[Adenosine deaminase 1 deficiency, an inborn error of metabolism underlying a severe form of combined immunodeficiency]. / Déficit complet en adénosine-désaminase-1 : une erreur innée du métabolisme responsable d'un déficit immunitaire combiné sévère.

Severe combined immune deficiencies (SCIDs) are a heterogeneous group of severe cellular immunodeficiencies. Early diagnosis is essential to allow adapted care before life-threatening systemic infections or complications associated with live vaccines. Adenosine deaminase 1 deficiency (ADA1) is an inborn error of metabolism leading to severe lymphopenia and characteristic bone lesions. Herein, we present the typical case of a child in whom ADA SCID was diagnosed at 2 months of life, revealed by lung involvement and extreme lymphopenia. Immune restoration in terms of peripheral lymphocyte count with enzyme replacement therapy, namely pegylated bovine ADA, is satisfactory so far. The search for a compatible donor is underway. Correcting the genetic defect by gene transfer is also being considered. The phenotype of this very rare condition is described. A severe peripheral lymphopenia in a young child is a finding of utmost importance for the diagnosis of a primary cellular immunodeficiency.

How reliable are models for malaria vaccine development? Lessons from irradiated sporozoite immunizations.

Models occupy a key position in the development of anti-parasitic vaccines, yet their relevance has been seldom addressed. It is customary to admit that malaria vaccine development requires easy-to-handle, laboratory models. Animal models involving predominantly inbred rodents and primates as parasite hosts are currently the basic tools for the study of host-parasite interactions. Literature however indicates that the induction of host protection is more difficult in natural host-parasite pairs than in experimental models of parasite infection. Moreover different models delineate a wide range of host-pathogen relationship profiles providing a mosaic of contradictory informations, yet there is little incentive to delineate their relevance or to exploit recent advances to develop improved model systems. In this context the analysis of natural host-parasite interactions between Plasmodium berghei and its mammalian host and reservoir, the tree rat Grammomys surdaster could ge of relevance in the study of host-parasite interactions.

Premunition against Plasmodium falciparum in a malaria hyperendemic village in Myanmar.

Premunition, naturally acquired protective immunity against Plasmodium falciparum, has been described in hyperendemic areas of Africa and Papua New Guinea. However, its occurrence in Asia is debatable. In order to elucidate this question, a longitudinal study was undertaken in Oo-Do, a malaria endemic village in Myanmar [Burma] in 1995-97. Only 2 species, Plasmodium falciparum and P. vivax, were detected, with the former predominating. Data from 116 subjects showed that all were infected at one time or another, over a period of 3 years, with a 38% reinfection rate after eradication of patent parasitaemia. The high rate of prevalence (90-100%) of parasite-specific antibodies in the indirect immunofluorescence antibody test and the presence of the primary vector (Anopheles minimus) and 15 other species of Anopheles throughout the year indicated a high level of transmission. The spleen rate was 70% in 5-9 years old children and was inversely related with age. The incidence of parasitaemia was maximal (49%) in children aged 2-4 years, and then declined marginally with age. There was a significant difference (P = 0.001) between the asymptomatic and febrile parasitaemia levels. Also, malarial episodes occurred more frequently in children than in adults (P = 0.001). Taken together, all these facts indicated that the inhabitants of Oo-Do had progressively developed non-sterile partial protective immunity against P. falciparum malaria, or premunition. To our knowledge, this is the first detailed clinico-epidemiological study to document the occurrence of premunition in Myanmar.

Mutations in ribosomal protein S19 gene and diamond blackfan anemia: wide variations in phenotypic expression.

Mutations of the ribosomal protein S19 (RPS19) gene were recently identified in 10 patients with Diamond Blackfan anemia (DBA). To determine the prevalence of mutations in this gene in DBA and to begin to define the molecular basis for the observed variable clinical phenotype of this disorder, the genomic sequence of the 6 exons and the 5' untranslated region of the RPS19 gene was directly assessed in DBA index cases from 172 new families. Mutations affecting the coding sequence of RPS19 or splice sites were found in 34 cases (19.7%), whereas mutations in noncoding regions were found in 8 patients (4.6%). Mutations included nonsense, missense, splice sites, and frameshift mutations. A hot spot for missense mutations was identified between codons 52 and 62 of the RPS19 gene in a new sequence consensus motif W-[YFW]-[YF]-x-R-[AT]-A-[SA]-x-[AL]-R-[HRK]-[ILV]-Y. No correlation between the nature of mutations and the different patterns of clinical expression, including age at presentation, presence of malformations, and therapeutic outcome, could be documented. Moreover, RPS19 mutations were also found in some first-degree relatives presenting only with isolated high erythrocyte adenosine deaminase activity and/or macrocytosis. The lack of a consistent relationship between the nature of the mutations and the clinical phenotype implies that yet unidentified factors modulate the phenotypic expression of the primary genetic defect in families with RPS19 mutations.

