Questo lavoro è stato finanziato da borse di studio personale del Wellcome Trust di Beate Kampmann (056.608, GRO77273).

1. Preparazione del giornalista BCG magazzino lux micobatteri <ol><li> Crescere BCG lux (M. bovis BCG ceppo Montreal trasformato con il plasmide pSMT1 navetta) con agitazione a 200gpm a metà logaritmica fase Middlebrook 7H9 brodo glicerolo contenente 0,2%, 0,05% Tween 80 e il 10% di arricchimento ADC. </li><li> Preparare 1 ml di una diluizione 1:10 di cultura BCG lux in PBS sterile in un tubo luminometro (900μl PBS + 100μl BCG cultura lux) in duplice copia. </li><li> Carico provetta contenente N-decile aldeide (substrato dell&#39;enzima luciferasi) sul retro della macchina e garantire il coperchio è assicurato e tubi in posizione. </li><li> Primo luminometro iniettore con substrato tramite il programma principale, insieme a iniettare 100 UCL x 3 in ciascuno dei 3 tubi adescamento vuoto posto nel luminometro. </li><li> Caricare il 2 tubi in luminometro e le letture tramite un programma fissato a iniezione di 100 UCL di substrato in ogni provetta e leggere per 20 secondi in intervalli di 1 sec. </li><li> Quando lo stock ha raggiunto 1x10 8 RLU / ml a 2x10 8 RLU / ml (questo dura circa 3-4 giorni di crescita), aggiungere un uguale volume di glicerolo sterile al 30% alla cultura in un tubo da 50 ml falco e mescolare delicatamente. </li><li> Aliquota 1,5 ml volumi in etichetta 2 ml con tappo a vite microtubo e conservare a -80 ° C. </li></ol> 2. Determinazione magazzino RLU / CFU correlazione e contenuti di aliquote <ol><li> Aggiungere 15 ml di media cultura Middlebrook 7H9 con supplemento del 10% ADC ad una beuta da 200 ml con un tappo ventilato. </li><li> Aggiungere 15μl di Hygromycin e 30μl di Tween 20%. </li><li> Rimuovere una fiala di BCG lux dal freezer e scongelare a temperatura ambiente (RT) nel gabinetto di sicurezza. Aggiungere il contenuto della fiala al mezzo e serrare il tappo. </li><li> Incubare con agitazione a 200gpm a 37 ° C per 4 giorni. </li><li> Ogni giorno, make up di serie 10-diluizioni (1:10, 1:100, 1:1000, 1:10.000 e 1:100.000) per la determinazione CFU. </li><li> Impostare le diluizioni stesso in parallelo e duplicare per determinare luminescenza (cfr. parte 1,2-1,6). </li><li> Preparare due 3-vano piastre di agar Middlebrook 7H11 (1 l di liquido contenente 0,5% glicerolo, supplemento del 10% OADC, 1 ml di Tween 20% e 1 ml di Hygromycin). </li><li> Piastra in 100μl di ciascuna diluizione su un segmento di ogni piatto con crocette separati, distribuendo il liquido equamente tra la camera singola. </li><li> Sigillare ogni piatto con parafilm, mettere in un sacchetto di plastica sterile, sigillare con nastro autoclave, e incubare a 37 ° C per 2 settimane con i coperchi rivolti verso il basso. </li><li> Ispezionare regolarmente fino CFU apparire (2-3 settimane). </li><li> Per contare le colonie, rimuovere le piastre dal termostato e posto su un bancone colonia. Calcolare la media per ogni diluizione dalle piastre duplicato. </li><li> Calcolare la RLU / CFU rapporto con equivalente RLU e conta CFU per ogni diluizione. Il rapporto dovrebbe essere tra 3 e 5 RLU / CFU. </li></ol> 3. Preparazione della cultura BCG lux per inoculazione in sangue intero <ol><li> Aggiungere 15 ml di media cultura Middlebrook 7H9 con supplemento del 10% ADC ad una beuta da 200 ml con un tappo ventilato. </li><li> Aggiungere 