83 Another study conducted within Finnish population found that G

83 Another study conducted within Finnish population found that Gly in TLR4 299, in both infants and mothers, was associated with preterm labor,84 and same trend was observed in a study in Uruguay.85 Bacterial vaginosis (BV), Selleck Talazoparib known to induce preterm birth, is also reported to be associated with TLR4 polymorphisms. One study found Thr for TLR4 399 was significantly less common in women with BV compared with women without BV.86

Another study showed Gly for TLR4 299, which is known to impair responses to LPS, was associated with an increase in vaginal pH, Gardnerella vaginalis levels and concentration of anaerobic gram-negative rods.87 TLRs polymorphisms also affect on the susceptibility to pre-eclampsia. Recently, van Rijn et al.88 suggested that maternal TLR4 polymorphisms alter susceptibility to early-onset pre-eclampsia and elevated liver enzymes and low platelets (HELLP) syndrome. Hirschfeld et al. also found that the presence of TLR2 Arg753Gln and two TLR4 SNPs (Asp299Gly and Thr399Ile) was associated with normal pregnancy controls.89

These clinical observations indicate an important role for the TLR systems in pregnancy disorders, although further investigations are required to determine the specific find more mechanism underlying in each condition. The spatial and temporal pattern of TLR expression at the maternal–fetal interface has been described in physiological and pathological conditions. There is growing evidence that these TLRs recognize pathogens and react to them, not only in immune cells but also in non-immune cells such as the trophoblast. This implies clinical applications in pregnancy disorders, i.e., using TLR agonists as a therapeutic and/or prophylactic treatment, or detection of TLR expression as a diagnostic tool. There are several points that still need to be elucidated. While we have recognized the importance of the TLRs in the defense against pathogens, the role of these receptors in establishing tolerance to the growing fetus is still unknown. It is intriguing to speculate that TLRs at the maternal–fetal interface may play a role in establishing normal pregnancy, given

the fact that commensal bacteria, which may potentially be bound to the TLRs, are present in the reproductive tract, although further studies Methocarbamol are required to elucidate this hypothesis. It is also still unclear what regulates the expression pattern and functional activity of TLRs during pregnancy, either in physiological or in pathological conditions. Addressing this question may also help develop clinical applications. Recent research in the field of TLR shows that these receptors play so many important roles in various areas. Further studies on TLRs at the maternal–fetal interface will shed light on how the balance between tolerance to allergenic fetus and host defense against possible pathogens is maintained. The authors thank Mrs. JoAnn Bilyard for her assistance with the manuscript.

Media was replaced every 2 days for a total culture time of 9 day

Media was replaced every 2 days for a total culture time of 9 days. For CFSE (Molecular Probes, Eugene, OR, USA) labeling, 5 × 107/mL T cells were incubated in prewarmed PBS containing 1 μM CFSE for 10 min at 37°C followed by extensive washing and resuspension in PBS for adoptive transfer. Mixed BM chimeras in which only the αβ T cells lack 4–1BB were generated using TCRα−/− and 4–1BB−/− mice as described previously [49]. For the generation of the 4–1BBL−/− BM chimeras, 5 × 106 congenically marked 4–1BBL−/− or WT BM cells were used to reconstitute lethally irradiated WT or 4–1BBL−/− mice. All irradiated BM reconstituted mice were given water supplemented with 2 mg/mL of neomycin

sulfate (Bio-Shop, Burlington, Ontario,

Canada) during the first 4 weeks, and they were further rested for an additional 2 months before use. Three million OT-I T cells, prepared as above, were delivered i.v. to the mice and their recovery Selumetinib datasheet from spleen, LN, and BM analyzed 30 days later. Influenza A/PR8 and A/HKx31 viruses were grown in eggs and their tissue culture infectious dose determined by infection of MDCK cells [50]. Age- and sex-matched mice were used for infection. A dose of 100 HAU influenza A/X31 in 200 μL volume was used for primary intraperitoneal infection. Influenza A/X31 primed mice were rested for at least 30 days before challenge with influenza A/PR8 at a dose of 100 HAU in 200 μL intraperitoneally. Analysis of influenza NP366–374-specific CD8+ T cells using MHC tetramers as well as CD107a and intracellular cytokine staining Alpelisib research buy following a 6-hour restimulation was carried out as previously described [28]. H-2Db/NP366–374

