The rs4774 CIITA missense variant is associated with risk of systemic lupus erythematosus.

The major histocompatibility complex (MHC) class II transactivator gene (CIITA) encodes an important transcription factor required for human leukocyte antigens (HLA) class II MHC-restricted antigen presentation. MHC genes, including the HLA class II DRB1*03:01 allele, are strongly associated with systemic lupus erythematosus (SLE). Recently the rs4774 CIITA missense variant (+1632G/C) was reported to be associated with susceptibility to multiple sclerosis. In the current study, we investigated CIITA, DRB1*03:01 and risk of SLE using a multi-stage analysis. In stage 1, 9 CIITA variants were tested in 658 cases and 1363 controls (N=2021). In stage 2, rs4774 was tested in 684 cases and 2938 controls (N=3622). We also performed a meta-analysis of the pooled 1342 cases and 4301 controls (N=5643). In stage 1, rs4774(*)C was associated with SLE (odds ratio (OR)=1.24, 95% confidence interval (95% CI)=1.07-1.44, P=4.2 × 10(-3)). Similar results were observed in stage 2 (OR=1.16, 95% CI=1.02-1.33, P=8.5 × 10(-3)) and the meta-analysis of the combined data set (OR=1.20, 95% CI=1.09-1.33, P(meta)=2.5 × 10(-4)). In all three analyses, the strongest evidence for association between rs4774(*)C and SLE was present in individuals who carried at least one copy of DRB1*03:01 (P(meta)=1.9 × 10(-3)). Results support a role for CIITA in SLE, which appears to be stronger in the presence of DRB1*03:01.

CIITA is not associated with risk of developing rheumatoid arthritis.

The major histocompatibility complex (MHC) class II transactivator gene (CIITA) encodes an important transcription factor regulating genes required for human leukocyte antigen (HLA) class II MHC-restricted antigen presentation. MHC genes, particularly HLA class II, are strongly associated with risk of developing rheumatoid arthritis (RA). Given the strong biological relationship between CIITA and HLA class II genes, a comprehensive investigation of CIITA variation in RA was conducted. This study tested 31 CIITA single-nucleotide polymorphisms in 2542 RA cases and 3690 controls (N=6232). All individuals were of European ancestry, as determined by ancestry informative genetic markers. No evidence for association between CIITA variation and RA was observed after a correction for multiple testing was applied. This is the largest study to fully characterize common genetic variation in CIITA, including an assessment of haplotypes. Results exclude even a modest role for common CIITA polymorphisms in susceptibility to RA.

A candidate gene study of CLEC16A does not provide evidence of association with risk for anti-CCP-positive rheumatoid arthritis.

CLEC16A, a putative immunoreceptor, was recently established as a susceptibility locus for type I diabetes and multiple sclerosis. Subsequently, associations between CLEC16A and rheumatoid arthritis (RA), Addison's disease and Crohn's disease have been reported. A large comprehensive and independent investigation of CLEC16A variation in RA was pursued. This study tested 251 CLEC16A single-nucleotide polymorphisms in 2542 RA cases (85% anti-cyclic citrullinated peptide (anti-CCP) positive) and 2210 controls (N=4752). All individuals were of European ancestry, as determined by ancestry informative genetic markers. No evidence for significant association between CLEC16A variation and RA was observed. This is the first study to fully characterize common genetic variation in CLEC16A including assessment of haplotypes and gender-specific effects. The previously reported association between RA and rs6498169 was not replicated. Results show that CLEC16A does not have a prominent function in susceptibility to anti-CCP-positive RA.

Analysis of maternal-offspring HLA compatibility, parent-of-origin and non-inherited maternal effects for the classical HLA loci in type 1 diabetes.

