial cells (HUVEC) and human pericytes from placenta (hPC-PL) were mixed in a fibrin suspension. The mild effect of sound-induced hydrodynamic forces condensed cells into architected geometries showing good fidelity to the numerical simulation of the physical process. Local cell density increased significantly within the patterned areas and the capillary-like structures formed following the cell density gradient. Over five days, these patterns were well-maintained, resulting in concentric circles and honeycomb-like structures. Proteomic analysis of the pre-condensed cells cultured for 5 days, revealed over 900 differentially expressed proteins when cells were preassembled through mild-hydrodynamic forces. Gene ontology (GO) enrichment analysis identified cellular components, molecular functions, and biological processes that were up- and down-regulated, providing insights regarding molecular processes influenced by the local density enhancement. Furthermore, we employed Ingenuity Pathway Analysis (IPA) to identify altered pathways and predict upstream regulators. Notably, VEGF-A emerged as one of the most prominent upstream regulators. Accordingly, this study initiates the unraveling of the changes in microcapillary networks at both molecular and proteins level induced by cell condensation obtained through sound patterning. The findings provide valuable insights for further investigation into sound patterning as a biofabrication technique for creating more complex microcapillary networks and advancing in vitro models.

Condensed tannins fed to dairy goats: effects on digestibility, milk production, blood parameters, methane emission, and energy and nitrogen balances.

Condensed tannins (CT) are plant polyphenols that can affect feed digestibility and are potentially able to reduce enteric methane emissions in ruminants. In this in vivo trial with 8 lactating goats, we investigated the effects of 4 levels of inclusion of a commercial CT extract from quebracho (0, 2, 4, 6% on DM basis; C, Q2, Q4, Q6, respectively). The experimental design was a repeated 4 × 4 Latin square with 28-d periods (24 d of diet adaptation and 4 d of sample collection) using metabolic cages and 4 open circuit respiration chambers. The inclusion of CT in the diets did not affect the dry matter intake (DMI) but caused a linear decrease in diet digestibility, with reductions up to -11% for dry matter (DM), -21% for crude protein (CP), -23% for neutral detergent fiber (aNDFom), and -13% for gross energy, when comparing the Q6 and C diets. However, ruminal total volatile fatty acids (VFA) concentration was not affected by CT, although there were changes in VFA proportions. Milk yield (g/d) was highest for Q4 (3371) and lowest for Q6 (3066). In terms of milk composition, CT induced a linear reduction of fat and CP concentrations. The reduction in CP digestibility resulted in a linear reduction in the milk urea level, up to -37% with Q6. Positively, CT linearly reduced the somatic cells count expressed as linear score. The feed efficiency was linearly decreased by CT inclusion. Furthermore, a shift from urinary to fecal nitrogen excretion was observed with CT. The retained nitrogen was always negative (on average -1.93 g/d). The methane yield (on average 19.2 g CH4/kg DMI) was linearly reduced by CT inclusion, up to -18% with Q6. Regarding the CH4 intensity, CT induced a linear reduction when expressed per kg of milk, but not per kg of fat and protein corrected milk. Moreover, the CH4 production per kg of digestible aNDFom was linearly increased by CT. The metabolizable energy intake (MEI) was not affected by the treatments, but the metabolizability (q = MEI/gross energy intake) was reduced as CT inclusion increased. From the results of the present study, it turned out that CT have a negative impact on feed digestibility and feed use efficiency. Condensed tannins can lower CH4 emissions from ruminants; however, the main mechanism of action is likely the decrease in feed digestibility. Furthermore, CT did not improve the N use efficiency. According to these findings, the positive environmental impacts of CT are only related to the shift from urinary to fecal N excretion.

Vespa velutina nigrithorax venom allergy: inhibition studies approach for the choice of specific immunotherapy.