tetramers were provided by the National Institute for Allergy and Infectious Diseases tetramer facility (Emory University, Atlanta, GA, USA). Uninfected mice were used as negative controls for Db/NP366–374 tetramer staining. Isotype or fluorescence minus one controls were used as negative controls for cytokine staining. Congenically marked OT-I TCR transgenic cells were tracked using PE- or allophycocyanin-anti-mouse CD45.2 (eBioscience), Pacific Blue-anti-CD45.1 (Biolegend), FITC-anti-Vβ5.1 (BD Biosciences), biotin-anti-Vα2 and PerCP-anti-mouse CD8+ (BD Biosciences). Other antibodies used in this study included Cediranib (AZD2171) allophycocyanin-anti-mouse IFN-γ, FITC-anti-CD107a, PE- or PE-Cy5- or allophycocyanin-anti-CD44, FITC-anti-Ter119, Pe-Cy7- or PE-anti-mouse CD3, biotinylated-anti-mouse-4–1BB (clone 3H3), Alexa Fluor450- or PE-anti-B220, PE-, PE-Cy7- or allophycocyanin-anti-CD11c, Alexa Fluor488 anti-Gr1, PE-anti-Ly-6C, PE-anti-MHC-II, PE-Cy7-anti-F4/80, PerCP- or PE-Cy7-anti-CD11b, PE-Cy5.5-anti-mouse TCRβ, PE-Cy5.5-anti-mouse CD19, and FITC-anti-PDCA-1. The 4–1BB-deficient mouse was used as a negative control for analysis of 4–1BB expression on CD8+ T cells. Detection of 4–1BBL was done by i.v.

The stimulation of NK cytotoxicity by continuous CD27-CD70 intera

The stimulation of NK cytotoxicity by continuous CD27-CD70 interaction correlates with the reported enhanced CD8+ T-cell response of CD70-Tg mice to influenza virus infection and upon EL-4 tumour challenge. In this model continuous CD70 triggering initially enhances expansion

of the CD8+ T-cell population, combined with a higher cytotoxicity on a per cell basis 43. It is important to note that all evidenced changes for NK cells of CD70-Tg mice compared with WT mice, both phenotypical and functional, are dependent on CD27–CD70 interaction, as none of them is witnessed in CD70-Tg×CD27−/− mice. Since CD70 is up-regulated on activated B cells after antigenic stimulation, the CD70-Tg mice used in this study might provide a model for chronic CD70 expression, possibly resulting from continuous stimulation of the immune system during GSI-IX chemical structure JNK inhibitor purchase persistent infections. Our results clearly indicate that, as previously demonstrated for the CD8+ T-cell population, continuous CD70 triggering strongly reduces the NK cell number, however inducing

higher cytotoxicity capacities on a per cell basis. CD70-Tg (eight times backcrossed to C57BL/6) 29, IFN-γ−/−×CD70-Tg and CD70-Tg×CD27−/− 29 mice were used. Because the CD70 transgene, which is under the control of the human CD19 promotor, was located on the Y chromosome, female littermates were used as WT mice. All mice were housed under specific pathogen-free conditions in our animal facility and were treated and used in agreement with the guidelines of the local ethical committee. Spleen and liver from 4- to 15-wk-old

