The role of Product Development Partnerships in research and development for neglected diseases.

Product Development Partnerships (PDPs) are playing an increasingly important role in the development of new medicines for neglected diseases of the developing world; however, there has been limited information on their funding and expenditure patterns. This paper analyses funding for the 14 PDPs working on neglected disease research and development (R&D) by using unpublished data from the Global Funding of Innovation for Neglected Diseases (G-FINDER) project, which surveyed 2007 global investments into R&D of products for neglected diseases. PDPs captured US$469 million or 23% of 'external' R&D funding for neglected diseases, i.e. funding granted by donors to research organisations, as opposed to internal investments by donors. PDP's funding sources were highly concentrated with the Gates Foundation providing nearly half of PDPs' combined income (49%) and four public funders (the US Agency for International Development (USAID), the UK Department for International Development (DFID), the Dutch government and Irish Aid) providing 28%. PDPs collectively spent US$262 million on R&D activities in 2007, with 88% of this expenditure going to academic institutions, contract research organisations and companies in the developed world. Our analysis confirms the central role played by PDPs in R&D for neglected diseases, but highlights the need to diversify their funding sources.

The recombination-activating gene 1 of Pleurodeles waltl (urodele amphibian) is transcribed in lymphoid tissues and in the central nervous system.

The recombination-activating gene 1 (RAG1) product is required for the somatic rearrangement of immunoglobulin and T-cell receptor genes. We cloned and sequenced the large continuous open reading frame coding for the salamander Pleurodeles waltl RAG1 protein. Semi-quantitative RT-PCR experiments were performed to quantify the expression of RAG1 in different tissues. The strongest signal was observed in the thymus of juvenile animals, confirming the primary lymphoid nature of that organ. Weaker expression was observed in the spleen, brain, and eyes of adults. Signals in these tissues represented 5.5%, 4.6%, and 2.0%, respectively, of the signal detected in the thymus. Expression in brain was confirmed by in situ hybridization. Similarly, low amounts of RAG1 transcripts were previously detected in the mouse brain. Moreover, the transcription of RAG1 begins as early as the neurula stages of development. These data suggest that the RAG1 protein could play a role in the central nervous system of vertebrates.

Phenothiazine-induced increase in thyroid autoantigens and costimulatory molecules on thyroid cells: a pathophysiological mechanism for drug-induced autoimmunity?

We have previously demonstrated (J Immunol 1995; 154:3593) that MHC class II antigens can be induced on thyroid epithelial cells (TEC) by alimemazine, a member of the phenothiazine group. Although this expression of MHC class II antigens on TEC confers the theoretical ability to behave as antigen-presenting cells (APC), the simultaneous expression of self antigens and co-receptor(s) must also occur for efficient presentation of self antigens. Therefore, we investigated whether alimemazine applied at pharmacologic doses would modify the expression of thyroid antigens, and simultaneously, the expression of intercellular adhesion molecule-1 (ICAM-1), B7, and LFA-1 co-receptors in human TEC in culture. Using polymerase chain reaction (PCR) amplification and Northern blot analysis, we showed that alimemazine induces increases in thyroglobulin (Tg) and thyroid-stimulating hormone receptor (TSH-R) cDNA, within the first 2 h following its addition. This phenomenon is followed 48 h later by an increase of Tg and TSH-R protein expression on the surface of TEC. Furthermore, increases in the expression of ICAM-1 and B7 co-receptors were concomitantly observed. These results suggest that alimemazine, a drug currently used in paediatrics, could play a role in the induction and perpetuation of thyroid autoimmune disorders by transforming TEC into functional APC.

Grafts of ovaries in males and females of Pleurodeles waltl (urodele amphibia): evidence of a long-term tolerance of allografts; application following a space biology experiment.

