Tecnica e prospettive di utilizzo della citometria a flusso per la tipizzazione linfocitaria del liquido sinoviale di cane

RIASSUNTO L’utilizzo della citometria a flusso, o fluorimetria, per l’immunofenotipizzazione linfocitaria è una metodica in uso in medicina umana soprattutto nel settore reumatologico, dove riveste un’importanza notevole nella comprensione dei meccanismi degenerativi cartilaginei in corso di artrite reumatoide, mentre è poco utilizzata nella più comune artrosi. In medicina veterinaria la tecnica non è a tutt’oggi utilizzata nella diagnostica collaterale dei problemi ortopedici e reumatologici. Sono disponibili solo due segnalazioni di lavori sperimentali in argomento. Sono state fatte numerose prove su campioni di liquido sinoviale di cane, prelevato sia durante la visita clinica che in sede intraoperatoria, in corso di patologie articolari diverse. Il liquido è stato sottoposto a conta cellulare con contaglobuli elettronico; per essere processato a tal fine e per l’analisi citofluorimetrica, è stato sottoposto a pretrattamento con jaluronidasi. La componente cellulare mononucleata è stata quindi separata e concentrata mediante centrifugazione in gradiente di densità su LymphoprepTM (Ficoll-Isopaque) e studiata con citofluorimetro. Contemporaneamente l’immunofenotipizzazione linfocitaria è stata condotta anche su un campione di sangue periferico. Prove successive finalizzate a rendere il campione un fluido acquoso hanno permesso di evidenziare l’assoluta necessità di pretrattare il campione con 2 gocce di jaluronidasi alla concentrazione di 150UI/ml per 0,25 cc di liquido sinoviale. La quantità di liquido prelevabile è in generale scarsa nei fatti degenerativi cronici, più abbondante nei fenomeni infiammatori acuti, e questo può rappresentare un limite nell’applicazione di questa metodica nella pratica clinica. Per ottenere una buona lettura è necessario avere infatti almeno 1cc di liquido articolare e/o almeno 1000 cellule/μl. La distribuzione delle sottopopolazioni linfocitarie CD3+ e CD21+, ed in particolare del rapporto fra i linfociti CD4+ e CD8+ nel liquido sinoviale e nel sangue, si presenta indicativamente diversa nei fenomeni prevalentemente degenerativi rispetto a quelli squisitamente infiammatori. La tipizzazione linfocitaria nel liquido sinoviale del cane può aprire la strada ad ulteriori indagini per studiare più a fondo i meccanismi eziopatogenetici alla base della condrodegenerazione e della risposta locale e sistemica nei fenomeni osteoartritici e immunitari, oltreché fornire una ausilio nell’individuazione e nel monitoraggio delle terapie. SUMMARY The use of flow cytometry, also called cytofluorimetry, for lymphocyte immunophenotyping is a technique adopted in uman medicine, especially in the rheumatoid field, where is of paramount importance to understand the cartilage degenerative mechanisms during rheumatoid arthritis, while is less used in degenerative joint disease. Till now in veterinary medicine this technique is not included in the ancillary diagnostic methods for orthopaedic and rheumatologic problems. To date only two papers are available about its experimental use. Several tests were done on canine synovial fluid samples, collected during the clinical examination or intraoperatively, in subjects with different joint diseases. The synovial fluid was submit for total cell count with electronic cell-counter; for this purpose and for cytofluorimetric examination too was pre-treated with jaluronidasis. The mononuclear cell phase was then separated and concentrated by centrifugation on LymphoprepTM (Ficoll-Isopaque) and studied with cytofluorimetry. In the mean time the lymphocyte immunophenotyping was performed also on a peripheral blood sample. Further tests aimed to transform the sample in an aqueous fluid has shown the absolute necessity to pre-treat the sample with two drops of jaluronidasis 150 UI/ml in 0,25 cc of synovial fluid. In chronic degenerative joint disease only few drops of synovial fluid can be collected from joints, while more abundant quantities could came from acute inflammatory cases, and this could represent a limit for the applications of the technique in the clinical practice. To perform a good examination we need at least 1cc of synovial fluid and/or at least 1000 cells/μl. Lymphocyte CD3+ and CD21+ subpopulation distribution, and especially CD4+/CD8+ ratio in blood and synovial fluid, shows differences in prevalently degenerative and mainly inflammatory phenomena. The lymphocyte immunophenotyping of canine synovial fluid can represent an alternative instrument to deeply analyze the aetiopathogenetic mechanisms settled at the basis of chondrodegeneration and of the local and systemic response to osteoarthritic and immunological phenomena, and to help in therapeutic choice and monitoring

