Social stress results in altered glucocorticoid regulation and shorter survival in simian acquired immune deficiency syndrome

From early in the AIDS epidemic, psychosocial stressors have been proposed as contributors to the variation in disease course. To test this hypothesis, rhesus macaques were assigned to stable or unstable social conditions and were inoculated with the simian immunodeficiency virus. Animals in the unstable condition displayed more agonism and less affiliation, shorter survival, and lower basal concentrations of plasma cortisol compared with stable animals. Early after inoculation, but before the emergence of group differences in cortisol levels, animals receiving social threats had higher concentrations of simian immunodeficiency virus RNA in plasma, and those engaging in affiliation had lower concentrations. The results indicate that social factors can have a significant impact on the course of immunodeficiency disease. Socially induced changes in pituitary–adrenal hormones may be one mechanism mediating this relationship.

Defect in IgV gene somatic hypermutation in Common Variable Immuno-Deficiency syndrome

Common Variable Immuno-Deficiency (CVID) is the most common symptomatic primary antibody-deficiency syndrome, but the basic immunologic defects underlying this syndrome are not well defined. We report here that among eight patients studied (six CVID and two hypogammaglobulinemic patients with recurrent infections), there is in two CVID patients a dramatic reduction in Ig V gene somatic hypermutation with 40–75% of IgG transcripts totally devoid of mutations in the circulating memory B cell compartment. Functional assays of the T cell compartment point to an intrinsic B cell defect in the process of antibody affinity maturation in these two cases.

A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense.

In this paper we report a recessive mutation, immune deficiency (imd), that impairs the inducibility of all genes encoding antibacterial peptides during the immune response of Drosophila. When challenged with bacteria, flies carrying this mutation show a lower survival rate than wild-type flies. We also report that, in contrast to the antibacterial peptides, the antifungal peptide drosomycin remains inducible in a homozygous imd mutant background. These results point to the existence of two different pathways leading to the expression of two types of target genes, encoding either the antibacterial peptides or the antifungal peptide drosomycin.

Analysis of the Drosophila host defense in domino mutant larvae, which are devoid of hemocytes

We have analyzed the Drosophila immune response in domino mutant larvae, which are devoid of blood cells. The domino mutants have a good larval viability, but they die as prepupae. We show that, on immune challenge, induction of the genes encoding antimicrobial peptides in the fat body is not affected significantly in the mutant larvae, indicating that hemocytes are not essential in this process. The hemocoele of domino larvae contains numerous live microorganisms, the presence of which induces a weak antimicrobial response in the fat body. A full response is observed only after septic injury. We propose that the fat body cells are activated both by the presence of microorganisms and by injury and that injury potentiates the effect of microorganisms. Survival experiments after an immune challenge showed that domino mutants devoid of blood cells maintain a wild-type resistance to septic injury. This resistance was also observed in mutant larvae in which the synthesis of antibacterial peptides is impaired (immune deficiency larvae) and in mutants that are deficient for humoral melanization (Black cells larvae). However, if domino was combined with either the immune deficiency or the Black cell mutation, the resistance to septic injury was reduced severely. These results establish the relevance of the three immune reactions: phagocytosis, synthesis of antibacterial peptides, and melanization. By working in synergy, they provide Drosophila a highly effective defense against injury and/or infection.

An essential role for Bruton's [corrected] tyrosine kinase in the regulation of B-cell apoptosis.

Mutations of the Bruton's tyrosine kinase (btk) gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immune deficiency (Xid) in mice. To establish the BTK role in B-cell activation we examined the responses of wild-type and Xid B cells to stimulation through surface IgM and CD40, the transducers of thymus independent-type 2 and thymus-dependent activation, respectively. Wild-type BTK was necessary for proliferation induced by soluble anti-IgM (a prototype for thymus independent-type 2 antigen), but not for responses to soluble CD40 ligand (CD40L, the B-cell activating ligand expressed on T-helper cells). In the absence of wild-type BTK, B cells underwent apoptotic death after stimulation with anti-IgM. In the presence of wild-type but not mutated BTK, anti-IgM stimulation reduced apoptotic cell death. In contrast, CD40L increased viability of both wild-type and Xid B cells. Importantly, viability after stimulation correlated with the induced expression of bcl-XL. In fresh ex vivo small resting B cells from wild-type mice there was only barely detectable bcl-XL protein, but there was more in the larger, low-density ("activated") splenic B cells and peritoneal B cells. In vitro Bcl-XL induction following ligation of sIgM-required BTK, was cyclosporin A (CsA)-sensitive and dependent on extracellular Ca2+. CD40-mediated induction of bcl-x required neither wild-type BTK nor extracellular Ca2+ and was insensitive to CsA. These results indicate that BTK lies upstream of bcl-XL in the sIgM but not the CD40 activation pathway. bcl-XL is the first induced protein to be placed downstream of BTK.

