Identification of candidate disease genes in patients with common variable immunodeficiency

Background: Common variable immunodeficiency (CVID), the most prevalent form of primary immunodeficiency (PID), is characterized by hypogammaglobulinemia and recurrent infections. Understanding protein-protein interaction (PPI) networks of CVID genes and identifying candidate CVID genes are critical steps in facilitating the early diagnosis of CVID. Here, the aim was to investigate PPI networks of CVID genes and identify candidate CVID genes using computation techniques. Methods: Network density and biological distance were used to study PPI data for CVID and PID genes obtained from the STRING database. Gene expression data of patients with CVID were obtained from the Gene Expression Omnibus, and then Pearson’s correlation coefficient, a PPI database, and Kyoto Encyclopedia of Genes and Genomes were used to identify candidate CVID genes. We then evaluated our predictions and identified differentially expressed CVID genes. Results: The majority of CVID genes are characterized by a high network density and small biological distance, whereas most PID genes are characterized by a low network density and large biological distance, indicating that CVID genes are more functionally similar to each other and closely interact with one other compared with PID genes. Subsequently, we identified 172 CVID candidate genes that have similar biological functions to known CVID genes, and eight genes were recently reported as CVID-related genes. MYC, a candidate gene, was down-regulated in CVID duodenal biopsies, but up-regulated in blood samples compared with levels in healthy controls. Conclusion: Our findings will aid in a better understanding of the complex of CVID genes, possibly further facilitating the early diagnosis of CVID.     

Long-term follow up of patients with common variable immunodeficiency treated with intravenous immunoglobulin: Reevaluation of intravenous immunoglobulin replacement therapy

Five patients with common variable immunodeficiency treated in our hospital between December 1979 and December 1990 were given six kinds of intravenous immunoglobulin preparations (pepsin treated, S-sulfonated, polyethylene glycol treated, pH4 treated, alkylated, and pH4.25 formulation preparation) for replacement therapy. Duration of the therapy ranged from 7.6 to 11 years. Incidences of fever and acute infections were variable among patients, but no significant differences were seen in the incidences among periods given each preparation. Three cases revealed abnormal pulmonary functions in tests. Adverse reactions were rarely seen in our study periods, and no severe reactions were observed. No significant differences were seen in incidences of adverse reactions. Postinfusion levels of serum complement slightly decreased from preinfusion levels. However, the decrease in complement was not related to any adverse reaction. No long-term complications such as transmission of hepatitis have been observed. Our data suggest that no obvious differences exist between the efficacy and safety of each IVIG preparation. Differences of efficacy of IVIG replacement therapy may be due to the variable pathophysiology of each patient.Abbreviations CVID common variable immunodeficiency - IVIG intravenous immunoglobulin     

Asparagine-linked sugar chains of plasma membrane glycoproteins from healthy and common variable immunodeficiency B lymphoblastoid cell lines

Asparagine-linked sugar chains of plasma membrane glycoproteins, which are formed by glycosylation during B cell maturation, were examined with B lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus derived from healthy controls and patients with common variable immunodeficiency (CVI). Both two patients with CVI showed hypogammaglobulinemia and impaired B cell functions. LCLs from healthy controls and the patients showed CD19+ and HLA/DR+ in the cell surface and secreted IgM. In both healthy controls and the patients, the main oligosaccharide in asparagine-linked sugar chains of the membrane glycoproteins of LCLs was biantennary sugar chain with bisected GlcNAc (Gal₂-GlcNAc₂-Man₃-GlcNAc-GlcNAc-Fuc-GlcNAc_OT). Biantennary sugar chain with an-fucosyl residue linked at the proximal GIcNAc was seen but biantennary sugar chain without an-fucosyl residue at the proximal GlcNAc was little detected in each LCL. There was no difference in quality and quantity of asparagine-linked sugar chains between healthy controls and the patients. These results suggest that glycosylation during B cell maturation may not be impaired in patients with CVI.     

