Characterization of single chain antibody targets through yeast two hybrid

Background  

Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain.     

The Trw Type IV Secretion System of Bartonella Mediates Host-Specific Adhesion to Erythrocytes

Bacterial pathogens typically infect only a limited range of hosts; however, the genetic mechanisms governing host-specificity are poorly understood. The α-proteobacterial genus Bartonella comprises 21 species that cause host-specific intraerythrocytic bacteremia as hallmark of infection in their respective mammalian reservoirs, including the human-specific pathogens Bartonella quintana and Bartonella bacilliformis that cause trench fever and Oroya fever, respectively. Here, we have identified bacterial factors that mediate host-specific erythrocyte colonization in the mammalian reservoirs. Using mouse-specific Bartonella birtlesii, human-specific Bartonella quintana, cat-specific Bartonella henselae and rat-specific Bartonella tribocorum, we established in vitro adhesion and invasion assays with isolated erythrocytes that fully reproduce the host-specificity of erythrocyte infection as observed in vivo. By signature-tagged mutagenesis of B. birtlesii and mutant selection in a mouse infection model we identified mutants impaired in establishing intraerythrocytic bacteremia. Among 45 abacteremic mutants, five failed to adhere to and invade mouse erythrocytes in vitro. The corresponding genes encode components of the type IV secretion system (T4SS) Trw, demonstrating that this virulence factor laterally acquired by the Bartonella lineage is directly involved in adherence to erythrocytes. Strikingly, ectopic expression of Trw of rat-specific B. tribocorum in cat-specific B. henselae or human-specific B. quintana expanded their host range for erythrocyte infection to rat, demonstrating that Trw mediates host-specific erythrocyte infection. A molecular evolutionary analysis of the trw locus further indicated that the variable, surface-located TrwL and TrwJ might represent the T4SS components that determine host-specificity of erythrocyte parasitism. In conclusion, we show that the laterally acquired Trw T4SS diversified in the Bartonella lineage to facilitate host-restricted adhesion to erythrocytes in a wide range of mammals.     

Interactions between CD47 and thrombospondin reduce inflammation

CD47 on the surface of T cells was shown in vitro to mediate either T cell activation or, in the presence of high amounts of thrombospondin (TSP), T cell apoptosis. We report here that CD47-deficient mice, as well as TSP-1 or TSP-2-deficient mice, sustain oxazolone-induced inflammation for more than four days, whereas wild-type mice reduce the inflammation within 48 h. We observe that prolonged inflammation in CD47-, TSP-1-, or TSP-2-deficient mice is accompanied by a local deficiency of T cell apoptosis. Finally, we show that upon activation normal T cells increase the expression of the proapoptotic Bcl-2 family member BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein) and undergo CD47-mediated apoptosis. This finding is consistent with our previous demonstration of a physical interaction between BNIP3 and CD47 that inhibits BNIP3 degradation by the proteasome, sensitizing T cells to CD47-induced apoptosis. Overall, these results reveal an important role in vivo for this new CD47/BNIP3 pathway in limiting inflammation by controlling the number of activated T cells.     

Neuroprotection and stroke: time for a compromise

In April 2007, there existed a repertory of 286 trials concerned with acute ischemic stroke on the Stroke Trials Registry ( http://www.strokecenter.org/trials/ ), of which 209 trials were considered as complete (with no evidence of patient benefit unless one considers the much hard fought for and modest results of the tPA studies). Among other questions arising from such failures, one can wonder whether the plethora of pharmacological agents that exhibited neuroprotective properties in pre-clinical studies were selected for clinical trials entirely based upon their experimental efficacy. This mini-review will try to point out some of the weaknesses that could underline the failure of both researchers and clinicians involved in the field of stroke to obtain their ultimate goal – brain protection.     

Cytomegalovirus infection in Gambian infants leads to profound CD8 T-cell differentiation

Cytomegalovirus (CMV) infection is endemic in Gambian infants, with 62% infected by 3 months and 85% by 12 months of age. We studied the CD8 T-cell responses of infants to CMV following primary infection. CMV-specific CD8 T cells, identified with tetramers, showed a fully differentiated phenotype (CD28(-) CD62L(-) CD95(+) perforin(+) granzyme A(+) Bcl-2(low)). Strikingly, the overall CD8 T-cell population developed a similar phenotype following CMV infection, which persisted for at least 12 months. In contrast, primary infection was accompanied by up-regulation of markers of activation (CD45R0 and HLA-D) on both CMV-specific cells and the overall CD8 T-cell population and division (Ki-67) of specific cells, but neither pattern persisted. At 12 months of age, the CD8 T-cell population of CMV-infected infants was more differentiated than that of uninfected infants. Although the subpopulation of CMV-specific cells remained constant, the CMV peptide-specific gamma interferon response was lower in younger infants and increased with age. As the CD8 T-cell phenotype induced by CMV is indicative of immune dysfunction in the elderly, the existence of a similar phenotype in large numbers of Gambian infants raises the question of whether CMV induces a similarly deleterious effect.     

Structural and thermodynamic bases for the design of pure prolactin receptor antagonists: X-ray structure of Del1-9-G129R-hPRL

Competitive antagonists of the human prolactin (hPRL) receptor are a novel class of molecules of potential therapeutic interest in the context of cancer. We recently developed the pure antagonist Del1-9-G129R-hPRL by deleting the nine N-terminal residues of G129R-hPRL, a first generation partial antagonist. We determined the crystallographic structure of Del1-9-G129R-hPRL, which revealed no major change compared with wild type hPRL, indicating that its pure antagonistic properties are intrinsically due to the mutations. To decipher the molecular bases of pure antagonism, we compared the biological, physicochemical, and structural properties of numerous hPRL variants harboring N-terminal or Gly(129) mutations, alone or combined. The pure versus partial antagonistic properties of the multiple hPRL variants could not be correlated to differences in their affinities toward the hPRL receptor, especially at site 2 as determined by surface plasmon resonance. On the contrary, residual agonism of the hPRL variants was found to be inversely correlated to their thermodynamic stability, which was altered by all the Gly(129) mutations but not by those involving the N terminus. We therefore propose that residual agonism can be abolished either by further disrupting hormone site 2-receptor contacts by N-terminal deletion, as in Del1-9-G129R-hPRL, or by stabilizing hPRL and constraining its intrinsic flexibility, as in G129V-hPRL.     

Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.