Related U.S. Application Data 20 страница

US 9, 539, 210 B2

specific active ingredient employed; the specific composi­tion employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

The pharmaceutical compositions of the present invention may be administered by any route. In some embodiments, the pharmaceutical compositions of the present invention are administered by a variety of routes, including oral, intrave­nous, intramuscular, intra-arterial, intramedullary, intrathe­cal, subcutaneous, intraventricular, transdermal, interder- mal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), transdermal, mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous injection, intramuscular injection, and/or subcutaneous injection. In some embodiments, inventive vaccine nanocar­riers are administered parenterally. In some embodiments, inventive vaccine nanocarriers are administered intrave­nously. In some embodiments, inventive vaccine nanocar­riers are administered orally.

In general the most appropriate route of administration will depend upon a variety of factors including the nature of the vaccine nanocarrier (e. g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e. g., whether the subject is able to tolerate oral administra­tion), etc. The invention encompasses the delivery of the inventive pharmaceutical composition by any appropriate route taking into consideration likely advances in the sci­ences of drug delivery.

In certain embodiments, the vaccine nanocarriers of the invention may be administered in amounts ranging from about 0. 001 mg/kg to about 100 mg/kg, from about 0. 01 mg/kg to about 50 mg/kg, from about 0. 1 mg/kg to about 40 mg/kg, from about 0. 5 mg/kg to about 30 mg/kg, from about 0. 01 mg/kg to about 10 mg/kg, from about 0. 1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e. g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).

In some embodiments, the present invention encompasses “therapeutic cocktails” comprising populations of inventive vaccine nanocarriers. In some embodiments, all of the vaccine nanocarriers within a population of vaccine nano­carriers comprise a single species of targeting moiety which can bind to multiple targets (e. g. can bind to both SCS-Mph and FDCs). In some embodiments, different vaccine nano­carriers within a population of vaccine nanocarriers com­prise different targeting moieties, and all of the different targeting moieties can bind to the same taiget. In some embodiments, different vaccine nanocarriers comprise dif­ferent targeting moieties, and all of the different targeting moieties can bind to different targets. In some embodiments, such different targets may be associated with the same cell type. In some embodiments, such different targets may be associated with different cell types.

Combination Therapies

It will be appreciated that vaccine nanocarriers and phar­maceutical compositions of the present invention can be employed in combination therapies. The particular combi­nation of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will be appreciated that the therapies employed may achieve a desired effect for the same purpose (for example, an inventive vaccine nanocar­rier useful for vaccinating against a particular type of microbial infection may be administered concurrently with another agent useful for treating the same microbial infec­tion), or they may achieve different effects (e. g., control of any adverse effects attributed to the vaccine nanocarrier).

In some embodiments, pharmaceutical compositions of the present invention may be administered either alone or in combination with one or more other therapeutic agents. By “in combination with, ” it is not intended to imply that the agents must be administered at the same time and/or for­mulated for delivery together, although these methods of delivery are within the scope of the invention. The compo­sitions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be admin­istered at a dose and/or on a time schedule determined for that agent. Additionally, the invention encompasses the delivery of the inventive pharmaceutical compositions in combination with agents that may improve their bioavail­ability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.

The particular combination of therapies (therapeutics and/ or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and/or the desired therapeutic effect to be achieved. It will be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive vaccine nanocarrier may be admin­istered concurrently with another therapeutic agent used to treat the same disorder), and/or they may achieve different effects (e. g., control of any adverse effects attributed to the vaccine nanocarrier). In some embodiments, vaccine nano­carriers of the invention are administered with a second therapeutic agent that is approved by the U. S. Food and Drug Administration.

In will further be appreciated that therapeutically active agents utilized in combination may be administered together in a single composition or administered separately in dif­ferent compositions.

In general, it is expected that agents utilized in combina­tion with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodi­ments, the levels utilized in combination will be lower than those utilized individually.

In some embodiments, inventive vaccine nanocarriers may be administered in combination with an agent, includ­ing, for example, therapeutic, diagnostic, and/or prophylac­tic agents. Exemplary agents to be delivered in accordance with the present invention include, but are not limited to, small molecules, organometallic compounds, nucleic acids, proteins (including multimeric proteins, protein complexes, etc. ), peptides, lipids, carbohydrates, hormones, metals, radioactive elements and compounds, drugs, vaccines, immunological agents, etc., and/or combinations thereof.

