Imaging transgene expression in live animals.

Monitoring the expression of therapeutic genes in targeted tissues in disease models is important to assessing the effectiveness of systems of gene therapy delivery. We applied a new light-detection cooled charged-coupled device (CCCD) camera for continuous in vivo assessment of commonly used gene therapy delivery systems (such as ex vivo manipulated cells, viral vectors, and naked DNA), without the need to kill animals. We examined a variety of criteria related to real-time monitoring of luciferase (luc) gene expression in tissues including bone, muscle, salivary glands, dermis, liver, peritoneum, testis, teeth, prostate, and bladder in living mice and rats. These criteria included determination of the efficiency of infection/transfection of various viral and nonviral delivery systems, promoter specificity, and visualization of luciferase activity, and of the ability of luciferin to reach various organs. The exposure time for detection of luc activity by the CCCD camera is relatively short (approximately 2 minutes) compared with the intensified CCD camera photon-counting method (approximately 15 minutes). Here we transduce a variety of vectors (such as viruses, transfected cells, and naked DNA) by various delivery methods, including electroporation, systemic injection of viruses, and tail-vein, high-velocity-high-volume administration of DNA plasmids. The location, intensity, and duration of luc expression in different organs were determined. The distribution of luciferin is most probably not a barrier for the detection of in vivo luciferase activity. We showed that the CCCD photon detection system is a simple, reproducible, and applicable method that enables the continuous monitoring of a gene delivery system in living animals.

Rapid seroprotection against hepatitis B following the first dose of a Pre-S1/Pre-S2/S vaccine.

BACKGROUND/AIMS: Will immunization with an experimental Pre-S1/Pre-S2/S hepatitis B vaccine (Bio-Hep-B) induce faster seroprotection using fewer doses as compared with a yeast derived S vaccine (Engerix B). METHODS: Healthy volunteers, n = 36, mean age 23 y, randomized to receive 2 or 3 doses of both vaccines given months 0 and 6, or 0, 1 and 6. RESULTS: Following primary immunization, seroprotection occurred in 6, 39, 53 and 60% in the Bio-Hep-B group at weeks 1, 2, 3 and 4, compared with 0, 12, 18 and 12.5% in the Engerix-B vaccinees, respectively. Six months following injection of the first dose, seroprotection was 70 and 25% in Pre-S/S and S vaccinees respectively. Area under the curve in vaccinees of Bio-Hep-B; versus Engerix-B showed mean anti-HBs level of 365 +/- 166 and 85 +/- 48 mIU/ml x day respectively (P = 0.012). At month 7, 100% seroprotection was achieved in both groups while anti-HBs rose from 81 to 28,800 mIU/ml and from 12 to 923 mIU/ml in recipients of Bio-Hep-B and Engerix-B respectively (P < 0.025). CONCLUSIONS: Bio-Hep-B induces rapid seroprotection against hepatitis B in 60-70% of vaccinees, within 4-24 weeks after the first dose. Two instead of the conventional three doses of the Pre-S/S vaccine may be sufficient to induce adequate seroprotection.

Liver regeneration induced by a designer human IL-6/sIL-6R fusion protein reverses severe hepatocellular injury.

The cytokine IL-6 plays a significant role in liver regeneration in conjunction with additional growth factors (HGF, TNF-alpha, and TGF-alpha). Many IL-6 effects depend on a naturally occurring soluble IL-6 receptor (sIL-6R). Here, the chimeric protein hyper-IL-6, constructed from the human IL-6 protein fused to a truncated form of its receptor, was found to have superagonistic IL-6 properties, and as such, enhanced liver cell regeneration. Hyper-IL-6 reversed the state of hepatotoxicity and enhanced the survival rates of rats suffering from fulminant hepatic failure after D-galactosamine administration. The hyper-IL-6 protein has a significant potential for use in the treatment of severe human liver diseases.

Maintenance of immune memory to the hepatitis B envelope protein following adoptive transfer of immunity in bone marrow transplant recipients.