mice were removed, Y27632 disrupted and passed through a 40 μm cell strainer (Falcon, NJ, USA). Hepatic leukocytes were prepared using two-step discontinuous Percoll gradients (GE Healthcare, IL, USA). BM cells were isolated by irrigation of femurs and tibias. Erythrocytes from spleen and BM were lysed with 0.17 M NH4Cl. For functional assays, splenocytes were enriched with DX5 Microbeads (Miltenyi Biotec, CA, USA). mAb used were anti-NK1.1 (clone PK136), anti-CD3 (clone 145-2C11), anti-CD49b (clone DX5), anti-Ly49D (clone 4E5), anti-CD314 (clone CX5), anti-CD43 (clone S7), anti-CD95 (clone Jo2), anti-CD69 (clone H1.2F3), anti-granzyme B (clone GB11), anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-IFN-γ (clone XMG1.2), annexin-V and 7-AAD (BD Pharmingen, CA, USA). Anti-CD122 (clone TM-β1; kindly provided by Dr. T. Tanaka, Tokyo, Japan), anti-Ly49E/C (clone 4D12) 32, anti-Ly49A (clone JR9-318; kindly provided by Dr. J. Roland, Paris, France), anti-Ly49H (clone 3D10; kindly provided by Dr. W. Yokoyama, MO, USA), anti-Ly49G2 (clone 4D11; American Type Culture Collection, MD, USA), anti-CD11b (clone M1/70), anti-NKG2A/C/E (clone 3S9) 32, anti-CD27 (clone LG.7F9, eBioscience, CA, USA) and anti-CD16/CD32 (unconjugated, clone 2.4G2; kindly provided by Dr. J. Unkeless, NY, USA).

IL-17 is another major subset of CD4+ T cells that have been

IL-17 is another major subset of CD4+ T cells that have been selleckchem linked to host immune responses to extracellular bacteria and fungi. IL-17 is recognized

as stimulating many cells of the innate immune system particularly recruiting and activating neutrophils to sites of inflammation as well as stimulating endothelial and epithelial cells to synthesize inflammatory cytokines IL-1, IL-6 and TNF-α (7). However, the host immune defence against infectious diseases has many multiple overlapping systems for avoidance of immunopathology, and pathogens have evolved many interference mechanisms for immune evasion and survival. It may therefore be more appropriate to define combinations of cytokines and effector cells at particular stages of the response when describing the immunopathology of scabies and attempts by the host immune response to clear the mite. Presentation Fludarabine nmr with a primary infestation of scabies usually occurs 4–6 weeks after infection and is characterized by a generalized itching often more intense at night. The pruritic papules in human scabies are typically restricted to the webs of the fingers, followed by wrists,

elbows, periumbilical skin, buttocks, ankles, the penis in men and the periareolar region in women. Total mite numbers in humans are usually self limiting, in the region of 10–12 mites per patient (8). Spontaneous recovery of scabies in humans has been described to only occur with subsequent selleck chemicals llc reinfestations. Immunological memory

to mite antigens has been demonstrated with an induction time of only 24 h for hypersensitivity with patients infested for a second time (8). Additionally, parasite numbers were significantly reduced, and in approximately 60% of the cases reinfestation of sensitized hosts was unsuccessful. The clinical appearance of scabies can be wide ranging, but the classical clinical sign for diagnosis is the burrow, found in the horny layer of the epidermis. Diagnosis can be problematic, (9) and in some situations the rash and itch of scabies can persist for up to several weeks after curative treatment, possibly attributed to dead mites or mite products remaining within the skin layers. In chronic infestations, atypical excoriation and eczematization of the skin may develop. Patients taking topical or oral steroids or who are immunosuppressed because of other disease also present uncharacteristically. In some cases, nodular scabies can develop, which can persist for several months after successful treatment. These firm red-brown nodules are often extremely itchy and are commonly found in the groin, buttocks and periumbilical area.

