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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., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

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.

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.

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.

Hypermethioninemia and absolute methionine intolerance were observed in three siblings. These patients had several peculiar clinical features comprising failure to thrive, mental and motor retardation, facial dysmorphy with abnormal hair and teeth, and myocardiopathy. Hepatic S-adenosylhomocysteine hydrolase activity was decreased by 80% in the three children. These clinical and biochemical features differ from those of hypermethioninemias previously described, and thus represent a new form of inherited disorder of methionine metabolism. Whether S-adenosylhomocysteine hydrolase deficiency is primary or secondary to an unknown metabolic defect remains to be determined.

An experimental model of adenosine deaminase deficiency was established on the human T cell line Jurkat by using 2'-deoxycoformycin, a strong specific inhibitor of the enzyme. When deoxyadenosine was added to the inhibited cells, the nucleotide profile was modified reproducing that found in lymphocytes from adenosine deaminase-deficient children. The metabolism of phosphoinositides, analyzed by either the release of [3H]inositol phosphates or the breakdown of 32P-prelabeled phosphatidyl inositides, was compared in normal and modified cells where dATP was accumulated. No modification in 32P labeling of phosphoinositides was detectable within the 32P-loading period. However, when the cells were stimulated by phytohemagglutinin or anti-CD3 monoclonal antibody, the phosphoinositide hydrolysis was strongly reduced in the dATP-containing lymphoblasts. This decrease was correlated with the intracellular dATP concentration.

Xanthinuria, described in 1954 by Dent and Philpot, is a rare metabolic disorder, characterised by a deficiency in xanthine-oxidase, a key enzyme in the synthesis of uric acid. It results in hypouricaemia and hypouricuria, the urinary excretion of products of purine synthesis taking place in the form of uric acid precursors: hypoxanthine and xanthine. By virtue of the very slight solubility of xanthine, this xanthinuria may cause urinary lithiasis, in general occurring early. More often, however, the disease is asymptomatic and diagnosed following the chance discovery of hypouricaemia. We report 6 recent cases.

A severe genetic deficiency of adenosine deaminase is causally associated with an autosomal recessive form of severe combined immunodeficiency disease, while subjects with absent erythrocyte but partial lymphocyte enzyme activity remain immunocompetent. The genetic expression of adenosine deaminase in B-lymphoblast cell lines derived from four unrelated subjects with the "partial" enzyme deficiency was examined. Enzymatic activity among these cell lines ranged from 5 to 50% of normal with the level of immunoreactive adenosine deaminase protein either proportional to enzyme activity or elevated in two of the cases. Northern blot analysis using a cDNA probe showed that adenosine deaminase mRNA in each of these cell lines was of normal expected size (1.6-1.8 kilobases) and was present in normal to above normal amounts. Rates of enzyme synthesis varied from 165 to 15% of normal. Adenosine deaminase protein degradation rates in these cell lines were 1.5 to almost 3 times faster than normal, consistent with the observed absence of the enzyme in erythrocytes. From these analyses apparent abnormalities in mRNA regulation, translation, and protein degradation can be identified among the partially adenosine deaminase-deficient cell lines studied. Ultimately, it will be essential to determine the nature of the protein mutation and the gene defect to define the structural alterations and functional abnormalities of enzyme variants isolated from subjects with partial adenosine deaminase deficiency.

We studied the ability of purine compounds to restore the proliferation of concanavalin-A-stimulated rat T-lymphocytes under conditions of purine de novo synthesis inhibition and, on the other hand, the inhibition by purine nucleosides of the response of these cells to a mitogenic stimulation under conditions of normal purine de novo synthesis. The use of 50 microM azaserine, a potent inhibitor of purine de novo synthesis, allowed us to define the physiologically active salvage pathways of purine bases, ribo- and deoxyribonucleosides in concanavalin-A-stimulated rat T-lymphocytes. Except for guanylic compounds, all purines completely restored cell proliferation at a concentration of 50 microM. Guanine, guanosine and 2'-deoxyguanosine at concentrations up to 500 microM did not allow us to restore more than 50% of the cell proliferation. In conditions of normal purine de novo synthesis, the addition of 1000 microM adenine, adenosine, 2'-deoxyadenosine or 100 microM 2'-deoxyguanosine inhibited rat T-lymphocyte proliferation. The differences between the degree of inhibition of cell proliferation could be explained only in part by the differences between the capacities of salvage of these compounds. Furthermore, the fact that 2'-deoxyguanosine toxicity was dependent and 2'-deoxyadenosine toxicity independent on the activation state of the cells provided more evidence that the biochemical mechanisms of inhibition of cell proliferation should be different for these two nucleosides.