15μl di Hygromycin e 30μl di Tween 20%. </li><li> Rimuovere una fiala di BCG lux dal freezer e scongelare a temperatura ambiente nel gabinetto di sicurezza. Aggiungere il contenuto della fiala al mezzo e serrare il tappo. </li><li> Incubare con agitazione a 200gpm a 37 ° C per 2 a 4 giorni. </li><li> Preparare 1 ml di una diluizione 1:10 di cultura BCG lux in PBS sterile in un tubo luminometro (900μl PBS + 100μl BCG cultura lux) in duplice copia. </li><li> Luminometro primo con il substrato (come descritto sopra), delicatamente vortice i 2 tubi e caricare nel luminometro e le letture (come descritto sopra). </li><li> Utilizzare questa lettura per diluire in basso la cultura in PBS per dare un equivalente di 7x10 6 RLU (questo darà un inoculo di circa 2x10 5 CFU / ml di sangue e un rapporto di BCG a monociti di circa 1:1, assumendo un conteggio dei monociti di circa 2x10 4x10 5 a 5 monociti per ml di sangue). </li></ol> 4. Preparazione di sangue intero <ol><li> Da 3 a 5 ml di sangue venoso in una provetta contenente eparina senza conservanti. </li><li> Trasferire il sangue in una provetta da 50 ml falco e diluire con un uguale volume di RPMI 1640 contenente HEPES glutammina e 25 mm (senza pen / strep). </li><li> 900μl aliquota di sangue diluito in triplice copia in tubi sterili bijou per ogni punto temporale (6 tubi in totale:. 0ore e 3 per 3 per 96hrs Istituito tubi aggiuntivi per timepoints aggiuntive, se necessario. </li><li> 900μl aliquota di terreno di coltura Middlebrooks 7H9 con il 10% ADC + supplemento 50μg/ml Hygromycin (lo stesso mezzo utilizzato per la cultura lux BCG) in duplice copia per i controlli di crescita. </li><li> Aggiungere 100μl della diluito BCG lux a ciascuna provetta di sangue, e per il controllo 2 crescitatubi. </li><li> Mescolare bene e posto le 3 bijous per la 96hr time-point e le 2 controlla la crescita al loro fianco in incubatrice a dondolo a 37 ° C, 20 giri / min (CO 2 non richiesto). </li></ol> 5. Luminescenza di misura (a 0hr e 96hrs) <ol><li> Centrifugare i 3 bijous a 2000g per 10 min. </li><li> Rimuovere con cura 300μl di sopranatante senza disturbare il pellet e il luogo in 2 ml con tappo a vite microtubi di congelare a -80 ° C per l&#39;analisi delle citochine successive. </li><li> Aggiungere 300μl di PBS in ogni bijou per sostituire il volume del surnatante eliminato. </li><li> Aspirare il contenuto di ogni bijou nel corrispondente tubo 50ml falco e aggiungere 8 ml di acqua distillata ad ogni provetta. Iniziare un timer per 10 minuti. Nb: Questo è un tempo sensibile al passo, e questo incubazione non deve essere più di 10 minuti, a partire da quando le prime cellule entrano in contatto con l&#39;acqua. </li><li> Sciacquare ogni bijou con 2 ml di acqua distillata e vortex per 5 secondi prima di ribaltamento nel tubo corrispondente falco. </li><li> Al termine della incubazione di 10 minuti, centrifugare i tubi falco a 2000g per 10 minuti. </li><li> Surnatante decantare in disinfettante Surfanios. </li><li> Aggiungere qualche granello di ogni pellet e vortice. </li><li> Aggiungere 1 ml di PBS sterile per ciascuna provetta e vortex. </li><li> In tubi luminometro, fare diluizioni 1:10 per ogni campione in duplicato con PBS (PBS 900μl campione + 100μl) e al 96hr time-point. Effettuare le diluizioni stesso per ciascuno dei controlli crescita. </li><li> Delicatamente vortice