Summary: Vespa velutina nigrithorax (VVN), commonly known as Asian wasp because endemic in Asia, represents an alien species in Europe. VVN can induce allergic reactions similar to those caused by other Hymenoptera and death after VVN stings, presumably due to fatal allergic reactions, has been reported. In the treatment of Hymenoptera venom hypersensitivity, specific immunotherapy (VIT) is highly effective. Currently, there is no specific available VIT for VVN, so it is relevant to assess if patients stung by VVN and showing allergic reactions could be treated with the Hymenoptera commercially available extracts Vespa crabro (VC) and Vespula spp (Vspp) or if they need the specific VIT with VVN venom extract. Methods. Four patients with a clinical history of systemic reactions after VVN sting were evaluated. Serum specific IgE were assayed quantitatively with an automated fluoro-enzyme immunoassay ImmunoCAP™ Specific IgE by Phadia™ 1000 System (Thermo Fisher Scientific, Uppsala, Sweden) for VC, Vspp and VVN. Cap inhibition assays were performed incubating serum samples with 200 µl of each venom at increasing concentrations and subsequently specific IgE against each of the venoms were determined in the samples by Phadia™ 250 System (Thermo Fisher Scientific, Uppsala, Sweden). Results. Our results suggested that both Vspp and VC venoms were able to inhibit the specific IgE for VVN, although the VC compared to the Vspp venom showed a higher inhibition. Conclusions. Our inhibition studies suggested that VIT with VC venom, nowadays when there is not specific available VIT for VVN, may be more effective than Vspp VIT in patients with VVN sting reactions.

Percutaneous puncture during PCNL: new perspective for the future with virtual imaging guidance.

CONTEXT: Large and complex renal stones are usually treated with percutaneous nephrolithotomy (PCNL). One of the crucial steps in this procedure is the access to the collecting system with the percutaneous puncture and this maneuver leads to a risk of vascular and neighboring organs' injury. In the last years, the application of virtual image-guided surgery has gained wide diffusion even in this specific field. OBJECTIVES: To provide a short overview of the most recent evidence on current applications of virtual imaging guidance for PCNL. EVIDENCE ACQUISITION: A non-systematic review of the literature was performed. Medline, PubMed, the Cochrane Database and Embase were screened for studies regarding the use virtual imaging guidance for PCNL. EVIDENCE SYNTHESIS: 3D virtual navigation technology for PCNL was first used in urology with the purpose of surgical training and surgical planning; subsequently, the field of surgical navigation with different modalities (from cognitive to augmented reality or mixed reality) had been explored. Finally, anecdotal preliminary experiences explored the potential application of artificial intelligence guidance for percutaneous puncture. CONCLUSION: Nowadays, many experiences proved the potential benefit of virtual guidance for surgical simulation and training. Focusing on surgery, this tool revealed to be useful both for surgical planning, allowed to achieve a better surgical performance, and for surgical navigation by using augmented reality and mixed reality systems aimed to assist the surgeon in real time during the intervention.

Human Papillomavirus (HPV) oral lesions surgical management with Nd:YAG laser versus blade and QMR scalpel: a single center experience in 251 cases.

HPVs are a large and varied group of viruses capable of infecting both animals and humans. They have evolved together with their respective hosts and are isolable in different and numerous species of birds, reptiles, marsupials and mammals. The present study aims to analyze and compare the epidemiological aspects of 251 HPV-associated benign lesions treated with three different surgical approaches. Between January 2004 and December 2019, 251 HPV-associated benign lesions (papillomas and / or condylomas) were treated at the Center of Medicina e Patologia Orale Laser Assistita of the University of Parma. After loco-regional anesthesia, the excisional biopsy was performed with A): 15C scalpel blade; B): molecular quantum resonance scalpel (RQM); C): Nd: YAG laser (1064 mn, 3.5W, 70Hz). The epidemiological analysis considered: gender, age, site of the lesion, surgical technique and recurrence. Our study did not show substantial differences in gender prevalence and the mean age of 50 years (minimum 8 years, maximum 85 years). Our study showed that HPV-associated lesions are localized in almost half of patients (47.9%) on the tongue or hard palate, less frequently on the cheeks (16.3%) and on the upper or lower lip (14,4) and in less than 10% of patients in the soft palate. Our study showed a low relapse rate (3%) for all three therapeutic approaches used, meaning that the cold blade, RQM scalpel and Nd: YAG laser can be successfully used to treat lesions associated with HPV, the laser approach has proved to be slightly more effective: probably thanks to the radicalization obtained through carbonization of the biopsy surrounding tissues. Since a small group of these viruses are the responsible agent of several types of human cancers (including squamous cell carcinoma of the oral cavity), early screening and treatment of HPV-associated lesions is essential to prevent the development of oncological diseases.