Immunologic basis of transplant-associated arteriosclerosis.

Although immunosuppressive therapy minimizes the risk of graft failure due to acute rejection, transplant-associated arteriosclerosis of the coronary arteries remains a significant obstacle to the long-term survival of heart transplant recipients. The participation of specific inflammatory cell types in the genesis of this lesion was examined in a mouse model in which carotid arteries were transplanted across multiple histocompatibility barriers into seven mutant strains with immunologic defects. An acquired immune response--with the participation of CD4+ (helper) T cells, humoral antibody, and macrophages--was essential to the development of the concentric neointimal proliferation and luminal narrowing characteristic of transplant arteriosclerosis. CD8+ (cytotoxic) T cells and natural killer cells were not involved in the process. Arteries allografted into mice deficient in both T-cell receptors and humoral antibody showed almost no neointimal proliferation, whereas those grafted into mice deficient only in helper T cells, humoral antibody, or macrophages developed small neointimas. These small neointimas and the large neointimas of arteries grafted into control animals contained a similar number of inflammatory cells; however, smooth muscle cell number and collagen deposition were diminished in the small neointimas. Also, the degree of inflammatory reaction in the adventitia did not correlate with the size of the neointima. Thus, the reduction in neointimal size in arteries allografted into mice deficient in helper T cells, humoral antibody, or macrophages may be accounted for by a decrease in smooth muscle cell migration or proliferation.

Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus.

The gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) has been proposed recently as a candidate gene for the mouse severe combined immune deficiency (scid) locus. We have used a partial cDNA clone for human DNA-PKcs to map the mouse homologue using a large interspecific backcross panel. We found that the mouse gene for DNA-PKcs does not recombine with scid, consistent with the hypothesis that scid is a mutation in the mouse gene for DNA-PKcs.

Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: A dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia

Severe jaundice leading to kernicterus or death in the newborn is the most devastating consequence of glucose-6-phosphate dehydrogenase (EC 1.1.1.49; G-6-PD) deficiency. We asked whether the TA repeat promoter polymorphism in the gene for uridinediphosphoglucuronate glucuronosyltransferase 1 (EC 2.4.1.17; UDPGT1), associated with benign jaundice in adults (Gilbert syndrome), increases the incidence of neonatal hyperbilirubinemia in G-6-PD deficiency. DNA from term neonates was analyzed for UDPGT1 polymorphism (normal homozygotes, heterozygotes, variant homozygotes), and for G-6-PD Mediterranean deficiency. The variant UDPGT1 promoter allele frequency was similar in G-6-PD-deficient and normal neonates. Thirty (22.9%) G-6-PD deficient neonates developed serum total bilirubin ≥ 257 μmol/liter, vs. 22 (9.2%) normals (P = 0.0005). Of those with the normal homozygous UDPGT1 genotype, the incidence of hyperbilirubinemia was similar in G-6-PD-deficients and controls (9.7% and 9.9%). In contrast, in the G-6-PD-deficient neonates, those with the heterozygous or homozygous variant UDPGT1 genotype had a higher incidence of hyperbilirubinemia than corresponding controls (heterozygotes: 31.6% vs. 6.7%, P < 0.0001; variant homozygotes: 50% vs. 14.7%, P = 0.02). Among G-6-PD-deficient infants the incidence of hyperbilirubinemia was greater in those with the heterozygous (31.6%, P = 0.006) or variant homozygous (50%, P = 0.003) UDPGT1 genotype than in normal homozygotes. In contrast, among those normal for G-6-PD, the UDPGT1 polymorphism had no significant effect (heterozygotes: 6.7%; variant homozygotes: 14.7%). Thus, neither G-6-PD deficiency nor the variant UDPGT1 promoter, alone, increased the incidence of hyperbilirubinemia, but both in combination did. This gene interaction may serve as a paradigm of the interaction of benign genetic polymorphisms in the causation of disease.

Carbohydrate-deficient glycoprotein syndrome type V: Deficiency of dolichyl-P-Glc:Man9GlcNAc2-PP-dolichyl glucosyltransferase

Deficiency of dolichyl-P-Glc:Man9GlcNAc2-PP-dolichyl glucosyltransferase is the cause of an additional type of carbohydrate-deficient glycoprotein syndrome (CDGS type V). Clinically this type resembles the classical type Ia of CDGS caused by the deficiency of phosphomannomutase. As a result of the glucosyltransferase deficiency in CDGS type V nonglucosylated lipid-linked oligosaccharides accumulate. The defect is leaky and glucosylated oligosaccharides are found on nascent glycoproteins. The limited availability of glucosylated lipid-linked oligosaccharides explains the incomplete usage of N-glycosylation sites in glycoproteins. This finding is reflected in the presence of transferrin forms in serum that lack one or both of the two N-linked oligosaccharides and the reduction of mannose incorporation to about one-third of control in glycoproteins of fibroblasts.

