An AAV2/5 vector enhances safety of gene transfer to the mouse salivary gland.

This study was designed to improve AAV-mediated gene transfer to the murine submandibular salivary glands. Our first aim was to utilize AAV pseudotype vectors, containing the genetic elements of the canonical AAV2, packaged within capsids of AAV serotypes 5, 8, and 9. Having determined that this pseudotyping increased the efficiency of gene transfer to the glands by several orders of magnitude, we next asked whether we could reduce the gene transfer inoculum of the pseudotype while still achieving gene transfer comparable with that achieved with high-dose AAV2. Having achieved gene transfer comparable with that of AAV2 using a pseudotype vector (AAV2/5) at a 100-fold lower dose, our final objective was to evaluate the implications of this lower dose on two pre-clinical parameters of vector safety. To evaluate systemic toxicity, we measured AAV vector sequestration in the liver using qPCR, and found that the 100-fold lower dose reduced the vector recovered from the liver by 300-fold. To evaluate salivary gland function, we undertook whole-proteome profiling of salivary gland lysates two weeks after vector administration and found that high-dose (5 × 109) AAV altered the expression level of ~32% of the entire salivary gland proteome, and that the lower dose (5 × 107) reduced this effect to ~7%.

Ultrasound-assisted non-viral gene transfer to the salivary glands.

We report a non-viral gene transfer method using ultrasound induced microbubble destruction to allow the uptake of plasmid gene transfer vectors to the cells of the mouse salivary gland. The Luciferase (Luc) reporter gene, driven by a cytomegalovirus (CMV) promoter, was delivered unilaterally to the submandibular salivary gland via retroductal cannulation and Luc expression was monitored with in vivo imaging. The CMV-Luc plasmid was delivered to the salivary gland in a carrier solution containing microbubbles composed of lipid-encased perfluoropropane gas, with two different concentrations of microbubbles used (100 and 15% volume/volume). An Adenoviral (Ad) vector using an identical CMV-Luc expression cassette was used as a positive control at two different dosages. Whereas ultrasound-assisted gene transfer (UAGT) with 100% microbubbles was weak and rapidly extinguished, UAGT with the 15% microbubble solution was robust and stable for 28 days. UAGT seems to be a practicable and promising method for non-viral gene delivery to the salivary glands.

Evidence for a possible role of Asialo-GM1-positive cells in the graft-versus-leukemia repression of a murine type-C retroviral leukemia.

Studies were performed to examine whether, in addition to T cells, there might be other immune cells also capable of exerting a graft-versus-leukemia (GVL) response following allogeneic marrow transplant. Using an MHC-matched mouse model, consisting of normal B10.S donors and SJL/J Rauscher-retroviral-leukemic recipients, the donor cells were selectively depleted of their Asialo-GM1+ component prior to being infused into the leukemic recipients. The incidence of relapse was then compared against that for matched leukemic control recipients of undepleted cells from the same donors. FCM analysis of the depletion protocol indicated that exposure to anti-Asialo-GM1 antibody eliminated more than half of the donor NK1.1+ cells, but caused no significant losses among the Thy-1+, CD3+, or CD8+ cells. Nevertheless, fatal relapse among the leukemic recipients of the depleted cells was nearly double that found among the leukemic control recipients of undepleted cells, 47.5 vs 25.4% (P = 0.01). In a parallel study, using normal SJL/J recipients, this same depletion protocol was found to have no significant effect on the incidence of graft-versus-host disease (GVHD). These results therefore suggest that Asialo-GM1+ NK cells may be capable of contributing to the suppression of relapse in this type of leukemic recipient of allogeneic marrow, and that this suppression may occur independently of GVHD.

Comparison of the effects of CD3 and CD5 donor T cell depletion on graft-versus-leukemia in a murine model for MHC-matched unrelated-donor transplantation.