High adenosine deaminase level among healthy probands of Diamond Blackfan anemia (DBA) cosegregates with the DBA gene region on chromosome 19q13. The DBA Working Group of Société d'Immunologie Pédiatrique (SHIP).

Phenotypic characterization of Diamond Blackfan Anemia (DBA) patients and their relatives was performed in 54 families. Complete blood count, fetal hemoglobin level, erythrocyte i antigen expression, and erythrocyte adenosine deaminase (eADA) activities were quantitated in patients and relatives. eADA was elevated in 28 of 34 transfusion-independent DBA patients, whereas persistence of erythrocyte i antigen was noticed in only 10 of 20 DBA patients. High eADA activities were also found in 14 of 149 healthy family members, allowing us to identify an isolated high eADA phenotype in these families. In contrast, increase in erythrocyte i antigen expression, elevated fetal hemoglobin levels, and macrocytosis were much less frequently noted in nonaffected members of the DBA families studied. Importantly, isolated high eADA phenotype was found to be significantly associated with genetic markers on chromosome 19 that segregate with the DBA phenotype. Isolated high eADA phenotype thus seems to reflect a silent phenotype of DBA in affected families. These findings suggest that elevated eADA activity in unaffected individuals needs to be taken into account during genetic assessment of DBA families and could be used for accurate assessment of mode of inheritance.

Regulation of adenylyl cyclase activity in human peripheral blood mononuclear cells: effects of protein kinase inhibitors and of a calcium ionophore.

In a previous paper we presented evidence for a negative regulation of adenylyl cyclase activity by tyrosine protein kinase(s) in the human leukemic T cell line Jurkat. In order to examine this point in non malignant cells, we conducted the present study in human peripheral blood mononuclear cells (PBMC). In these cells, staurosporine, a broad spectrum protein kinase inhibitor, enhanced not only the receptor-mediated, induced by prostaglandin E2 (PGE2), but also the direct (forskolin-induced) stimulation of adenylyl cyclase activity. Herbimycin A, a specific protein tyrosine kinase inhibitor, reproduced only in part the effect of staurosporine, whereas bisindolylmaleimide, the most specific protein kinase C (PKC) inhibitor known at present time, was ineffective. All these observations were made both in the absence and presence of isobutylmethylxanthine, a phosphodiesterase inhibitor, indicating that the effects of staurosporine and herbimycin A on cAMP accumulation were not due to phosphodiesterase inhibition. The calcium ionophore A 23187 also enhanced the PGE2-induced cAMP accumulation, and this effect was not additive to that of staurosporine, but additive to that of herbimycin A. These results confirm and extend those obtained in Jurkat cells. Taken together, they indicate that in human PBMC the adenylyl cyclase activity is negatively regulated by tyrosine kinase(s) and not by PKC, and positively regulated by Ca2+. They also suggest that the major enhancement by staurosporine of the PGE2-induced cAMP accumulation, although chiefly mediated by protein tyrosine kinase inhibition, also depends on another, presently undetermined, effect of the drug simulating that of Ca2+.

A hypothesis about the chronicity of malaria infection.

It is generally accepted that malaria evolves as a chronic blood infection by escaping the immune responses directed against a series of antigens that express variable epitopes and/or by selecting parasite populations with distinct polymorphic antigens. However, exacting in vitro studies, performed with clinically well-defined biological material, have correlated the state of protection of African adults (in whom low-grade infection persists) with an indirect defence mechanism where the antibodies are effective owing to their ability to cooperate with blood monocytes. Further studies showed that the antibody bridges the parasite (at the merozoite stage) with a monocyte and triggers the release of mediators which have a parasitistatic, reversible and non-antigen-specific effect. The fact that the parasite directly triggers the antiparasite effect leads Pierre Druilhe and Jean-Louis Pérignon to formulate here an alternative hypothesis for the chronicity of malaria infection, which would rely on conserved antigenic targets and, in contrast with direct mechanisms, would not select emerging mutated parasites. The above two mechanisms are discussed in the context of their fitness with clinical and parasitological observations. It is proposed that they are not mutually exclusive but, rather, may come into play successively as patients gradually evolve from high-grade symptomatic to low-grade asymptomatic parasitic infection.

