Anne Wijkhuisen

Identification in the NK1 tachykinin receptor of a domain involved in recognition of neurokinin A and septide but not of substance P

The three mammalian tachykinins, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), exert their physiological effects through specific receptors, NK1, NK2 and NK3, respectively. However, homologous binding studies have recently demonstrated that, contrary to the generally accepted belief, NKA could bind NK1 receptor with high affinity (Hastrup and Schwartz, 1996). Using COS‐7 cells expressing the human NK1 receptor, we show that two simultaneous point mutations (E193L and V195R) in a restricted five amino acid sequence (the (193–197) region), selected because of its hydropathic complementarity with the common C‐terminal extremity of tachykinins, abolish both the high‐affinity binding and highly potent biological activity of NKA, without affecting those of SP. In addition, the same mutations also suppressed the high functional activity of septide, a synthetic SP atypical agonist ([pGlu6‐Pro9] SP 6–11). These results suggest that the (193–197) region, located at the end of the second extracellular loop of the receptor, could be part of a common high‐affinity binding domain for both NKA and septide, distinct from the SP binding site.

Use of DNA immunization to produce polyclonal antibodies against the native human neurokinin-1 receptor for substance P

Antibodies against the native form of the human NK1 receptor (hNK1R) for the neuropeptide substance P (SP), an important immunoregulator, are difficult to produce using classical immunization techniques. We show here that mice immunized with a plasmid harboring hNK1R cDNA developed antibodies recognizing extracellular epitopes of native hNK1R expressed on CHO cell membranes, as shown by FACS and immunofluorescence analysis, some antibodies being specifically directed against the second extracellular loop (E2) of the receptor. This original strategy, DNA immunization, thus efficiently generated new immunological tools to further analyse the role of SP in the regulation of immune cell functions.

Distribution and ultrastructure of tachykinin-like immunoreactivity in the frog (rana esculenta) spinal cord, notably, the dorsal horn

Tachykinins are involved in pain transmission at the spinal level. In frog, at least four tachykinins [TK] have been isolated from the brain, but their organization in the dorsal horn of the spinal cord is still poorly known. We have reexamined TK distribution by immunocytochemistry using an antibody recognizing the sequence common to all tachykinins in the spinal cord and dorsal root ganglia of the green frog Rana esculenta. A dense tachykinin‐like immunoreactivity (TK‐LI) was observed in the dorsolateral fasciculus or Lissauers tract running ventromedial to the entry of the dorsal root and in numerous small and medium‐sized dorsal root ganglion cells showing a primary afferent origin for part of TK‐LI of the dorsal horn. The observation of numerous cell bodies in the dorsal horn, in addition, suggested a local or propriospinal origin. One group of cells was localized at the entrance of the Lissauers tract TK‐LI fibers into the dorsal horn, and another group was localized in the upper dorsal horn, a region with a low density of TK‐LI fibers. It was suggested that the latter group may correspond to neurokinin B. Electron microscopic examination of the Lissauers tract showed numerous immunoreactive axons, some located at the center of glomerular‐like arrangements, suggesting that the information brought by these fibers may be transmitted and most probably modulated before their entry in the dorsal horn. In conclusion, the functional organization of tachykinins in the frog spinal cord seems to be similar to that of mammals, albeit with a different morphological organization. J. Comp. Neurol. 433:183–192, 2001.

Pharmacological in vitro evaluation of new substance P-cyclodextrin derivatives designed to drug targeting towards NK1-receptor bearing cells.

Some biological properties of new bifunctional conjugates designed for drug targeting were evaluated through in vitro experiments. Eight peptidylcyclodextrin compounds were used, which correspond to modified beta- or gamma-cyclodextrin (CD) grafted on neuropeptide substance P (SP) or a shorter derivative (SP(4-11)). Using anti-SP and anti-CD antibodies as molecular probes, we showed that the main structural features of the two moieties of these adducts were preserved. Binding experiments, using CHO cells expressing the human SP-specific NK1 receptor, demonstrated the functionality of all peptidylcyclodextrin derivatives, which exhibited IC50 values in a 10(-9)-10(-7) M range. All compounds were able to induce a pharmacological response, triggering phosphatidylinositol turnover with EC50 values in the same range as the natural ligand. Moreover, autoradiography analysis of rat spinal corn sections proved that [125I]SP binding was dose-dependently displaced by one selected compound (a gamma-CD-SP), showing a similar affinity of this adduct for the rat neurokinin 1 receptor. Our observations demonstrate that these peptidylcyclodextrins efficiently target NK1 receptor-expressing cells.

Pharmacological properties of peptides derived from an antibody against the tachykinin NK1 receptor for the neuropeptide substance P

Two peptides were derived from the structural analysis of a previously described monoclonal antibody [Mol. Immunol. 37 (2000) 423] against the tachykinin NK(1) receptor for the neuropeptide substance P. Here we show that these two peptides were able to inhibit the inositol phosphate transduction pathway triggered both by substance P and neurokinin A, another high-affinity endogenous ligand for the tachykinin NK(1) receptor. They also reduced the cAMP production induced by substance P. By contrast, only one antagonist peptide was able to prevent substance P and neurokinin A from binding the receptor, as revealed both by biochemical and autoradiographic studies. First, these results illustrate the generality of the antibody-based strategy for developing new bioactive peptides. Second, they indicate that antagonists, even exhibiting very close amino acid composition, can interact with the tachykinin NK(1) receptor at different contact sites, some of them clearly distinct from the contact domains for endogenous agonists.

