William J. Yount

Mannan antigenemia in the diagnosis of invasive Candida infections

Because it is often difficult to diagnose invasive Candida infections, a sensitive hemagglutination inhibition assay to detect the surface antigen, mannan, was developed. Mannan antigenemia was detected early in the course of infection in 4 of 14 patients with systemic candidiasis and 2 of 5 patients with invasive gastrointestinal candidiasis. Mannan was not detected in 48 patients with noninvasive Candida or other systemic mycotic infections or in 99% of 234 patients in other control groups. Mannan antibodies were almost universally present in both candidiasis and control groups. In four patients with systemic candidiasis, an early period of mannan antigenemia was followed by a rapid rise in mannan antibody titer. These findings suggest that antemortem diagnosis would be improved in one-third of cases of invasive Candida infection detected by the hemagglutination inhibition assay. A positive test for serum mannan would be an early and specific signal of invasive disease.

Imbalances of gamma globulin subgroups and gene defects in patients with primary hypogammaglobulinemia

Analysis of immunoglobulin classes, gammaG subgroups, and Gm genetic markers from 59 patients with various types of immune deficiencies was undertaken to assess the function of the several cistrons concerned with synthesis of gamma globulins. 13 patients including two sibling pairs were found to have gammaG subgroup imbalances. All of these patients had non sex-linked disease. 11 of the 13 had preponderance of the gammaG3 subgroup. In most instances of gammaG3 preponderance it was the Gm(b) type of gammaG3 that was selectively retained; the Gm(g) type, controlled by the allelic gene was markedly depressed but not absent in the cases where it could be studied. Other imbalances, either seen concomitantly with gammaG3 preponderance or independently, included predominance of the gammaG2 subgroup and selective absence of single gammaG subgroups.One family was encountered with probable structural gene abnormalities in the autosomal Gm loci. Both parents had different abnormal gene complexes detectable by absence of specific Gm markers and the propositus received both types from the parents. Similar gene complexes have been seen previously in rare instances through population screening but only in the heterozygous state and were not associated with clinically evident hypogammaglobulinemia. Of several other families of patients with subgroup imbalance, two were informative in that structural gene defects could be excluded. Studies on 22 first degree relatives of patients with subgroup imbalances indicated that the most common abnormality detected was in gammaA which was absent in 3 and markedly decreased in 2 others; other abnormalities included decreased levels of specific genetic types of gammaG globulin. It is concluded that gammaG subgroup imbalances are frequently found in non sex-linked immunoglobulin deficiency disorders and in some instances may be associated with family abnormalities suggesting either regulator or structural gene defects.

STUDIES OF THE Vi (γ2c) SUBGROUP OF γ-GLOBULIN A RELATIONSHIP BETWEEN CONCENTRATION AND GENETIC TYPE AMONG NORMAL INDIVIDUALS

Further delineation of the antigens characteristic of the Vi or gamma(2c) subgroup of gamma-globulin was carried out utilizing a number of rabbit and primate antisera. Two genetic antigens characteristic of this subgroup, Gm(b) and Gm(g), were also detected by precipitation techniques with certain of the antisera. These were clearly differentiated from antigens common to all proteins of this subgroup. The concentration, of Vi protein in normal and pathological sera from several population groups was measured quantitatively utilizing a variety of immunological procedures. All sera studied showed measurable levels. The mean value for Caucasian sera was 1.06 mg/ml, representing approximately 8% of gammaG-globulin. This agreed closely with a figure of 8.4% for the incidence of myeloma proteins of the Vi subgroup among all gammaG-myeloma proteins in Caucasians. A relationship was found between the Vi subgroup concentration and the specific genetic type of a given individual. Measurements of the Gm(b) genetic determinants, which are found solely in Vi-type proteins, brought forward this relationship. Gm(b+) individuals showed higher concentrations of Vi-type gamma-globulin than those who were Gm(b-), and this difference was statistically significant for both the homozygous and heterozygous states. It appeared that the structural genes for Gm(b+) polypeptide chains showed a greater synthetic capacity than those for Gm(b-) types. The possible significance of such effects in governing the relative composition of the antibody population in a given individual is discussed.

Subclass restriction and polyclonality of the systemic lupus erythematosus marker antibody anti-Sm

Anti-Sm antibodies are highly specific markers for the diagnosis of systemic lupus erythematosus (SLE). This specificity suggests that the immunoregulation of these autoantibodies would reflect fundamental immune abnormalities in this disorder. As a clue to this immunoregulation, we have investigated the isotype distribution of anti-Sm antibodies by enzyme-linked immunosorbent assays. We have found that the anti-Sm response is markedly restricted to the IgG1 heavy chain isotype. On the other hand, the light chain distribution reflects that in normal serum, while isoelectric focusing analysis fails to show an oligoclonal pattern. The related specificity, anti-ribonucleoprotein, is also restricted to IgG1, while the SLE-specific antibody anti-double-stranded DNA is mostly IgG1 with a lesser contribution by IgG3. These results suggest that antinuclear antibodies that are strongly associated with SLE are produced by a T cell-dependent response, probably driven by antigen. The immunoregulation of the response to several autoantigens may be quite similar.

Subclass restriction of anti-SS-B (La) autoantibodies.

Antibodies to the SS-B (La) nuclear ribonucleoprotein particle are relatively specific for the diagnoses of Sjögrens syndrome or systemic lupus erythematosus. The formation of such autoantibodies is likely, then, to reflect the basic immunopathogenesis of these disorders. We have studied the isotype distribution of anti-SS-B antibodies as a clue to their immunoregulation. Using specific ELISA assays, we found that nearly all anti-SS-B antibodies in 39 patients were IgG, and, of these, only the IgG1 and, to a much lesser extent, IgG3 subclasses were represented. Both kappa and lambda light chain antibodies were found in most sera, and the overall kappa/lambda ratio approximated that of normal serum immunoglobulin. These results suggest that the formation of anti-SS-B antibodies is T-cell dependent and that the response is polyclonal in most patients.