Studies were designed to prospectively evaluate the effects of selective depletion for donor T cells strongly expressing the CD3 and CD5 pan-T antigens on the incidence of leukemia relapse following bone marrow transplantation. This evaluation was performed under controlled conditions in a mouse model for MHC-matched unrelated-donor transplantation, employing Rauscher leukemic SJL/J mice as the recipients and leukemia-resistant B10.S mice as the donors. Selective donor cell depletion for CD3 and CD5 was accomplished ex vivo prior to transplantation by incubation with the appropriate monoclonal antibody plus complement. When untreated, Rauscher leukemia resulted in a 97% fatality incidence. This was reduced to 30% by the transplant of non-depleted B10.S cells, with another 37% recipients dying from GVHD and graft failure. CD3 depletion reduced the GVHd deaths to 6% but increased relapse to 62%. Conversely, CD5 depletion had no effect on relapse or on GVHD but did significantly increase graft failure, thus negatively affecting survival. Evaluation of the results, done in conjunction with flow cytometry analysis of the effects of CD3 versus CD5 depletion on the donor cells, suggests that the T cells involved in suppressing leukemic relapse in these studies, and hence contributing to the GVL response, most probably had a phenotype of CD3+, CD5-.

Effect of donor and recipient gender disparities on fatal graft-vs.-host disease in a mouse model for major histocompatibility complex-matched unrelated-donor bone marrow transplantation.

Sex chromosome-linked minor histocompatibility determinants have been shown to affect the incidence and severity of graft-vs.-host disease (GVHD) in both humans and animals. On the basis of earlier studies done in mice and humans, it has often been assumed that this effect is due to a simple response of female donor cells recognizing recipient male HY antigens as foreign and reacting against them. However, the data of various clinical groups have not always supported this assumption. Moreover, since most of the earlier mouse studies focused only on the single transplant direction of female into male and/or were done under totally syngeneic conditions, the possibility of a GVHD response based on donor recognition of the recipient female HX antigen as foreign was never fully addressed. We have therefore reexamined the question in a more clinically relevant allogeneic transplantation setting, using a major histocompatibility complex (MHC)-matched, unrelated-donor mouse model. Five different donor/recipient sets were paired in all four possible gender combinations. The results indicated that, in addition to GVHD reaction against male HY, reaction against female HX was also possible. The results also showed that when the total level of GVHD due to autosomal chromosome minor histocompatibility disparities is extensive, it may masks the influence of gender-related factors on GVHD. Finally, the data also suggest the possibility that the sex chromosome-linked minor histocompatibility determinants may be polymorphic and thus capable of multiple allele expression.

Effect of the YCD3-1 anti-mouse monoclonal on murine GvHD.

The potential for applying the YCD3-1 rat-anti-mouse IgG2b anti-CD3-epsilon monoclonal antibody (MAB) to the study of graft-versus-host disease (GvHD) in mouse models has been examined. This MAB, unlike the previously developed hamster-anti-mouse-CD3 MABs, had been reported to exhibit strong cytolytic properties when applied in vitro in the presence of complement. Therefore, it was of interest to determine whether it could be effectively used for T-cell depletion in mice to suppress GvHD in the same manner as the anti-human-CD3 MABs have been applied in clinical transplantation. Evaluation of the effectiveness of this antibody was carried out both under fully allogeneic MHC-mismatched and under unrelated-donor MHC-matched marrow transplant conditions. For both types of transplantation, depletion of the donor cells with YCD3-1 plus complement prior to their injection into lethally irradiated recipients significantly suppressed GvHD, resulting in survivals of 75-79%, as compared to 8-13% in the controls that received undepleted cells from the same donors (p < 0.0001). These results suggest that the YCD3-1 MAB may have a potential for use as a negative selection agent in the further definition of the roles of the various T-cell subtypes, as well as the possible roles of natural-killer cells, in future studies into the mechanisms of GvHD, and of the graft-versus-leukemia effect.

The role of CD4 and CD8 T cells in the graft-versus-leukemia response in Rauscher murine leukemia.