Ovary grafts were investigated in the salamander Pleurodeles using juveniles and adults as donors and hosts. Ovaries were provided by standard or histo-compatible strains and by standard females which had been submitted to a space flight. Laparotomy of the hosts was used to control viability of grafts. Entire juvenile ovaries transplanted into castrated juvenile females or males were tolerated and developed. Ovarian parts of adult females, which contained a majority of oogonies, could also be tolerated by juvenile animals. In addition, ovarian parts supported a better recovery and differentiation than parts that mainly included mature oocytes. About 24 months after the ovary grafts, some hosts (genetical females or males and standard or spatialized females) crossed with standard males provided progenies originating from oocytes of the grafted ovaries. The protocols applied offer a new range of potentialities, adapted to various experimental purposes such as life science research in space or sex differentiation studies.

Molecular heterogeneity of antigen- or idiotype-induced anti-thyroglobulin monoclonal autoantibodies.

To define the molecular basis of the cognitive interaction in experimental autoimmune thyroiditis (EAT), we sequenced the variable regions of monoclonal autoantibodies to thyroglobulin (Tg), specific or not for the F40D peptide, a Tg peptide capable of inducing EAT in CBA/J mice. Three MoAbs were obtained by immunization with syngeneic Tg of CBA/J (3B8G9, 2F6F2) or C57Bl/6 (4D11F4) mice. 3B8G9 was specific for F40D peptide, whereas 2F6F2 and 4D11F4 were not. Two others were raised in CBA/J mice by manipulation of idiotypic pathways: B12 resulted from the immunization with one Ab2 beta, bearing the internal image of one F40D epitope, and TA2 from the immunization with F40D-specific cytotoxic HTC2 T cells. B12 and TA2 were both specific for F40D. All hybridomas expressed different members of the J558 VH family, except 3B8G9 which expressed a Q52 VH gene segment. These data led us to hypothesize that regulatory anti-id autoantibodies used members of one VH family located in the 5'-end of the VH locus, whereas EAT-associated autoantibodies used a member of one of the most D-proximal VH family. As expected, no homologies were found when anti-F40D monoclonal autoantibodies were compared with two other monoclonal autoantibodies displaying a different epitopic specificity. Among the anti-F40D monoclonal autoantibodies, one histidine residue located in position 35 of the CDR1 region was constantly found. Moreover, TA2 and B12 exhibited two common amino acids in their CDR3 regions, one glycine and one tyrosine, in positions 98 and 99, respectively. Striking homologies were found between TA2 and one anti-polyGAT MoAb, and between 3B8G9 and some anti-phenyloxazolone (phOx) monoclonal autoantibodies. Lastly, the VK sequence from 4D11F4 was identical at the amino acid level to the VK sequence from another monoclonal autoantibody, 81B1, which was previously raised towards syngeneic Tg in CBA/J mice. Our data imply that anti-idiotypic regulatory circuits in EAT might be generated by a heterogeneous population of B cells rather than obtained by a single dominant B cell population.

Phenothiazine induces de novo MHC class II antigen expression on thyroid epithelial cells. A new mechanism for drug-induced autoimmunity.

Autoimmune responses are initiated by MHC class II-restricted T cell responses directed against tissue-specific autoantigens. Furthermore, HLA-DR expression in thyroid epithelial cells is a prominent feature of autoimmune thyroid disease. In the present work, we were particularly interested in a phenothiazine, a neuroleptic and anti-depressant drug of pharmacologic importance named alimemazine. Our interest in this compound stems from previous findings of immune effects of this and other phenothiazines. We demonstrate that MHC class II Ags can be experimentally induced on thyroid cells by pharmacologic concentrations of alimemazine, a drug commonly used in psychiatry. In contrast, MHC class II Ags were not induced on the lymphoid cell lines Raji and Jurkat. Expression of MHC class II Ag on the surface of the cloned human thyroid cell hybridoma, GEJ, was demonstrated by flow cytometry. Moreover, by using Northern blot and Southern blot analyses, this finding was confirmed at the molecular level in GEJ and in murine thyroid epithelial cell cultures, respectively. The functional role of phenothiazine-, de novo-induced MHC class II Ags on thyroid cells was assessed by both syngeneic murine thyroglobulin-specific and allogeneic proliferative T cell responses. These results suggest that antidepressant drugs of the phenothiazine type could play a role in the induction and the perpetuation of thyroid autoimmune disorders, through induction of class II restriction elements on normally class II-negative thyroid epithelial cells.