Adoptive transfer of immunity against hepatitis B surface antigen (HBsAg) has been documented in mice and humans. In the present study, we report long-term follow-up of antibodies to HBsAg in humans who received allogeneic bone marrow transplantation (BMT) from donors immunized with HBsAg. BM donors were immunized with recombinant HBsAg. BM or PB cells were transplanted to HLA matched recipients. Recipients were followed for anti-HBs seroconversion. Control groups included non-immunized or rHBsAg immunized healthy adults as well as individuals that had had hepatitis B and recovered spontaneously. PBLs were stimulated in vitro with rHBsAg and stimulation was expressed as stimulation index. Adoptive transfer of immunity to HBsAg was initially documented in 12 recipients of BM from anti-HBc+/anti-HBs+ donors. An almost 4 year follow-up showed detectable protective anti-HBs levels (>10 mIU/ml) in 50% of patients. Immunity to HBV was also documented in 22/35 BMT recipients (62%), who received their bone marrow from actively immunized donors. In 7/9 of these BMT recipients, anti-HBs antibodies levels were documented 25 months following BMT. In 6/8 (75%) of patients who received only PBLs from HBV immune donors, adoptive transfer of immunity to HBV, and seroconversion to HBsAg+, were documented within 2 months of i.v. injection. Evidence for specific cellular immune response with increased SIs was documented for healthy vaccinees, and BMT recipients, and in none of the healthy non-vaccinated controls. These results suggest that adoptive transfer of immunity to HBV is a useful method for providing long-lasting protection for BM recipients.

Aqueous humor contains transforming growth factor-beta and a small (less than 3500 daltons) inhibitor of thymocyte proliferation.

The anterior chamber of the eye is an immunologically privileged site in which allografts survive longer than at other body sites. In this regard, it is relevant that aqueous humor (AH) inhibits lymphocyte proliferation. In order to analyze AH for specific substances that inhibit thymocyte proliferation, samples of human AH, murine AH, and rhesus monkey AH were added to cultures of thymocytes stimulated by IL-1 or IL-2 in the presence of PHA. All samples of AH tested had potent inhibitory activity on thymocyte proliferation in this system. Inhibitory activity was lost by heating AH to 80 degrees C for 1 h. Dialysis of murine AH indicated that species smaller than 3500 Da were capable of mediating this activity; we have termed the factor(s) responsible for this "small inhibitory factor(s) of AH." Retentate, containing species larger than 3500 Da, retained inhibitory activity, but less than nondialyzed AH. Assays for PGE2 demonstrated that murine and human AH contained small quantities of PGE2. These quantities were insufficient to inhibit thymocyte proliferation in our assay system. Furthermore, AH from mice treatend with indomethacin had full inhibitory activity. Assays for transforming growth factor beta (TGF-beta) after acid activation demonstrated significant quantities of latent TGF-beta within human and murine AH which could be largely neutralized by antisera to TGF-beta. Active TGF-beta "activity" was also present without acid activation in samples of AH at a level approximately 20% that of latent TGF-beta. However, most of this "activity" could not be neutralized by antisera to TGF-beta. AH contains factors capable of limiting thymocyte proliferation.

Preliminary characterization of functional residual host-type T lymphocytes following conditioning for allogeneic HLA-matched bone marrow transplantation (BMT).