AIM: Type 1 diabetes (T1D) is a complex trait for which variation in the classical human leucocyte antigen (HLA) loci within the Major Histocompatibility Complex (MHC) significantly influences disease risk. To date, HLA class II DR-DQ genes confer the strongest known genetic effect in T1D. HLA loci may also influence T1D through additional inherited or non-inherited effects. Evidence for the role of increased maternal-offspring HLA compatibility, and both parent-of-origin (POO) and non-inherited maternal HLA (NIMA) effects in autoimmune disease has been previously established. The current study tested hypotheses that classical HLA loci influence T1D through these mechanisms, in addition to genetic transmission of particular risk alleles. METHODS: The Type 1 Diabetes Genetics Consortium (T1DGC) cohort was of European descent and consisted of 2271 affected sib-pair families (total n = 11 023 individuals). Class I genes HLA-A, Cw and B, and class II genes HLA-DRB1, DQA1, DQB1, DPA1 and DPB1 were studied. The pedigree disequilibrium test was used to examine transmission of HLA alleles to individuals with T1D. Conditional logistic regression was used to model compatibility relationships between mother-offspring and father-offspring for all HLA loci. POO and NIMA effects were investigated by comparing frequencies of maternal and paternal transmitted and non-transmitted HLA alleles for each locus. Analyses were also stratified by gender of T1D-affected offspring. RESULTS: Strong associations were observed for all classical HLA loci except for DPA1, as expected. Compatibility differences between mother-offspring and father-offspring were not observed for any HLA loci. Furthermore, POO and NIMA HLA effects influencing T1D were not present. CONCLUSIONS: Maternal-offspring HLA compatibility, POO and NIMA effects for eight classical HLA loci were investigated. Results suggest that these HLA-related effects are unlikely to play a major role in the development of T1D.

Linkage and association study of late-onset Alzheimer disease families linked to 9p21.3.

A chromosomal locus for late-onset Alzheimer disease (LOAD) has previously been mapped to 9p21.3. The most significant results were reported in a sample of autopsy-confirmed families. Linkage to this locus has been independently confirmed in AD families from a consanguineous Israeli-Arab community. In the present study we analyzed an expanded clinical sample of 674 late-onset AD families, independently ascertained by three different consortia. Sample subsets were stratified by site and autopsy-confirmation. Linkage analysis of a dense array of SNPs across the chromosomal locus revealed the most significant results in the 166 autopsy-confirmed families of the NIMH sample. Peak HLOD scores of 4.95 at D9S741 and 2.81 at the nearby SNP rs2772677 were obtained in a dominant model. The linked region included the cyclin-dependent kinase inhibitor 2A gene (CDKN2A), which has been suggested as an AD candidate gene. By re-sequencing all exons in the vicinity of CDKN2A in 48 AD cases, we identified and genotyped four novel SNPs, including a non-synonymous, a synonymous, and two variations located in untranslated RNA sequences. Family-based allelic and genotypic association analysis yielded significant results in CDKN2A (rs11515: PDT p = 0.003, genotype-PDT p = 0.014). We conclude that CDKN2A is a promising new candidate gene potentially contributing to AD susceptibility on chromosome 9p.

The MHC2TA -168A/G polymorphism and risk for rheumatoid arthritis: a meta-analysis of 6861 patients and 9270 controls reveals no evidence for association.

BACKGROUND: An association between major histocompatibility complex (MHC) genes, particularly those within the class II HLA region, and rheumatoid arthritis (RA) is well established, and accounts for an estimated 30% of the genetic component in RA. The MHC class II transactivator gene (MHC2TA) on chromosome 16p13 has recently emerged as the most important transcription factor regulating genes required for class II MHC-restricted antigen presentation. Previous studies of a promoter region polymorphism (-168A/G, rs3087456) in the MHC2TA gene and RA have yielded conflicting results. OBJECTIVE: To assess the association of the MHC2TA -168A/G polymorphism (rs3087456) and risk for RA by meta-analysis. METHODS: Meta-analysis was performed for 6861 patients with RA and 9270 controls from 10 case-control studies. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for each study. Summary ORs and 95% CIs were calculated for random effects models. RESULTS: No effect was observed for the G risk allele (OR 1.02, 95% CI 0.93 to 1.12, p = 0.70) or the GG risk genotype (OR 1.14, 95% CI 0.95 to 1.36, p = 0.16). CONCLUSIONS: Our results indicate that the MHC2TA -168A/G polymorphism (rs3087456) is not associated with RA yet underscore the importance of including shared epitope allele carrier status, secondary phenotypes and more complete characterisation of MHC2TA variation in future studies.

Exploring the association of glyceraldehyde-3-phosphate dehydrogenase gene and Alzheimer disease.

