Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein.One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.Severe acute respiratory syndrome (SARS) was first identified in 2002 as a newly emerging disease in Guangdong Province, China. The disease, associated with unusual atypical pneumonia, spread in 2003 to over 30 countries worldwide with more than 8,000 reported cases and an estimated 55% mortality among the elderly (9). A virus was isolated from tissues of SARS patients (10, 21, 23, 32) and a SARS-associated coronavirus (SARS-CoV), a new member in the family of Coronaviridae, was identified as the causative agent fulfilling Koch's postulates (12).The clinical course of SARS is highly variable (31) after a relatively short 6-to 10-day incubation period (9). In ca. 20% of the patients, SARS-CoV infection progresses to a stage of respiratory failure requiring ventilation support. Overall, 10% of the patients, ca. 6.8% of patients younger and 55% of patients older than 60 years of age (9), die as a consequence of immunopathological lung damage caused by a hyperactive antiviral immune response (29).Antibodies to SARS-CoV become detectable in patient's serum between days 10 and 15 and correlate with a decline in viral loads. More than 93% of the patients were reported to have seroconverted by day 28 (31). The pattern of SARS-CoV replication and development of a neutralizing immune response observed in experimentally infected mice largely resembles...

The immortalised HBMEC cell lines have maintained their normal phenotype for the majority of characteristics examined. The expression of E-selectin and VCAM-1, which are not constitutively expressed on the cell lines, can be induced by stimulation of the endothelial cells with IL-1beta. The cell lines have furthermore maintained their capability to bind HPC. They will therefore be useful to investigate the interactions between HPC and HBMEC involved in homing of HPC.

In the process of homing, CD34 + hematopoietic progenitor cells migrate across the bone marrow endothelium in response to stromal cell-derived factor (SDF)-1. To develop more efficient stem cell transplantation procedures, it is important to define the adhesion molecules involved in the homing process. Here, we identified the adhesion molecules that control the migration of primary human CD34 + cells across human bone marrow endothelial cells.Migration of CD34 + cells is enhanced across interleukin 1β prestimulated bone marrow endothelium, suggesting an important role for the endothelium in adhesion and formation of the chemotactic gradient. Under these conditions, 30-100 ng/ml SDF-1 induced a rapid and efficient migration of CD34 + cells (± 46% migration in 4 h). In contrast, 600-1,000 ng/ml SDF-1 were required for optimal migration across fibronectin-coated filters. Subsequent studies revealed that transendothelial migration of CD34+ cells is mediated by β1-and β2-integrins and PECAM-1 (CD31) but not by CD34 or E-selectin. Whereas these antibodies individually blocked migration for 25%-35%, migration was reduced by 68% when the antibodies were combined. Thus, these adhesion molecules play specific and independent roles in the transmigration process. Finally, O-glycosylated proteins appeared to play a role, since SDF-1-induced migration of CD34 + cells (treated with a glycoprotease from Pasteurella haemolytica) across endothelial cells was clearly inhibited.In conclusion, we show that efficient SDF-1-induced migration of primary human CD34 + cells across bone marrow endothelium is mediated by β1-integrins, β2-integrins, CD31 and O-glycosylated proteins.

The SH2 domain-containing transforming Shc protein has been implicated in mitogenic signaling via several surface receptors through p21ras. Following tyrosine phosphorylation by either receptor or non-receptor tyrosine kinases, Shc may interact with the adaptor protein Grb2, which is linked to Sos1, a guanine nucleotide exchange factor for human ras. Ligation of the antigen receptor complex on B cells (BCR) is known to activate various intracellular signaling pathways, which may accumulate in mitogenic responses. With respect to the initial steps, the activation of BCR-associated non-receptor tyrosine kinases appears to be indispensible. In this report we show that Shc proteins become tyrosine phosphorylated after BCR ligation on both transformed and normal human B cells. This is accompanied by the association of Shc with Grb2 proteins and a yet unidentified 145-kDa tyrosine phosphorylated protein. Subcellular fractionation revealed that this activation-induced multimeric Shc complex rapidly translocates towards the plasma membrane. Co-ligation of the BCR with the CD19 molecule results in a marked increase of these events, whereas CD19 cross-linking alone does not induce Shc tyrosine phosphorylation or translocation. Thus, in B cells the Shc complex may represent a molecular junction between the BCR and the mitogenic p21ras cascade.

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Adhesion of CD34+ hematopoietic progenitor cells (HPCs) to sinusoidal endothelium probably plays a key role in homing of transplanted CD34+ HPCs to the bone marrow (BM). We have investigated the role of various adhesion molecules in the interaction of purified CD34+ HPCs derived from BM or peripheral blood (PB) and a human BM-derived endothelial cell line. Adhesion of CD34+ HPCs to endothelial cells was measured with the use of a double-color flow microfluorimetric adhesion assay. In this assay, adhesion is measured under stirring conditions, simulating blood flow in sinusoidal marrow vessels. Adhesion of PB CD34+ cells to human BM endothelial cells (HBMECs) was observed only after interleukin (IL)-1beta prestimulation of the endothelial cells. This adhesion was strongly increased after addition of phorbol-myristate acetate (PMA). Adhesion of PB CD34+ cells to IL-1beta-prestimulated HBMECs was inhibited by blocking monoclonal antibodies (mAbs) against E-selectin and by neuraminidase treatment of the PB CD34+ cells. mAbs against very late activation antigen (VLA)-4 inhibited adhesion only when the E-selectin-mediated interaction was prevented. No clear inhibiting effect was found with blocking mAbs against beta2-integrins. Stimulation with the beta1-integrin-activating mAb, 8A2, induced adhesion of CD34+ cells to endothelial cells. In conclusion, stimulation of both endothelial cells and CD34+ HPCs is necessary for adhesion of CD34+ HPCs to endothelial cells. We furthermore demonstrated that E-selectin and VLA-4 mediated this adhesion.

The activation of B-cell antigen receptor-associated protein tyrosine kinases is an early and crucial event in B-cell signaling. Apart from the B-cell antigen receptor (BCR), the B-cell-specific transmembrane glycoprotein CD19 has also been shown to directly activate intracellular signaling cascades. In addition, because CD19 and the BCR are associated on the surface of activated B-cells, it has been proposed that close approximation between these two entities is crucial for optimal B-cell triggering. To test this hypothesis, bispecific antibodies were generated that bind membrane IgM and CD19 simultaneously. Although CD19 bispecific antibodies strongly induced tyrosine phosphorylation, they were, in contrast to muF(ab)2 fragments, unable to induce a proliferative response. Detailed analysis of the early signaling events showed that compared with muF(ab)2 fragments CD19 bispecific antibodies potently raised the intracellular [Ca2+], which was correlated with an efficient tyrosine phosphorylation of syk. Strikingly, the assembly of Grb2 complexes that may couple the BCR to p21(ras) was clearly altered by the CD19 bispecific antibody. In addition to the reported Shc and 145-kDa phosphoproteins, a prominent 90-95-kDa phosphoprotein resembling CD19 was detected in the Grb2 complexes. Thus, studies with CD19 bispecific antibodies show that CD19 co-ligation both quantitatively and qualitatively alters BCR signaling.