Alfredo Corell

Primary Immunodeficiency Caused by Mutations in the Gene Encoding the CD3-γ Subunit of the T-Lymphocyte Receptor

PRIMARY immunodeficiency diseases are a heterogeneous group of disorders resulting from intrinsic defects of the immune system.1 They are frequently associated with repeated bacterial, fungal, or v...

Immunodeficiency associated with anorexia nervosa is secondary and improves after refeeding

Several studies have addressed the question of starvation effects on immune function by means of changes in lymphocyte subsets, cytokine induction or lymphocyte activation. Anorexia nervosa (AN) patients are severely malnourished and contradictory results have been obtained regarding the accompanying immunodeficiency, including its assignation as a part of the primary nervous disorder. In the present work, an extensive immunological function examination was carried out on 40 AN patients who were compared with a control group of 14 healthy girls. The AN patients were also classified according to their nutritional status (by the Body Mass Index: BMI), this being critical for a better understanding of these secondary immunodeficiency bases. Moreover, another immune system study was performed on five patients after refeeding. Lymphocyte subsets and function, cytokine induction and peripheral blood concentrations, and innate as well as humoral immunity were evaluated. Deregulation in the cytokine network, owing to the interaction of the central nervous (CNS) and immune systems, seems to be the initial immune alteration in AN immunodeficiency but it has not been disproved that the immunodeficiency is a direct consequence of the original psychiatric perturbation. Spontaneous high levels of circulating interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) have been observed; this is probably one of the causes of the anomalies found in the T‐cell subpopulations (mainly the naive CD4+CD45RA+ reduction and the cytotoxic CD8+ increase) and T‐cell activation status (mainly the down‐regulation of the CD2 and CD69 activation pathways). This finally leads to an impairment, not only in T‐cell function but also in T‐cell to B‐cell co‐operation. The AN specificity of these results is confirmed by the fact that these immune alterations improve after refeeding and when nutritional status becomes less critical, which also suggests that AN immunodeficiency is indeed secondary to malnutrition.

A Point Mutation in a Domain of Gamma Interferon Receptor 1 Provokes Severe Immunodeficiency

ABSTRACT Gamma interferon (IFN-γ) and the cellular responses induced by it are essential for controlling mycobacterial infections. Most patients bearing an IFN-γ receptor ligand-binding chain (IFN-γR1) deficiency present gross mutations that truncate the protein and prevent its expression, giving rise to severe mycobacterial infections and, frequently, a fatal outcome. In this report a new mutation that affects the IFN-γR1 ligand-binding domain in a Spanish patient with mycobacterial disseminated infection and multifocal osteomyelitis is characterized. The mutation generates an amino acid change that does not abrogate protein expression on the cellular surface but that severely impairs responses after the binding of IFN-γ (CD64 and HLA class II induction and tumor necrosis factor alpha and interleukin-12 production). A patients younger brother, who was also probably homozygous for the mutation, died from meningitis due toMycobacterium bovis. These findings suggest that a point mutation may be fatal when it affects functionally important domains of the receptor and that the severity is not directly related to a lack of IFN-γ receptor expression. Future research on these nontruncating mutations will make it possible to develop new therapeutical alternatives in this group of patients.

Allelic diversity at the primate major histocompatibility complex DRB6 locus

The HLA-DRB6 gene (also called DRBσ/V1) has been found only in about 26% of human HLA haplotypes, i.e.; DR1, DRw10, and DR2-bearing ones (Corell et al. 1991). In contrast, exon-2 DRB6 sequences have been obtained from all tested primates: nine chimpanzees (Pan troglodytes), three gorillas (Gorilla gorilla) and three orangutans (Pongo pygmaeus); other apes which had already been sequenced (one gorilla and one chimpanzee) also had the DRB6 gene. Thus, all apes tested from three different species, some of them evolutionary separated by at least 14–16 million years, bear the DRB6 gene. In addition, more than one gene copy per haplotype has been found in one chimpanzee; this, together with the apparent loss of this gene in some of the human DR haplotypes, may indicate that the DR genome has undergone evolutionary changes more recently and more actively than class I or III genes. In addition, ten different and presumably allelic DRB6 exon-2 sequences have been obtained, and some of them coming from different species are more similar to each other than the one from the same species; this finding goes in favor of the trans-species theory of major histocompatibility complex polymorphism generation. Also, data are presented supporting that DRB6 may be one of the eldest genes of the DRB family, thus one of the first to diverge from the ancestral DRB gene.