In certain embodiments, vaccine nanocarriers which delay the onset and/or progression of a particular microbial infec­tion may be administered in combination with one or more

US 9, 539, 210 B2

107 additional therapeutic agents which treat the symptoms of microbial infection. To give but one example, upon exposure to rabies virus, nanocarriers comprising immunomodulatory agents useful for vaccination against rabies virus may be administered in combination with one or more therapeutic agents useful for treatment of symptoms of rabies virus (e. g. antipsychotic agents useful for treatment of paranoia that is symptomatic of rabies virus infection).

In some embodiments, pharmaceutical compositions comprising inventive vaccine nanocarriers comprise less than 50% by weight, less than 40% by weight, less than 30% by weight, less than 20% by weight, less than 15% by weight, less than 10% by weight, less than 5% by weight, less than 1% by weight, or less than 0. 5% by weight of an agent to be delivered.

In some embodiments, vaccine nanocarriers are adminis­tered in combination with one or more small molecules and/or organic compounds with pharmaceutical activity. In some embodiments, the agent is a clinically-used drug. In some embodiments, the drug is an anti-cancer agent, anti­biotic, anti-viral agent, anti-HIV agent, anti-parasite agent, anti-protozoal agent, anesthetic, anticoagulant, inhibitor of an enzyme, steroidal agent, steroidal or non-steroidal anti­inflammatory agent, antihistamine, immunosuppressant agent, anti-neoplastic agent, antigen, vaccine, antibody, decongestant, sedative, opioid, analgesic, anti-pyretic, birth control agent, hormone, prostaglandin, progestational agent, anti-glaucoma agent, ophthalmic agent, anti-cholinergic, analgesic, anti-depressant, anti-psychotic, neurotoxin, hyp­notic, tranquilizer, anti-convulsant, muscle relaxant, anti­Parkinson agent, anti-spasmodic, muscle contractant, chan­nel blocker, miotic agent, anti-secretory agent, anti­thrombotic agent, anticoagulant, anti-cholinergic, P-adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, vasodilating agent, anti-hyperten- sive agent, angiogenic agent, modulators of cell-extracellu­lar matrix interactions (e. g. cell growth inhibitors and anti­adhesion molecules), inhibitors of DNA, RNA, or protein synthesis, etc.

In certain embodiments, a small molecule agent can be any drug. In some embodiments, the drug is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regula­tory body. For example, drugs approved for human use are listed by the FDA under 21 C. F. R. §§330. 5, 331 through 361, and 440 through 460, incorporated herein by reference; drugs for veterinary use are listed by the FDA under 21 C. F. R. §§500 through 589, incorporated herein by reference. All listed drugs are considered acceptable for use in accor­dance with the present invention.

A more complete listing of classes and specific drugs suitable for use in the present invention may be found in Pharmaceutical Drugs: Syntheses, Patents, Applications by Axel Kleemann and Jurgen Engel, Thieme Medical Publish­ing, 1999 and the Merck Index: An Encyclopedia of Chemi­cals, Drugs and Biologicals, Ed. by Budavari et al., CRC Press, 1996, both of which are incorporated herein by reference.

In some embodiments, vaccine nanocarriers are adminis­tered in combination with one or more nucleic acids (e. g. functional RNAs, functional DNAs, etc. ) to a specific loca­tion such as a tissue, cell, or subcellular locale. For example, inventive vaccine nanocarriers which are used to delay the onset and/or progression of a particular microbial infection may be administered in combination with RNAi agents which reduce expression of microbial proteins. Molecular

properties of nucleic acids are described in the section above entitled “Nucleic Acid Targeting Moieties. ”

In some embodiments, vaccine nanocarriers are adminis­tered in combination with one or more proteins or peptides. In some embodiments, the agent to be delivered may be a peptide, hormone, erythropoietin, insulin, cytokine, antigen for vaccination, etc. In some embodiments, the agent to be delivered may be an antibody and/or characteristic portion thereof. Molecular properties of which are described in the section above entitled “Protein Targeting Moieties. ”