Host T lymphocytes which escape the effects of chemoradiotherapy may proliferate and lead to graft rejection, particularly in recipients of T cell-depleted bone marrow (BM) allografts. We studied the efficacy of several conditioning regimens including a new immunosuppressive regimen--total lymphoid irradiation (TLI) plus conventional chemotherapy and total body irradiation (TBI)--in abrogating residual host T lymphocytes as assessed by their ability to grow in vitro. A total of 38 patients were evaluated, 29 with hematologic malignancies, six with severe aplastic anemia (AA) and three with beta-thalassemia major (TM), of whom 32 were transplanted with HLA-identical T cell-depleted allogeneic BM from sibling donors. The median observation period was 15 months (range 3-21) posttransplant. Peripheral blood mononuclear cells (PBMC) taken from each patient on the day of transplant were cultured with interleukin 2 (IL-2) + phytohemagglutinin + irradiated donors' PBMC. Survival of cells for less than 2 weeks in vitro without proliferation was observed in 20 of 29 cases with malignancies and was considered negative. In this group only two leukemia (L) patients rejected the graft. Limited cell growth (less than or equal to 3 weeks) was seen in four L patients, two of whom showed early graft failure. Vigorous T cell growth (greater than 5 weeks, 62-96% CD4+ cells) was noted in eight cases (two L, four AA, two TM; none received TBI). In this group, sustained engraftment was observed in 7/7 patients who were treated with cyclosporin A (CSA) post grafting. Overall, we could demonstrate no clear correlation between graft failure and cell growth in vitro. The proliferating cells exhibited considerable cytotoxic activity in vitro against several tumor cell lines and were susceptible to pharmacological doses of CSA. The low incidence of continuous T cell proliferation in vitro in PBMC of L patients suggests that a combination of TLI, TBI and cyclophosphamide (CY) is highly effective in abrogating the host T cells and subsequent graft rejection. Since a rather small number of patients was included in this study, further studies are needed to determine the possible value of the in vitro T cell proliferation assay as a means for predicting graft failure.

Characterization of a tumorigenic murine T-lymphoid-cell line spontaneously derived from an IL-2-dependent T-cell line.

The establishment of IL-2-independent T-cell lines spontaneously derived from long-term IL-2-dependent cytotoxic T-cell lines is described. Two lines (cloned and uncloned) studied in detail have shown the following characteristics: (1) Permanent loss of IL-2 dependence. (2) Partial or complete loss of both cytotoxic activity and the IL-2 receptor. (3) Increased expression of T-cell membrane markers (Thy1.2, Lyt1.2) compared with the parental line. (4) Lower level of DNA methylation than in freshly obtained lymphoid cells. (5) Different karyotypic pattern from the parental IL-2-dependent line, with a mean number of 39-40 chromosomes and a resemblance to T leukemic lines. (6) Leukemia caused in normal syngeneic C57BL/6 mice by the uncloned line, in contrast to the cloned IL-2-independent line or the parental dependent line. Unlike established leukemic lines, however, the independent line gave rise to tumors which regressed in some mice within a few days of their appearance. These findings suggest that T-cell lines maintained with IL-2 for prolonged periods of time (greater than 3 months) can undergo transformation and, therefore, should not be utilized for immunotherapeutic purposes.

Immunotherapy of murine and human tumors in mice with lymphokines and interleukin-2-propagated lymphocytes.

The therapeutic efficacy of crude interleukin-2 (IL-2) preparations and of IL-2-propagated lymphocytes (cultured T cells; CTC) was assessed, with and without chemotherapy, in conventional mice and in athymic nude mice implanted with murine or human neoplasms. Treatment of conventional mice implanted with lung and mammary carcinomas by repeated administration of low-dose cyclophosphamide (CY), IL-2, and tumor-sensitized CTC resulted in delayed tumor onset, retarded tumor growth rate, prolonged survival, and a higher cure rate as compared with mice receiving only CY. In some experiments, nonsensitized CTC (but not fresh lymphocytes) were therapeutically effective almost to the same extent as tumor-sensitized CTC. In most instances, the combination of chemotherapy and IL-2 was significantly more curative than chemotherapy alone, and the addition of indomethacin improved the outcome of both chemotherapy and chemoimmunotherapy. Multiple administrations of IL-2 into athymic mice, before or after implantation of human tumors, delayed or completely inhibited tumor growth. IL-2 injections into normal, untreated, as well as X-irradiated or CY-treated mice, resulted in enhanced natural cytotoxicity and cytotoxic responsiveness in vitro to syngeneic tumors, greater proliferative response to phytohemagglutinin, and a tendency toward depressed proliferative responses to concanavalin A and to allogeneic leukocytes. It is concluded that the appropriate application of lymphokines and IL-2-propagated lymphocytes can be effective in the immunotherapy of experimental tumors and in immunorestoration of immunosuppressed mice. However, since crude lymphokine preparations have been employed in these studies, further analysis with purified lymphokines is required to confirm these observations.