Using a mouse model for MHC-matched unrelated donor transplantation, the relative influences of the CD4 and CD8 T cell subtypes on graft-versus-leukemia (GVL) were examined in a murine erythroleukemia induced in SJL/J mice by the injection of Rauscher virus. Following leukemia induction, the mice were given 9.5 Gy of total body irradiation (TBI) and injected with mixed marrow and spleen cells from normal MHC-matched--but minor histocompatibility mismatched--B10.S donors. Prior to their injection these donor cells were selectively depleted ex vivo for either CD4, CD8 or Thy-1 by exposure to the appropriate monoclonal antibody (MoAb) plus complement. Following transplant the recipients were observed for 20 weeks, along with parallel control groups, for survival, leukemia relapse, graft failure and graft-versus-host disease; 98% of the controls receiving no transplantation therapy died of leukemia. Among the controls that received TBI plus undepleted B10.S cells 30.9% died of leukemia relapse, but another 34.2% survived free of any clinical evidence of their leukemia. Donor cell depletion for Thy-1 increased the relapse to 68.8%, while survival fell to 10.4%. CD8 depletion resulted in a relapse of 55.6%, with a survival of 19.4%. By contrast, CD4 depletion had no effect on relapse, but did significantly increase the incidence of graft failure. At the end of the 20 weeks additional tests were run to determine whether those transplant survivors that had remained leukemia-free were also free of any residual Rauscher virus. Those tests showed that they were not.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical evidence for IgE involvement in Graves' orbitopathy.

Orbital muscle and adipose tissues from seven Graves' orbitopathy patients were studied with in situ assays for IgE. The cases varied in disease severity and site biopsied. Two monoclonal and one polyclonal anti-IgE reagents produced similar results. Identically prepared monoclonal anti-IgM and anti-IgG antibodies and tissues obtained from five patients treated for unrelated orbital disorders were used as controls. Graves' tissues exhibited extravasated leukocytes and leukocyte-rich vessels. These leukocytes were mostly lymphoid. Some basophils and mast cells were identified and polymorphonuclear cells were abundant within vessels of adipose tissue. IgE-positive material was found in association with the majority of leukocytes and with muscle fibers. Parallel sections reacted with anti-IgM antibody were negative, whereas anti-IgG produced diffuse staining with no specific structures highlighted. Control, non-Graves' tissues had no evidence of immune cell activity and were either negative or displayed reactions with anti-IgE reagents that were in most cases different from those of Graves' tissue. Serum IgE was measured in six of the seven patients and was elevated in the two patients with fulminating disease.

Interleukin-2-induced lymphoproliferative responses.

Interleukin-2 (IL-2) is capable of both stimulating an in vitro lymphoproliferative response and augmenting non-major-histocompatibility-complex-(MHC)-restricted cytotoxicity. However, there are conflicting reports about the phenotypes of responding cells. In the present studies, we determined phenotypes of Ficoll/Hypaque-separated peripheral blood mononuclear cells stimulated with 50, 100 or 1000 U/ml IL-2; analyses were performed after 1, 3 and 5 weeks. With all concentrations, there was a progressive increase in CD3+ cells: after 3-5 weeks more than 90% of the cells reacted with this antibody. However, the proportions of CD4+ and CD8+ cells proved to be a function of the IL-2 concentration. Cultures containing 50 U/ml or 100 U/ml favored the expansion of the CD4+ subset. By contrast, in cultures stimulated with 1000 U/ml. CD8+ cells predominated. At baseline, CD8+ cells comprised 28 +/- 2%; after 3 weeks, this value increased to 51 +/- 5%. In addition, the proportion of CD56+ (Leu19, NKH-1) cells depended on the amount of IL-2. At 50 U/ml, there was no appreciable change in CD56+ cells. However, at 1000 U/ml, CD56+ cells increased from 17 +/- 1% (day 0) to 39 +/- 4% (3 weeks). This increase was primarily due to an expansion of the CD3+ CD56+ subset (non-NMC restricted cytotoxic T cells). By contrast, natural killer (NK) cells, as measured by the CD16 antibody, steadily declined at all IL-2 concentrations.