i tubi e caricare nel luminometro e le letture (come descritto sopra) Nb: Letture tra triplica dello stesso campione dovrebbe essere entro il 15% l&#39;uno dall&#39;altro. </li></ol> 6. Rappresentante dei risultati: E &#39;importante usare i batteri in fase logaritmica di crescita per le analisi del sangue intero lux, cioè cresciuta negli 48-72 ore prima di inoculare i campioni, in quanto l&#39;attività metabolica è sub-ottimale se uno fuori direttamente dal freezer o in fase stazionaria. La crescita preventiva deve essere standardizzato per una serie di esperimenti sia a 48 o 72 ore per evitare la variabilità. La figura 1 mostra la curva di crescita di una cultura rappresentante della BCG lux. Il tempo di raddoppio è di circa 24 ore fino a quando la fase stazionaria è raggiunto. <img alt="Figura 1" src="/files/ftp_upload/3332/3332fig1.jpg" /> Figura 1. Curva di crescita rappresentante di BCG lux oltre 96 ore in media 7H9 e la correlazione di RLU e CFU. Anche se i valori RLU sempre correlate con CFU, il numero di RLU CFU corrispondenti ad un singolo può variare a seconda del magazzino, che è il motivo per cui è buona norma utilizzare le azioni stesse congelato tutta una serie di esperimenti per garantire una molteplicità consistente di infezione ( MOI). La tabella 1 mostra un esempio di data crudo al momento della vaccinazione (T 0) e in 96 ore. I rapporti di crescita sono calcolati utilizzando la formula T 96 / T 0, ma intervalli di tempo gli altri possono naturalmente anche essere misurata. E &#39;, tuttavia, non consiglia di utilizzare le culture oltre 96 ore, come la morte cellulare si verifica una significativa. <img alt="Tabella 1" src="/files/ftp_upload/3332/3332table1.jpg" /> Tabella 1. Esempio di dati grezzi al momento dell&#39;inoculazione (T0) ed a 96 ore (T96) e rapporti di crescita calcolato da 3 donatori adulti. A seconda della disposizione antigene-specifiche risposte della memoria e forse conta anche dei neutrofili, i rapporti di crescita variano da individuo a individuo, come mostrato nella Figura 2 nel sangue dei bambini con e senza infezione da HIV. In media, i bambini piccoli hanno rapporti di crescita maggiori rispetto agli adulti, test cutaneo alla tubercolina (TST) + ve individui hanno rapporti di crescita inferiore rispetto TST-ve gli individui, e pazienti affetti da HIV hanno rapporti di forte crescita dovuta al deficit della popolazione di cellule T CD4, uno dei mediatori chiave della risposta immunitaria cellulare per micobatteri. <img alt="Figura 2" src="/files/ftp_upload/3332/3332fig2.jpg" /> Figura 2 Inter-donatore variabilità:. Crescita del rapporto T 0 contro 96 ore per una serie di pazienti, a seconda del sottostante stato di sieropositività. Riproducibilità dei rapporti di crescita in un periodo di tempo è mostrato in Figura 3, che riassume i risultati da 64 donatori sanguinato due volte in un periodo di 12 mesi e da un singolo donatore sanguinato ripetutamente per esperimenti di controllo. Potenziali cause di variabilità possono essere cambiamenti nella sensibilizzazione micobatteriche o variabilità all&#39;interno dei livelli di citochine di accoglienza, come osservato in molti saggi biologici. <img alt="Figura 3" src="/files/ftp_upload/3332/3332fig3.jpg" /> Figura 3. (A) donatore singolo a 12 volte nell&#39;arco di 12 mesi. (B) multiple (64) i donatori in 2 occasioni su 12 mesi.