As already mentioned, the preliminary results obtained for KIR ge

As already mentioned, the preliminary results obtained for KIR genes at the worldwide scale suggest similar patterns to those found for HLA, but await confirmation through more thorough analyses. In this article, we have summarized our current knowledge of the polymorphism Wnt mutation of three immunogenetic complexes, GM, HLA and KIR, in relation to their diversity in human populations and the interpretation of that knowledge. Actually, these three genetic complexes represent a small fraction of our genome restricted to three different chromosomes. Likewise, studies of

mtDNA and Y-chromosome markers, which have proved to be highly informative to reconstruct gender-specific molecular phylogenies of the human species (refs 142, 143, among many others) also correspond to minor DNA fractions (∼ 0·0005% of the total haploid

genome, for mtDNA, and ∼ 2%, for the Y chromosome). By contrast, analyses of microsatellites and single nucleotide polymorphisms have provided relevant information on the entire genome (e.g. refs 144, 145). Impressive technical improvements now also allow high throughput DNA sequencing with promising genome-wide application to the study of human genetic variation worldwide, although this is still in the early stages. Therefore, the study of immunogenetic complexes may be seen as a limited contribution to our knowledge of human genome diversity. Another possible drawback of the analysis of immunogenetic markers in the field of anthropology JNK inhibitor mouse is the fact that they are prone to natural selection, as discussed in the present review. As IgG, HLA and KIR molecules are instrumental in immune responses, their evolution is clearly influenced by environmental factors, which may

be a disadvantage when one tries to reconstruct the peopling history of modern humans. Indeed, of when selection is at work, the observed genetic relationships among human populations may not reflect their degree of historical relatedness, as can also be concluded for some highly variable phenotypic traits like human pigmentation.26,27 This would speak for neutral markers corresponding to non-coding regions of the genome, like microsatellites and single nucleotide polymorphisms, being preferred for genetic studies in anthropology. On the other hand, general conclusions drawn by analysing the patterns of genetic diversity of widely studied immunogenetic markers, like GM and HLA, are shown to be congruent with those found for other genetic markers. This is the case for at least five major results. 1  Of the total genetic diversity of the human species, the highest level of variation is found within populations, whereas inter-population variation represents only a minor proportion of the total genetic variance.

, 2009) Briefly, the culture medium (550 μL) was placed into a r

, 2009). Briefly, the culture medium (550 μL) was placed into a rotor, and the viscosity was measured as shearing stress between a rotor and a rotor shaft at 50 r.p.m., 20 °C, using a rotary viscometer (Tokimec Inc., Tokyo, Japan). Five independent cultures of each strain were measured and statistical differences between the two Ferrostatin-1 molecular weight groups were determined using an unpaired t-test with the level of significance set at P<0.05. To examine cell surface structures, scanning electron microscopy (SEM) was performed. Bacteria grown on TSAY for 24 h were collected on a piece of filter paper (Glass fiber GA55,

Toyo Roshi, Tochigi, Japan), fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 2 h and 1% OsO4 in 0.1 M phosphate

buffer for 1 h at 4 °C, and dehydrated through an ethanol series and 2-methyl-2-propanol, followed by platinum ion coating (E-1030, Hitachi, Tokyo, Japan). Specimens were examined using an SEM (S-4800, Hitachi) at an accelerating voltage of 3 kV. The exopolysaccharide was prepared from culture supernatants as described previously (Yamanaka et al., 2009). In brief, YS-11 was grown at 37 °C in TSBY for 24 h. Supernatants were separated by centrifuging the liquid culture at 12 000 g for 30 min, and sodium acetate was added to a final concentration of 5%. The mixture was stirred for 30 min at 22 °C, and the exopolysaccharide Endocrinology antagonist was isolated by ethanol precipitation from the reaction mixture. The ethanol-precipitated material was collected by centrifugation (18 200 g for 15 min at 22 °C), dissolved in 5% sodium acetate, and treated with chloroform : 1-butanol (1 : 5 by volume). Water-soluble and chloroform-butanol layers were separated by centrifugation. An equal amount of ethanol was added to the water-soluble fraction to isolate the exopolysaccharides. This procedure was repeated

