Patients suffering from acute graft-versus-host disease after bone-marrow transplantation have functional CD4+CD25hiFoxp3+ regulatory T cells.

Acute Graft-Versus-Host Disease (aGVHD), mediated by CD4(+) and CD8(+) effector T cells, is a life-threatening complication in hematopoietic stem cell (HSC) transplantation. Naturally-occurring CD4(+)CD25(hi)(Foxp3(+)) regulatory T cells (T(reg)) have been shown to modulate tolerance to aGVHD in murine graft models. In this report, we investigated their role in the prevention of aGVHD in patients transplanted with bone-marrow-derived HSC. When CD4(+)CD25(hi)Foxp3(+) T cells were isolated from bone-marrow grafts, they showed no suppressive activity. The analysis of their function in patients suffering from aGVHD after transplantation revealed a gain of suppressive activity indicating their inability to control the aGVHD induction. Thus, our findings clearly demonstrate that CD4(+)CD25(+) and CD4(+)CD25(hi)Foxp3(+) T cells, when administered in steady-state physiological conditions, do not influence the outcome of aGVHD after bone-marrow transplantation.

Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response.

Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-gamma), protein-specific lymphoproliferative responses, IFN-gamma, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.

Hepatitis C virus non-structural protein 3-specific cellular immune responses following single or combined immunization with DNA or recombinant Semliki Forest virus particles.

The capacity of recombinant Semliki Forest virus particles (rSFV) expressing the hepatitis C virus non-structural protein 3 (NS3) to induce, in comparison or in combination with an NS3-expressing plasmid, specific cellular and humoral immune responses in murine models was evaluated. In vitro studies indicated that both types of vaccine expressed the expected size protein, albeit with different efficacies. The use of mice transgenic for the human HLA-A2.1 molecule indicated that the rSFV-expressed NS3 protein induces, as shown previously for an NS3 DNA vaccine, NS3-specific cytotoxic lymphocytes (CTLs) targeted at one dominant HLA-A2 epitope described in infected patients. All DNA/rSFV vaccine combinations evaluated induced specific CTLs, which were detectable for up to 31 weeks after the first injection. Overall, less than 1 log difference was observed in terms of the vigour of the bulk CTL response induced and the CTL precursor frequency between all vaccines (ranging from 1:2.6x10(5) to 1:1x10(6)). Anti-NS3 antibodies could only be detected following a combined vaccine regimen in non-transgenic BALB/c mice. In conclusion, rSFV particles expressing NS3 are capable of inducing NS3-specific cellular immune responses targeted at a major HLA-A2 epitope. Such responses were comparable to those obtained with a DNA-based NS3 vaccine, whether in the context of single or combined regimens.

Different hepatitis C virus nonstructural protein 3 (Ns3)-DNA-expressing vaccines induce in HLA-A2.1 transgenic mice stable cytotoxic T lymphocytes that target one major epitope.

The immunogenicity of the Hepatitis C virus (HCV) nonstructural protein 3 (NS3) was investigated using different DNA-based strategies and a preclinical mouse model transgenic for the HLA-A2.1 molecule. Plasmids expressing NS3 either as a wild-type protein, as a fusion with murine lysosome-associated-membrane protein-1 specific sequences, or under the control of the Semliki Forest virus replicase were evaluated in vitro and in vivo. All plasmids were shown to express the expected size protein. These 3 NS3-expressing vaccines induced overall comparable levels of CTLs when measured at different times postvaccination although mice injected with the NS3-LAMP expressing plasmid showed a particularly homogeneous and overall vigorous response (specific lysis ranged from 60% to 90 % for an E:T ratio of 33.3:1 with a mean CTL precursor frequency of 1:2.10(5) cells). Out of the four HLA-A2.1-restricted NS3 epitopes previously described in HCV infected patients (aa 1073-1081, aa 1406-1415; aa 1169-1177 and aa 1287-1296), the NS3-DNA generated CTLs were predominantly targeted at the aa 1073-1081 epitope. Peptide-based immunization showed that the mouse repertoire was intact for all epitopes tested except one (aa 1287-1296). In conclusion, the 3 NS3-DNA vaccines although based on different mode of action, shared a comparable efficacy at inducing CTL. Surprisingly, the breadth of such response was restricted to a single, major epitope.

Transgenic mice produced by retroviral transduction of male germ-line stem cells.

Male germ-line stem cells are the only cell type in postnatal mammals that have the capability to self-renew and to contribute genes to the next generation. Genetic modification of these cells would provide an opportunity to study the biology of their complex self-renewal and differentiation processes, as well as enable the generation of transgenic animals in a wide range of species. Although retroviral vectors have been used as an efficient method to introduce genes into a variety of cell types, postnatal male germ-line stem cells have seemed refractory to direct infection by these viruses. In addition, expression of genes transduced into several types of stem cells, such as embryonic or hematopoietic, is often attenuated or silenced. We demonstrate here that in vitro retroviral-mediated gene delivery into spermatogonial stem cells of both adult and immature mice results in stable integration and expression of a transgene in 2-20% of stem cells. After transplantation of the transduced stem cells into the testes of infertile recipient mice, approximately 4.5% of progeny from these males are transgenic, and the transgene is transmitted to and expressed in subsequent generations. Therefore, there is no intrinsic barrier to retroviral transduction in this stem cell, and transgene expression is not extinguished after transmission to progeny.

DNA vaccines for hepatitis C virus.