<ol>
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Test battericida nel contesto della ricerca tubercolosi vaccino sono ben stabiliti nei modelli animali, dove sono applicati a schermo vaccini candidati potenzialmente promettenti. Riduzione della carica batterica nei vari organi funzioni come il principale lettura di immunogenicità. La prima generazione di umani test battericida sono stati progettati utilizzando macrofagi da solo, infettati con M. tubercolosi. CFU servita come principale lettura di sopravvivenza dei micobatteri dop...

<table border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Nessun gatto </td></tr><tr><td> BCG lux </td><td> Vari </td><td> n / a </td></tr><tr><td> Middlebrook 7H11 agar </td><td> Becton Dickinson </td><td> 283810 </td></tr><tr><td> Middlebrook 7H9 brodo </td><td> BD Biosciences </td><td> 271310 (500g) </td></tr><tr><td> Middlebrook ADC arricchimento </td><td> BD Biosciences </td><td> 212352 </td></tr><tr><td> Middlebrook OADC supplemento </td><td> Becton Dickinson </td><td> 212240 </td></tr><tr><td> Tween 80 </td><td> Sigma Aldrich </td><td> 274364 </td></tr><tr><td> Glicerina </td><td> Sigma Aldrich </td><td> G5150 </td></tr><tr><td> Hygromycin B </td><td> Roche </td><td> 10843555001 (20ml) </td></tr><tr><td> N-decile aldeide </td><td> Sigma </td><td> D7384-100G </td></tr><tr><td> Etanolo (&gt; 99,7%) </td><td> VWR </td><td> 101077 </td></tr><tr><td> PBS sterile </td><td> In Casa </td><td> n / a </td></tr><tr><td> RPMI-1640 con L-Glutamina, HEPES 25mM </td><td> Sigma </td><td> R0883 </td></tr><tr><td> Acqua distillata sterile </td><td> In Casa </td><td> n / a </td></tr><tr><td colspan="3"> Attrezzatura </td></tr><tr><td> Tubo luminometro (porta iniettabili obbligatorio) </td><td> Berthold AutoLumat LB953 o monotubo luminometro Sirius </td><td></td></tr><tr><td> Tubi luminometro, 12 x 75mm, pyrex senza montatura </td><td> Corning VWR </td><td> 99445-12 </td></tr><tr><td> Beute con tappi ventilati, sterile in policarbonato 250ml </td><td> Corning via Fisher Scientific </td><td> 2150329 </td></tr><tr><td> BD Vacutainer il prelievo di sangue Tubi (senza conservanti eparina) </td><td> BD </td><td> 367676 </td></tr><tr><td> Provette Falcon da 50 ml, conico </td><td> BD </td><td> 352077 </td></tr><tr><td> 7ml Sterilin campione tubi in polipropilene bijou </td><td> VWR </td><td> 99445-12 </td></tr><tr><td> Sterilin Contenitori universali (30 ml) </td><td> Laboratorio di Analisi LTD </td><td> 128C </td></tr><tr><td> Perle di vetro diametro di 2mm </td><td> SigmaAldrich </td><td> Z143928 </td></tr><tr><td> 10ml pipette e ragazzo pipetta </td><td> qualsiasi </td><td></td></tr><tr><td> 1000μl e 200μl pipette e puntali con filtro </td><td> qualsiasi </td><td></td></tr><tr><td colspan="3"> Attrezzatura generale di laboratorio: </td></tr><tr><td> Classe II di sicurezza Armadio </td><td></td><td></td></tr><tr><td> Frigorifero (4 ° C) </td><td></td><td></td></tr><tr><td> Centrifuga </td><td></td><td></td></tr><tr><td> Rocking shaker piattaforma (180 0) miscelare tubi </td><td></td><td></td></tr><tr><td> Shaker orbitale incubatore a 37 ° C per crescere colture batteriche </td><td></td><td></td></tr></tbody></table>

a functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged bcg or m.tb (bcg lux/m.tb lux)