BACKGROUND: Previous linkage studies have shown that chromosome 12 harbors susceptibility genes for late-onset Alzheimer disease (LOAD). However, association studies of several candidate genes on this chromosome region have produced ambiguous results. A recent study reported the association between the glyceraldehyde-3-phosphate dehydrogenase (GAPD) gene on chromosome 12p and the risk of LOAD. METHODS: The authors conducted family-based and case-control association studies in two independent LOAD data sets on 12 single-nucleotide polymorphisms (SNPs) in the GAPD gene and its paralogs. RESULTS: No association was found of the GAPD gene with LOAD in the family-based data set, but marginal evidence of association was seen in the later-onset subgroup when age at onset was stratified. The SNP rs2029721 in one GAPD pseudogene was also found to be associated with risk for LOAD in the unrelated case-control data set (p = 0.003). CONCLUSIONS: The GAPD gene and its pseudogene may play a role in the development of late-onset Alzheimer disease. However, the effect, if any, is likely to be limited.

Lack of association between UBQLN1 and Alzheimer disease.

Alzheimer disease (AD) is heterogeneous and complex with a strong genetic diathesis. It is the most common cause of dementia affecting the elderly. Linkage studies [Kehoe et al., 1999; Hum Mol Genet 8: 237-245]; [Pericak-Vance et al., 2000; Exp Gerontol 35: 1343-1352]; [Myers et al., 2002; Am J Med Genet 114: 235-244]; [Blacker et al., 2003; Hum Mol Genet 12: 23-32] identified chromosome 9q as a region containing a possible AD candidate gene. Functional protein studies [Mah et al., 2000; J Cell Biol 151: 847-862]; [Ko et al., 2002; J Biol Chem 277: 35386-35392] identified the UBQLN1 gene on chromosome 9q that encodes ubiquilin as a likely candidate for a role in late-onset AD pathogenesis. A recent family-based study by [Bertram et al., 2005; N Engl J 352: 884-894] reported genetic association and expression evidence for a putative AD risk allele of an intronic single nucleotide polymorphism (SNP) within the UBQLN1 gene. In this study, we comprehensively assessed whether any of seven polymorphisms located across the UBQLN1 gene are associated with AD in another large family-based data set and an independent case-control data set. We found no significant association of AD risk with any of the seven SNPs genotyped (including those SNPs previously reported by Bertram et al.) in either the family-based or case-control data set. Age-specific analyses and analyses conditional on Apolipoprotein E (ApoE) genotype and sex also revealed no significant associations to AD risk in either data set. Using age at onset (AAO) as a quantitative trait revealed a modest age modifying association; however, the results were inconsistent between the data sets. Our results suggest that UBQLN1 variants do not increase risk for AD in these data.

Covariate analysis of late-onset Alzheimer disease refines the chromosome 12 locus.

Alzheimer disease (AD) is a progressive neurodegenerative disorder of later life with a complex etiology and a strong genetic component. Several genomic screens have suggested that a region between chromosome 12p13 and 12q22 contains at least one additional locus underlying the susceptibility of AD. However, localization of this locus has been difficult. We performed a 5 cM microsatellite marker screen across 74 cM on chromosome 12 with 15 markers in 585 multiplex families consisting of 994 affected sibpairs and 213 other affected relative pairs. Analyses across the entire data set did not reveal significant evidence of linkage. However, suggestive linkage was observed in several subsets. In the 91 families where no affected individuals carry an ApoE varepsilon4 allele, an HLOD score of 1.55 was generated at D12S1042. We further examined the linkage data considering the proposed linkages to chromosome 9 (D9S741) and chromosome 10 (alpha-catenin gene). There was a modest (P=0.20) increase in the LOD score for D12S368 (MLOD=1.70) when using the D9S741 LOD scores as a covariate and a highly significant (P<0.001) increase in the MLOD score (4.19) for D12S1701 in autopsy-confirmed families (n=228) when using alpha-catenin LOD scores as a covariate. In both cases, families with no evidence of linkage to D9S741 or alpha-catenin demonstrated most of the evidence of linkage to chromosome 12, suggesting locus heterogeneity. Taken together, our data suggest that the 16 cM region between D12S1042 and D12S368 should be the subject of further detailed genomic efforts for the disease.

Interaction between the alpha-T catenin gene (VR22) and APOE in Alzheimer's disease.