The clinical features and biological results in a second patient with a metabolic defect of the molybdenum cofactor are described. The first case was reported in 1978 by Duran et al. Their clinical description was similar with early encephalopathy and myoclonial and dislocation of the lens. Biologically, this condition is characterised by secondary hypo-uricemia and hypo-uricuria due to xanthine oxidase deficiency and by sulphituria, resulting from sulphite oxidase deficiency. These two enzymes have a common hepatic molybdenum cofactor, the structure and metabolism of which are only partially known.

Complete lack of adenine phosphoribosyltransferase (APRT) is a not uncommon cause of urinary lithiasis in young children. The calculi are made up of a very poorly soluble substance, 2,8-dihydroxyadenine, which results from the oxidation of adenine by xanthinoxidase. A study of the 7 cases of APRT deficiency hitherto published (including 5 cases with lithiasis) shows that the diagnosis is rarely made, since the conventional methods of urinary stone analysis are unable to distinguish dihydroxyadenine from uric acid. This form of lithiasis can be prevented by inhibiting xanthinoxidase with allopurinol. The remarkable efficacy of this treatment and the frequent severity of the disease should raise the possibility of 2,8-dihydroxyadenine lithiasis in very case of alleged uric acid lithiasis in young children.

Inherited deficiency of the enzyme adenosine deaminase (ADA) results in a syndrome of severe combined immunodeficiency (SCID). Children with ADA- -SCID lack ADA in all cells and tissues. In contrast, a "partial" deficiency of ADA has been described in six immunologically normal children from four different "families." These children lack ADA in their erythrocytes but retain variable amounts of activity in their lymphoid cells. We have examined ADA activity in lymphoid line cells from four of these children, who are unrelated, for evidence of genetic heterogeneity. One child, who is Caucasian, has an enzyme with increased electrophoretic mobility, a diminished isoelectric point (pI 4.8 vs. Nl = 4.9) and very low activity (2.3 vs. Nl = 82.9 +/- 12.9 nmol/mg protein per min); as a second child has an enzyme with normal electrophoretic mobility but increased isoelectric point (pI = 5.0), markedly diminished heat stability at 56 degrees C (t1/2 = 4.2' vs. Nl = 40') and low activity (12.1); a third has an enzyme with only diminished heat stability (t1/2 = 6.5'), no detectable abnormality in charge and almost normal activity (41.9); while the fourth exhibits only diminished ADA activity (25.0) with no striking qualitative abnormalities. Thus, we have found evidence for three different mutations at the structural locus for ADA in three of these individuals, (a) an acidic, low activity heat stable mutation (b) a basic, somewhat higher activity, heat labile mutation, and (c) a relatively normal activity heat labile mutation. In the fourth, there is as yet no compelling evidence for a mutation at the structural locus for ADA and a mutation at a regulatory locus cannot be excluded.