High frequency of the HLA-DRB1*0405-(Dw15)-DQw8 haplotype in Spaniards and its relationship to diabetes susceptibility.

A study of DR4 subtypes has been done in Spanish unrelated controls and insulin-dependent diabetics by using dot blot hybridization with specific DR4B1 exon-2 oligonucleotides and automated dideoxy DNA sequencing. Dw15-DQw8 is the predominant DR4 subtype present in our normal population (37%); this DR4 frequency characteristic singles out our population from all other Caucasoids tested so far and may also be a marker of the original Iberian paleo-North African population. Dw15-DQw8 is not significantly increased in our insulin-dependent diabetics sample and despite its relative high frequency in the control population it does not have a bearing in lowering insulin-dependent diabetes mellitus frequency of DR4-positive Spaniards. In addition, no particular DR4 split is by itself significantly increased in Spanish diabetics; this may indicate that selective diabetogenic environmental factors may be working upon DR4-positive individuals, but on genes (or gene products) other than DR or at least not upon the polymorphic sites of DRB1 exon-2 products.

A novel CD18 genomic deletion in a patient with severe leucocyte adhesion deficiency: a possible CD2/lymphocyte function‐associated antigen‐1 functional association in humans

Leucocyte adhesion deficiency (LAD) is an autosomal‐recessive genetic disease that is characterized clinically by severe bacterial infections and caused by mutations in the CD18 gene that codes for the β2 integrin subunit. A patient with a severe LAD phenotype was studied and the molecular basis of the disease was identified as a single homozygous defect in a Herpes virus saimiri (HVS)‐transformed T‐cell line. The defect identified involves a deletion of 171 bp in the cDNA that encodes part of the proteic extracellular domain. This genetic abnormality was further studied at the genomic DNA level and found to consist of a deletion of 169 bp (from − 37 of intron 4 to + 132 of exon 5), which abolishes the normal splicing and results in the total skipping of exon 5. The 171‐bp shortened ‘in‐frame’ mRNA not only resulted in the absence of CD18 expression on the cell surface but also in its absence in the cytoplasm of HVS T‐cell lines. Functionally, the LAD‐derived HVS T‐cell lines showed a severe, selective T‐cell activation impairment in the CD2 (but not in the CD3) pathway. This defect was not reversible when exogenous interleukin‐2 (IL‐2) was added, suggesting that there is also a functional interaction of the lymphocyte function‐associated antigen‐1 (LFA‐1) protein in the CD2 signal transduction pathway in human T cells, as has been previously reported in mice and in the human Papillon–Lefèvre syndrome. Thus, HVS transformation is not only a suitable model for T‐cell immunodeficiency studies and characterization, but is also a good system for investigating the immune system in pathological conditions. It may also be used in the future in cellular models for in vitro gene‐therapy trials.

Mutations of CD40 ligand in two patients with hyper-IgM syndrome

Two patients with the X-linked form of the hyper-IgM syndrome have been studied. Both patients present: 1. Mutations in the CD40L gene (a nonsense point mutation that introduces a termination codon at the extracellular domain of the protein, and a deletion that eliminates exon 4 as consequence of an abnormal splicing). 2. Lack of CD40L expression on the lymphocyte surface after stimulation with ionomycin and PMA. 3. Altered lymphocytic proliferation in response to anti-CD3. 4. Hyper IgM, low IgG and IgA levels and neutropenia. One of the patients shows, in addition, low Natural Killer cell numbers and severe herpetic infections, which distinguishes this case from the common hyper-IgM syndrome phenotype. Finally, a hyper-IgM stable phenotype has been immortalized by Herpes virus Saimiri for the first time.