Time Course of Paclitaxel-Induced Apoptosis in an Experimental Model of Virus-Induced Breast Cancer

Early assessment of the efficacy of treatment is important in patients with breast cancer, whose routine adjuvant regimen frequently includes chemotherapy. Irrespective of the exact mechanisms involved in induction, the common early phenotypic marker of apoptosis is the expression on the outer cell membrane surface of phosphatidylserine, which avidly binds annexin V. 99mTc-labeled annexin V has been proposed for in vivo scintigraphic detection of apoptosis, albeit with contradicting results. This study was performed to define the time course of apoptosis induced by the chemotherapeutic agent paclitaxel in a model of virus-induced murine breast cancer. Methods: The RIII virus induces an estrogen-dependent, slow-growing breast cancer; BALB-c/cRIII female mice with breast tumors averaging 10 mm were studied, both in baseline conditions and at various times after the intravenous administration of paclitaxel (equivalent to a human dose of 20 mg/70 kg of body weight). The biodistribution of 99mTc-annexin V was evaluated at baseline and then at 1, 3, 6, and 24 h after paclitaxel administration. Apoptotic and antiapoptotic markers were also evaluated in tumor samples obtained at the same time points: DNA breaks (terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling [TUNEL]), active caspase-3, apoptosis-inducing factor, and Bcl-2 protein. Results: Baseline uptake of 99mTc-annexin V in breast tumors was about 2-fold higher than the uptake in normal breast tissue (demonstrating some ongoing apoptosis); tracer uptake increased at 1 and 3 h after paclitaxel administration (to almost double the baseline value) and then declined to levels even lower than baseline. Although no activation of the apoptosis-inducing factor mechanism was detected, a peak in TUNEL-positive tumor cells was reached 3 h after paclitaxel administration (to more than 6-fold the baseline level). The antiapoptotic marker Bcl-2 exhibited a biphasic pattern, with a maximum drop at 3 h, followed by return toward baseline levels at 6 h. Conclusion: These results define the time course of various biologic events taking place in this model of murine breast cancer after a proapoptotic insult (single-dose paclitaxel). Although confirming that in vivo uptake of 99mTc-annexin V reflects the degree of apoptosis, the study also suggests that the apoptotic response to antitumor therapy may differ from tumor type to tumor type. Therefore, contradicting results previously reported may depend on an inadequate time window chosen for imaging with 99mTc-annexin V

Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus

BACKGROUND:Severe acute respiratory syndrome (SARS) emerged in China in 2002 and spread to other countries before brought under control. Because of a concern for reemergence or a deliberate release of the SARS coronavirus, vaccine development was initiated. Evaluations of an inactivated whole virus vaccine in ferrets and nonhuman primates and a virus-like-particle vaccine in mice induced protection against infection but challenged animals exhibited an immunopathologic-type lung disease. DESIGN:Four candidate vaccines for humans with or without alum adjuvant were evaluated in a mouse model of SARS, a VLP vaccine, the vaccine given to ferrets and NHP, another whole virus vaccine and an rDNA-produced S protein. Balb/c or C57BL/6 mice were vaccinated i.m. on day 0 and 28 and sacrificed for serum antibody measurements or challenged with live virus on day 56. On day 58, challenged mice were sacrificed and lungs obtained for virus and histopathology. RESULTS:All vaccines induced serum neutralizing antibody with increasing dosages and/or alum significantly increasing responses. Significant reductions of SARS-CoV two days after challenge was seen for all vaccines and prior live SARS-CoV. All mice exhibited histopathologic changes in lungs two days after challenge including all animals vaccinated (Balb/C and C57BL/6) or given live virus, influenza vaccine, or PBS suggesting infection occurred in all. Histopathology seen in animals given one of the SARS-CoV vaccines was uniformly a Th2-type immunopathology with prominent eosinophil infiltration, confirmed with special eosinophil stains. The pathologic changes seen in all control groups lacked the eosinophil prominence. CONCLUSIONS:These SARS-CoV vaccines all induced antibody and protection against infection with SARS-CoV. However, challenge of mice given any of the vaccines led to occurrence of Th2-type immunopathology suggesting hypersensitivity to SARS-CoV components was induced. Caution in proceeding to application of a SARS-CoV vaccine in humans is indicated