The metabolism of the two methionine precursors, L-homocysteine and 5'-deoxy-5'-methylthioadenosine was compared to the ability of these compounds to support cell growth in a Met-free medium, in the following mammalian cell lines: Raji, CCL 39 and BHK cells. These three cell lines metabolized L-homocysteine and 5'-deoxy-5'-methylthioadenosine into methionine, S-adenosyl-L-methionine and proteins. However there was a discrepancy between metabolic and growth studies: Raji cells could grow on L-homocysteine and on 5'-deoxy-5'-methylthioadenosine, BHK cells could grow on L-homocysteine but not on 5'-deoxy-5'-methylthioadenosine, and CCL 39 cells could not grow either on L-homocysteine or on 5'-deoxy-5'-methylthioadenosine. The metabolism of exogenous methionine, and of methionine endogenously synthesized from 5'-deoxy-5'-methylthioadenosine was studied in CCL 39 and Raji cells, incubated with 25 microM [methyl-14c] methionine + 25 microM 5'-deoxy-5'-methylthioadenosine or 25 microM [methyl-14c] 5'-deoxy-5'-methylthioadenosine + 25 microM methionine: there was no difference between the metabolism of exogenous and endogenous methionine in either type of cell. Our results indicate that i) "methionine dependence" initially described for L-homocysteine [Hoffman, R.M. and Erbe, R.W. (1976) Proc. Natl. Acad. Sci. USA 73, 1523], can also be observed with the other precursor of methionine, ie 5'-deoxy-5'-methylthioadenosine; ii) "methionine-dependence" can not be considered as the inability of a cell to grow on methionine endogenously synthesized from a precursor, but depends on the precursor used, and possibly, on a toxic effect of the precursor in the absence of methionine.

5'-Deoxy-5'-methylthioadenosine inhibits Concanavalin A induced rat T lymphocyte proliferation in a dose dependent manner (50 microM to 1000 microM). The extent of inhibition by MTA of lymphocyte proliferation was greatest when MTA was added to the cells at the same time as Concanavalin A. The determination of cyclic AMP level from 30 min to the 6th hour shows that 5'-Deoxy-5'-methylthioadenosine inhibition is correlated with an elevation of cyclic AMP at this mitogen recognition phase. 5'-Deoxy-5'-methylthioadenosine concentrations that inhibit rat T lymphocyte proliferation also inhibit phosphodiesterase activity. This biochemical mechanism could be specific to 5'-Deoxy-5'-methylthioadenosine inhibition since in an another model of inhibition of rat T lymphocyte proliferation (2'-Deoxyadenosine 10 microM, in adenosine deaminase deficiency conditions: 2'-Deoxycoformycin 10 microM), no significant modification of cyclic AMP level can be demonstrated.

5'-Deoxy-5'-methylthioadenosine, a by-product of polyamine metabolism, is a potent inhibitor of cell proliferation. MTA phosphorylase cleaves MTA into adenine and 5'-methylthioribose-1-P. We studied MTA inhibition and salvage into purine compounds and methionine in concanavalin A-stimulated rat T lymphocytes and in Raji cells. When de novo purine synthesis was inhibited by azaserine (20 microM), low concentrations of MTA, (less than or equal to 20 microM), were able to completely restore cell proliferation in both types of cells. When cells were cultured in a methionine-free medium, MTA (15 microM) completely fulfilled the methionine requirement of Raji cells but only 50% of that of rat T lymphocytes. MTA displayed a dose-dependent inhibition of the proliferation of both types of cells, but in the case of MTA salvage into purines or methionine, the curves were shifted to higher MTA concentrations. In vitro studies by Backlund et al. (Backlund, P.S., Chang, C.P. and Smith, R.A. (1982) J. Biol. Chem. 257, 4196-4202) on rat liver homogenates, suggested that the last step of MTA salvage into methionine may be the transamination of 2-keto-4-methylthiobutyrate to methionine. We present evidence that this is a step physiologically efficient in intact cells.

Concanavalin A-induced proliferation of rat T-lymphocytes is completely inhibited by 10(-5) M pyrazofurin, a potent inhibitor of pyrimidine de novo synthesis, as judged by cell viability and [3H]thymidine incorporation. Proliferation is completely restored by 5 X 10(-5) M uridine. Cytidine, deoxycytidine, deoxyuridine and thymidine 10 X 10(-5) M each, fail to re-establish proliferation but produce an isotropic dilution of [3H]thymidine uptake in DNA. Bases (cytosine, uracil and thymine) neither restore proliferation nor induce isotopic dilution. The unexpected inability of cytidine to reverse de novo pyrimidine synthesis inhibition suggests a lack of cytidine deaminase activity in rat T-lymphocytes. This is confirmed by a direct sensitive radioisotopic assay (less than 0.001 nmol X min-1 X 10(-6) cells).