Correlation between temperature rise and auto-fluorescence: ex vivo study with Er:YAG laser, Quantum Molecular Resonance scalpel and Nd:YAG laser on hard and soft tissues.

Surgical techniques of soft and hard oral tissues highly benefited from new technologies such as the Quantic Molecular Resonance (QMR) lancet, the Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser and the Erbium-doped Yttrium Aluminum Garnet (Er:YAG) laser. Increasingly, these technologies replace scalpel, conventional electrosurgery and traditional rotary surgery instruments due to their proven advantages. Features such as reduction of the surgical time, more efficient bleeding control resulting in higher intra-operative visibility and improvement of postoperative course with better Quality of Life score (QoL) are highlighted in numerous studies published in the literature. The thermal rise of tissues during surgical incision, performed with other instruments rather than traditional cold blade scalpels, is not to be ignored by the operator and it must take into consideration first when choosing the surgical instrument and then throughout all the surgical act. Auto-fluorescence (AF) is a property possessed by every cell that exposed to a specific wavelength can absorbance or reflect with peculiar characteristics and its direct examination has been proposed as a non-invasive visual tool for investigation of suspicious changes in oral mucosa. At the limit of our knowledge, few studies have been published in the literature regarding tissue's temperature variations and the interest in Infra-Red temperature detection has been shown in various medicine fields and none of published studies investigated the possible correlation between temperature raise and AF variations. This ex vivo study aims to analyse and compare through the use of a thermal imaging camera and simultaneous detection of AF, the possible correlation between temperature increase and auto-fluorescence.

Enterococcus faecium NCIMB10415 responds to norepinephrine by altering protein profiles and phenotypic characters.

The long-term established symbiosis between gut microbiota and humans is based upon a dynamic equilibrium that, if unbalanced, could lead to the development of diseases. Despite the huge amount of data concerning the microbiota-gut-brain-axis, little information is available on what happens at the molecular level in bacteria, when exposed to human signals. In the present study, the physiological effects exerted by norepinephrine (NE), a human hormone present in significant amounts in the host gut, were analyzed using the commensal/probiotic strain Enterococcus faecium NCIMB10415 as a target. The aim was to compare the protein profiles of treated and untreated bacteria and relating these proteome patterns to some phenotypic modifications important for bacteria-host interaction. Actually, to date, only pathogens have been considered. Combining a gel-free/label-free proteomic analysis with the evaluation of bile salts resistance, biofilm formation and autoaggregation ability (as well as with the bacterial growth kinetics), allowed to detect changes induced by NE treatment on all the tested probiotic properties. Furthermore, exposure to the bioactive molecule increased the abundance of proteins related to stress response and to host-microbe interaction, such as moonlight proteins involved in adhesion and immune stimulation. The results of this investigation demonstrated that, not only pathogens, but also commensal gut bacteria are affected by host-derived hormones, underlining the importance of a correct cross-signalling in the maintenance of gut homeostasis. SIGNIFICANCE: The crucial role played by the human gut microbiota in ensuring host homeostasis and health is definitively ascertained as suggested by the holobiome concept. The present research was intended to shed light on the endocrinological perturbations possibly affecting microbiota. The microbial model used in this study belongs to Enterococcus faecium species, whose controversial role as gut commensal and opportunistic pathogen in the gut ecosystem is well recognized. The results obtained in the present investigation clearly demonstrate that E. faecium NCIMB10415 can sense and respond to norepinephrine, a human hormone abundant at the gut level, by changing protein profiles and physiology, inducing changes that could favor survival and colonization of the host tissues. To our knowledge, this is the first proteomic report concerning the impact of a human hormone on a commensal/probiotic bacterium, since previous research has focused on exploring the effects of neuroendocrine molecules on growth and virulence of pathogenic species.

