Human cytomegalovirus (HCMV) infection is common and usually well controlled. Immunocompromised patients such as those undergoing bone marrow transplantation and infected newborns are especially vulnerable to HCMV disease (5,20,23).The immune control of HCMV replication appears to be mainly mediated by cellular immune responses. CD4 ϩ and CD8 ϩ T lymphocytes have been proposed to play a major role in the control of viral replication and in protection from disease. The contribution of anti-IE1-and anti-pp65-specific Tcell precursors to the total anti-HCMV immunity is now well established (7,8,15,19,34). IE1 is the major protein produced in the immediate-early phase of the HCMV replication cycle, and the matrix protein pp65 has been shown to be internalized immediately after the viral input without de novo synthesis and then to be available for presentation to specific CD8 ϩ cytotoxic T lymphocytes (CTL) (2, 19). The establishment of a rapid T-cell response before the synthesis of new infectious virions could provide an efficient means to avoid spreading of the virus. This strongly supports the idea that both CD4 ϩ and CD8 ϩ T cells directed against IE1 and pp65 could be critical for the generation of effective vaccines against HCMV and in anti-HCMV cell therapy.The transfer of anti-HCMV effector T cells to allogeneic bone marrow recipients results in protection from HCMV diseases associated with transplantation. The procedure is based on the use of HCMV-infected autologous fibroblasts to stimulate anti-HCMV-specific T cells in vitro (33). The authors showed that persistence of cytotoxic CD8 ϩ T cells in recipients was facilitated by a simultaneous recovery of CD4 ϩ helper T cells after bone marrow transplantation (33). More recently, the use of Epstein-Barr virus (EBV)-transformed B cells transduced with a recombinant retrovirus expressing pp65 has been suggested to allow the concomitant expansion of both anti-EBV-and anti-HCMV-specific T cells (31). A similar strategy used autologous B lymphoblastoid cells stably transfected with cDNA coding for either pp65 or IE1 for the generation of specific CD8 ϩ T-cell clones (26). Our approach to circumvent the use of infectious virus in ex vivo expansion protocols for cellular immunotherapy is based on a procedure allowing the simultaneous triggering of the anti-IE1 and -pp65 responses by means of a recombinant chimeric protein, IE1-pp65.In this paper, we report the construction and purification of a recombinant IE1-pp65 protein from insect cells. We demonstrate that an important proportion of anti-HCMV CD4 ϩ T cells was directed against IE1-pp65 in HCMV-seropositive donors and that the protein induced activation of HLA-DR3-restricted anti-IE1 CD4 ϩ T-cell clones, as assessed through gamma interferon (IFN-␥) secretion and cytotoxicity. Moreover, soluble IE1-pp65 was able to stimulate and to expand anti-pp65 CD8 ϩ T cells from peripheral blood mononuclear

Vascularized proximal fibular epiphyseal transfer in children enables reconstruction of long-bone epiphyseal defect, while conserving axial growth potential. This technique was applied in two children for diaphyseal-epiphyseal reconstruction of the proximal humerus and distal radius respectively, using a graft vascularized only by the anterior tibial artery. There were no major complications during harvesting. Both cases showed transplant growth, of a mean 0.5cm/year. Joint function in the proximal humerus reconstruction was satisfactory, with functional range of motion. In the distal radius reconstruction, range of motion was almost zero; insufficient transplant growth induced radial club hand, requiring partial correction by progressive lengthening using an external fixator. In case of severe bone loss, fibular epiphyseal-diaphyseal graft vascularized only by the anterior tibial artery is a feasible attitude.

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