The Senate Committee on Human Resources and Facilities was called to order by Chairman Raymond D. The HCF‐1 polypeptides resulting from site‐specific proteolyses form a non‐covalent heterogenous complex made up of N‐ and C‐terminal fragments of the protein ranging in size from 68 to 180 kDa (Wilson et al., 1993, 1995; Kristie et al., 1995). Seeks $1.5 million from Raptor star A private-school teacher and part-time model is suing Raptors forward Morris Peterson for $1.5 million for allegedly infecting her with genital herpes. Within two months, on Feb. I would have felt amazing if I had someone to talk about it with who had gone through the same thing at that first time. HIV – Early Symptoms – Duration: 4. You can use this if you already have those sores on your body.
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Plaque-purified HSV-1 strains McKrae (wild-type) or KOS and HSV-1 recombinant viruses expressing IL-12p35 and IL-12p40 were grown in rabbit skin cell monolayers in minimal essential medium (MEM) containing 5% fetal calf serum, as previously described.22 McKrae virus is virulent at an infectious dose of 2 × 105 plaque-forming units (PFU)/eye, whereas KOS, DM (the parental virus for HSV-IL-12p35 and HSV-IL-12p40 viruses, which lacks both the LAT and γ34.5 transcripts), HSV-IL-12p35, and HSV-IL-12p40 viruses are attenuated strains. Female BALB/c, BALB/c-IL-12p35−/−, BALB/c-IL-12p40−/−, BALB/c-CD19−/− (B-cell deficient), BALB/c-SCID, BALB/c-EBI3−/−, C57BL/6, C57BL/6-CD4−/−, C57BL/6-CD8−/−, C57BL/6-IL-12p35−/−, C57BL/6-IL-12p40−/−, C57BL/c-IL-23p19−/−, C57BL/6-FoxP3DTR, and C57BL/6-IL-12rα−/− (CD25-deficient) mice aged 6 weeks were purchased from the Jackson Laboratory (Bar Harbor, ME). : The Association; 1989 Feb15. Rudensky.23 Female hemizygous C.FVB-Tg (Itgax-DTR/GFP) 57Lan/J mice on a BALB/c background24 and C57BL/6-FoxP3DTR were bred at Cedars-Sinai Medical Center, whereas C57BL/6-IL-23p19−/− were bred at the University of Tennessee at Knoxville. Mice were infected ocularly with 2 × 105 PFU McKrae, KOS, HSV-IL-12p35, HSV-IL-12p40, or DM virus. Each virus was suspended in 5 μL tissue culture media and administered as an eye drop. All mice on a BALB/c background were infected with KOS, HSV-IL-12p35, HSV-IL-12p40, or DM virus, whereas all C57BL/6 mice were infected with McKrae or DM virus.
In this study to show that demyelination is not HSV-1 strain specific, we have used wild-type virulent HSV-1 strain McKrae, wild-type avirulent HSV-1 strain KOS, and highly avirulent HSV-1 strain DM that lacks both the LAT and the γ34.5 transcripts as challenge viruses. No ocular pathology was detected in ocularly infected mice by day 14 after infection. Liposome encapsulation of dichloromethylene diphosphonate (Cl2MDP) was performed as previously described.25,26 To deplete macrophages, each mouse received 100 μL of the mixture intraperitoneally and subcutaneously. Macrophage depletion was carried out on days −5, −2, +1, +4, +7, and +10 relative to ocular infection with HSV-1. This study used the National Health Insurance Research Database (NHIRD) from 1996 to 2007, which is derived from the Taiwan National Health Insurance (NHI) program and provides a sample of 1,000,000 random subjects to scientists in Taiwan for research purposes. Briefly, the first depletion was performed 24 hours before ocular infection, followed by four additional depletions on days +1, +4, +7, and +10 relative to ocular infection. In Interscience Conference on Antimicrobial Agents and Chemotherapy, Minneapolis, 1985.
FoxP3DTR mice were depleted of their FoxP3 by treatment with DT, as described previously.23 Briefly, the mice were administered DT 72 and 24 hours before ocular infection, followed by five additional treatments on days +1, +3, +5, +7, and +9 after infection. Each mouse received an intraperitoneal injection of 100 μg purified GK1.5 (anti–CD4), 2.43 (anti–CD8), or PC61.5.3 (anti–CD25) monoclonal antibodies (NCCC, Minneapolis, MN) in 100 μL PBS 5 and 2 days before ocular challenge. The injections were repeated on days +1, +4, +7, and +10 relative to ocular infection. Mock-depleted control mice were injected similarly with the irrelevant mAb of the same isotype. benthamiana × N. In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The complete open reading frame for IL-12p35 and IL-12p40 was cloned into the VR-1055 expression vector, whereas the complete open reading frame for IL-12p70 was purchased from InvivoGen (San Diego, CA).