Gastrointestinal nematode infections in goats: differences between strongyle faecal egg counts and specific antibody responses to Teladorsagia circumcincta in Nera di Verzasca and Alpine goats.

Strongylida are gastrointestinal nematodes (GIN) of greatest importance in small ruminants throughout the world. Differences in resistance and resilience to GIN among goat breeds were reported. This study aims to investigate the mechanism underlying the breed-associated differences using a cosmopolitan (Alpine, AB) and an autochthonous (Nera di Verzasca, NV) goat breed. At first, fifteen goats from the same herd (NV = 7, AB = 8) at day 0 were infected with infective larvae (L3) of mixed GIN. From the 15th day post-infection (DPI), individual parasite egg excretion (faecal egg counts, FEC) was performed on all goats, once per week, until the 63rd DPI. Afterwards, in goats under field conditions (30 AB and 30 NV reared on the same farm), individual faecal and blood samples were collected; FEC-specific antibody and PCV levels were explored. In goats with experimental GIN infection, mean eggs per gram of faeces (EPG) values were consistently lower in NV goats. In goats with natural GIN infection, EPG and prevalence values showed high variability in both breeds; among individual variables, breed had a significant influence on EPG. Further, PCV and anti-T. circumcincta IgA levels were influenced by the breed. Lower PCV values were also associated with higher strongyle EPG in AB goats, and anti-T. circumcincta IgA levels were influenced by both strongyle EPG and breed, with IgA levels being higher in AB vs. NV goats and positively associated with EPG. Neither EPG nor breed had any influence on IgE levels. Both studies on experimental and natural infection confirmed that goats of NV are more resistant to infection with gastrointestinal nematodes.

Immunoglobulin A antibodies to oxidized collagen type II as a potential biomarker for the stratification of spondyloarthritis from rheumatoid arthritis.

OBJECTIVES: The discovery of diseased tissue-specific neoantigens offers the opportunity to develop important disease tissue-specific biomarkers that can help in the prediction, diagnosis, and stratification of diseases. This opportunity is specifically significant for autoimmune diseases where diagnostic biomarkers are not available. Inflammatory autoimmune diseases are commonly associated with local generation of large amounts of reactive oxidants. We have previously identified oxidative post-translationally modified (oxPTM) tissue-specific neoantigens in rheumatoid arthritis (RA) and type 1 diabetes that elicit an immune response. In the current study, we studied the presence and clinical significance of antibodies to oxPTM collagen type II (CII) in patients with spondyloarthritis (SpA). METHOD: Levels of antibodies specific to native CII and oxPTM-CII were assessed by enzyme-linked immunosorbent assay. RESULTS: Immunoglobulin G (IgG) binding to oxPTM-CII was observed in 52%, 83%, and 28% of serum samples from patients with axial spondyloarthritis (axSpA), RA, and psoriatic arthritis (PsA), respectively. Importantly, while strong IgA anti-oxPTM-CII responses were detected in axSpA and PsA patients, with 47% and 84% respective binders, no IgA anti-oxPTM-CII was detected in RA patients. IgA anti-oxPTM-CII reactivity in axSpA patients treated with biologics was higher and more frequent, with 85% binders compared to 9% binders in patients treated with synthetic disease-modifying anti-rheumatic drugs. CONCLUSION: Our data imply that SpA and PsA are associated with the presence of antibodies to oxPTM-CII, suggesting that there may be a humoral component that may distinguish patients with SpA from RA. Our approach could be adapted to other diseases, particularly to inflammatory autoimmune diseases.

Identification of a caleosin associated with hazelnut (Corylus avellana L.) oil bodies.

