Allotype analysis to distinguish the origin of varicella-zoster virus immunoglobulin G after allogeneic stem cell transplantation.

Varicella-zoster virus (VZV) reactivation is a frequent complication after allogeneic hematopoietic stem cell transplantation (HSCT). Although previous studies have revealed that cellular immunity is important for suppressing reactivation, the role of humoral immunity against VZV has been poorly evaluated. We analyzed inherited polymorphisms in the immunoglobulin G (IgG) heavy chain constant regions of 50 HSCT recipient-donor pairs to distinguish donor-derived and recipient-derived antibodies. Twelve pairs were informative regarding the origin of IgG, since either the donors (n = 3) or recipients (n = 9) were homozygous null for the IgG1m(f) allotype. In these 9 homozygous-null recipients, allotype-specific IgG against VZV were measured by enzyme-linked immunosorbent assay and compared with measles-IgG. All 9 homozygous-null recipients were monitored for more than 1 year after HSCT, with (n = 4, localized zoster) or without (n = 5) clinical VZV disease. In 3 patients with VZV disease, donor-derived IgG against VZV was elevated between 500 to 700 days after HSCT after the episode of VZV disease. In 1 patient who suffered from VZV disease just before HSCT, donor-derived VZV IgG was elevated within 3 months after HSCT. On the other hand, 2 patients who received reduced-intensity conditioning (RIC) transplantation from an IgG1m(f) null donor maintained recipient-derived IgG against VZV for more than 1 year, whereas it was decreased within 3 months in 1 recipient who received conventional conditioning. In conclusion, the production of anti-VZV IgG by recipient plasma cells persists long after RIC. In patients without symptomatic VZV reactivation, donor-derived anti-VZV IgG did not reach titers comparable to those measured in healthy virus carriers.

Genetic regulation of antibody responses to human epidermal growth factor receptor 2 in breast cancer.

Host genetic factors responsible for the interindividual differences in naturally occurring antibody responses to the human epidermal growth factor receptor 2 (HER-2) in human beings have not been identified. The aim of the present investigation was to determine whether immunoglobulin gamma heavy-chain marker (GM) and kappa light-chain marker (KM) allotypes, which are genetic markers of IgG heavy chains and κ-type light chains, respectively, contribute to these differences. A total of 152 Estonian women with breast cancer were characterized for IgG antibodies to HER-2 and allotyped for several GM and KM markers. IgG3 determinant GM 13 was significantly associated with higher HER-2 IgG levels (median IgG titer 800 vs 400, p = 0.007). Other GM allotypes, known to be in linkage disequilibrium with GM 13, were also associated with higher anti-HER-2 antibody levels, albeit not as strongly. These results show that GM allotypes are associated with humoral immunity to HER-2, a finding with potentially significant implications for immunotherapy of breast cancer.

Epistatic effects of genes encoding immunoglobulin GM allotypes and interleukin-6 on the production of autoantibodies to 60- and 65-kDa heat-shock proteins.

Immunoglobulin GM and KM genes have been associated with antibody responses to a variety of antigens. A promoter-region polymorphism of interleukin-6 (IL-6) gene (-174 G/C) has been shown to be associated with antibody responses to heat-shock proteins (hsp) 60 and hsp65. To examine the possible epistatic effects of these unlinked genetic systems on the autoimmune responses to hsp60 and hsp65, 176 healthy Caucasian subjects from Finland were genotyped for several allelic determinants of GM, KM, and IL-6 genes by PCR-RFLP methods. IgG antibodies to hsp60 and hsp65 were measured by an ELISA. Significant interactive effects of GM f,z and IL-6-174 genotypes were noted for both anti-hsp60 (P=0.002) and anti-hsp65 (P=0.038) antibody levels. Since these autoantibodies have been implicated in susceptibility to coronary heart disease and carotid atherosclerosis, the associations reported here might be relevant to the etiology of these diseases.

Gel test assay for IgG subclass detection by GM typing: application to hemolytic disease of the newborn.

The gel test assay was evaluated for IgG subclass detection by GM typing of antibodies and compared to the classical inhibition agglutination method on slides or microtiter plates. We used a panel of 5 murine monoclonal antibodies directed against G1M(1), G1M(3), G1M(17), G2M(23), and G3M(21) and 1 human polyclonal anti-G3M(5) antibody. Eleven polyclonal antisera (of immunized women) directed against red blood cells were tested for the GM allotypes carried by their alloantibodies. We controlled the specificity of the gel test reaction using a panel of anti-RH(D) monoclonal antibodies. All reagents exhibited a good reactivity and specificity. They can be used for routine typing. The gel test assay for IgG subclass detection is a specific, simple, and low-cost technique for the detection and management of severe forms of diseases in alloimmunized pregnancies.

Serum IgG2 level, Gm(23) allotype and FcgammaRIIa and FcgammaRIIIb receptors in refractory periodontal disease.