Plasmid DNA encoding each gene was purified on a cesium chloride gradient. Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). As a negative control, we used mock-vaccinated mice that were similarly injected with vector DNA alone. Brains and spleens from macrophage- or mock-depleted mice were harvested on days 8 and 11 postinfection (PI) with KOS. Single-cell suspensions from individual mouse brains and spleens were prepared as described previously.20 Monoclonal antibodies used for flow cytometry were purchased from BD Biosciences (San Diego, CA) and eBioscience (San Diego, CA). Cell surface and intracellular staining of single-cell suspensions of mononuclear cells (MNCs) and splenocytes from individual mice was accomplished according to the manufacturer’s instructions. Antibodies included anti–CD3-PERCP (clone 145–2C11), anti–CD4-FITC (clone L3T4), anti–CD8a-PE (clone 53–6.7), anti–CD25-PE-CY7 (clone PC61.5.3), and anti–FoxP3-Pacific blue (clone FJK-16s).
Penta-color flow cytometric analyses of MNCs or splenocytes was performed using a flow cytometer (FACScan; BD Biosciences). Percentages of cells stained with mAbs were calculated by forward or side scatter gating of MNCs or splenocyte preparations. Controls included nonrelevant isotype-matched antibody, no primary antibody, or no secondary antibody alone. A minimum of 1 × 104 events was acquired based on a live cell gate. As previously mentioned, HSK is at its core an inflammatory disease with chemokines involved in migration of leukocytes to sites of infection and inflammation [25, 26] and cytokines responsible for the activation of cells which mediate the cellular destruction following their activation. CD4+CD25+ or CD4+CD25− T cells from pooled suspensions were isolated magnetically as described by the manufacturer (Miltenyi Biotec, Auburn, CA). Each recipient SCID mouse was injected once intraperitoneally with 1 × 105 CD4+CD25+ or CD4+CD25− T cells in 300 μL MEM, and a subset of SCID mice received 1 × 106 CD4+CD25− T cells.
Recipient SCID mice were ocularly infected 4 hours after transfer with KOS. ONs, brains, and SCs of experimental and control mice were removed at necropsy on days 7, 10, and 14 PI, embedded in optimal cutting temperature compound (OCT; Tissue-Tek; Sakura, Torrance, CA) for cryosectioning, and stored at −80°C. Transverse sections of brains, SCs, and ONs, each 10-μm thick (spaced 50 μm apart), were sectioned using a cryostat (CM3050S; Leica, Wetzlar, Germany), air dried overnight, and fixed in acetone for 3 minutes at 25°C.28 The presence or absence of demyelination in infected mice was evaluated using Luxol fast blue (LFB) staining of formalin-fixed sections of ONs, as we described previously.20 Demyelination in each section was confirmed by monitoring adjacent sections. Transverse sections of SCs, 10 μm thick (spaced 50 μm apart), were sectioned and mounted on slides (Superfrost Plus Gold; Fisher Scientific), then air dried for 10 minutes and stored at −80°C. Many of these conditions may leave permanent delays, however, early recognition can lead to early intervention with improvement in outcome.” Ms. Similar results were obtained when the co‐localization experiments were performed using anti‐HCF‐1 antibodies to detect endogenous HCF‐1 in untransfected cells (data not shown). After three rinses for 5 minutes each in PBS, we incubated slides for 1 hour at 25°C with appropriate Alexa Fluor 488–, Alexa Fluor 594–, or Alexa Fluor 647–conjugated secondary antibodies (Invitrogen-Molecular Probes).
After an additional three rinses for 5 minutes each with PBS at 25°C, we air dried slides in the dark and mounted them with antifade reagent (Prolong Gold with DAPI; Invitrogen–Molecular Probes). Fluorophores were imaged in separate channels (ApoTome-equipped Axio Imager Z1; Carl Zeiss Microimaging, Thornwood, NY). We variously used the following antibodies for immunohistochemistry: mouse HSV-1 (FITC-conjugated, 1:100; GenWay, San Francisco, CA), rabbit GFAP (1:1000; Dako), rat CD11b (1:200; Serotec, Raleigh, NC), and rat F4/80 (1:50; Serotec). Three images each of white matter and gray matter were captured for three sections through each SC, and we obtained a threshold optical density that best distinguished staining from background. For HSV-1, GFAP, CD11b, and F4/80 burden analyses, data are reported as the percentage of labeled area captured (positive pixels) divided by the full area captured (total pixels). Threshold optical densities and burden analyses were measured using ImageJ software, release 1.40g (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). To analyze the presence or absence of demyelination in experimental and control groups, Fisher’s exact tests were performed (Instat; GraphPad, San Diego, CA).
Statistical analyses for immunohistochemistry photomicrographs were performed using data analysis software (Excel; Microsoft, Redmond, WA). For single comparisons of the means, we used t-tests for independent samples to assess significance. An examiner blinded to sample identities performed all analyses, and code was not broken until analyses were completed. For all analyses, we set alpha levels at 0.05. To assess the impact of innate and adaptive immunity in protection from CNS demyelination in HSV-1–infected mice, female BALB/c mice were depleted of macrophages, DCs, NK cells, CD4+ T cells, CD8+ T cells, or CD4+ plus CD8+ T cells, as described in Materials and Methods. Mice were ocularly infected with the avirulent HSV-1 KOS strain. Includes references.