Defective peripheral B cell selection in common variable immune deficiency patients with autoimmune manifestations

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Defective B cell response to TLR9 ligand (CpG-ODN), Streptococcus pneumoniae and Haemophilus influenzae extracts in common variable immunodeficiency patients

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and antibody deficiency to both T dependent and independent antigens. Patients suffer from recurrent sinopulmonary infections mostly caused by Streptococcus pneumoniae and Haemophilus influenzae, but also gastrointestinal or autoimmune symptoms. Their response to vaccination is poor or absent. In this study we investigated B cell activation induced by the TLR9 specific ligand (CpG-ODN) and bacterial extracts from S. pneumoniae and H. influenzae known to stimulate several TLR. We found that B cells from CVID patients express lower levels of CD86 after stimulation with CpG-ODN, S. pneumoniae and H. influenzae extracts in combination with anti-IgM antibody and also display a lower proliferative index when stimulated with bacterial extracts. Our results point to a broad TLR signalling defect in B lymphocytes from CVID patients that may be related to the hypogammaglobulinaemia and poor response to vaccination characteristic of these patients.     

Cadmium exposure of rainbow trout, Salmo gairdneri Richardson: effects on immune functions

Differential leucocyte counts, phagocytosis, humoral antibody response and the in vitro blasto-genetic response to mitogens (lipopolysaccharide and Concanavalin A) and to an antigen ( Vibrio anguillarum ) were studied in rainbow trout exposed to 0,0.7 or 3.6 μg Cd 1⁻¹ for 12 weeks.
Although the fish did not exhibit any clinical or histological changes, cadmium exposure was found to affect two of the immune parameters measured. The cellular response of fish immunized with V. anguillarum to the homologous antigen was significantly lower for splenocytes obtained from fish exposed to cadmium for 9 weeks (3.6 μg Cd 1⁻¹ group) than for splenocytes obtained from non-exposed fish. Conversely, the humoral antibody response to V. anguillarum O-antigen was higher in the 3.6 μg Cd 1⁻¹ group than in the non-exposed group. Protective immunity of fish vaccinated against V. anguillarum was equally as good in the cadmium-exposed group as in the non-exposed group. No cadmium-induced changes in differential leucocyte counts or in the proportions of phagocytic cells were observed.     

B cells from common variable immunodeficiency patients fail to differentiate to antibody secreting cells in response to TLR9 ligand (CpG-ODN) or anti-CD40+IL21

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterised by hypogammaglobulinaemia and antibody deficiency to T dependent and independent antigens. Patients suffer from recurrent respiratory infections and poor response to vaccination. Although the underlying molecular defect is unknown, most CVID patients show impaired late B cell differentiation. We investigated B cell differentiation and immunoglobulin secretion induced by two different stimuli: TLR9 specific ligand (CpG-ODN) and anti-CD40 combined with IL21. The contribution of BCR signalling (anti-IgM stimulation) was also evaluated. B cells from CVID patients produced low levels of IgG and IgA in response to both kinds of stimuli that was not restored by anti-IgM. Production of IgM was conserved when cells were stimulated with anti-CD40 and IL21. These results point to a wide signalling defect in B lymphocytes from CVID patients that may be related to their hypogammaglobulinaemia and poor response to vaccination.     

Subcutaneous immunoglobulin therapy in a patient with myopathic dropped head syndrome and common variable immunodeficiency

Prominent neck extension weakness is an uncommon clinical entity, also termed dropped-head syndrome, that may be part of a generalized neuromuscular disorder. We report here the case of a woman with dropped-head syndrome and pulmonary arterial hypertension secondary to systemic sclerosis. Subsequently, she developed common variable immunodeficiency and subcutaneous immunoglobulin therapy was started. After two months from the start of therapy we did not observe any improvement in the degree of flexion of the head, although the clinical examination shows an improvement in neck extensor muscle strength. Subcutaneous immunoglobulin therapy could be a possible therapeutic option for the treatment of myopathic neck extensor weakness.     