In some embodiments, vaccine nanocarriers are adminis­tered in combination with one or more carbohydrates, such as a carbohydrate that is associated with a protein (e. g. glycoprotein, proteogycan, etc. ). A carbohydrate may be natural or synthetic. A carbohydrate may also be a deriva- tized natural carbohydrate. In certain embodiments, a car­bohydrate may be a simple or complex sugar. In certain embodiments, a carbohydrate is a monosaccharide, includ­ing but not limited to glucose, fructose, galactose, and ribose. In certain embodiments, a carbohydrate is a disac­charide, including but not limited to lactose, sucrose, malt­ose, trehalose, and cellobiose. In certain embodiments, a carbohydrate is a polysaccharide, including but not limited to cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), methylcellulose (MC), dextrose, dextran, glycogen, xanthan gum, gellan gum, starch, and pullulan. In certain embodiments, a carbohydrate is a sugar alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, malitol, and lactitol. Molecular properties of carbohydrates are described in the section above entitled “Vaccine Nanocarriers Comprising Carbohydrates. ”

In some embodiments, vaccine nanocarriers are adminis­tered in combination with one or more lipids, such as a lipid that is associated with a protein (e. g. lipoprotein). Exem­plary lipids that may be used in accordance with the present invention include, but are not limited to, oils, fatty acids, saturated fatty acid, unsaturated fatty acids, essential fatty acids, cis fatty acids, trans fatty acids, glycerides, mono­glycerides, diglycerides, triglycerides, hormones, steroids (e. g., cholesterol, bile acids), vitamins (e. g. vitamin E), phospholipids, sphingolipids, and lipoproteins. Molecular properties of lipids are described in the section above entitled “Lipid Vaccine Nanocarriers. ”

Those skilled in the art will recognize that this is an exemplary, not comprehensive, list of therapeutic, diagnos­tic, and/or prophylactic agents that can be delivered in combination with the vaccine nanocarriers of the present invention. Any therapeutic, diagnostic, and/or prophylactic agent may be administered with vaccine nanocarriers in accordance with the present invention.

Kits

The invention provides a variety of kits comprising one or more of the nanocarriers of the invention. For example, the invention provides a kit comprising an inventive vaccine nanocarrier and instructions for use. A kit may comprise multiple different vaccine nanocarriers. A kit may comprise any of a number of additional components or reagents in any combination. All of the various combinations are not set forth explicitly but each combination is included in the scope of the invention.

According to certain embodiments of the invention, a kit may include, for example, (i) a vaccine nanocarrier com­prising at least one immunomodulatory agent, wherein the at least one immunomodulatory agent is capable of stimulating both a T cell and В cell response; (ii) instructions for administering the vaccine nanocarrier to a subject in need thereof.

US 9, 539, 210 B2

In certain embodiments, a kit may include, for example, (i) a vaccine nanocarrier comprising at least one immuno­modulatory agent, wherein the at least one immunomodu­latory agent is capable of stimulating both a T cell and В cell response, at least one targeting moiety, and/or at least one immunomodulatory agent; (ii) instructions for administering the vaccine nanocarrier to a subject in need thereof.

In certain embodiments, a kit may include, for example, (i) at least one immunomodulatory agent, wherein the at least one immunomodulatory agent is capable of stimulating both a T cell and В cell response; (ii) at least one targeting moiety; (iii) at least one immunostimulatory agent; (iv) a polymeric matrix precursor; (v) lipids and amphiphilic enti­ties; (vi) instructions for assembling inventive vaccine nano­carriers from individual components (i)-(v).

In some embodiments, the kit comprises an inventive nanocarrier and instructions for mixing. Such kits, in some embodiments, also include an immuno stimulatory agent and/or an immunomodulatory agent (e. g., a В cell or T cell antigen) The nanocarrier of such kits may comprise an immunomodulatory agent (e. g., a T cell antigen, such as a universal T cell antigen) and/or a targeting moiety. The T cell antigen and/or the targeting moiety may be on the surface of the nanocarrier. In some embodiments, the immu­nomodulatory agent and the antigen are the same. In some embodiments, they are different.

Kits typically include instructions for use of inventive vaccine nanocarriers. Instructions may, for example, com­prise protocols and/or describe conditions for production of vaccine nanocarriers, administration of vaccine nanocarriers to a subject in need thereof, etc. Kits generally include one or more vessels or containers so that some or all of the individual components and reagents may be separately housed. Kits may also include a means for enclosing indi­vidual containers in relatively close confinement for com­mercial sale, e. g., a plastic box, in which instructions, packaging materials such as styrofoam, etc., may be enclosed. An identifier, e. g., a bar code, radio frequency identification (ID) tag, etc., may be present in or on the kit or in or one or more of the vessels or containers included in the kit. An identifier can be used, e. g., to uniquely identify the kit for purposes of quality control, inventory control, tracking, movement between workstations, etc.