One monoclonal antibody to human thyrotropin (TSH) receptor agonist of TSH hormone stimulates the proliferation of human thyroid cells.

The question of thyroid cell growth induction by monoclonal antibodies (mAbs) to human thyrotropin receptor (hTSH-R), which stimulate increases in cAMP thyroid cells, is under debate and their implication in Graves' disease (GD) is controversial. In order to address this issue, we used characterized reagents, i.e., mAbs to hTSH-R, and cloned human thyroid hybridoma cells (GEJ). Cell counting on Days 1, 2, and 3 after addition of mAb and Northern blot analysis of mRNA specific for the c-fos oncogene were used to assess the proliferation of the thyroid cells. MAbs to hTSH-R, obtained by immunization of DBA/1 mice with affinity-purified hTSH-R, were added to GEJ cells in concentrations varying from 0.66 to 660 nM. Their effects on GEJ cell growth were compared to those of human TSH, of hLH, and of control mAb. Cell counting and evaluation of GEJ c-fos transcripts showed that mAbs to hTSH-R induce significant GEJ cell growth, whereas they were ineffective on the control cell line. Among them, one mAb, 34A, exhibited an impressive activity on thyroid cell growth and induced cAMP production comparable to that induced by bovine or human TSH. Our data demonstrate the existence of immunoglobulin which stimulates thyroid growth and elevates cAMP. The higher the cAMP production, the greater the thyroid cell proliferation. Furthermore, the data suggest a possible role for agonistic anti-hTSH-R autoantibodies in the immunopathogenesis of GD.

Antibodies specific for human thyrotropin receptor induce MHC antigen expression in thyroid cells.

Autoantibodies (AAbs) to hormone receptors are found in autoimmune diseases such as Graves' disease (GD) or myasthenia gravis. A structural link between hormone receptor and MHC genes has been documented, suggesting a possible co-regulation of MHC and hormone receptor genes. Thus, in vitro experiments were designed to search for a pathologic role for AAbs. In a model study, we investigated whether adding murine anti-human thyrotropin receptor mAbs would affect MHC gene expression in either cloned human thyroid epithelial cell or primary murine thyroid epithelial cell cultures. We found that two anti-human thyrotropin receptor monoclonal AAbs, 11E7 and 34A, induced, with an intensity comparable to that of IFN-gamma, transcription and expression of class I and class II/Ii chain proteins in human and murine thyroid epithelial cells. Two other anti-human thyrotropin receptor mAbs, 12E3 and 243-3, were ineffective. These data suggest a new role for autoantibodies in the pathology of autoimmune endocrinopathies.

Induction of autoimmunity by immunization of mice with human thyrotropin receptor.

The development of autoimmunity was investigated after repeated immunizations with human thyrotropin receptor (hTSH-R) of five congenic strains of female and male mice. After each immunization, free T3 levels and antibodies to hTSH-R and to six peptides of the hTSH-R were assayed. Our results showed that H-2s and H-2q female mice developed features of autoimmunity such as antibody responses to hTSH-R and to hTSH-R peptides, transient variations in the levels of free T3 thyroid hormone, and lymphocytic infiltrations in their thyroid glands. Concerning the antibody responses to hTSH-R peptides, we found that peptide P1 (352-366) contained a major B cell epitope. Furthermore, strain-specific B cell epitope was exemplified by peptide 92 (12-30) and two male- and female-specific B cell epitopes were located in peptides 91 (32-46) and 93 (316-330), respectively. These features appeared rather related to hyperthyroidism.

Production of Ab2 beta, anti-idiotypic monoclonal antibodies, specific for human thyrotropin receptor.