Caleosins are involved in several cellular and biological processes that are closely associated with the synthesis, degradation and stability of oil bodies (OB). Because of the importance and the multiple roles of these OB-associated proteins, in silico identification of sequences corresponding to putative caleosins in the hazelnut genome has been performed, and the association with seed OB was verified using a proteomic approach. Five full-length sequences (CavCLO-H1, CavCLO-H2, CavCLO-H3, CavCLO-L1, CavCLO-L2), belonging to the two groups of caleosins (H and L), have been identified in the hazelnut genome. The number of identified caleosins is in agreement with that previously observed in other plant species, confirming that caleosins comprise small gene families in plants. A proteomic approach allowed us to verify only the presence of CavCLO-H1 in hazelnut OB, suggesting that several members inside this family could have different roles during plant growth and development. In silico analysis also suggests that CavCLO-H1 may act as a peroxygenase.

Combining immunofluorescence with immunoblot assay improves the specificity of autoantibody testing for myositis.

OBJECTIVE: Immunoblot (IB) methods are widely used to detect myositis-specific autoantibodies (MSAs); however, false-positive results are common. In this study, we aimed to determine whether associating the anti-nuclear antibody (ANA) IIF pattern may help to improve the specificity of MSA detection by IB in patients with idiopathic inflammatory myositis (IIM). METHODS: Serum samples from 104 patients presenting with muscle weakness/myalgia and positive to at least one MSA by IB (MYOS12 Diver and MIOS7 Diver, D-tek) were tested for ANAs on HEp-2000 cells (Immuno Concepts). The chi-square test was used to analyse the concordance of the MSA result and its corresponding pattern by ANA testing between patients with and without IIM. RESULTS: Eighty-three of the 104 patients had a diagnosis of definite IIM, while in 21 cases, patients were affected by other autoimmune diseases or various non-systemic diseases. Forty nine of 83 (59%) patients in the IIM group and 4/21 (19%) in the non-IIM group showed a concordance between ANA pattern and MSAs by IB (P < 0.001). MSA monopositivity was significantly associated with IIM (91.6%) compared with 61.9% in the non-IIM group (P = 0.0005). CONCLUSIONS: Considering both the MSA result and its corresponding pattern by ANA testing may help to improve the specificity of MSA detection by IB and to confirm the diagnosis of MSA-associated IIM. The monopositivity of MSAs is an important additional tool to validate IB results.

Improved accuracy in DFS pattern interpretation using a novel HEp-2 ELITE system.

INTRODUCTION/OBJECTIVES: Accurate interpretation of DFS70 (dense fine speckled 70) and mixed antinuclear antibodies (ANAs) patterns can be challenging using conventional HEp-2 immunofluorescence (IIF) method. We evaluated a novel HEp-2 IIF substrate (HEp-2 ELITE/DFS70-KO) composed of a mixture of engineered HEp-2 devoid of the DFS70 autoantigen and conventional HEp-2 cells. The study assessed the utility of the new substrate in ANA screening and its advantages. METHOD: One thousand and five consecutive routine samples sent for ANA screening were tested on both standard HEp-2 and the HEp-2 ELITE DFS70 KO substrates (ImmuGlo ANA HEp-2 and HEp-2 ELITE/DFS70-KO, Trinity Biotech, Buffalo, NY). Anti-DFS70 antibody specificity was additionally determined by immunoblot (IB). Clinical and serological data were included in the analysis of the overall impact of the novel HEp-2 substrate on DFS pattern interpretation. RESULTS: Of the 22 cases suspected as positive for DFS pattern alone or in combination with homogeneous or speckled patterns on conventional HEp-2 cells, 17 were interpreted with a higher accuracy using the new HEp-2 ELITE method as positive for DFS70 (monospecific DFS70 (10), mixed DFS70 (7)), speckled (3), and DFS (2) patterns. CONCLUSIONS: The new substrate was not only useful in deciphering unclear mixed ANA patterns but also highly sensitive in detecting DFS70 pattern in comparison to the DFS70 positivity obtained using IB.

Validation of an easy questionnaire on the assessment of salt habit: the MINISAL-SIIA Study Program.