EXEMPLIFICATION

Example 1

Subcapsular Sinus Macrophages in Lymph Nodes
Clear Lymph-Borne Viruses and Present them to
Antiviral В Cells

Materials and Methods

Method Summary

VSV-IND and VSV-NJ virions were purified from culture supernatants of infected BSRT7 cells and used either unmodified or fluorescently labeled with Alexa-568 (red) or Alexa-488 (green). Fluorescent viruses used for tissue imag­ing were UV-irradiated to prevent generation of non-fluo- rescent progeny. Fluorescent labeling or UV-irradiation of VSV-IND particles did not affect their antigenicity or their ability to elicit a calcium flux in VI10YEN cells (not shown). Following fluorescent virus injection into footpads, draining popliteal LNs were harvested for analysis by electron microscopy or to generate frozen sections for immunostain­ing and confocal microscopy. To image adoptively trans­ferred В cells in LNs, VI10YEN and wildtype В cells were

fluorescently labeled and со-transferred by i. v. injection into wildtype or mutant recipient mice. 18 hours later, when В cells had homed to В cell follicles, mice were injected with labeled or unlabeled VSV in the right footpad. At different time intervals thereafter, the draining popliteal LN was observed by MP-IVM or harvested for confocal microscopy or for flow cytometry to analyze the activation state of virus-specific and control В cells. In some experiments, macrophages in the popliteal LN were depleted by sc injections of CLL, and animals were used for experiments 7-10 days later. MP-IVM, electron microscopy, immunohis­tochemistry and flow cytometry for various markers was performed on LNs with and without prior CLL treatment. VSV propagation from the footpad injection site to the blood and other organs was assessed by injecting a defined amount of live VSV into footpads followed by tissue harvest at two hours or six hours after VSV injection. To measure viral titers, tissues were homogenized and used in plaque assays. Some viral propagation experiments were performed after cannulation of the thoracic duct.

Mice and Antibodies

C57BL/6 and BALB/c mice were purchased from Taconic Farms (Germantown, N. Y. ). VI10YEN (Hangartner et al.,

2003, Proc. Natl. Acad. Sci., USA, 100: 12883; incorporated herein by reference), C3^_/_ (Wessels etal., 1995, Proc. Natl. Acad. Sci., USA, 92: 11490; incorporated herein by refer­ence), MHCII-EGFP (Boes et al., 2002, Nature, 418: 983; incorporated herein by reference), Act-EGFP (Wright et al., 2001, Blood, 97: 2278), and DH-LMP2Amice (Casola et al.,

2004, Nat. Immunol., 5: 317; incorporated herein by refer­ence) were bred in barrier animal facilities at Harvard Medical School and the Immune Disease Institute (IDI). Radiation chimeras were generated by irradiation of Act (EGFP) mice with two doses of 650 rad and reconstitution with C57BL/6 bone marrow, and were allowed to reconsti­tute for 8 weeks prior to use. In some experiments, SCS macrophages were depleted by footpad injections of 30 pl clodronate liposomes (CLL), 7-10 days prior to the experi­ment.

Clodronate was a gift of Roche Diagnostics GmbH, Mannheim, Germany. Other reagents for preparation of liposomes were: Phosphatidylcholine (LIPOID E PC, Lipoid GmbH, Ludwigshafen, Germany) and cholesterol (Sigma- Aldrich).

Mice were housed under specific pathogen-free and anti­viral antibody-free conditions in accordance with National Institutes of Health guidelines. All experimental animal procedures were approved by the Institutional Animal Com­mittees of Harvard Medical School and the IDI.