Experimental hemochromatosis due to MHC class I HFE deficiency: Immune status and iron metabolism

The puzzling linkage between genetic hemochromatosis and histocompatibility loci became even more so when the gene involved, HFE, was identified. Indeed, within the well defined, mainly peptide-binding, MHC class I family of molecules, HFE seems to perform an unusual yet essential function. As yet, our understanding of HFE function in iron homeostasis is only partial; an even more open question is its possible role in the immune system. To advance on both of these avenues, we report the deletion of HFE α1 and α2 putative ligand binding domains in vivo. HFE-deficient animals were analyzed for a comprehensive set of metabolic and immune parameters. Faithfully mimicking human hemochromatosis, mice homozygous for this deletion develop iron overload, characterized by a higher plasma iron content and a raised transferrin saturation as well as an elevated hepatic iron load. The primary defect could, indeed, be traced to an augmented duodenal iron absorption. In parallel, measurement of the gut mucosal iron content as well as iron regulatory proteins allows a more informed evaluation of various hypotheses regarding the precise role of HFE in iron homeostasis. Finally, an extensive phenotyping of primary and secondary lymphoid organs including the gut provides no compelling evidence for an obvious immune-linked function for HFE.

Innate and acquired humoral immunities to influenza virus are mediated by distinct arms of the immune system

“Natural” Igs, mainly IgM, comprise part of the innate immune system present in healthy individuals, including antigen-free mice. These Igs are thought to delay pathogenicity of infecting agents until antigen-induced high affinity Igs of all isotypes are produced. Previous studies suggested that the acquired humoral response arises directly from the innate response, i.e., that B cells expressing natural IgM, upon antigen encounter, differentiate to give rise both to cells that secrete high amounts of IgM and to cells that undergo affinity maturation and isotype switching. However, by using a murine model of influenza virus infection, we demonstrate here that the B cells that produce natural antiviral IgM neither increase their IgM production nor undergo isotype switching to IgG2a in response to the infection. These cells are distinct from the B cells that produce the antiviral response after encounter with the pathogen. Our data therefore demonstrate that the innate and the acquired humoral immunities to influenza virus are separate effector arms of the immune system and that antigen exposure per se is not sufficient to increase natural antibody production.

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.

Animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors.

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is the major target for antiretroviral therapy of the acquired immunodeficiency syndrome (AIDS). While some inhibitors exhibit activity against most retroviral RTs, others are specific for the HIV-1 enzyme. To develop an animal model for the therapy of the HIV-1 infection with RT inhibitors, the RT of the simian immunodeficiency virus (SIV) was replaced by the RT of HIV-1. Macaques infected with this SIV/HIV-1 hybrid virus developed AIDS-like symptoms and pathology. The HIV-1-specific RT inhibitor LY300046.HCl, but not zidovudine [3'-azido-3'-deoxythymidine (AZT)] delayed the appearance of plasma antigenemia in macaques infected with a high dose of the chimeric virus. Infection of macaques with the chimeric virus seems to be a valuable model to study the in vivo efficacy of new RT inhibitors, the emergence and reversal of drug resistance, the therapy of infections with drug-resistant viruses, and the efficacy of combination therapy.

Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged “enhanced” green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 → Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.

Immune regulation by the ST6Gal sialyltransferase

The ST6Gal sialyltransferase controls production of the Siaα2-6Galβ1-4GlcNAc (Sia6LacNAc) trisaccharide, which is the ligand for the lectin CD22. Binding of CD22 to Sia6LacNAc is implicated in regulating lymphocyte adhesion and activation. We have investigated mice that lack ST6Gal and report that they are viable, yet exhibit hallmarks of severe immunosuppression unlike CD22-deficient mice. Notably, Sia6LacNAc-deficient mice display reduced serum IgM levels, impaired B cell proliferation in response to IgM and CD40 crosslinking, and attenuated antibody production to T-independent and T-dependent antigens. Deficiency of ST6Gal was further found to alter phosphotyrosine accumulation during signal transduction from the B lymphocyte antigen receptor. These studies reveal that the ST6Gal sialyltransferase and corresponding production of the Sia6LacNAc oligosaccharide are essential in promoting B lymphocyte activation and immune function.