BACKGROUND: APOE is the only gene that has been consistently replicated as a risk factor for late onset Alzheimer's disease. Several recent studies have identified linkage to chromosome 10 for both risk and age of onset, suggesting that this region harbours genes that influence the development of the disease. A recent study reported association between single nucleotide polymorphisms (SNPs) in the VR22 gene (CTNNA3) on chromosome 10 and plasma levels of Abeta42, an endophenotype related to Alzheimer's disease. OBJECTIVE: To assess whether polymorphisms in the VR22 gene are associated with Alzheimer's disease in a large sample of Alzheimer's disease families and an independent set of unrelated cases and controls. RESULTS: Several SNPs showed association in either the family based or case-control analyses (p<0.05). The most consistent findings were with SNP6, for which there was significant evidence of association in both the families and the unrelated cases and controls. Furthermore, there was evidence of significant interaction between APOE-4 and two of the VR22 SNPs, with the strongest evidence of association being concentrated in individuals carrying APOE-4. CONCLUSIONS: This study suggests that VR22 or a nearby gene influences susceptibility to Alzheimer's disease, and the effect is dependent on APOE status.

Opsonization of Actinobacillus actinomycetemcomitans by immunoglobulin G antibodies to the O polysaccharide of lipopolysaccharide.

Sera of localized juvenile periodontitis (LJP) patients colonized by Actinobacillus actinomycetemcomitans serotype b often contain markedly elevated levels of immunoglobulin G (IgG) antibodies to serospecific determinants in the O polysaccharide of lipopolysaccharide (LPS), as well as to outer membrane proteins of this species. IgG antibodies in LJP sera are known to opsonize A. actinomycetemcomitans for subsequent phagocytosis and killing by human neutrophils. The objective of this study was to determine whether outer membrane proteins or serospecific determinants in LPS are the primary target for opsonic IgG antibodies in LJP sera. An A. actinomycetemcomitans serotype b O-polysaccharide affinity column was constructed and subsequently used to purify LPS-specific IgG antibodies from LJP serum. The affinity-purified anti-LPS IgG antibodies were enriched in content of IgG2 (66.2%, compared with 37.0% in the total IgG fraction) and were immunospecific for A. actinomycetemcomitans serotype b LPS. In an opsonophagocytic assay using neutrophils from donors who were homozygous for the H131 allotype of Fcy receptor IIa (CD32), it was found that LPS-specific IgG antibodies exhibited substantially greater opsonic activity toward A. actinomycetemcomitans serotype b than an LJP IgG fraction that was depleted of LPS-reactive antibodies but contained antibodies against outer membrane proteins of this species. The results of this study indicate that serospecific determinants in the O polysaccharide of A. actinomycetemcomitans serotype b are a principal target for opsonic antibodies in sera of LJP subjects.

Immunoglobulin G subclass response to major outer membrane proteins of nontypable Haemophilus influenzae in children with acute otitis media.

Children with acute otitis media as the result of nontypable Haemophilus influenzae often develop serum bactericidal and/or opsonic IgG antibodies to this organism during convalescence. Outer membrane proteins appear to be the principal targets for such antibodies. In this study we characterized the IgG subclass responses to major outer membrane proteins of nontypable H. influenzae in otitis-prone children in whom this organism had colonized. Three of the major outer membrane proteins (P2, P5, and P6) were isolated from the homologous nontypable H. influenzae strain recovered from the middle ear at the time of acute infection. Sera were obtained during the acute phase and at 1 and 6 months thereafter. The outer membrane proteins, which were isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis, were used as test antigens in a quantitative IgG subclass enzyme immunoassay. The results of this analysis indicate that the temporal characteristics and distribution of IgG subclass antibodies were found to differ for each of the outer membrane proteins. Moreover, substantial variation between patients was observed with respect to both temporal characteristics and subclass distribution of the IgG response to the three outer membrane proteins. Significantly, sera from two of three otitis-prone subjects contained detectable levels of IgG antibody to the conserved P6 outer membrane protein at the time of acute infection, with serum from one subject also containing detectable levels of IgG3 antibody to this same protein. Nevertheless, the organism persisted in the middle ears of these patients. The results of this study indicate that otitis-prone children manifest a highly variable IgG subclass response to both conserved (P6) and variable (P2) outer membrane proteins of nontypable H. influenzae. Further study is required to ascertain whether these IgG subclass antibodies are biologically efficacious and whether otitis-prone children possess the immunologic maturity to respond to nontypable H. influenzae outer membrane protein-based vaccines in a predictable manner.

Immunoglobulin G2 antibodies promote neutrophil killing of Actinobacillus actinomycetemcomitans.