Antibodies were purchased from BD Biosciences (San Jose, Calif. ), except anti-B220-Alexa647 (Invitrogen- Caltag), anti-LYVE-1 (Millipore-Upstate), goat-anti-rabbit- APC (Invitrogen), goat-anti-GFP-FITC (Rockland), anti- FITC-Alexa488 (Invitrogen), and Fab anti-IgM-FITC (Jackson Immunoresearch). The following antibodies were purchased from AbD-Serotec: anti-CD68-Alexa647, anti- CDllb-Alexa647, F4/80-Alexa647, anti-CD169-FITC (3D6). The anti-idiotypic antibody 35. 61 for detection of the VI10 BCR in VI10YEN mice (Hangartner et al., 2003, Proc. Natl. Acad. Sci., USA, 100: 12883; incorporated herein by reference) was produced from hybridoma supernatants according to standard methods.

Flow Cytometry

Flow cytometric analysis of blood samples was performed after retro-orbital phlebotomy of mice and lysis of erythro­cytes with ACK buffer (0. 15 M NH₄C1, 1 mM KHCO₃, 0. 1 mM EDTA (disodium salt), pH 7. 2). Single-cell suspensions

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of LNs and spleens for flow cytometry were generated by careful mincing of tissues and subsequent digestion at 37° C. for 40 minutes in DMEM (Invitrogen-Gibco) in the presence of 250 pg/ml liberase CI (Roche) plus 50 pg/ml DNase-I (Roche). After 20 minutes of digestion, samples were vig­orously passed through an 18G needle to ensure complete organ dissociation. All flow cytometric analyses were per­formed in FACS buffer containing PBS with 2 mM EDTA and 2% FBS (Invitrogen-GIBCO) on a FACScalibur (BD Pharmingen), and analyzed by FlowJo software (Treestar Inc., Ashland, Oreg. ). For calcium flux, cells were labeled with 4 iiM Fluo-LOJO (Teflabs) in DMEM containing 10% FCS for 90 minutes at 37° C. Cells were spun through FCS and used immediately.

Viruses and VSV Plaque Assay

VSV serotypes Indiana (VSV-IND, Mudd-Summers derived clone, in vitro rescued (Whelan et al., 1995, Proc. Natl. Acad. Sci., USA, 92: 8388; incorporated herein by reference) and plaque purified) or New Jersey (VSV-NJ, Pringle Isolate, plaque purified) were propagated at a MOI of 0. 01 on BSRT7 cells. Supernatants of infected cells were cleared from cell debris by centrifugation at 2000xg, filtered through 0. 45 pm sterile filters and subjected to ultracentrifu­gation at 40, 000xg for 90 minutes. Pellets were resuspended in PBS and purified by ultracentrifugation (157, 000xg, 60 minutes) through a cushion of 10% sucrose in NTE (0. 5 mM NaCl, 10 mM Tris-HCl pH 7. 5, 5 mM EDTA pH 8). After resuspension in PBS overnight, virus protein was quantified by BCA assay (Pierce), and infectivity was quantified by plaque assay. Some batches were labeled with carboxylic acid succinimidyl esters of AlexaFluor-488 or AlexaFluor- 568 (Invitrogen-Molecular Probes) at a 104-105-fold molar excess of Alexa dye over virus particles. Unconjugated dye was removed by ultracentrifugation through 10% sucrose in NTE, pellets resuspended in PBS and stored frozen. Infec­tivity of VSV preparations was quantified by plaque assay on green monkey kidney cells (Vero). VSV titers from organs of infected mice were determined similarly, after homogenization of the organs with a Potter-Elvejhem homogenizer. When necessary, during viral preparation, the approximately 4 ml supernatants from the 157, 000xg ultra­centrifugation were collected and concentrated with a 10, 000 MWCO Amicon Ultra (Millipore). In order to account for residual infectivity in concentrated supernatants, VSV stocks were diluted to levels of infectivity equal to that of the concentrated supernatants and calcium flux in VI10YEN В cells was compared over further 100 fold dilutions of VSV and supernatant. UV-inactivated, Alex- aFluor-568 labeled Adenovirus 5 (AdV5) was generated following standard procedures (Leopold et al., 1998, Human Gene Therapy, 9: 367; incorporated herein by reference). All infectious work was performed in designated BL2+ work­spaces, in accordance with institutional guidelines, and approved by the Harvard Committee on Microbiological Safety.

