We recently demonstrated that a human recombinant scFv, L19, reacting with the ED-B domain of fibronectin, a marker of angiogenesis, selectively targets tumoral vasculature in vivo. Using the variable regions of L19, we constructed and expressed a human "small immunoprotein" (SIP) and a complete human IgG1 and performed biodistribution studies in tumor-bearing mice to compare the blood clearance rate, in vivo stability and performance in tumor targeting of the 3 L19 formats [dimeric scFv (scFv) 2 , SIP and IgG1]. The accumulation of the different antibody formats in the tumors studied was a consequence of the clearance rate and in vivo stability of the molecules. Using the SIP, the %ID/g in tumors was 2-5 times higher than that of the (scFv) 2 , reaching a maximum 4 -6 hr after injection. By contrast, the accumulation of IgG1 in tumors constantly rose during the experiments. However, due to its slow clearance, the tumor-blood ratio of the %ID/g after 144 hr was only about 3 compared to a ratio of 10 for the (scFv) 2 and 70 for the SIP after the same period of time. The different in vivo behavior of these 3 completely human L19 formats could be exploited for different diagnostic and/or therapeutic purposes, depending on clinical needs and disease. Furthermore, the fact that ED-B is 100% homologous in human and mouse, which ensures that L19 reacts equally well with the human and the murine antigen, should expedite the transfer of these reagents to clinical trials. © 2002 Wiley-Liss, Inc. Key words: antibody formats; tumor vasculature; tumor targeting; clinical applications; cancer diagnosis and therapyDespite their enormous potential as therapeutic agents, monoclonal antibodies (mAbs) of nonhuman origin have not performed as well as expected in clinical trials as a result of their immunogenicity, 1,2 poor pharmacokinetic properties 3,4 and inefficiency in recruiting effector functions. 5,6 The recent prospect of isolating human antibody fragments from phage display libraries 7-10 transcends these problems, revitalizing studies and rekindling hopes of using these reagents to treat major diseases. Indeed, these molecules should serve as ideal building blocks for novel diagnostic and therapeutic tools. 11,12 Furthermore, these antibodies can be "matured" to reach affinities in the picomolar range, 13 desirable, if not necessary, for their clinical use. 14,15 Clinical applications of human antibody fragments for the selective delivery of diagnostic or therapeutic agents nonetheless require highly specific targets. In the case of tumors, the most popular targets are cell-surface antigens, which are usually neither abundant nor stable. On the other hand, during tumor progression the microenvironment surrounding tumor cells undergoes extensive modification that generates a "tumoral environment" that could ultimately represent a suitable target for antibody-based tumor therapy. 16 In fact, the concept that the altered tumor microenvironment is itself a carcinogen that can be targeted is increasingly gaining consensus. Mol...

In rotavirus-infected cells, the non-structural proteins NSP5 and NSP2 localize in complexes called viroplasms, where replication and assembly occur. Recently, we have demonstrated direct interaction of NSP5 with NSP2, and as a consequence of that, up-regulation of NSP5 hyperphosphorylation. To investigate a possible structural role for the NSP2-NSP5 interaction, we analysed the cytoplasmic distribution of the two proteins in transfected cells by immunofluorescence using specific antibodies. Here we report that NSP2 and NSP5 can drive the formation of viroplasm-like structures (VLS) in the absence of other rotaviral proteins and rotavirus replication. Several NSP5 deletion mutants were constructed and expressed in combination with NSP2. Both the N-and C-terminal domains of NSP5 were found to be essential for VLS formation. Only one mutant, with an internal deletion of residues 81-130, was able to interact with NSP2 to form VLS. Analysis of the phosphorylation capacity of the different mutants in vivo indicated that hyperphosphorylation of NSP5 is necessary, but not sufficient, for VLS formation. Our results suggest a role for the non-structural protein NSP5 in the structure of viroplasms mediated by its interaction with NSP2.

Viroplasms are discrete structures formed in the cytoplasm of rotavirus-infected cells and constitute the replication machinery of the virus. The non-structural proteins NSP2 and NSP5 localize in viroplasms together with other viral proteins, including the polymerase VP1, VP3 and the main inner-core protein, VP2. NSP2 and NSP5 interact with each other, activating NSP5 hyperphosphorylation and the formation of viroplasm-like structures (VLSs). We have used NSP2 and NSP5 fused to the enhanced green fluorescent protein (EGFP) to investigate the localization of both proteins within viroplasms in virus-infected cells, as well as the dynamics of viroplasm formation. The number of viroplasms was shown first to increase and then to decrease with time post-infection, while the area of each one increased, suggesting the occurrence of fusions. The interaction between NSP2 and a series of NSP5 mutants was investigated using two different assays, a yeast two-hybrid system and an in vivo binding/immunoprecipitation assay. Both methods gave comparable results, indicating that the N-terminal region (33 aa) as well as the C-terminal part (aa 131-198) of NSP5 are required for binding to NSP2. When fused to the N and C terminus of EGFP, respectively, these two regions were able to confer the ability to localize in the viroplasm and to form VLSs with NSP2.

CD57(+) expression in T lymphocytes has been recognized for decades as a marker of in vitro replicative senescence. In recent years, accumulating evidences have pointed on the utility of this marker to measure functional immune deficiency in patients with autoimmune disease, infectious diseases, and cancers. We review here the relevant literature and implications in clinical settings.

Rotavirus viroplasms are cytosolic, electron-dense inclusions corresponding to the viral machinery of replication responsible for viral template transcription, dsRNA genome segments replication and assembly of new viral cores. We have previously observed that, over time, those viroplasms increase in size and decrease in number. Therefore, we hypothesized that this process was dependent on the cellular microtubular network and its associated dynamic components. Here, we present evidence demonstrating that viroplasms are dynamic structures, which, in the course of an ongoing infection, move towards the perinuclear region of the cell, where they fuse among each other, thereby gaining considerably in size and, simultaneouly, explaining the decrease in numbers. On the viral side, this process seems to depend on VP2 for movement and on NSP2 for fusion. On the cellular side, both the temporal transition and the maintenance of the viroplasms are dependent on the microtubular network, its stabilization by acetylation, and, surprisingly, on a kinesin motor of the kinesin-5 family, Eg5. Thus, we provide for the first time deeper insights into the dynamics of rotavirus replication, which can explain the behavior of viroplasms in the infected cell.