Representative photomicrographs of ON sections from infected or control mice are shown in and . Conspicuous demyelination was present in ONs of infected macrophage-depleted mice (A, arrows) but not in either mock depleted (B) or uninfected macrophage-depleted mice (C). A similar pattern of results was observed in brain and SC after macrophage depletion and infection (D, F, arrows). Demyelination was not detected in mock-depleted brains (E) or SCs of infected mice (G). Induction of demyelination in CNS of macrophage-depleted BALB/c mice. BALB/c mice were macrophage or mock depleted and infected or mock infected ocularly with the avirulent HSV-1 strain KOS. Representative ON, brain, and SC sections on day 14 PI from …
Role of NK, DCs, T cells, or B cells in CNS demyelination. BALB/c or BALB/c-DTR mice were depleted of their NK cells, DCs, CD4+ T cells, CD8+ T cells, or both CD4+ and CD8+ T cells and infected ocularly with the avirulent HSV-1 strain KOS, whereas CD19-deficient … In contrast to macrophage depletion results described in , mice that were depleted of NK cells, DCs, CD4+ T cells, CD8+ T cells, or both CD4+ and CD8+ T cells did not show evidence of ON demyelination (). Similarly, demyelination was undetectable in brains or SCs of treated mice (data not shown). Furthermore, to determine whether B cells play any role in CNS demyelination, BALB/c-CD19−/− mice were infected with the HSV-1 KOS strain as described. No demyelination was detected in ONs of infected mice (). However, as with wild-type BALB/c mice, CD19−/− mice that were depleted of macrophages and infected with KOS developed ON demyelination (, Mθ-depleted, arrows).
Thus, our results show that macrophages protect against infection-induced CNS demyelination, whereas NK cells, DCs, B cells, and T cells were dispensable in our model. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Similar to results for BALB/c mice shown in , C57BL/6 mice that were macrophage-depleted and infected with McKrae virus showed ON, brain, and SC demyelination (, arrows). Demyelination was not detected in ONs, brains, or SCs of mock-depleted mice (). Weight gain resumed after 72 hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. As expected, no demyelination was detected in ONs, brains, or SCs of mock-depleted mice (data not shown).
These data suggest that demyelination induced in the absence of macrophages is not specific to individual mouse or virus strains. Detection of demyelination in CNS of macrophage-depleted C57BL/6 mice. C57BL/6 mice were macrophage or mock depleted and infected ocularly with the virulent HSV-1 strain McKrae or the avirulent DM virus that lacks the LAT and γ34.5 transcripts. … To determine the time course of demyelination in the CNS of macrophage-depleted mice, we depleted female BALB/c mice of macrophages, infected them with KOS, and harvested ONs at 8 and 11 days PI. On day 8 PI, demyelination was absent in macrophage- or mock-depleted mice (A, C). However, a different pattern of results was evident on day 11 PI in macrophage-depleted mice, which manifested prominent ON demyelination (B; see arrows).
By contrast, demyelination was not detected in mock-depleted mice (D). It was first reported that mice lacking IL-17 receptor displayed reduced neutrophil infiltrate and less corneal disease . Thus, our results show that demyelination is copious in infected macrophage-depleted mice as early as day 11 PI. Time course of appearance of demyelination in macrophage-depleted mice. BALB/c mice were macrophage or mock depleted and infected ocularly with the avirulent HSV-1 strain KOS. ONs were obtained on days 8 and 11 PI, fixed, sectioned, and stained with LFB. …
To investigate potential differences in HSV-1 infectivity between macrophage- and mock-depleted mice, we performed immunostaining on the same SC samples used to assess demyelination (). Dr. Thus, experiments were performed where instead of immunodepleting HCF‐1 from HeLa nuclear extracts, anti‐HCF‐1 peptide antibodies (Kristie et al., 1995; Ajuh et al., 2000) were added directly to pre‐mRNA splicing reactions to determine whether the presence of antibody molecules bound to HCF‐1 in nuclear extract will block splicing. Quantitative image analyses revealed significant increases in HSV-1 and GFAP burden in SC white matter (P < 0.001) and gray matter (P < 0.001) of macrophage-depleted versus mock-depleted mice (B, C). We observed significant decreases in signal intensity for the macrophage cell surface markers CD11b (P < 0.01 in white matter; P < 0.001 in gray matter) and F4/80 antigen [P < 0.01 in white matter and gray matter) in macrophage-depleted versus mock-depleted mice (B, C), providing confirmatory evidence that macrophage depletion was successful. Increased HSV-1 infection and glial activation in SCs of infected mice after macrophage depletion. BALB/c mice were macrophage or mock depleted and infected ocularly with the avirulent HSV-1 strain KOS, as in . SCs were removed on day 14 PI and ...