Cellular immune competence of a Drosophila mutant with neoplastic hematopoietic organs

In the Tum^l mutant of Drosophila melanogaster, the larval hematopoietic organs undergo neoplastic changes and release into circulation large numbers of blood cells. The lamellocytes, and to a lesser extent the plasmatocytes from which they are derived, are the cells that encapsulate various endogenous tissues and form melanotic tumors. The mutation is temperature sensitive, with maximum gene expression manifested at 29°C. The ability of Tum^l larvae to encapsulate eggs of the wasp parasite Leptopilina heterotoma is dependent not only on temperature, with host larvae much more immune reactive at 29°C than at lower temperatures (15° or 21°C), but also on the interval of time following infection when temperature shift experiments are performed. When the shift of parasitized larvae from 21° to 29°C is delayed by 18 hr the hosts are not as immune reactive as those shifted immediately after infection. Since Tum^l larvae are potentially highly immune reactive at the time of infection (with sufficient numbers of lamellocytes in circulation to encapsulate parasites), the low degree of immune competence in hosts shifted to 29°C after 18 hr or maintained at lower temperatures suggests that the increased capacity of blood cells to react against foreign surfaces is dependent on the cells acquiring new or altered recognition and adherence properties at 29°C. The 18-hr delay may provide the parasite with an opportunity to interfere with the acquisition of these specific cellular alterations. Differential hemocyte counts from parasitized larvae show abnormally low lamellocyte counts in susceptible hosts, indicating that successfully developing parasites interfere with the differentiation of hemocytes.     

Dynamics of Simian immunodeficiency virus-specific cytotoxic T-cell responses in tissues

Although the dynamics of human immunodeficiency virus and Simian immunodeficiency virus (SIV)-specific cytotoxic T cells (CTLs) have been well documented in the blood, little is known regarding CTL development in other tissues. In this study, seven Mamu-A*01+ macaques were inoculated with SIVmac. Two macaques were killed at 21 days of infection, and SIV gag p11C tetramer responses were measured in the blood, axillary and mesenteric lymph nodes, spleen, bone marrow, and thymus. Three with clinical signs of disease were killed and similarly examined. Four macaques were followed throughout disease progression, and intestinal biopsies and blood were examined at regular time points after inoculation. In animals followed prospectively, peak early tetramer responses were detected in the blood (3.9-19% of CD3+ CD8+ T cells) between day 14-21 post-inoculation (p.i.). After day 49, tetramer responses in the blood diminished and remained relatively stable through day 200, ranging from 0.7-6.5% of CD3+ CD8+ T cells. In contrast, tetramer-positive T cells increased in the intestine in later stages of infection (100-200 days p.i.) in all four infected animals (peak values from 5.3 to 28.8%). Percentages of tetramer-positive cells were consistently higher in the intestine than in the blood in all four animals after day 100. In animals with acquired immunodeficiency syndrome, percentages of CTL in tissues were variable, but were consistently higher in the intestine and spleen compared with blood. These data suggest that while high CTL responses develop at a similar rate, and magnitude in both peripheral and mucosal lymphoid tissues in primary SIV infection, mucosal CTL responses may predominate later in the course of the disease.     

诺如病毒感染宿主免疫应答机制及疫苗研究进展

诺如病毒是引起人类急性胃肠炎的重要食源性病原之一。由于体外复制系统和感染模型的缺乏,研究人员对其宿主保护性免疫的理解始终有限,导致控制病毒感染方面的研究也受到较大阻碍。近年来,随着病毒衣壳蛋白外源表达、替代病毒的使用、志愿者实验的开展,尤其是细胞培养模型的突破,使得体液免疫和细胞免疫的研究取得较大进展。因此,本文针对诺如病毒感染宿主的先天性免疫、体液免疫和细胞免疫应答机制等进行了综述,并对后续其在诺如病毒候选疫苗研制等领域的应用进行了展望。     

