Bacterial Toxin Fusion Proteins Elicit Mucosal Immunity against a Foot-and-Mouth Disease Virus Antigen When Administered Intranasally to Guinea Pigs

Peptides corresponding to the foot-and-mouth disease virus VP1 G-H loop are capable of inducing neutralizing antibodies in some species but are considered relatively poor immunogens, especially at mucosal surfaces. However, intranasal administration of antigens along with the appropriate delivery vehicle/adjuvant has been shown to induce mucosal immune responses, and bacterial enterotoxins have long been known to be effective in this regard. In the current study, two different carrier/adjuvant approaches were used to augment mucosal immunity to the FMDV O1 BFS G-H loop epitope, in which the G-H loop was genetically coupled to the E. coli LT-B subunit and coexpressed with the LTA2 fragment (LTA2B-GH), or the nontoxic pseudomonas exotoxin A (ntPE) was fused to LTA2B-GH at LT-A2 to enhance receptor targeting. Only guinea pigs that were inoculated intranasally with ntPE-LTA2B-GH and LTA2B-GH induced significant anti-G-H loop IgA antibodies in nasal washes at weeks 4 and 6 when compared to ovalbumin or G-H loop immunized animals. These were also the only groups that exhibited G-H loop-specific antigen-secreting cells in the nasal mucosa. These data demonstrate that fusion of nonreplicating antigens to LTA2B and ntPE-LTA2B has the potential to be used as carriers/adjuvants to induce mucosal immune responses against infectious diseases

Evaluation of New York State's Mandatory Occupant Restraint Law: volume III - observational surveys of safety restraint use by children in New York State. Final report.

National Highway Traffic Safety Administration, Washington, D.C.Mode of access: Internet.COP: 2Author corporate affiliation: New York State, AlbanyReport covers the period Oct 1984 - Sept 1985Subject code: EASubject code: ENBBSubject code: ENBCSubject code: PDDSCSubject code: RCCECSubject code: WT

Evaluation of New York State's Mandatory Occupant Restraint Law. Volume I - observational surveys of safety restraint use in New York State. Final report.

National Highway Traffic Safety Administration, Washington, D.C.Mode of access: Internet.COP: 2Author corporate affiliation: Institute for Traffic Safety Management and Research, Albany, N.Y.Subject code: ENBBSubject code: LCCSubject code: PDDSSubject code: RCCESubject code: WT

Recombinant IL-7/HGFβ Hybrid Cytokine Enhances T Cell Recovery in Mice Following Allogeneic Bone Marrow Transplantation

<div><p>T cell immunodeficiency is a major complication of bone marrow (BM) transplantation (BMT). Therefore, approaches to enhance T cell reconstitution after BMT are required. We have purified a hybrid cytokine, consisting of IL-7 and the β-chain of hepatocyte growth factor (HGFβ) (IL-7/HGFβ), from a unique long-term BM culture system. We have cloned and expressed the IL-7/HGFβ gene in which the IL-7 and HGFβ genes are connected by a flexible linker to generate rIL-7/HGFβ protein. Here, we show that rIL-7/HGFβ treatment enhances thymopoiesis after allogeneic BMT. Although rIL-7 treatment also enhances the number of thymocytes, rIL-7/HGFβ hybrid cytokine was more effective than was rIL-7 and the mechanisms by which rIL-7 and rIL-7/HGFβ increase the numbers of thymocytes are different. rIL-7 enhances the survival of double negative (DN), CD4 and CD8 single positive (SP) thymocytes. In contrast, rIL-7/HGFβ enhances the proliferation of the DN, SP thymocytes, as well as the survival of CD4 and CD8 double positive (DP) thymocytes. rIL-7/HGFβ treatment also increases the numbers of early thymocyte progenitors (ETPs) and thymic epithelial cells (TECs). The enhanced thymic reconstitution in the rIL-7/HGFβ-treated allogeneic BMT recipients results in increased number and functional activities of peripheral T cells. Graft-versus-host-disease (GVHD) is not induced in the rIL-7/HGFβ-treated BMT mice. Therefore, rIL-7/HGFβ may offer a new tool for the prevention and/or treatment of T cell immunodeficiency following BMT.</p> </div

The number of total and naïve T cells in the spleen of allo-BMT recipients was significantly increased after rIL-7/HGFβ treatment.

<p>Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice and treated with cytokines as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082998#pone-0082998-g001" target="_blank">Figure 1</a>. On day 30 after BMT, the number of total (A) CD4 and (C) CD8 splenic T cells; and donor-origin naive (CD62L^hi CD44^lo) (B) CD4 and (D) CD8⁺ splenic T cells was evaluated by flow cytometry. The data are representative of 2 independent experiments with 4-6 mice per group. * P<0.05 compared with PBS-treated mice. ** P<0.05 compared with rIL-7 and/or rHGFβ-treated mice.</p

The number of ETPs and TECs was significantly increased in allo-BMT recipients after rIL-7/HGFβ treatment.