Herpes virus saimiri transformation of T cells in CD3γ immunodeficiency: phenotypic and functional characterization

The characterization of T cell immunodeficiencies could in part be supported by using stable cell lines in which biochemical and molecular studies of the defect could be carried out thereby omitting frequent bleeding of patients. First attempts to obtain such cell lines included HTLV-I transformation and exogenous IL-2 administration, but both models have important disadvantages. Recently, a virus isolated from the squirrel monkey, Herpes virus saimiri (HVS), has been reported to have the ability to transform T cells. A stable IL-2-dependent HVS-transformed T cell line from a CD3 gamma deficient patient has been obtained; and this cell line displays both the phenotypic and the functional characteristics of the patients lymphocytes. Moreover, the line down-modulates TCR/CD3 surface expression upon CD3 engagement, as do the patients lymphocytes, showing that CD3 gamma and its phosphorylation are not necessary for TCR/CD3 internalization. In addition, the abnormal staining pattern of different anti-TCR/CD3 monoclonal antibodies is preserved in the HVS-patient line. Since HVS is capable of transforming CD3 gamma- T cells, the CD3 gamma chain does not seem to be involved in the HVS receptor process. The fact that it is not possible to obtain a CD8+ HVS line from the CD3 gamma- patient supports the existence of a functional anomaly in his scanty CD8+ peripheral lymphocytes. Thus, HVS transformation is a suitable model for T cell immunodeficiency studies and characterization. It may also be used in the future in cellular models for in vitro gene therapy trials.

FREQUENCIES OF HLA-A24 AND HLA-DR4-DQ8 ARE INCREASED AND THAT OF HLA-B BLANK IS DECREASED IN CHRONIC TOXIC OIL SYNDROME

The cause of toxic oil syndrome (TOS) has not yet been definitively determined, but some genetic susceptibility factors (certain HLA antigens and female sex) have been identified in 236 patients. Similarities with genetic factors for scleroderma and hydralazine‐induced lupus (i.e. in TOS female sex and HLA‐A24, Pcorrected= 0.00001 and DR4, Pcorrected= 0.04, respectively) may provide a clue to the responsible xenobiotic and its pathogenesis, and may also help in understanding the basis of the related eosinophilia‐myalgia syndrome associated with tryptophan ingestion. In this paper it is also established that a human class I antigen (HLA‐A24) and, independently, an HLA class II haplotype (DR4‐DQ8, Pcorrected= 0.04) and arginine 52 in the α‐DQ chains (Pcorrected= 0.03) are associated with TOS susceptibility, similarly to insulin‐dependent diabetes. This further supports the classification of TOS as an autoimmune disease. Also, the increased frequency of a particular set of low‐frequency HLA class I antigens in chronic TOS patients (i.e. B27, B37, B38 and B49) and the probable decrease in the frequency of HLA‐B homozygotes in surviving patients (Pcorrected= 0.008) may provide an objective model to explain the maintenance of the HLA polymorphism: less frequent HLA alleles may be more advantageous in the event of unexpected human contact with unusual xenobiotics (not only microbes); however, other mechanisms working together to preserve and generate HLA polymorphism may coexist.

Differential contribution of C4 and HLA-DQ genes to systemic lupus erythematosus susceptibility

The particular histocompatability antigen (HLA) gene(s) that may confer systemic lupus erythematosus (SLE) susceptibility remains unknown. In the present study, 58 unrelated patients and 69 controls have been analyzed for their class I and class II serologic antigens, class II (DR and DQ) DNA restriction fragment length polymorphism, their deduced DQA1 and B1 exon 2 nucleotide sequences and their corresponding amino acid residues. By using the etiologic fraction (δ) as an almost absolute measure of the strongest linkage disequilibrium of an HLA marker to the putative SLE susceptibility locus, it has been found that the strength of association of the HLA marker may be quantified as follows: DQA1*0501 (associated to DR3) or DQB1*0201 (associated to DR3) > non Asp 57 βDQ/Arg 52 αDQ > DR3 > non Asp 57 βDQ. Thus, molecular HLA DQ markers tend to be more accurate as susceptibility markers than the classical serologic markers (DR3). However, dominant or recessive non Asp 57 βDQ susceptibility theories, as previously postulated for insulin-dependent diabetes mellitus, do not hold in our SLE nephritic population; indeed, three patients bear neither Arg 52 αDQ nor Asp 57 βDQ susceptibility factors. On the other hand, nonsusceptibility factors are included in our population in the A30B18CF130-DR3DQ2(Dw25) haplotype and not in A1B8CS01-DR3DQ2(Dw24); this distinctive association has also been recorded in type I diabetes mellitus and may reflect the existence of common pathogenic HLA-linked factors for both diseases only in the A30B18CF10DR3DQ2-(Dw 25) haplotype. Finally, the observed increase of deleted C4 genes (and not ‘null’ C4 proteins) in nephritic patients shows that C4 genes are disease markers, but probably without a pathogenic role.