昆虫天然免疫反应分子机制研究进展

昆虫体内缺乏高等脊椎动物所具有的获得性免疫系统, 只能依赖发达的天然免疫系统抵抗细菌、 真菌、 病毒等外源病原物的侵染。本文概括了昆虫天然免疫反应发生和作用的分子机制相关进展, 重点阐述了重要免疫相关因子在昆虫天然免疫反应中的功能和作用机制。昆虫天然免疫反应分为体液免疫和细胞免疫两种, 二者共同作用完成对病原物的吞噬 (phagocytosis)、 集结 (nodulation)、 包囊 (encapsulation)、 凝结 (coagulation)和黑化（melanization）等。当昆虫受到外界病原物的侵染时, 首先通过体内的模式识别蛋白（pattern recognition proteins/receptor, PRPs）识别并结合病原物表面特有的模式分子（pathogen-associated molecular pattern, PAMPs）, 继而一系列包括丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂在内的级联激活反应被激活和调控, 产生抗菌肽、 黑色素等免疫效应分子, 清除或杀灭外源物。抗菌肽是一类小分子量的阳离子肽, 具有广谱抗菌活性, 针对不同类型的病原物, 抗菌肽的产生机制也不尽相同。昆虫体内存在着两种信号转导途径调节抗菌肽的产生： 一是由真菌和大部分革兰氏阳性菌激活的Toll途径; 二是由革兰氏阴性菌激活的Imd途径（immune deficiency pathway）。这两个途径通过激活不同转录因子调控不同抗菌肽基因的表达参与昆虫体内的天然免疫反应。     

Induction of the immune response to cell surface antigens in vitro

Summary Two tissue culture incubation systems are described in which immune responses to cell surface antigens have been demonstrated In the one-way “mixed lymphocyte interaction” system, a specific stimulation of thymidine uptake was induced by a particulate membrane antigen fraction, the microsomal lipoproteins (MLP)when low levels (0.01 to 0.001 μg per ml) were incubated with spleen or lymph node cells from nonsensitized mice. No stimulation was seen when allogeneic MLP was used at high levels, 10 μg per ml, nor at any level with syngeneic MLP. Specific effectors were demonstrated after 72-hr incubation with stimulatory levels of allogeneic MLP in three separate in vitro assays, a plaque-forming cell reduction assay, a tumor target assay, and an antigen-binding cell assay. In the latter assay, [¹²⁵I]MLP was used as the source of antigen. This system has limited potential inasmuch as mouse spleen cells do not survive in it beyond the 4th day of culture. The second tissue culture system, the Marbrook system, has much greater possibilities because at least 25% of the inoculum is recovered 7days later. In this culture system a cell-free sheep erythrocyte membrane preparation can induce, plaque-forming cells in the absence of macrophages. Using a sensitive radioimmunoassay, frees specific antibody was detected in culture supernatant fluids. With the same culture system, allogeneic lymphocytotoxic cells (killer) have been induced with spleen cells from unprimed mice in strains differing at the major histocompatibility locus (H-2). Allogeneic MLP induced very significant “killer” cell activity with spleen cells from primed mice. In a syngeneic tumor systems, significant amounts of killer cell activity were induced with unprimed spleen cell inocula, and much larger amounts induced with spleen cells from immunized mice. Presented in the formal symposium on Carcinogenesis in Vitro, at the 25th Annual Meeting of the Tissue Culture Association, Miami Beach, Florida, June 3–6, 1974. This work was supported by Public Health Service Rescarch grants CA 07973 and CA 10815 from the National Cancer Institute.     