<p>Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice and treated with cytokines as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082998#pone-0082998-g001" target="_blank">Figure 1</a>. On day 30 after BMT, ETPs and TECs were analyzed. (A) Representative flow cytometric profiles showing the percentage of donor-origin lineage^- c-kit⁺ IL-7Rα^- CD44⁺CD25^- ETPs in total thymocytes. (B) Number of donor-origin ETPs in the cytokine-treated BMT mice. (C) Representative flow cytometric profiles showing the percentage of CD45^-EpCAM⁺MHC II⁺Ly51⁺ cTECs and CD45^-EpCAM⁺MHC II⁺Ly51^- mTECs in total TECs of the rIL-7/HGFβ-treated BMT mice. (D) Number of total TECs, cTEC and mTECs in the cytokine-treated BMT mice. The data are representative of 2 independent experiments with 4-6 mice per group. * P<0.05 compared with PBS-treated mice. ** P<0.05 compared with rIL-7 and/or rHGFβ-treated mice. </p

The number of thymocyte subsets was significantly increased in allo-BMT recipients after rIL-7/HGFβ treatment.

<p>Lethally irradiated mice (BALB/c, 4-10 week old) were injected i.v. with 2 X10⁶ TCD-BM from B6 mice. Groups of mice were then injected i.p. with the equimolar amounts of rIL-7/HGFβ (15 μg), rIL-7 (5 μg) and/or rHGFβ (10 μg), or PBS at 2-day intervals from days 1 to 26 after BMT. The number of (A) total thymocytes, donor-origin (B) CD4 and CD8 DN, (C) CD4 and CD8 DP, (D) CD4 SP, and (E) CD8 SP thymocytes was analyzed on day 30 after BMT. Means <u>+</u> S.D. are presented. The data are representative of 2 independent experiments with 4-6 mice per group. * P<0.05 compared with PBS-treated mice. ** P<0.05 compared with rIL-7 and/or rHGFβ-treated mice. </p

The survival of pre-selection DP and the proliferation of DN and SP thymocytes were enhanced by rIL-7/HGFβ.

<p>(A-F, I) Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice and treated with cytokines as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082998#pone-0082998-g001" target="_blank">Figure 1</a>. On day 30 after BMT, (A) The percentages of Annexin V⁺ cells in each thymocyte subset were analyzed by flow cytometry. (B) Representative histograms of intracellular staining for Bcl-2 by DN thymocytes. (C) Relative mean fluorescence intensity (MFI) <u>+</u> SD of Bcl-2 by DN thymocytes from the cytokine-treated BMT mice. (D) Representative histograms of intracellular staining for Bcl-xL by DP thymocytes. (E) Relative MFI of Bcl-xL by DP thymocytes. (F) rIL-7/HGFβ treatment enhances the survival of CD69^- pre-selection DP thymocytes. (G, H) rIL-7/HGFβ maintains the survival and the expression of Bcl-xL of DP thymocytes <i>in </i><i>vitro</i>. Purified DP thymocytes were cultured in the presence of equimolar amounts of rIL-7/HGFβ (30 ng/ml), rIL-7 (10 ng/ml) and/or rHGFβ (20 ng/ml), or PBS for 24 and 48 hours. Cells were analyzed for (G) annexin V⁺ cells by flow cytometry and (H) the expression of Bcl-xL by Western blot. (I) the cytokine-treated allo-BMT mice were injected i.p. with BrdU. The percentages of BrdU⁺ cells by thymocyte subsets were then analyzed by flow cytometry 4 hour later. The data are representative of 2 independent experiments with 4-6 mice per group. * P<0.05 compared with PBS-treated mice.</p

rIL-7/HGFβ-treated allo-BMT recipients do not develop GVHD.

<p>Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice. Syngeneic BMT (both BM and recipients from B6 mice) were used as controls. Both syngeneic and allo-BMT recipients were treated with rIL-7/HGFβ (15 μg) or PBS at 2-day intervals from days 1 to 26 after BMT. (A) weights and (B) histopathological analysis of signs of GVHD in liver, small bowel (SB) and large bowel (LB) were conducted on day 75 after BMT. (C) Splenocytes harvested from the allo-BMT recipients were used as effector cells for MLRs. Splenocytes from normal non-BMT B6 mice were used as controls. The effector cells were cultured with irradiated splenocytes (as stimulators) from normal non-BMT BALB/c, B6, and CBA mice, respectively. Cell proliferation was determined by BrdU incorporation. Mean OD in MLRs/OD in spontaneous proliferation with splenocytes from all-BMT recipients or normal B6 mice as effectors and splenocytes from BALB/c, B6, and CBA mice as stimulators were 0.21/0.20, 0.20/0.20, 2.97/0.21, as well as 2.95/0.21, 0.21/0.20, and 3.15/0.22, respectively. Data are shown as stimulation index. (A-C) The data are representative of 2 independent experiments of 5 mice each group. </p