Defective T cell colony formation and IL-2 receptor expression in HIV-infected homosexuals: relationship between functional abnormalities and CD4 cell numbers.

HIV infection is known to induce a progressive T helper/inducer (CD4) lymphopenia and to impair the functional activities of residual cells. The present studies examined the relationship between the CD4 cell count and three functional assays: T cell colony formation in semisolid media, the capacity of PHA-stimulated cells to express IL-2 receptors, and their ability to synthesize and secrete IL-2. Cells from antibody-positive homosexuals with normal numbers of CD4 cells (greater than 700/microliters) showed defective reactivity in two assays, colony growth, and IL-2 receptor expression. These defects became progressively more pronounced in homosexuals with moderate (400-700 cells/microliters) and severe (less than 400/microliters) reductions in assays for IL-2 production by PHA-stimulated lymphocytes. Mixing experiments suggest that cells from HIV-infected men nonspecifically inhibit the colony growth of normal cells; this abnormality could be reversed by addition of exogenous IL-2. These data suggest that defective colony growth and reduced IL-2 expression are functional abnormalities directly resulting from HIV infection. Furthermore, these changes can precede the CD4 lymphopenia induced by this viral infection.

Defective T-cell colony formation and IL-2 receptor expression at all stages of HIV infection.

The T-cell colony assay is a highly sensitive measure of immunological dysfunction. The present study evaluated this in vitro response in asymptomatic HIV-infected homosexuals, those with chronic adenopathy as their only clinical manifestation and patients with either ARC or AIDS. The mean colony count in antibody-positive asymptomatic individuals was significantly reduced when compared to either heterosexual controls or antibody-negative homosexuals. Furthermore, there were no differences in the responses of these antibody-positive individuals and those with chronic lymphadenopathy as their only clinical manifestation. By contrast, patients with AIDS or ARC showed a profound defect; this suggests that the colony assay can detect a functional gradient across the spectrum of HIV infections. Colony growth was correlated with the absolute number of T-helper cells and the ability of PHA-stimulated lymphocytes to express IL-2 receptors; no correlation was found with the number of suppressor/cytotoxic cells or in vitro production of IL-2. Recent HIV seroconverters had normal colony counts but impaired ability to express IL-2 receptors. These data suggest a sequential loss of T-cell function as a result of HIV infection; the earliest manifestations are impaired expression of IL-2 receptors and reduced proliferative responses, as measured in the colony assay.

The effects of interleukins and other soluble factors on T-lymphocyte colony formation.

When plated in semi-solid media, PHA-stimulated human peripheral blood mononuclear cells (PBMC) form discrete T-cell colonies. By contrast, Sephadex G-10 non-adherent (NA) cells (greater than 96% T lymphocytes) show virtually no clonal growth unless cocultured with soluble factors derived from either normal adherent cells or tumour cell lines. Purified IL-1 was able to initiate colony growth of mitogen-stimulated NA cells; cultures containing 20 U of human IL-1 yielded colony counts that were only slightly less than those with PBMC. In addition, recombinant IL-2, free of measurable IL-1, was able to provide the initiating signal required for clonal expansion. Both recombinant and lymphocyte-derived IL-2 were able to enhance the clonal growth of PBMC. Colony growth could be initiated by supernatants derived from short-term cultures of either monocytic (U937, HL60) or B-cell (Raji, Daudi) tumour cell lines. The abilities of these tumour cell lines to promote clonal responses did not correlate with their contents of either IL-1 or IL-2. By contrast, supernatants derived from either K562 (an erythroleukaemic line) or MOLT 4 (a T-cell lymphoma) cells did not provide the initiating signal.