Sera from patients with localized juvenile periodontitis (LJP) often contain markedly elevated levels of immunoglobulin G2 (IgG2) antibodies reactive to cell envelope constituents of Actinobacillus actinomycetemcomitans. The objective of this study was to determine if these IgG2 antibodies are capable of supporting phagocytosis and killing of A. actinomycetemcomitans by human neutrophils. Polyclonal IgG2 antibodies were prepared from high-titer LJP serum by affinity chromatography, yielding a preparation which was > 99% subclass restricted and retained immunoreactivity to A. actinomycetemcomitans antigens. Affinity-purified IgG2 antibodies were evaluated by an in vitro opsonophagocytic assay that employed neutrophils obtained from donors who were homozygous for the H131 allotype of Fc gamma receptor type IIa (CD32), which efficiently binds human IgG2 antibodies. Affinity-purified IgG2 antibodies from LJP serum but not from sera of periodontally healthy individuals promoted phagocytosis and killing of A. actinomycetemcomitans. The expression of IgG2-dependent opsonic activity required the presence of complement. Incubation of A. actinomycetemcomitans with neutrophils in the presence of an optimal concentration of LJP IgG2 (50 micrograms/ml) and 5% hypogammaglobulinemic serum (as a complement source) resulted in a > 1 log10 reduction in bacterial viability within 30 min. The opsonic activity of IgG2 antibodies was found to be comparable to that observed with affinity-purified IgG1 antibodies. Moreover, IgG1 antibodies interacted synergistically with IgG2 antibodies in promoting opsonophagocytosis of A. actinomycetemcomitans. The results of this study indicate that LJP serum contains IgG2 antibodies which, when employed in conjunction with neutrophils that express Fc gamma receptors capable of recognizing this subclass, are opsonic for A. actinomycetemcomitans.

Development of a rapid latex agglutination test for periodontal pathogens.

The studies reported here describe the development, characterization, and initial application of latex agglutination assays for periodontal pathogens. Latex reagents were prepared by sensitization of latex microspheres with rabbit IgG antibodies to either Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, or Prevotella intermedia. The protein concentration utilized for sensitization and microsphere size were optimized. One reagent was prepared to A. actinomycetemcomitans and a second combination reagent was prepared by mixing latex sensitized with antibodies to P. gingivalis and latex sensitized with antibodies to P. intermedia. The sensitivity of both latex reagents in the traditional wet and a dried format was evaluated. In addition, sensitivity and specificity with homologous and heterologous bacterial suspensions were evaluated. The reagents were found to demonstrate both specificity and sensitivity. Initial studies with subgingival human plaque demonstrated the ability of these reagents to detect the specific organisms in plaque.

Capnocytophaga species: increased resistance of clinical isolates to serum bactericidal action.

Capnocytophaga, a newly recognized genus of capnophilic gram-negative bacilli, is part of the normal oral flora. The capacity of Capnocytophaga to cause sepsis and local infections in both immunocompromised and nonimmunocompromised hosts has been documented. Given the recognition that serum resistance may contribute to the virulence of some gram-negative bacteria, we attempted to define the serum sensitivity of clinical isolates of Capnocytophaga from blood and other sites of infection. Whereas nine of nine isolates from human subgingival plaque showed greater than 95% loss of viability under standardized assay conditions, nonoral isolates exhibited variable serum sensitivity. Six of six isolates from blood showed considerable serum resistance (mean survival, 59.7% +/- 38.3%; range, 14.4%-113.3%). Comparison of the electrophoretic mobilities of lipopolysaccharides (LPSs) from sensitive and resistant strains revealed reduced LPS heterogeneity and lower apparent molecular weight among serum-resistant strains. Thus, serum resistance, possibly influenced by LPS structure, may be an important factor contributing to the pathogenic potential of Capnocytophaga spp.

Human oviductal fluid proteins. II. Preparation of an antiserum to a human oviductal fluid protein: existence of autoantibodies against it in some sera.

Rabbit antisera elicited by injection of human oviductal fluid (HOF) and rendered specific by absorption with human kidney and human serum revealed the presence of two possibly HOF-specific antigen(s). One antigen was detected by immunodiffusion in 14 of the 14 fluids tested with one of these antisera. HOF-specific antigen was shown to be a beta-globulin by immunoelectrophoresis. A second antigen was seen in two samples. That autoantibodies might exist directed against HOF-specific antigen in the donor's serum was suggested by immunodiffusion experiments in which 4 of the 13 sera tested gave lines of precipitation when reacted against the corresponding oviductal fluid. In one instance, this line was the same as one of the two lines seen in tests using rabbit antiserum.