twice, and the ethanol-precipitated material was freeze-dried and stored at −80 °C until use (Campbell & Pappenheimer, 1966). Contaminated Histone demethylase lipopolysaccharides were removed from preparations using the method described by Adam et al. (1995). The sugar composition of the purified viscous material was determined by means of HPLC for neutral and amino sugars and colorimetry for uronic acid as detailed elsewhere (Yamanaka et al., 2009). To examine the biological effect of extracellular viscous materials and cell surface-associated structures, mutants lacking these phenotypes were constructed by transposon mutagenesis. Fifteen milliliters of an overnight culture of YS-11 was used to inoculate 800 mL of TSBY. The culture was incubated at 37 °C until the OD600 nm of the bacterial culture measured reached 0.6–0.7. The cells were harvested by centrifugation at 5700 g for 20 min at 4 °C and washed three times with ice-cold 10% glycerol. The cells were resuspended with 4 mL of 10% ice-cold glycerol, divided into small aliquots, frozen with dry ice-100% cold ethanol, and stored at −80 °C until use.

Data was analysed using SPSS software,

p < 0 05 is signif

Data was analysed using SPSS software,

p < 0.05 is significant. Data is expressed as median (Interquartile range). Results: Only 8 of the 30 patients had data to date for hsTnT post-transplant. Group 1 (n = 5) had a hsTnT of 8.2 ± 4.27 ng/L which was lower compared to Group 2 (n = 25, 53.40 ± 36.85). Median ages in Group 1 were 43.39 ± 16.17 years and 52.45 ± 15.52 years in Group 2. Group 2 hsTnT significantly decreased post transplant mTOR inhibitor by 40.25 ± 40.14 (P = 0.036). Group 1 had no cardiac events post-transplant. However, 16% of Group 2 suffered a cardiac event in the post-transplant period. Conclusions: Basally elevated hsTnT alters significantly following transplantation and possibly identifies patients at high risk for cardiovascular events following transplantation. Larger studies need to be done to confirm this effect and consideration should

be made for a normal or low hsTnT level as an entry criterion to the decreased donor transplant waitlist. 266 SUPPORTING LEAVE FOR LIVING ORGAN DONORS SCHEME – AN INNOVATIVE FEDERAL POLICY SOLUTION TO FINANCIAL Selleck FDA approved Drug Library BARRIERS L TOY, T MATHEW, A WILSON, M LUDLOW Kidney Health Australia, Canberra, ACT, Australia Aim: Financial hardship for live donors is an issue that Kidney Health Australia (KHA) has been advocating for, both on behalf of and with, living donors, those with kidney disease, their families and carers. Background: More than 200 otherwise healthy people choose to undergo an invasive surgical procedure to become a live kidney donor every year. Those donating a kidney are subjected to out of pocket expenses for the cost of the procedure and unpaid leave often compounds their financial situation. Methods: Some international approaches focussed

on reimbursement for out of pocket medical costs – a difficult model in very Australia noting the differing responsibilities of Federal, State and Territory Governments. KHA focussed on a federal response by utilising an existing policy precedent from outside the health portfolio (maternity and defence force leave) and demonstrated workable budget costings for consideration. Results: In April 2013 the Federal Minister for Health, with KHA, announced a two year pilot of the scheme, commencing 1 July 2013. It covers live kidney and partial liver donation, providing access to 6 weeks paid leave at minimum wage rates. Up until 28 February 2014 there have been 90 registrations, with 36 claims already reimbursed following the donation procedure. Conclusions: Success depends on a comprehensive communication and support strategy to ensure potential donors, recipients, employers, and hospital staffs are confident in accessing the Scheme. Although modelling suggests the Scheme may pay for itself over time, the strongest justification is its potential in correcting the current burdens borne by live donors.

Ritonavir also may induce the activities of CYP1A2, CYP2C9, CYP2C

Ritonavir also may induce the activities of CYP1A2, CYP2C9, CYP2C19 and glucuronosyl transferase.126 Two small studies demonstrated that the co-administration of fluconazole produced little or no effect on the pharmacokinetics of ritonavir.123,124