Evaluation of qualitative and semi-quantitative cut offs for rapid diagnostic lateral flow test in relation to serology for the detection of SARS-CoV-2 antibodies: findings of a prospective study

There is limited information to compare the qualitative and semi-quantitative performance of rapid diagnostic tests (RDT) and serology for the assessment of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the objective of the study was (a) to compare the efficacy of SARS-CoV-2 antibody detection between RDT and laboratory serology, trying to identify appropriate semi-quantitative cut-offs for RDT in relation with quantitative serology values and to (b) evaluate diagnostic accuracy of RDT compared to the NAAT gold standard in an unselected adult population

Experimental Infection of Prairie Dogs with Monkeypox Virus

Infected prairie dogs can transmit monkeypox virus by respiratory and mucocutaneous contact with susceptible animals and humans

Experimental Infection of Ground Squirrels (Spermophilus tridecemlineatus) with Monkeypox Virus

A new, small-animal model of severe orthopoxvirus infection (monkeypox) is described

MR-proADM as prognostic factor of outcome in COVID-19 patients

17siMid Regional pro-ADM (MR-proADM) is a promising novel biomarker in the evaluation of deteriorating patients and an emergent prognosis factor in patients with sepsis, septic shock and organ failure. It can be induced by bacteria, fungi or viruses. We hypothesized that the assessment of MR-proADM, with or without other inflammatory cytokines, as part of a clinical assessment of COVID-19 patients at hospital admission, may assist in identifying those likely to develop severe disease. A pragmatic retrospective analysis was performed on a complete data set from 111 patients admitted to Udine University Hospital, in northern Italy, from 25th March to 15th May 2020, affected by SARS-CoV-2 pneumonia. Clinical scoring systems (SOFA score, WHO disease severity class, SIMEU clinical phenotype), cytokines (IL-6, IL-1b, IL-8, TNF-α), and MR-proADM were measured. Demographic, clinical and outcome data were collected for analysis. At multivariate analysis, high MR-proADM levels were significantly associated with negative outcome (death or orotracheal intubation, IOT), with an odds ratio of 4.284 [1.893–11.413], together with increased neutrophil count (OR = 1.029 [1.011–1.049]) and WHO disease severity class (OR = 7.632 [5.871–19.496]). AUROC analysis showed a good discriminative performance of MR-proADM (AUROC: 0.849 [95% Cl 0.771–0.730]; p < 0.0001). The optimal value of MR-proADM to discriminate combined event of death or IOT is 0.895 nmol/l, with a sensitivity of 0.857 [95% Cl 0.728–0.987] and a specificity of 0.687 [95% Cl 0.587–0.787]. This study shows an association between MR-proADM levels and the severity of COVID-19. The assessment of MR-proADM combined with clinical scoring systems could be of great value in triaging, evaluating possible escalation of therapies, and admission avoidance or inclusion into trials. Larger prospective and controlled studies are needed to confirm these findings.openopenSozio E.; Tascini C.; Fabris M.; D'Aurizio F.; De Carlo C.; Graziano E.; Bassi F.; Sbrana F.; Ripoli A.; Pagotto A.; Giacinta A.; Gerussi V.; Visentini D.; De Stefanis P.; Merelli M.; Saeed K.; Curcio F.Sozio, E.; Tascini, C.; Fabris, M.; D'Aurizio, F.; De Carlo, C.; Graziano, E.; Bassi, F.; Sbrana, F.; Ripoli, A.; Pagotto, A.; Giacinta, A.; Gerussi, V.; Visentini, D.; De Stefanis, P.; Merelli, M.; Saeed, K.; Curcio, F