BACKGROUND/OBJECTIVES: The aim of the present study was to validate a short questionnaire on habitual dietary salt intake, to quickly and easily identify individuals whose salt consumption exceeds recommended levels. SUBJECTS/METHODS: A total of 1131 hypertensive subjects participating in the MINISAL-SIIA study were included in the analysis. Anthropometric indexes, blood pressure, and 24-h urinary sodium excretion (NaU) were measured. A fixed-sequence questionnaire on dietary salt intake was administered. RESULTS: NaU was significantly associated with scores, with a linear association across categories (p for trend <0.0001). In addition, participants who achieved a total score above the median value (eight points) had significantly higher NaU than those whose score was below median (p < 0.0001). In the total sample, the prevalence of "high NaU" (NaU > 85 mmol/day) and "very high NaU" (NaU > 170 mmol/day) was 86 and 35%, respectively. The score of the questionnaire had a significant ability to detect both "high NaU"-with a specificity of 95% at the score of 10 points-and "very high NaU"-with a specificity of 99.6% at score of 13 points. CONCLUSIONS: The main results of the study indicates that a higher score of this short questionnaire is distinctive of habitual high salt consumption in hypertensive patients.

Metaproteomic characterization of theVitis vinifera rhizosphere.

The rhizosphere is a hotspot of microbial activity where the release of root exudates stimulates bacterial density and diversity. The majority of the bacterial cells in soil are viable, unculturable, but active. Proteomic tools could be useful in gaining information about microbial community activity and to better understand the real interactions between roots and soil. The aim of this work was to characterize the bacterial community associated with Vitis vinifera cv. Pinot Noir roots using a metaproteome approach. Our results confirmed the large potential of proteomics in describing the environmental microbial communities and their activities: in particular, we showed that bacteria belonging to Streptomyces, Bacillus, Bradyrhizobium, Burkholderia and Pseudomonas genera are the most active in protein expression. Concerning the biological activity of these genera in the rhizosphere, we observed the exclusive presence of the phosphorus metabolic process and the regulation of primary metabolic processes. To our knowledge, this is the first study reporting the rhizosphere proteome of V. vinifera, describing the bacterial community structure and activity of an important ecosystem for the Italian landscape, agriculture and economy.

Ixodid ticks on wild donkeys in a Mediterranean nature reserve (Asinara National Park): diversity and risk factors.

The Sardinian coloured donkey Equus asinus (Perissodactyla: Equidae) and its albino colour morph represent the wildlife species most typical of the island of Asinara. This Mediterranean island represents a favourable context for ticks and tick-borne diseases; however, knowledge of the tick fauna on Asinara is scarce. A total of 106 Sardinian donkeys were inspected for tick infestation from June to November 2015. All ticks found were collected, classified by stage and sex, and identified to species level. The level of infestation of each donkey was determined; both the overall tick infestation and infestations of each detected species were classified on a scale of 1-3 to give an infestation score (IS). Overall, 256 hard ticks were collected from 60 of 106 donkeys (56.6%). Rhipicephalus bursa, Haemaphysalis punctata and Hyalomma marginatum (all: Ixodida: Ixodidae) infested 26.4%, 28.3% and 6.6% of donkeys, respectively. Different variables affected the IS. With reference to overall tick infestation, a higher IS was observed in donkeys grazing on grassland and Mediterranean shrubland and in albino donkeys compared with coloured donkeys. The collected ticks included species involved in the transmission of pathogens to humans, which highlights the risks for public health in a tourist destination such as Asinara National Park.

A better definition of the anti-DFS70 antibody screening by IIF methods.