Test funzionali hanno a lungo avuto un ruolo chiave nella misurazione di immunogenicità di un vaccino somministrato. Questo è convenzionalmente espresso in titoli battericida del siero. Studi di siero titoli battericida in risposta a vaccini per l&#39;infanzia ci hanno permesso di sviluppare e convalidare i livelli di cut-off per la risposta immunitaria protettiva e tale cut-off sono in uso di routine. Nessun tali analisi sono state prese in avanti nella valutazione di routine dei vaccini che inducono soprattutto immunità cellulo-mediata in forma di risposte delle cellule T effettrici, come i vaccini TB. Nel modello animale, le prestazioni di un candidato vaccino somministrato di routine è valutato in test standardizzati battericida, e tutti gli attuali romanzo TB-vaccino candidati sono stati sottoposti a questo passo nella loro valutazione prima della fase 1 sperimentazione umana. La valutazione di immunogenicità e quindi probabilità di efficacia protettiva del romanzo di vaccini anti-TBC dovrebbe idealmente subire una simile graduale valutazione nei modelli umani oggi, comprese le misurazioni in saggi battericida. Test battericida nel contesto della ricerca tubercolosi vaccino sono già ben radicate nei modelli animali, dove sono applicati a schermo vaccini candidati potenzialmente promettenti. Riduzione della carica batterica nei vari organi funzioni come il principale lettura di immunogenicità. Tuttavia, non tali dosaggi sono stati inseriti nella sperimentazione clinica per il romanzo anti-TB vaccini fino ad oggi. Anche se c&#39;è ancora incertezza circa i meccanismi esatti che portano alla morte dei micobatteri all&#39;interno dei macrofagi umani, l&#39;interazione dei macrofagi e delle cellule T con micobatteri è chiaramente necessario. Il dosaggio descritti in questo documento rappresenta una nuova generazione di test battericida che consente studi di tali componenti chiave cellulari con tutti gli altri fattori cellulari e umorali presenti nel sangue intero senza fare ipotesi sulla loro contributo relativo individuali. Il test descritto dal nostro gruppo utilizza piccoli volumi di sangue intero ed è già stato impiegato in studi di adulti e bambini in TB è endemica impostazioni. Abbiamo dimostrato immunogenicità del vaccino BCG, aumento della crescita dei micobatteri in pazienti sieropositivi, così come l&#39;effetto della terapia anti-retrovirale e vitamina D sulla sopravvivenza dei micobatteri in vitro. Qui riassumiamo la metodologia, e presentare i nostri dati riproducibilità utilizzando questo modello relativamente semplice, a basso costo e di campo-friendly. Nota: Definizioni / Abbreviazioni BCG lux = M. bovis BCG, ceppo Montreal, trasformato con il plasmide pSMT1 navetta che trasportano i geni luxAB dal Vibrio harveyi, sotto il controllo dei micobatteri GroEL (hsp60) promotore. UFC = unità formanti colonia (una misura della vitalità dei micobatteri).

Watch this Scientific Journal Video about A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux at JoVE.com

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

Abbiamo descritto un approccio alternativo alla enumerazione dei micobatteri<em&gt; In vitro</em&gt;, Che utilizza giornalista-gene tag micobatteri invece di unità formanti colonie (CFU). &quot;Sopravvivenza&quot; degli organismi così come risposta dell&#39;ospite-marcatori vengono misurati contemporaneamente, fornendo un basso costo, sistema versatile e funzionale per gli studi di host / patogeno interazioni nel contesto della tubercolosi.

Un test funzionale di sangue intero per misurare la vitalità dei micobatteri, utilizzando Reporter-Gene Tagged BCG o M. Tb (BCG Lux / M. Tb Lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

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Immunology and Infection

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

13.8K Views.  Imperial College London. Abbiamo descritto un approccio alternativo alla enumerazione dei micobatteri In vitro, Che utilizza giornalista-gene tag micobatteri invece di unità formanti colonie (CFU). "Sopravvivenza" degli organismi così come risposta dell'ospite-marcatori vengono misurati contemporaneamente, fornendo un basso costo, sistema versatile e funzionale per gli studi di host / patogeno interazioni nel contesto della tubercolosi.

Video: A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Espansione, di purificazione, e la valutazione funzionale delle cellule del sangue umano periferico NK

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

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Immunologia e infezioni

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Enumerazione delle popolazioni dei leucociti del sangue periferico per le principali studi clinici multicentrici utilizzando un intero saggio Fenotipizzazione Sangue

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Gli antigeni Protetto funzionali globuli rossi dalla membrana innesto di Compact Poligliceroli Hyperbranched

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Utilizzando il Overlay saggio misurare Qualitativamente produzione batterica di sensibilità e di pneumococcico batteriocine

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Un semplice citometria a flusso metodo per misurare il glucosio assorbimento e glucosio Expression Transporter per monociti sottopopolazioni nel sangue intero

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

L&#39;efficacia e la protezione polmonare risposta immunitaria seguito sottocutanea e intranasale BCG Amministrazione nei topi

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

L&#39;uso di un monociti monostrato test per valutare fagocitosi Fcy recettore-mediata

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Espressione di antigeni esogeni nel Mycobacterium bovis BCG vaccino via genetica decorazione superficiale con il sistema avidina-biotina

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

Ottimizzazione del interferone-gamma ELISpot analisi di risposte a cellula T misura nel modello cavia dopo la vaccinazione

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...