Il Fenomeno delle Dipendenze nella Regione Liguria. Anno 2010

-Il Report analizza il fenomeno delle dipendenze nella regione Liguria. La descrizione del fenomeno si sviluppa intorno all\u27analisi degli indicatori individuati dall\u27Osservatorio Europeo delle Dipendenze di Lisbona (OEDT): 1-uso di sostanze nella popolazione generale (questo indicatore va a rilevare i comportamenti nei confronti di alcol e sostanze psicoattive da parte della popolazione generale); 2-prevalenza d\u27uso problematico delle sostanze psicoattive; 3-domanda di trattamento degli utilizzatori di sostanze; 4-mortalit? degli utilizzatori di sostanze; 5-malattie infettive. Altri due importanti indicatori che si stanno sviluppando, e che vengono qui illustrati, sono l\u27analisi delle Schede di Dimissione Ospedaliera (SDO) e gli indicatori relativi alle conseguenza sociali dell\u27uso di droghe (criminalit? droga correlata). Inoltre sono state applicate diverse metodologie standard di stima sia per quantificare la quota parte sconosciuta di utilizzatori di sostanze che non afferiscono ai servizi, sia per identificarne alcune caratteristiche

Sardines at a junction: seascape genomics reveals ecological and oceanographic drivers of variation in the {NW} Mediterranean Sea

By evaluating genetic variation across the entire genome, one can address existing questions in a novel way while raising new ones. The latter includes how different local environments influence adaptive and neutral genomic variation within and among populations, providing insights into local adaptation of natural populations and their responses to global change. Here, under a seascape genomic approach, ddRAD data of 4609 single nucleotide polymorphisms (SNPs) from 398 sardines (Sardina pilchardus) collected in 11 Mediterranean and one Atlantic site were generated. These were used along with oceanographic and ecological information to detect signals of adaptive divergence with gene flow across environmental gradients. The studied sardines constitute two clusters (FST = 0.07), a pattern attributed to outlier loci, highlighting putative local adaptation. The trend in the number of days with sea surface temperature above 19°C, a critical threshold for successful sardine spawning, was crucial at all levels of population structuring with implications on the species' key biological processes. Outliers link candidate SNPs to the region's environmental heterogeneity. Our findings provide evidence for a dynamic equilibrium in which population structure is maintained by physical and ecological factors under the opposing influences of migration and selection. This dynamic in a natural system warrants continuous monitoring under a seascape genomic approach that might benefit from a temporal and more detailed spatial dimension. Our results may contribute to complementary studies aimed at providing deeper insights into the mechanistic processes underlying population structuring. Those are key to understanding and predicting future changes and responses of this highly exploited species in the face of climate change

Il Fenomeno delle Dipendenze nella Regione Abruzzo. Rapporto Anno 2009

Report on the state of legal and illegal substances use in the territory of Abruzzo RegionIl Report analizza il fenomeno delle dipendenze nella Regione Abruzzo. La descrizione del fenomeno si sviluppa intorno all\u27analisi degli indicatori individuati dall\u27Osservatorio Europeo delle Dipendenze di Lisbona (OEDT): 1-uso di sostanze nella popolazione generale (questo indicatore va a rilevare i comportamenti nei confronti di alcol e sostanze psicoattive da parte della popolazione generale); 2-prevalenza d\u27uso problematico delle sostanze psicoattive; 3-domanda di trattamento degli utilizzatori di sostanze; 4-mortalit? degli utilizzatori di sostanze; 5-malattie infettive. Altri due importanti indicatori che si stanno sviluppando, e che vengono qui illustrati, sono l\u27analisi delle Schede di Dimissione Ospedaliera (SDO) e gli indicatori relativi alle conseguenza sociali dell\u27uso di droghe (criminalit? droga correlata). Inoltre sono state applicate diverse metodologie standard di stima sia per quantificare la quota parte sconosciuta di utilizzatori di sostanze che non afferiscono ai servizi, sia per identificarne alcune caratteristiche