Affinity diffusion. I. Method for measuring dissociation constants of precipitating antibodies.

Dissociation constant (Kd) of antigen-antibody reactions can be obtained from the rates (measured by the progression of the precipitate vs. time) with which antigens diffuse into antibody-containing gels, as a function of antibody-concentration. A bovine serum albumin vs. rabbit anti-bovine serum albumin system was studied with whole antiserum and with its purified IgG fraction. A value was found for Kd of approximately 1.0 x 10-5 moles per liter. It is note- worthy that in monodimensional single diffusion gel precipitation systems of this type, the rate of progression of the precipitate front is significantly faster than the molecular diffusion coefficient of the antigen.

Affinity diffusion II. Comparison between thermodynamic data obtained by affinity diffusion and precipitation in tubes.

Association constants (Ka) of the precipitating system bovine serum albumin (BSA) goat anti-BSA were obtained at different temperatures via affinity diffusion (taking l/Kd - Ka) as well as via precipitation in tubes at optimal ratios. With affinity diffusion values of Ka of 0.6 to 1.1 x 10(5) l/M were found, whilst with precipitation in tubes Ka was from 3.3 to 11.2 X 10(7) l/M, using the same BSA and anti-BSA preparations. Via affinity diffusion binding energies delta F of approximately -6 to -7 kcal/M were found, with values of delta H close to zero, and a delta S of +23 entropy units. With precipitation in tubes these values were delta F -10.2 to -10.7 kcal/M, delta H -4.6 to -7.6 kcal/M and delta S +10 to +20 entropy units. The differences found with the two different methods must be ascribed to the fact that with affinity diffusion of precipitating antigen-antibody systems one just measures the interaction between the precipitating components with the highest dissociation constants, whilst with precipitation in tubes one measures the total energy of association of the system. With affinity diffusion and with precipitation in tubes, the same degree of positive entropy is observed. The system measured with affinity diffusion is approximately isothermic, whilst the total system, measured by precipitation in tubes, is strongly exothermic. Affinity diffusion still takes place at pH 9.5, at which pH no precipitation in the liquid phase takes place at optimal ratio; one may conclude from this that affinity diffusion mainly involves van der Waals interactions, as electrostatic bonding between BSA and anti-BSA is virtually abolished at that pH. This agrees well with the observation that the affinity diffusion reaction is isothermic.

Two methods for the removal of erythrocytes from buffy coats for the production of human leukocyte interferon.

Two different methods of removing erythrocytes from buffy coats, with the ultimate aim of inducing the white blood cells for the production of leukocyte interferon, i.e., erythrocyte lysis with ammonium chloride, and erythrocyte agglomeration with hydroxyethyl starch, are compared. After two treatments with 0.83% NH4Cl all erythrocytes are lysed and most granulocytes are damaged, although the lymphocytes appear mostly unaffected (as judged by microphotography of Wright-stained smears). Treatment with 3% hydroxyethyl starch causes most erythrocytes to agglomerate and sediment to the bottom of a vessel in less than one hour; the leukocytes (lymphocytes as well as granulocytes), which remain in suspension, are morphologically intact. NH4Cl-treated leukocytes yield, on an average, 3.6 times less interferon than hydroxyethyl starch-treated leukocytes. Leukocyte viability, as judged by trypan blue exclusion does not appear much influenced by either NH4Cl or hydroxyethyl starch treatment. The advantage of hydroxyethyl starch apparently lies in the fact that its use leaves the granulocytes intact and thus obviates the undue release of proteolytic enzymes into the culture medium, with their concomitant deleterious effect on interferon production.

Preparative electrophoresis of human lymphocytes. II. Use of non-immunoglobulin bearing lymphocytes obtained by electrophoretic levitation for the elicitation of a rabbit anti-human "T-cell" serum.

By intradermal injection (in Freund's complete adjuvant) of rabbits with only a few million human T-lymphocytes, obtained by preparative electrophoresis, an anti-human "T-cell" serum can be obtained. Although the antiserum is best used in a fairly concentrated form, its anti-human "T-cell" specificity (by immunofluorescence and E-rosetting) appears excellent, without the need to resort to absorption with various unwanted antigens. There may however, also be other specificities in the antiserum, as it also reacts with human granulocytes.