Staurosporine and herbimycin A augment agonist-induced elevation of cAMP in Jurkat T-lymphoblasts.

Cross-talk between signalling pathways appears to play an important role in T-lymphocyte activation. In the present work, we have studied the effects of different inhibitors of protein tyrosine kinases or protein serine/ threonine kinases on the agonist-induced cAMP accumulation in the human T-lymphoblast cell line Jurkat. Staurosporine, a potent but nonspecific inhibitor of protein kinases, produced a ten-fold enhancement of the response to PGE2. No significant effect was obtained with two specific protein kinase C inhibitors (GF 109203X and H7), whereas herbimycin A, a specific protein tyrosine kinase inhibitor, markedly enhanced the PGE2-induced cAMP accumulation: its effect was approximately 60% that of staurosporine. It was confirmed that both staurosporine and herbimycin A inhibited by more than 90% the release of IP3 induced by ligation of the T-cell receptor, a known protein tyrosine kinase-dependent mechanism. To our knowledge, this study provides the first indication of a protein tyrosine kinase-mediated inhibition of agonist-induced cAMP accumulation. The possible targets of this inhibition are discussed.

Bone marrow gene transfer in three patients with adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency results in severe combined immune deficiency disease (SCID), which is fatal without treatment. Allogeneic bone marrow transplantation (BMT) is the treatment of choice if an HLA-identical sibling bone marrow donor is available, resulting in almost 100% cure rate. BMT-related mortality is high in patients lacking such a donor. For these patients, efficient transfer of a recombinant ADA gene into hematopoietic stem cells is a therapeutic option if it results in the outgrowth of a 'genetically repaired' lymphoid system. Based on successful gene transfer studies in monkeys, we performed retrovirus-mediated gene transfer into CD34+ bone marrow cells of three patients with ADA deficiency. Two patients received bovine ADA conjugated to polyethylene glycol (PEG-ADA); in the third patient, PEG-ADA was started 4 months after gene transfer. Gene transfer resulted in a 5-12% transduction frequency of in vitro colony forming cells (CFU-Cs). No toxicity was observed during and after infusion of the graft. Following infusion of the transduced CD34+ cells, transduced granulocytes and mononuclear cells persisted in the circulation for 3 months. In addition, the gene was present in the marrow of one of the patients at 6 months after gene transfer. Expression of the gene was not detected. After this period, the gene could not be detected. In monkey studies we showed that myeloablation, which was not performed in the patients, may enhance engraftment of genetically modified cells. We hypothesize that lack of myeloablation, administration of bovine ADA and low numbers of transduced progenitor cells all may have contributed to the relative low numbers of transduced cells in the patients. Under these conditions, no selective advantage of the genetically corrected progenitor cells was observed.

Four new adenosine deaminase mutations, altering a zinc-binding histidine, two conserved alanines, and a 5' splice site.

Three new missense mutations (H15D, A83D, and A179D) and a new splicing defect (573 + IG-->A) in the 5' splice site of intron 5 were among six mutant adenosine deaminase (ADA) alleles found in three unrelated patients with severe combined immunodeficiency disease, the most common phenotype associated with ADA deficiency. When expressed in vitro, the H15D, A83D, and A179D proteins lacked detectable ADA activity. The splicing defect caused skipping of exon 5, resulting in premature termination of translation and a reduced level of mRNA. H15D is the first naturally occurring mutation of a residue that coordinates directly with the enzyme-associated zinc ion. Molecular modeling based on the atomic coordinates of murine ADA suggests that the D15 mutation would create a cavity or gap between the zinc ion and the side chain carboxylate of D15. This could alter the ability of zinc to activate a water molecule postulated to play a role in the catalytic mechanism. A83 and A179 are not directly involved in the active site, but are conserved residues located respectively in alpha helix 4 and beta strand 4 of the alpha/beta barrel. Replacement of these small hydrophobic Ala residues with the charged, more bulky Asp side chain may distort ADA structure and affect enzyme stability or folding.