人类免疫缺陷病毒感染者免疫功能重建不全的研究进展

约20%的人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染者在接受抗病毒治疗后,外周血CD4^＋ T细胞水平无法有效恢复,即免疫功能重建不全,但发生机制目前尚不明确。本文根据HIV感染者的临床和免疫学指标,分析发生免疫功能重建不全的风险因素;进一步从外周循环系统中T细胞损伤、细胞因子变化及肠道黏膜局部CD4^＋ T细胞删除等方面解析可能机制,其中肠道黏膜CD4^＋ T细胞删除可能与肠道微生物菌群诱导的细胞焦亡有关。虽然目前国际上有多项关于免疫功能重建不全患者治疗方案的临床试验,但由于缺乏一致的、有力的基础研究证据,导致疾病诊断指标缺乏,单独细胞因子给药治疗亦未取得突破性进展。因此,应深入探索免疫功能重建不全发生机制,进行大样本、长时间的前瞻性队列研究,制定免疫功能重建不全的临床与免疫学界定标准,从而为临床治疗提供科学依据。     

Up-regulation of IL-12 in monocytes: a fundamental defect in common variable immunodeficiency

We show that LPS-stimulated circulating CD14-positive monocytes from patients with common variable immunodeficiency (CVID) express a higher proportion of intracellular IL-12-positive cells than monocytes from patients with X-linked agammaglobulinemia or normal subjects. We used four-color flow cytometry and measured IL-12 with an Ab to the p40 subunit following stimulation with LPS. The raised IL-12 is associated with an increased frequency of IFN-gamma-positive T cells, but not of IFN-gamma-positive CD56+ NK cells. These increases in frequency of cytokine-positive cells are due to a decrease in the absolute numbers of circulating monocytes and T cells that are negative for IL-12 and IFN-gamma, respectively. The increased frequency of IL-12-positive monocytes appears to be selective because TNF-alpha was not increased, and is thus unlikely to reflect a general activation. Chronic infection is also unlikely to explain our data since cells from X-linked agammaglobulinemia patients with a similar Ig deficiency do not show these changes. Our data suggest a fundamental abnormality in the IL-12/IFN-gamma circuit in CVID, with up-regulation of IL-12 being the "primary" factor. This imbalance is likely to skew the immune response away from Ab production and also explains the failure of CVID T cells to make Ag-specific memory cells and the chronic inflammatory and granulomatous complications that are a feature of CVID. This disease appears to be a rare example of a polarized Th1-type response and may in part be due to a genetic defect in the control of IL-12 production.     

Synthesis of deacetylthymosin beta 11 and its effect on the impaired T-lymphocytes of a uremic patient with common variable immunodeficiency

The untetracontapeptide corresponding to the entire amino acid sequence of deacetylthymosin beta 11 was synthesized by assembling six peptide fragments via the azide followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole in trifluoroacetic acid in the presence of dimethylselenide. The synthetic peptide was tested for its effect on the impaired blastogenic response of phytohemagglutinin-stimulated T-lymphocytes of a uremic patient with common variable immunodeficiency. The synthetic peptide had some restoring activity on the impaired blastogenic response of T-lymphocytes in the one patient tested.     

Induction of immune response against NY-ESO-1 by CHP-NY-ESO-1 vaccination and immune regulation in a melanoma patient

BACKGROUND: NY-ESO-1 is a cancer/testis antigen highly immunogenic in cancer patients. Cholesterol-bearing hydrophobized pullulan (CHP) is a nanoparticle-forming antigen-delivery vehicle and CHP complexed with NY-ESO-1 protein (CHP-NY-ESO-1) efficiently activates CD4 and CD8 T cells in vitro. AIM: In this study we report on a 50-year-old male melanoma patient with multiple skin and organ metastases (T4N3M1c) who was vaccinated with CHP-NY-ESO-1 at biweekly intervals and who had an unusual disease course. We characterized in this patient humoral and cellular immune responses, immune regulatory cells, and cytokine profiles in the peripheral blood and at local tumor sites. RESULTS: Ten days after the second CHP-NY-ESO-1 vaccination (day 25), blisters appeared on the skin at the metastatic lesions associated with inflammatory changes. A skin biopsy showed the presence of many NY-ESO-1-expressing apoptotic melanoma cells as determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) test. However, the tumors continued to grow, and the patient died of pulmonary failure due to multiple metastases on day 48. Serum antibody responses were detected after the second CHP-NY-ESO-1 vaccination and antibody titer increased with subsequent vaccinations. Th1 dependent IgG1 was the predominant immunoglobulin subtype. Both, NY-ESO-1-specific CD4 and CD8 T cell responses were detected in PBMC by IFN-gamma secretion assays. After CHP-NY-ESO-1 vaccination a slight decrease in CD4(+)CD25(+)Foxp3(+) Tregs was observed in PBMC but significantly increased numbers of CD4(+)CD25(+)Foxp3(+) Tregs and CD68(+) immunoregulatory macrophages were detected at the local tumor sites. CD4(+)CD25(+)Foxp3(+) Tregs were also increased in the blister fluid. Cytokines in the serum suggested a polarization towards a Th1 pattern in the PBMC and those in the blister fluid suggested a Th2-type response at the tumor site. CONCLUSIONS: Our observations indicate induction of specific humoral and cellular immune responses against NY-ESO-1 after CHP-NY-ESO-1 vaccination in a melanoma patient. The concomitant appearance of regulatory T cells and of immune regulatory macrophages and cytokines at the local tumor sites in this patient may explain immune escape.     