The purpose of this investigation was to compare the levels of serum IgG2, the frequency of detection of Gm(23)-negative allotype and frequency of detection of FcgammaRIIa and FcgammaRIIIb receptor haplotypes in 32 refractory, 54 successfully treated and 27 periodontally healthy individuals. Refractory subjects showed mean full mouth attachment loss and/or >3 sites with attachment loss >2.5 mm within 1 year after both scaling and root planing, and surgery plus systemically administered tetracycline. Successfully treated subjects showed mean attachment level gain and no sites with attachment loss >2.5 mm 1 year post-therapy. Periodontally healthy subjects exhibited no pocket depth or attachment level >3 mm, and no evidence of progressing disease during 1 year of monitoring. Blood was obtained from each subject at baseline. Serum IgG2 and Gm(23) allotype were determined using radial immunodiffusion. DNA was extracted from whole blood and the FcgammaR genotypes determined using PCR and allele specific oligonucleotide probes. Significance of differences among clinical groups were sought using the Kruskal-Wallis or chi-square tests. Associations between 2 or more variables were tested using regression analysis. Refractory subjects exhibited higher mean attachment loss and pocket depth than successfully treated or periodontally healthy subjects. Smoking status did not differ significantly among groups. No significant differences in serum IgG2 levels and frequency of detection of Gm(23)-negative allotype were observed among the clinical groups. Serum IgG2 level was positively associated with the number of serum antibody responses to subgingival species (r=0.51, p<0.001). Subjects with the Gm(23)-negative allotype exhibited lower mean levels of serum IgG2 (3.06+/-0.3 versus 3.9+/-0.2, p<0.01) and mean number of serum antibodies to subgingival species (17.7+/-1.7 versus 23.3+/-1.4, p<0.05) than allotype positive individuals. No significant differences in FcgammaR haplotype distribution were observed among the 3 clinical groups. Associations of serum IgG2 level, Gm(23) allotype, FcgammaRIIa and FcgammaRIIIb receptor haplotypes and smoking status were weakly related or not related to clinical status. This lack of relationship may have been due to a reality of no relationship, or the inadvertent pooling of subjects where these factors were of primary importance with subjects in whom these factors played a less important role.

Cellular immuno-PCR. Detection of a carbohydrate tumor marker.

Carbohydrate tumor-antigens are important tumor markers for diagnosis and functional characteristics of human cancer cells. Detection of these carbohydrate tumor antigens on metastatic cancer cells in blood is a difficult task. We developed a highly sensitive method to detect a cell surface carbohydrate antigen using a hybrid technology referred to as cellular immuno-PCR. This technique uses the human monoclonal antibody (HumAb) L612, specific to a tumor-related antigen (ganglioside) GM3 that is expressed on the cell surface of human tumor cells and not normal cells. L612 coupled to a DNA oligonucleotide for exponential amplification by DNA polymerase chain reaction (PCR) can be used to enhance the detection signal. The DNA-HumAb conjugate was assessed for detection of a small number of human cancer cells after PCR amplification and Southern blot analysis. To assess the assay specificity human melanoma and other cancer cell lines, as well as healthy donor and melanoma patients, bloods were assessed. Cellular immuno-PCR requires < 1 ng/ml DNA-HumAb complex and was shown to have a detection level of < 10 cells in titration studies in which melanoma cells were diluted in 2 million healthy donor peripheral blood lymphocytes. The assay was shown to be very sensitive and could detect low levels of GM3 antigen expression by tumor cells. This novel approach for detecting a carbohydrate tumor antigen on tumor cells in blood provides a potential useful clinicopathological assay.

Autoimmunity to ryanodine receptor and titin in myasthenia gravis is associated with GM allotypes.

Myasthenia gravis (MG) is mediated by autoantibodies against the acetylcholine receptor at the muscle endplate. Some MG patients have in addition antibodies (Ab) to the skeletal muscle proteins ryanodine receptor (RyR) and titin. We have examined GM and KM allotypes, RyR and titin Ab in 44 MG patients (37 thymoma patients and 7 non-thymoma, late-onset patients) and 292 non-MG controls to see if GM/KM allotypes associate with differences in autoantibody production. All patients had titin Ab, and 15 thymoma patients had also RyR Ab. The phenotype GM 1, 2, 3 23 5, 21 was significantly increased in the patients with titin Ab compared with the non-MG controls (chi2 = 4.93, p < 0.05). Thymoma patients with RyR Ab had a higher frequency of the GM 3 23 5 phenotype compared with RyR Ab negative patients and controls (chi2 = 7.1, p < 0.05). KM allotypes did not differ between RyR Ab positive or titin Ab positive patients and controls. GM phenotypes may thus be associated with an autoimmune response against the muscle proteins titin and RyR in MG patients.

Serum Gm allotype levels in common variable immunodeficiency: preponderance of homozygous G2m(",") on IGHCG2.