Hepatitis C virus is an RNA encoded virus of the Flaviviridae family. In most cases, infections develop into a chronic carrier stage that can result in the onset of cirrhosis and hepatocellular carcinoma over a 20- to 30-year period. Because existing therapies are still of limited benefit and expensive, the development of a vaccine represents a priority to prevent further spreading of the infection. Immune correlates of protection remain poorly defined although increasing evidence suggests that both humoral and cellular immune responses are likely to contribute to protection and/or neutralization of the virus. Current DNA-based vaccines, while capable of generating the latter, appear limited in their capacity to induce a strong and long-lasting antibody response.

Expanded indications for transcervical thymectomy in the management of anterior mediastinal masses.

BACKGROUND: Transcervical thymectomy (TCT) is an accepted though controversial approach for thymectomy in myasthenia gravis (MG). The suggestion of thymoma on computed tomography (CT) has been considered a contraindication to TCT. We sought to determine whether the indications for TCT could be safely expanded to include selected patients with thymomas as well as other types of anterior mediastinal masses. METHODS: Between January 1992 and September 1999, we performed 121 TCTs: 98 in patients with MG and 23 in patients without MG. The patients' records were retrospectively reviewed. RESULTS: Among the 98 MG patients, 28 had CT scans suspicious for thymoma. Of these, 14 had a thymoma pathologically. These were classified as stage I (5), stage II (8), and stage III (1). Five patients required extension of the incision for completion of the procedure. There have been no thymoma recurrences to date with a mean follow-up of 48 months (range 3 to 96 months). In the 23 patients without MG, 12 had new anterior mediastinal masses, 4 had a history of treated lymphoma, 1 had a history of treated germ cell tumor, and 6 had suspected mediastinal parathyroid adenoma. Diagnostic tissue was obtained in all patients undergoing the procedure for diagnosis, and in 4 of 6 patients, a parathyroid adenoma was successfully resected. CONCLUSIONS: Transcervical exploration and thymectomy offers a less invasive approach to the diagnosis and/or definitive treatment of selected anterior mediastinal masses. We suggest that it is appropriate to expand its use to several clinical scenarios beyond the typical indication of thymectomy in MG patients without thymoma.

Use of conventional or replicating nucleic acid-based vaccines and recombinant Semliki forest virus-derived particles for the induction of immune responses against hepatitis C virus core and E2 antigens.

Replicating and nonreplicating nucleic acid-based vaccines as well as Semliki Forest-recombinant Viruses (rSFVs) were evaluated for the development of a vaccine against hepatitis C virus (HCV). Replicating SFV-DNA vaccines (pSFV) and rSFVs expressing HCV core or E2 antigens were compared with classical CMV-driven plasmids (pCMV) in single or bimodal vaccine protocols. In vitro experiments indicated that all vaccine vectors produced the HCV antigens but to different levels depending on the antigen expressed. Both replicating and nonreplicating core-expressing plasmids induced, upon injection in mice, specific comparable CTL responses ranging from 10 to 50% lysis (E:T ratio 100:1). Comparison of different injection modes (intramuscular versus intraepidermal) and the use of descalating doses of DNA (1-100 microgram) did not show an increased efficacy of the core-SFV plasmid compared with the CMV-driven one. Surprisingly, rSFVs yielded either no detectable anticore CTL or very low anti-E2 antibody titers following either single or bimodal administration together with CMV-expressing counterparts. Prime-boost experiments revealed, in all cases, the superiority of DNA-based only vaccines. The anti-E2 antibody response was evaluated using three different assays which indicated that all generated anti-E2 antibodies were targeted at similar determinants. This study emphasizes the potential of DNA-based vaccines for induction of anti-HCV immune responses and reveals an unexpected and limited benefit of SFV-based vaccinal approaches in the case of HCV core and E2.

Culture of mouse spermatogonial stem cells.

Spermatogenesis occurs within the seminiferous tubules of mammals by a complex process that is highly organized, extremely efficient and very productive. At the foundation of this process is the spermatogonial stem cell that is capable of both self-renewal and production of progeny cells, which undergo differentiation over a period of weeks to months in order to generate mature spermatozoa. It had been thought that germ cells survive only a brief period in culture, generally less than a few weeks. However, an accurate assessment of the presence of spermatogonial stem cells in any cell population has only recently become possible with development of the spermatogonial transplantation technique. Using this technique, we have demonstrated that mouse spermatogonial stem cells can be maintained in culture for approximately 4 months and will generate spermatogenesis following transplantation to the seminiferous tubules of an appropriate recipient. Extensive areas of cultured donor cell-derived spermatogenesis are generated in the host, and production of mature spermatozoa occurs. Cultivation of the testis cells on STO feeders is beneficial to stem cell survival. These results provide the first step in establishing a system that will permit spermatogonial stem cells to be cultivated and their number increased in vitro to allow for genetic modification before transplantation to a recipient testis.

Reconstitution of spermatogenesis from frozen spermatogonial stem cells.

Spermatozoa from a number of species can be cryopreserved and then subsequently used to fertilize eggs. However, this technique has several limitations. First, the freezing protocol varies for each species and must be determined empirically, and for some species appropriate methods have not yet been identified. Second, because these cells are fully differentiated, they will not undergo replication when thawed, and recombination of genetic information cannot occur. We now demonstrate, by using the recently developed spermatogonial transplantation technique, that male germline stem cells can be successfully cryopreserved. Donor testis cells isolated from prepubertal or adult mice and frozen from 4 to 156 days at -196 degrees C were able to generate spermatogenesis in recipient seminiferous tubules. Relatively standard preservation techniques were used, suggesting that male germ cells from other species can also be stored for long periods. Because transplanted testis stem cells will ultimately undergo replication and meiotic recombination during spermatogenesis, one might consider these preserved male germ lines as biologically immortal.