Fine mapping of IGAD1 in IgA deficiency and common variable immunodeficiency: identification and characterization of haplotypes shared by affected members of 101 multiple-case families

To limit the region containing a mutation predisposing to selective IgA deficiency (IgAD) and common variable immunodeficiency (CVID), 554 informative members of 101 multiple-case families were haplotyped at the IGAD1 candidate locus in the MHC. Microsatellite markers were placed onto the physical map of IGAD1 to establish their order and permit rapid haplotype analyses. Linkage analysis of this extended family set provided additional support for a strong susceptibility locus at IGAD1 with a maximum multipoint nonparametric linkage score in excess of 3. Although the transmission of maternal IGAD1 haplotypes from unaffected heterozygous parents to the affected offspring was in excess, this was not apparent in multiple-case families with a predominance of affected mothers, suggesting that this parental bias is influenced by the affection status of transmitting parents and supporting a maternal effect in disease susceptibility. Of 110 haplotypes shared by 258 affected family members, a single haplotype (H1) was found in 44 pairs of affected relatives, accounting for the majority of the IGAD1 contribution to the development of IgAD/CVID in our families. The H1 allelic variability was higher in the telomeric part of the class III region than in the distal part of the class II region in both single- and multiple-case families. Incomplete H1 haplotypes had most variant alleles in the telomeric part of the analyzed region in homozygous IgAD/CVID patients, whereas this was not observed in unaffected homozygotes. These data suggest that a telomeric part of the class II region or centromeric part of the class III region is the most likely location of IGAD1.     

Protective immune responses induced by a non-pathogenic simian/human immunodeficiency virus (SHIV) against a challenge of a pathogenic SHIV in monkeys

A simian/human immunodeficiency virus (SHIV)-NM3n containing the human nef, but not the monkey nef, and vpr genes of SIV was inoculated into two cynomolgus monkeys, resulting in systemic infection with a minimum level of transient virus load. In order to study the nature of immune responses associated with the prevention of a pathogenic SHIV, the SHIV-NM3n-inoculated monkeys and three naive monkeys were intravenously challenged with a pathogenic SHIV containing the envelope gene of HIV-1 89.6. After the heterologous virus challenge, all of the SHIV-NM3n-inoculated animals completely avoided the loss of CD4+ T lymphocytes in PBMC as well as lymphoid tissues compared to pathogenic SHIV-injected control animals. The inhibition of CD4+ cell depletion was associated with maintaining the proliferative response of helper T-cells against SIV p27 in the previously nonpathogenic virus-inoculated animals following the pathogenic virus challenge. Furthermore, the decline of CD28+ cells, the increase in CD95+ cells, and the enhancement of in vitro apoptosis in PBMC were inhibited in the non-pathogenic virus-inoculated animals. These results suggest that nonpathogenic SHIV-NM3n infection induces the protection of monkeys from heterologous pathogenic viruses that may be associated with blocking the change in immune responses and the cell loss induced by a pathogenic virus.