VSV Neutralization Assay

Serum of immunized mice was prediluted 40-fold in MEM containing 2% FCS. Serial two-fold dilutions were mixed with equal volumes of VSV (500 pfu/ml) and incu­bated for 90 minutes at 37° C. in 5% CO₂. 100 pl of serum-virus mixture was transferred onto Vero cell mono- layers in 96-well plates and incubated for 1 hour at 37° C. The monolayers were overlaid with 100 pl DMEM contain­ing 1% methylcellulose and incubated for 24 hours at 37° C. Subsequently, the overlay was discarded, and the monolayer was fixed and stained with 0. 5% crystal violet. The highest dilution of serum that reduced the number of plaques by

50% was taken as titer. To determine IgG titers, undiluted serum was pretreated with an equal volume of 0. 1 mM P-mercaptoethanol in saline.

Adhesion Assays

96-well plates (Coming) were coated overnight with dilutions of recombinant murine VCAM-l-Fc or ICAM-1- Fc (R& D systems), or purified VSV-IND in PBS in tripli­cates. Negative control wells were coated with 4% BSA, positive control wells were coated with 1 mg/ml poly-L- lysine. Plates were blocked for 1-2 h at 4° C. with Hanks Balanced Salt Solution (HBSS)/1% BSAand washed. Naive В cells from V110YEN or C57BL/6 mice were negatively selected by magnetic cell separation using CD43 magnetic beads (Miltenyi, Bergisch Gladbach, Germany) and added to the plates at 3xl0⁵/well in HBSS with 1% BSA, 1 mM Ca₂⁺ and 1 mM Mg₂⁺ in the presence or absence of UV-inacti- vated VSV-IND (MOI of 1000) for 30 minutes at 37° C. After gentle washing (3 times in HBSS with 1% BSA), plates were fixed for 10 minutes with PBS/10% glutaralde­hyde, stained for 45 minutes with 0. 5% crystal violet/20% methanol, and washed in water. Dye was eluted by addition of 1% SDS and absorbance at 570 nm was spectrophoto- metrically determined (SpectraMax340PC microplate reader and SoftmaxPro 3. 1. 2 software, Molecular Devices Corporation) after 30 minutes.

Confocal Microscopy

For some analyses, C57BL/6 mice were injected into both hind footpads with 20 pg AlexaFluor-568 or AlexaFluor-488 labeled VSV-IND or VSV-NJ for 30 minutes. For other experiments, mice were transfused with lxlO⁷ negatively selected naive В cells from VIlOYENxMHCII-EGFP mice one day prior to the experiment. At predetermined time points, popliteal LNs were fixed in situ by footpad injections of phosphate buffered L-lysine with 1% paraformaldehyde/ periodate (PLP). After removal of popliteal LNs and 3-5 hours incubation in PLP at 4° C., popliteal LNs were washed in 0. 1 M PBS, pH 7. 2 and cryoprotected by an ascending series of 10%, 20%, and 30% sucrose in PBS. Samples were snap-frozen in TBS tissue freezing liquid (Triangle Bio­medical Sciences, Durham N. C. ) and stored at -80° C. Sections of 40 pm thickness were mounted on Superfrost Plus slides (Fisherbrand) and stained with fluorescent anti­bodies in a humidified chamber after Fc receptor blockade with 1 pg/ml antibody 2. 4G2 (BD Pharmingen). Samples were mounted in FluorSave reagent solution (EMD-Calbio- chem) and stored at 4° C. until analysis. Images were collected with a BioRad confocal microscopy system using an Olympus BX50WI microscope and 10x/0. 4 or 60x/l. 2 W objectives. Images were analyzed using LaserSharp2000 software (BioRad Cell Science, Hemel Hempstead, Great Britain) and Photoshop CS (Adobe). Quantification of T/B border localized В cells was done by counting cells that were within 50 pm of the T/B border, as denoted by B220 counterstain, any cells localized in more central regions were considered follicular.

Electron Microscopy

Popliteal LNs were fixed in situ by footpad injection of 2% formaldehyde and 2. 5% glutaraldehyde in 0. 1 M sodium cacodylate buffer, pH 7. 4. The LNs were excised and immersed in the same buffer overnight at 4° C., washed in cacodylate buffer, and osmicated with 1% Osmium tetrox- ide/1. 5% Potassium ferrocyanide (in water) for 1 hour at room temperature in the dark. After washing in water, samples were washed 3-4 times in 0. 05 M malelate buffer pH 5. 15. Samples were counterstained for 2 hours in 1% uranyl acetate in maleate buffer and washed three times in water. Samples were dehydrated by incubation for 15 min­

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