https://www.selleckchem.com/products/PD-0332991.html Similarly, voriconazole (400 mg day−1) had no apparent effect on steady-state high-dose (800 mg day−1) ritonavir exposure.126 These findings are not surprising given that fluconazole or voriconazole are not potent CYP3A4 inhibitors and the studies employed a high dose of ritonavir (800–1200 mg day−1) that now is rarely used. In addition, the studies involving fluconazole employed a relatively low fluconazole dose (200 mg day−1).123,124 In a small study, fluconazole increased the AUC (50%) and Cmax (57%)

of saquinavir administered as a hard gel-cap. However, these changes were not deemed clinically significant.124 The non-nucleoside reverse transcriptase inhibitor efavirenz is primarily metabolised by CYP3A4 and CYP2B6 and undergoes subsequent glucuronidation. Efavirenz inhibits CYP2C9, CYP2C19 and CYP3A4 at concentrations that are achieved with standard dosing.127 In addition, efavirenz induces CYP3A4 activity in a concentration-dependent manner.128 Not surprisingly, a randomised placebo-controlled interaction study Volasertib price in healthy male volunteers demonstrated that co-administration of voriconazole (400 mg daily in divided) and efavirenz (400 mg daily) produced moderate increases in efavirenz exposure (43%) and maximum serum concentrations (37%).129 Rutecarpine A study in healthy volunteers using a higher voriconazole dose (800 mg daily in divided doses) and a lower efavirenz (300 mg daily) produced little or no change in efavirenz pharmacokinetic parameters.130 This mild to moderate interaction is likely caused by voriconazole inhibition of CYP3A4. However, as discussed below, efavirenz produced more significant changes in voriconazole

disposition.129,130 Interactions involving azoles and warfarin.  Warfarin is a racemic compound. The S enantiomer of warfarin (S-warfarin) is metabolised by CYP2C9 and accounts for the pharmacological activity of warfarin. Itraconazole inhibits only CYP3A4, but a case report indicates that it may interact with warfarin.131 However, this finding has not been evaluated in a larger, more rigorous analysis. Fluconazole interacts with warfarin in a highly predictable manner. This azole inhibits S-warfarin metabolism approximately 70%, which results in a 38% increase in the international normalised ratio (INR) in patients who were previously stabilised on warfarin.132 The interaction between fluconazole and warfarin will occur even if the fluconazole dose is reduced by 50%. Therefore, in practice, this combination increases the risk of significant bleeding and should be avoided if possible.133 If the two drugs are required to be used concomitantly, the INR must be closely monitored and the warfarin dose will need to be adjusted accordingly.

Functional plasticity in DCs allows these cells to present antige

Functional plasticity in DCs allows these cells to present antigen in an immunogenic or tolerogenic fashion, largely contingent on environmental factors [[39]]. Among those, costimulatory and coinhibitory interactions between DCs and T cells are pivotal in tipping the balance between immunity and tolerance in favor of either outcome. Originally thought selleckchem to selectively deliver inhibitory signals to T cells when engaged by CD80/CD86 molecules

on DCs, the surface T-cell receptor CTLA-4 (widely expressed by Treg cells) was later shown to behave as an activating ligand itself for CD80/CD86 “receptors” capable of transduction, resulting in intracellular signaling events. Through an as-yet-unidentified signaling cascade, DCs release type I and type II IFNs (depending on DC subsets) that act in an autocrine and paracrine fashion to induce strong IDO expression and function [[31]]. This might exemplify a mechanism whereby natural or induced Treg cells became engaged in controlling acute hyperinflammatory or allergic reactions in local tissue microenvironments [[40]]. Kynurenine-dependent, AhR-driven T-cell differentiation would then contribute to expand the pool of Treg cells [[6]]. However,

it became soon apparent that, in the long-term control of immune homeostasis and tolerance to self, IDO relies on different regulatory stimuli and cytokines, providing a basal function amenable to regulation by abrupt environmental changes [[41]]. The immunoreceptor tyrosine-based