BACKGROUND: Anti-DFS70 antibodies have been recently included in a new testing algorithm for patients with suspicion of connective tissue diseases (CTDs). This algorithm enables to assess the probability of having a CTD in patients with a positive antinuclear antibodies (ANA) result. The aim of the study was to analyze the the inter-method agreement between three different HEp-2 cell substrates for anti-DFS70 detection, focusing on two novel IIF methods that assess the presence of monospecific anti-DFS70 antibodies. METHODS: Immunological and clinical records of 29 patients who were double positive for anti-DFS70 autoantibodies using chemiluminescence assay (CIA) and Immunoblot (IB) were studied. The IIF on HEp-2 cells were determined using slides from Inova Diagnostics, Euroimmun and Immco. The capability to detect isolated anti-DFS70 antibodies was compared using immunoadsorption on NOVA Lite HEp-2 Select (Inova Diagnostics) and the HEp-2 ELITE/DFS70 knockout test (Immco). RESULTS: The three substrates had very good sensitivity for detecting patients with anti-DFS staining pattern (93.1%, 79.3% and 72.4% for Euroimmun, Immco and Inova respectively). Most of the patients had full inhibition of DFS pattern (65.5%) by immunoabsorption test. Also, the 55.2% of the subjects were positive for monospecific DFS pattern using HEp-2 ELITE/DFS70 knockout test. However, the correlation between the full inhibition by immunoadsorption and the monospecific DFS pattern in knockout cells was very low (kappa: 0.22). CONCLUSION: The evaluation of monospecific anti-DFS70 antibodies is clinically fundamental and challenging using traditional HEp-2 IIF. Results obtained in this study support the hypothesis that the lack of standardization across IIF kits along with the subjectivity of user interpretation among other factors contribute to the overall reduction in the agreement.

Toxoplasma gondii Antibodies in Bulk Tank Milk Samples of Caprine Dairy Herds.

A major public health issue, Toxoplasma gondii infection can affect humans mainly via the consumption of animal products from certain species, including small ruminants. Therefore, a regular monitoring of the infection in ovine and caprine populations is advisable for the control of human and animal toxoplasmosis. Antibody detection in individual and bulk tank milk (BTM) may represent a valid alternative to serological analysis, in that its collection is easy and does not affect animal welfare. Many serological tools for milk analysis have already been validated for several parasites, including Apicomplexa. Thus, the aim of the present study was to obtain epidemiological data on T. gondii infection through the detection of antibodies in BTM of dairy goat herds from an important area for caprine dairy production (northern Italy). The performance of a commercial ELISA was first evaluated for analysis of caprine milk samples, using a panel of serum-milk pairs of goats naturally infected by T. gondii. The analysis of BTM confirmed the presence of anti- T. gondii antibodies in 59% of the samples. Toxoplasma gondii antibody positivity was more frequently found in goats reared on farms under extensive (64.9%) or semi-intensive systems (68.7%) in comparison with intensive farms (51.1%). Analysis of milk was a valid alternative to serological tests, being easily applied in large-scale epidemiological surveys and for continuous monitoring of T. gondii infection.

Analytical variability in the determination of anti-double-stranded DNA antibodies: the strong need of a better definition of the old and new tests.

Anti-dsDNA antibodies are a heterogeneous group of antibodies, quite specific for SLE. Their variability is related to the assay used, the immunoglobulin class secondary antibody, and the dsDNA source. The standardization of measuring anti-dsDNA antibodies is still poor and different methods yield different results. Several novel technologies were developed during the last decades that represent viable alternatives to the traditional methods such as the chemiluminescent immunoassay (CIA) and multiplex flow immunoassay (MFI). Additionally, positive results for anti-dsDNA antibodies can be detected in patients with inflammatory arthritis (IA) treated with different biologics reducing its clinical specificity for SLE. Anti-dsDNA antibody levels were evaluated in 246 patient samples: 70 SLE and 176 disease control (including 96 IA during treatment with different biologics), using three enzyme immunoassays (indirect enzyme immunoassay, Bio-Rad Laboratories; chemiluminescent immunoassay, Inova Diagnostics; multiplex flow immunoassay, Bio-Rad Laboratories) and three Crithidia luciliae immunofluorescence tests (CLIFT) (Euroimmun AG, Bio-Rad Laboratories, INOVA Diagnostics). Diagnostic performances were assessed both including and excluding the IA patients. Agreements, measured by the Cohen's Kappa between all methods, ranged from moderate to substantial (0.47-0.68). The clinical sensitivities for the anti-dsDNA antibody tests varied from 5.7% by CLIFT A up to 33.3% provided by EIA while the clinical specificities varied from 89.8% by MFI to 98.9% provided by CLIFT B and C. Newer technologies, such as MFI and CIA, showed great potential as a diagnostic application. Significant variations among anti-dsDNA antibody assays were observed confirming the lack of standardization.