Common variable immunodeficiency (CVI) is one of the most frequent primary immunodeficiency diseases, characterized by defective antibody formation and associated with chronic sinopulmonary infections, autoimmunity and malignancies. The genes for the constant heavy chains of IgG are located on chromosome 14 and were further studied by identifying allelic, alternative Gm allotypes. These were defined by different epitopes for three of the IgG subclasses, G1m(a) and G1m(f) for IgG1, G2m(n) and G2m(") for IgG2 and G3m(g) and G3m(b) for IgG3. A sensitive competitive ELISA method for quantitation of the Gm allotypes G1m(a), G1m(f), G2m(n) and G3m(b) were used together with radial immunodiffusion IgG subclass quantitation. The dominating number of 25 of 33 patients (p < 0.001) expressed the homozygous G2m(",") allotype on IGHCG2 in combination with homozygous or heterozygous Gm allotypes on IGHCG1 and IGHCG3, namely Gm(f,f;",";b,b), Gm(a,a;",";g,g) and Gm(f,a;",";b,g). Studies of Gm allotype quantities revealed a progressive sequential impediment of the programmed cascade for downstream IGHCG gene rearrangements. According to the order of the IGHCG genes, the G3m allotype levels from the IGHCG3 were often normal, and G1m allotype levels from IGHCG1 were suppressed; G1m(a) was suppressed more than G1m(f), and most suppressed were G2m allotype levels from IGHCG2, both G2m(n) and G2m("). The susceptibility of CVI is associated to G2m(",") expression from the IGHCG2 locus on chromosome 14, which has also been found in IgA IgG subclass deficiency, conditions known among first-degree relatives.

Low concentrations of Gm allotypic subsets G3 mg and G1 mf in homozygotes and heterozygotes.

Serum concentrations of IgG3, IgG1, and of the Gm allotypic subsets of these two isotypes were measured in adult homozygotes and heterozygotes. Alleles G3 mb and G3 mst of the IgG3 locus, and alleles G1 ma and G1 max of the IgG1 locus were found to associate with a high concentration of the allotypic product. Alleles G3 mg (IgG3) and G1 mf (IgG1) were associated with a low concentration of the product. This was true regardless of the haplotype; for example, allele G3 mb was associated with a high concentration of the product in all haplotypes f;n+;b f;n-;b and fa;n+;b. One dose of allele G3 mg was associated with a characteristic mean concentration of the product (g-type IgG3). This rule was valid regardless of the other allele of the subject, thus, heterozygotes and G3 mg/g homozygotes had mean concentrations of 0.10 and 0.20 g/liter, respectively, of g-type IgG3. Products of the IgG1 alleles were also simply additive: one dose of allele G1 ma(x) or G1 mf was associated with mean concentrations of 3.63 and 2.84 g/liter, respectively, and two doses with twice these amounts. Only allele G3 mb did not completely follow this rule. We also studied the serum concentrations and the allotype distribution of 41 IgG1 and 31 IgG3 myeloma proteins. The results suggested that the allotype-associated differences in serum concentrations are caused by different numbers of B cells producing allotypic subsets of IgG1 or IgG3, not by different rates of synthesis per B cell.

Quantitation of Gm allotypes.

A method for quantitation of Gm allotypes is described. Alternative Gm allotypes of the three IgG subclasses, IgG1, IgG2 and IgG3, were investigated for the six most common Caucasian Gm phenotypes. Quantitation of G1m(a), G1m(f) of IgG1, G2m(n) of IgG2 and G3m(b) of IgG3 was performed with specific monoclonal antisera and purified myeloma proteins of different Gm allotypes. Mean +/- SD are given as percentage of a normal serum pool and in g/l for the Gm allotypes G1m(a), G1m(f), G2m(n) and G3m(b). For homozygous individuals the G2m(",") values are equal to the IgG2 levels and the G3m(g,g) values equal to the IgG3 levels. For heterozygous individuals the value for G2m(") is calculated as IgG2 minus G2m(n) and for G3m(g) as IgG3 minus G3m(b). Homozygous individuals have about double the amounts of the Gm allotype compared with heterozygous individuals. The gene activity of heterozygous individuals is given by quotients, mean +/- SD for G1m(a)/G1m(f) of IgG1, G2m(n)/G2m(") of IgG2 and G3m(b)/G3m(g) of IgG3 in different Gm phenotypes. Heterozygous individuals on all three IgG subclass loci have at least six different qualities of IgG molecules compared with three for homozygous individuals.

Genetic polymorphism of IgG in the mink. IX. High proportion of allotype-producing lymphocytes in individuals with minor level of allotypes H3 and H4 in serum.

The levels of mink C gamma-allotypes (H3, H4, H6 and H8) were determined in sera, and the proportion of the corresponding allotype-synthesizing B cells was estimated in peripheral blood, spleen and mesenteric lymph nodes. Individual differences in H6 levels and, possibly, those in H8 were entirely dependent on the proliferation degree of the corresponding clone of B cells and also determined by the dosage of the structural gene. There was no correspondence between the great numbers of H3+, H4+ cells and low levels of H3 and H4 allotypes in the sera of the majority of minks with their minor expression. A possible cause of this discrepancy may be a blockade of the secretion of IgG by H3+, H4+ cells. There exists most likely a gene (or genes) controlling the blockade of IgG secretion. The regulation of C gamma-allotype expression is presumably effected in a manner specific to each of the allotypes.