Suppression by tumor growth of T cell growth factor production in mouse lymphoid cell cultures.

Splenocytes of normal (NS) and tumor-bearing (TBS) mice (Balb/c, C57BL/6, C3H) were stimulated in vitro for 24 h with concanavalin A and the amount of T cell growth factor (TCGF) generated was measured. TBS of mice carrying subcutaneous implants (greater than 1 cm tumor diameter) of several T lymphomas, pulmonary and mammary carcinomas, and a melanoma, or intraperitoneal implants (greater than 10(8) cells) of ascitic lymphomas produced (per culture) 40-90% less TCGF than that generated by NS. TBS also contained a greater proportion of phagocytic cells and a higher ratio of Lyt 2+/Lyt 1+ T cells as compared with NS. In cocultures consisting of NS and TBS (1:1), TCGF production by NS was markedly suppressed. In contrast, addition of up to 30% tumor cells to NS decreased production only slightly. Removal from TBS of either phagocytes or Lyt 2+ T cells, or treatment in vitro with indomethacin [IND; an agent inhibiting prostaglandin (PG) synthesis], appreciably reduced their capacity to inhibit NS and improved TCGF production in TBS cultured alone. TCGF production by TBS could be completely restored by depletion of both phagocytes and Lyt 2+ T cells. Elevated quantities of TCGF (up to threefold) were generated by TBS of mice pretreated in vivo 1-4 days previously with low doses of either cyclophosphamide or X-irradiation, with or without IND. It is concluded that suppression of TCGF production in vitro by TBS is mediated by phagocyte-released PG and by Lyt 2+ T lymphocytes.

Methods for amplifying the induction and expression of cytotoxic response in vitro to syngeneic and autologous freshly-isolated solid tumors of mice.

Spontaneously arising tumors are frequently poorly immunogenic and exhibit a limited capacity to induce cytotoxic effector lymphocytes. In the present study, various approaches have been used to amplify the induction and expression of cytotoxic responses in vitro toward freshly isolated, autologous, and syngeneic solid neoplasms of spontaneous origin in mice. Cytotoxic lymphocytes were generated in one-way mixed lymphocyte-tumor cell cultures (MLTC) consisting of splenocytes or lymph node cells from normal and from tumor-bearing mice co-cultured with inactivated tumor cells. Optimal culture conditions have been established for the number of responder (R) cells, the method of inactivation of the stimulating (S) tumor cells, the responder/stimulator (R/S) cell ratio, and the duration of sensitization. Under optimal sensitization conditions only weak cytotoxic responses, as measured by the 51Cr-release assay, were generated. The antitumor cytotoxic activity could be augmented 2- to 12-fold by using each of the following procedures: (a) addition of crude or of partially purified interleukin-2 (IL-2) to the sensitization cultures; (b) depletion of nylon-adherent cells from the responding cell population; (c) enrichment of large lymphoblasts from the sensitized effector cell population by Percoll density gradient; and (d) treatment of mice donating the responder lymphocytes with low doses of either cyclophosphamide, adriamycin, or indomethacin. Although the highly reactive effector cells generated under the improved conditions also reacted appreciably with unrelated tumor target cells, only low levels of cytotoxicity could be demonstrated against normal target cells. The antitumor cytotoxic cells in sensitized splenocyte cultures were exclusively Thy1+, Lyt1-2+, whereas in lymph node cell cultures some cytotoxicity was also exerted by Thy1+, Lyt1+2+ cells.