Production of human antibodies by in vitro immunization using a fusion protein containing the transcriptional transactivator of HIV-1.

Antigen-specific activation of human B cells represents a key step for the production of monoclonal antibodies. Several approaches have been developed over the last thirty years in order to improve the process of lymphocyte activation in vitro. In the present study, we investigated whether the transcriptional transactivator (Tat) of human immunodeficiency virus, which possesses numerous biological activities, is able to trigger antibody secretion when incubated with human peripheral blood mononuclear cells. No such effect was observed when using Tat as a free protein. However, we found a significant IgM antibody production when Tat was previously fused to a double domain, called ZZ, derived from protein A of Staphylococcus aureus. The effect was also observed when the fusion protein, called ZZTat101, was incubated with purified B cells, indicating that the phenomenon does not require T-cell help. Antibody secretion was observed in the absence of cytokines that are usually used during in vitro immunization experiments, indicating that ZZTat101 provides the signals required for the initiation of the immune response. Antibody secretion was observed using a ZZTat mutant, containing only the Tat residues 22 to 57, called ZZTat22-57, indicating that this region is sufficient to initiate the immune response. In contrast, the effect was not found with a ZZTat22-57 mutant devoid of the seven Tat cysteines located between residues 22 and 37, demonstrating that these residues play a crucial role in the phenomenon. Our results pave the way to the development of a new in vitro immunization method based on antigens associated with ZZTat.

Obtaining anti-type 1 melatonin receptor antibodies by immunization with melatonin receptor-expressing cells

Antibodies (Abs) specific to cell-surface receptors are attractive tools for studying the physiological role of such receptors or for controlling their activity. We sought to obtain such antibodies against the type 1 receptor for melatonin (MT1). For this, we injected mice with CHO cells transfected with a plasmid encoding human MT1 (CHO-MT1-h), in the presence or absence of an adjuvant mixture containing Alum and CpG1018. As we previously observed that the immune response to a protein antigen is increased when it is coupled to a fusion protein, called ZZTat101, we also investigated if the association of ZZTat101 with CHO-MT1-h cells provides an immunogenic advantage. We measured similar levels of anti-CHO and anti-MT1-h Ab responses in animals injected with either CHO-MT1-h cells or ZZTat101/CHO-MT1-h cells, with or without adjuvant, indicating that neither the adjuvant mixture nor ZZTat101 increased the anti-cell immune response. Then, we investigated whether the antisera also recognized murine MT1 (MT1-m). Using cloned CHO cells transfected with a plasmid encoding MT1-m, we found that antisera raised against CHO-MT1-h cells also bound the mouse receptor. Altogether our studies indicate that immunizing approaches based on MT1-h-expressing CHO cells allow the production of polyclonal antibodies against MT1 receptors of different origins. This paves the way to preparation of MT1-specific monoclonal antibodies.

Production of antigen-specific human IgGs by in vitro immunization.

BackgroundWe previously developed in vitro immunization based on a fusion protein containing the transcriptional transactivator (Tat) of human immunodeficiency virus and a double domain, called ZZ, derived from protein A of Staphylococcus aureus. In this approach, naïve human peripheral blood mononuclear cells (PBMCs) trigger a specific IgM antibody (Ab) response in the presence of ZZTat. In the present study, we attempted to raise a specific IgG Ab response.ResultsWe found that PBMCs incubated with ZZTat and a mixture containing anti-CD40, IL4 and IL21 secrete anti-Tat IgG Abs in their supernatants, indicating that the cytokine cocktail provides an isotypic switch. Then, we deciphered the Tat determinant involved in the phenomenon and found that it is located in the region 22–57 and that, within this region, the cysteine-rich domain and the basic residues play a crucial role. Finally, we prepared a fusion protein containing a fragment derived from the NY-ESO-1 cancer/testis antigen (Ag) and showed that PBMCs incubated with ZZfNY-ESO-1Tat trigger a specific anti-fNY-ESO-1 IgG Ab response, which demonstrates the possibility of transferring immunizing ability to an Ag unrelated to Tat.ConclusionOur ZZTat-based in vitro immunization approach that offers the possibility to raise an IgG Ab response against NY-ESO-1 might represent a valuable first stage for the generation of fully human IgG specific Abs.

Pharmacological Investigations of New Peptido-Cyclodextrins

Cyclodextrins (CD’s) could be used as molecular carrier dedicated to drug targeting(1). In a previous work, we synthesized and characterized height new different peptido-cyclodextrins. They are composed with a s- or γ-CD part, and a peptidic part constituted of the neuropeptide Substance P (noted SP, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2)(2) or one of its derivatives, the SP 4-11. Products obtained were s- or γ-CD(Lys3)-SP, s- or γ-CD(Argl)-SP, di-s- or di-γ-CD(Argl, Lys3)-SP (noted CD-SP) and s- or γ-CD-SP 4-11. More, we demonstrated the preservation of the inclusion properties of CD’s part of these compounds. In this communication, we reporte in vitro pharmacological investigations of these CD-SP. We demonstrate the recognition properties of the diverse conjugates by SP receptor mimics (anti SP polyclonal antibodies), by recombinant human NK1 receptor (using binding experiments on CHO transfected cells) and the production of the second messengers inositolphosphates induced by fixation of the CD-SP on the NK1 receptor.