We had previously selected five monoclonal antibodies (mAb1) upon their specific binding to human thyrotropin (hTSH). These mAbs1, which are specific for two distinct epitopes of the hTSH beta-chain, also inhibit the hTSH binding to human thyroid membrane preparations. We then immunized BALB/c mice with the various mAbs1 in attempt to generate anti-idiotypic Abs (Ab2) directed to the hTSH receptor (hTSHR). Five hybridomas secreting monoclonal antibodies (mAb2) to the hTSHR were selected from one mouse immunized with the mixture of the five mAbs1. These monoclonal antibodies to the hTSHR were selected upon their ability to specifically bind to the hTSHR. Moreover, we showed that binding of mAbs2 to the individual mAbs1 used for immunization was specifically inhibited by hTSH and not by insulin or by human chorionic gonadotrophin, a highly related glycoprotein hormone. The five mAbs2 inhibited the binding of iodine-labeled hTSH to its receptor by 8 to 41%; moreover, two of them stimulated the adenylate-cyclase system of the thyroid cells. With respect to their properties, mAbs2 were classified as mAb2-beta.

Ig VH gene family usage in spleen cells of CBA/J mice immunized with experimental autoimmune thyroiditis (EAT) inducer antigens.

Experimental autoimmune thyroiditis (EAT) is an autoimmune disorder of the thyroid gland induced in susceptible strains of mice by thyroglobulin (Tg). We recently showed that low Mr (< 10 kDa) Tg tryptic fragments and a 40 amino-acid peptide (F40D) from Tg could induce EAT as well as native Tg. Because it has been reported that autoantibodies (A-Abs) express VH families preferentially located in the D-proximal VH gene segment, we investigated whether A-Abs specific for one pathogenic peptide from Tg were also skewed towards D proximal VH gene segment. In that respect, we immunized CBA/J mice with EAT inducer antigens of decreasing sizes: Tg (660 M(r)), < 10 kDa Tg trypic fragments or F40D peptide (4.9 kDa M(r)) from Tg. The VH gene segments utilized by immune spleen cells were determined by hybridization to total spleen cell RNA previously deposited onto nylon membranes and densitometric scans. This study was conducted on days 7 and 9 after determination of the maximum amounts of mRNA coding for immunoglobulins and on day 28 when A-Ab levels are the highest. Results were compared to VH gene segment expression both in normal and adjuvant-injected mice. We found that immunization of CBA/J mice with EAT inducer antigens stimulate B cells the restriction of which, in terms of VH family usage, depends on the size of the immunizing antigen: the larger the antigen, the higher the numbers of VH families used. Moreover, we found that B cell stimulation consecutive to immunization with the peptidic antigen inducing EAT occurs in VH Q52 family, a VH encoded by D-proximal gene segment.

Characterization of monoclonal antibodies to the human thyrotropin receptor.

We have produced four monoclonal antibodies (mAbs), 34A, 49G, 11E7, and 12E3, which bind the human TSH receptor (hTSH-R) when expressed on a human thyroid cell line (GEJ), freshly dissociated human and murine thyroid cells, or Chinese hamster ovary cells stably transfected with the hTSH-R gene. These mAbs were obtained after immunization of DBA/1 mice with affinity-purified TSH-binding sites from GEJ cells. Biochemical studies, including sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, Western blot, and immunoprecipitation of solubilized GEJ cell membranes or human thyroid cells showed that most of the mAbs recognized two bands: one located at 46-48 kilodaltons and the other at 86-88 kilodaltons. Inhibition of [125I]hTSH binding to solubilized porcine membranes (TSH-receptor auto-antikörper assay) or Chinese hamster ovary cell membranes previously transfected with hTSH-R gene showed that mAb 34A recognizes the hTSH-binding site of both receptors. In contrast, mAbs 49G, 11E7, and 12E3 recognize a structure located near the hTSH-binding site. Lastly, the ability of these mAbs to stimulate murine thyroid function was investigated by measuring cAMP production and iodide accumulation. The 34A mAb, which fully competes with [125I]TSH for binding to hTSH-R, was able to induce both functions. Conversely, the 12E3 mAb, which was the least potent inhibitor of [125I]TSH binding to hTSH-R-transfected cells had no effect. A relationship was, therefore, established between the capacity of mAb to hTSH-R to inhibit [125I]hTSH binding and their ability to induce thyroid functions.