inhibitory motifs (ITIMs) are known to signal via recruitment and activation of Src homology 2 domain phosphotyrosine phosphatase 1 (SHP-1), SHP-2, Inhibitor Library molecular weight and inositol polyphosphate-5-phosphatase D (SHIP), as shown in Fig. 1. A prototypic ITIM has the I/V/L/SxYxxL/V/F Alanine-glyoxylate transaminase sequence, where x denotes any amino acid and Y the phosphorylable tyrosine [[42, 43]]. In inflammation, phosphorylated ITIMs in IDO interact with suppressor of cytokine signaling 3 (SOCS3), resulting in proteasomal degradation of the enzyme [[30, 44]]. Two ITIMs are present in mouse and human IDOs, which, in the presence of proinflammatory IL-6, lead to SOCS3-dependent proteasomal degradation of the enzyme. This has been considered to be an important mechanism whereby the proinflammatory cytokine IL-6 interrupts tolerance in several acute responses to danger signals [[45]]. In contrast, in a TGF-β–dominated environment and in the absence of IL-6, Fyn-mediated phosphorylation of IDO activates a variety of downstream signaling effectors — including SHPs and noncanonical NF-κB — that further sustain TGF-β production, production of type I IFNs, and favor a bias of the pDCs toward a regulatory phenotype [[46-48]]. By means of this mechanism [[15, 49]], IDO enhances its own expression and stably tips the balance between canonical (i.e. proinflammatory) and noncanonical (antiinflammatory) NF-κB activation in favor of the latter [[50]].

After three

After three Alpelisib purchase 5-min washes in PBS, thin sections were exposed (2–4 h) to primary antibody (Table 1) diluted in 10% goat serum/PBS. Unbound primary antibody was removed with three 5-min washes in PBS and then exposed (2 h) to fluorophore-conjugated secondary antibody, all diluted 1 : 200 in 10% goat serum/0·1% Triton-X 100/PBS. After three 5-min washes in PBS, the slides were coverslipped

using ProLong® Gold antifade mounting media with DAPI (Molecular Probes, Inc., Eugene, OR, USA). DAPI staining aided in follicle localization, especially in the presence of a greatly expanded red pup postinfection. Immunohistochemistry (IHC) controls for these experiments included substitution of primary or secondary antibodies with antibody diluent,

and substitution of primary antibodies with isotype-matched irrelevant antibodies. Dual-labelling experiments were performed by co-incubation of primary antibodies followed by co-incubation of selective secondary antibodies. Nonspecific staining and cross-reactions between secondary antibodies or between a primary antibody and nonrelevant secondary antibody were not observed. Note: Attempts were made to localize CD8+ cells by IHC (primary antibody = BAQ111a, isotype = IgM; VMRD, Inc., find more Pullman, WA, USA). CD8 localization was precluded, however, by significant background mediated by anti-IgM secondary antibody. Immunohistochemistry (IHC) slides were viewed and photographed using an Axio Imager M1 microscope (Carl Zeiss Microimaging, Thornwood, NY, USA) equipped with an LED illuminator for bright field microscopy and an X-Cite 120 Fl Illuminating system (EXFO Photonic Solutions, Mississauga, ON, Canada) for epi-fluorescence microscopy. Digital images were captured using an AxioCam MRc5 digital camera connected to a desktop computer running AxioVision (version 4.7.1.0)

and prepared for presentation using Photoshop Elements (version 4.0; Adobe Systems Inc., San Jose, CA, USA). Figure images are representative, and variation dipyridamole within or between time points (dpi) is noted in the Results section. In particular, the term ‘progressive’ is used to indicate appreciation of an ordered change over time. Measurements of the splenic marginal zone included the region extending from its follicle junction (indicated in figures by a dashed curved line) to a width of ∼100 μm, and measurements of the red pulp included regions furthest away from neighbouring white pulp. IHC measurements must be considered approximate as uncontrolled changes in tissue dimensions are expected to have occurred during euthanasia and preparation of thin frozen sections. All data were tabulated in Microsoft Office Excel 2003 and are reported as mean ± standard error. Splenic volume (MRI) and differential cell count data were analysed for significant (P < 0·05) postinfection increases by paired T-test (SAS® for Windows 9.2; SAS Institute Inc., Cary, NC, USA).