Mechanisms of defense against P. falciparum asexual blood stages in humans.

Based on the observation that host-parasite immune relationships depend not only on the parasite species but also on the host, we consider that P. falciparum can be best studied in humans. In vivo observations suggest that there are in humans a series of immunities towards P. falciparum: besides innate non-antigen-specific defenses, which play a major role, additional defense mechanisms are acquired after consecutive malaria attacks. They contribute to realize such various states of immunity as acquired resistance to cerebral malaria, anti-disease immunity, strain-specific immunity, and, after a long time of exposure in hyper- or holoendemic areas, the host-parasite equilibrium characteristic of premunition. Passive transfer experiments have established that IgG play a major role in premunition. We review, on the one hand, in vitro evidences that protective antibodies have the characteristic capacity of promoting a monocyte-dependent inhibition of parasite growth (ADCI) and, on the other hand, the in vivo observations that suggest that this mechanism of defense could be one of the immune foundations of the state of premunition.

Immune mechanisms underlying the premunition against Plasmodium falciparum malaria.

The most unique characteristic of a parasite when it is in its normal host is the ability to make itself tolerated, which clearly indicates that it has sophisticated means to ensure the neutrality of its host. This is true also in the case of Plasmodium falciparum, since after numerous malaria attacks an equilibrium is reached with a chronic stage of infection, characterized by a relatively low parasitemia, and low or no disease (Sergent & Parrot 1935). We shall briefly review the main characteristics of this state of "premunition", and present data suggesting that the underlying mechanisms of defense rely on the cooperation between cell and antibodies, leading to an antibody dependent cellular inhibition of the intra-erythrocytic growth of the parasite.

Fast immunopurification of small amounts of specific antibodies on peptides bound to ELISA plates.

ELISA is widely used as a means to detect antibodies, but the potential of ELISA plates as an immunosorbent for the purification of specific antibodies does not seem to have been evaluated. In this study, ELISA plates coated with peptides representing short sequences of various antigens from Plasmodium falciparum, the etiologic agent of human malaria, have been successfully used as a means to purify small amounts of the corresponding antibodies. ELISA plates, identical to those used for antibody detection, also permitted the evaluation of various elution conditions for each pairing of peptide and serum; we tested four eluting buffers (0.2 M glycine, pH 2.5; 0.2 M lysine, pH 11.5; 3.0 M MgCl2, 0.075 M Hepes, 25% ethylene glycol, pH 7.1-7.2 and 4 M NH4SCN in 0.1 M NaH2PO4, pH 6.0) with four pairs of peptides and sera. The ELISA plates could also be used to estimate the affinity of the eluted antibodies by the technique of Pullen et al. (1986). The eluted antibodies were compared to those obtained by immunopurification on recombinant proteins adsorbed on nitrocellulose filters. In contrast to the latter, they were not contaminated by antibodies directed against the carrier moiety of the recombinant protein. When used in immunofluorescence assays with various stages of the parasite the antibodies immunopurified on peptides bound to ELISA plates were able to react with the native antigens in the parasite.

T-cell antigen receptor-mediated enhancement of the adenylate cyclase pathway depends on tyrosine protein kinases.

We have examined in the human T-cell line Jurkat the interaction between the activation through the T-cell receptor/CD3 complex and the adenylate cyclase pathway. OKT3, an anti-CD3 monoclonal antibody, did not activate by itself adenylate cyclase but produced a 3-7-fold increase of the cAMP accumulation induced by indirect (chloroadenosine, PGE2) or direct (forskolin) agonists of adenylate cyclase. A more detailed study with forskolin showed that OKT3 enhanced the effect of low concentrations of the agonist without affecting the maximal capacity of cAMP synthesis of the cells. The same concentrations of OKT3 produced both the enhancement of the adenylate cyclase pathway and the activation of phospholipase C. The enhancement by OKT3 of the adenylate cyclase pathway was inhibited by 0.5 microM staurosporine, a potent inhibitor of protein kinases, including tyrosine kinases and protein kinase C, whereas it was not inhibited by H7, a specific inhibitor of PKC. Staurosporine, at the same concentration, also inhibited the OKT3-induced activation of phospholipase C, a tyrosine kinase-dependent process. Taken together, these data indicate that activation of T-cell through the T-cell receptor enhances the adenylate cyclase pathway by a tyrosine protein kinase-dependent mechanism.