Allopurinol hypersensitivity: Granular deposition of IgM at the dermal-epidermal junction

A patient with allopurinol hypersensitivity, manifested by fever, lymphadenopathy and a severe erythematous, morbilliform, maculopapular rash was studied. On immunofluorescent staining of the patients skin, heavy granular deposits of immunoglobulin M (IgM) were found at the dermal-epidermal junction. Transformation of the patients lymphocytes could not be effected by a variety of combinations of allopurinol, allopurinol metabolites and serum. These data suggested that the hypersensitivity reaction caused by allopurinol had immune complex deposition as the central feature in pathogenesis. The predominance of IgM may provide a distinctive feature from the deposits generally seen in systemic and discoid lupus erythematosus.

Subpopulations of human lymphoblastoid cell lines. Correlation with the expression of surface receptors and content of Epstein-Barr virus genome.

Forty lymphoblast cell lines derived from normal subjects, patients with infectious mononucleosis, leukemia, and Burkitts lymphoma have been studied for surface receptors including surface Ig, complement receptors by the EAC rosette and fluorescent (Raji cell) techniques, and Fc (aggregate) receptor by direct and indirect immunofluorescence. Because of the B‐cell tropism of the Epstein‐Barr virus (EBV), an effort was made to correlate the expresion of various surface properties of lymphoblastoid cell lines with the content of EBV viral DNA as determined by complementary RNA‐DNA (cRNA‐DNA) hybridization on membrane filters or by DNA‐DNA renaturation kinetic analysis. The only correlation established was with the Fc receptor determined by direct immunofluorescence. No correlation of EBV genome equivalents per cell with complement receptor or surface Ig was noted, suggesting that the expression of these receptors is not influenced by EBV viral DNA content. Subgroups of lymphoblastoid cell lines were, on the basis of variable expression of surface receptors, designated B1, B2, B3, B4, and T. The distribution of lymphoblastoid cell lines into these subgroups were in the ratio of 14:4:1:4:1. The B1, B2, and B4cell lines (except Molt 4F) were found to contain EBV. The B3, subgroup, for which cell line 698 was the sole example, expressed surface immunoglobulins but no other B‐cell characteristics, and H.S.B., a T‐cell line, lacked detectable EBV.

Distribution of surface, cytoplasmic and secreted IgG subclasses in human lymphoblastoid cell lines and normal peripheral blood lymphocytes.

Twenty‐two IgG‐positive human lymphoblastoid cell lines and normal peripheral blood lymphocytes were studied for surface and cytoplasmic IgG, IgG subclasses, IgD and IgM, using monospecific fluorescein‐and rhodamine‐conjugated F(ab′)2 antibody fragments, and for secretion by double antibody radioimmunoassay. Several parallel observations and several differences in IgG subclass expression were noted between cell lines and normal lymphocytes. Surface IgG2 was frequently expressed in normal IgG‐positive lymphocytes but was seldom expressed in cell lines. Cell lines resembled normal IgG‐positive lymphocytes in the frequent expression of cytoplasmic IgG3 and lgG4, often without secretion. Cell lines and normal lymphocytes both showed more frequent distribution of IgG and IgG sub‐classes in cytoplasm than in surface immunoglobulin, and often a discrepancy of surface versus cytoplasmic IgG subclass. A good correlation was noted between surface, cytoplasmic and secreted IgG1. Despite a predominance of IgG2 and IgG4 surface IgG subclasses, and IgG3 and IgG1 in cytoplasm, secreted immunoglobulins from normal lymphocytes in short‐term culture showed a similar distribution of IgG subclasses to that seen in normal sera. Multiple expression of IgG subclasses was much more frequent in IgG‐positive cell lines than in normal peripheral blood lymphocytes, both in surface and cytoplasmic IgG.

Phytohemagglutinin Response in Systemic Lupus Erythematosus: RECONSTITUTION EXPERIMENTS USING HIGHLY PURIFIED LYMPHOCYTE SUBPOPULATIONS AND MONOCYTES

THE PHYTOHEMAGGLUTININ (PHA) RESPONSE OF LYMPHOCYTES FROM UNTREATED PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) WAS STUDIED USING HIGHLY PURIFIED SUBPOPULATIONS OF CELLS INVOLVED IN THE TRANSFORMATION RESPONSE: T lymphocytes, B lymphocytes, and monocytes. Cell transformation was quantitated using both tritiated thymidine ([(3)H]-TdR) incorporation into DNA and cytofluorographic determination of cellular DNA content. Dose-response curves using six concentrations of PHA and five concentrations of cells over 0-5 days revealed a decrease in [(3)H]TdR by stimulated lymphocytes from some SLE patients. This decrease in [(3)H]TdR was paralleled by a decreased percentage of cells in S, G(2), and M phases of the cell cycle. However, abnormal response occurred entirely in those SLE patients who were hypocomplementemic. The etiology of the impaired response was further examined. Lymphocyte receptors for concanavalin A were studied using cytofluorography of lymphocytes stained with fluorescein-conjugated concanavalin A. The frequency distribution of concanavalin A receptors was similar in the normocomplementemic and hypocomplementemic lupus patients and in normals. The latex phagocytic activity of lupus macrophages was similar to normals when allogeneic normal plasma was used in the culture medium. Phagocytic activity became abnormal in the presence of SLE plasma. However, there was no difference in the [(3)H]TdR response or the percentage of cells in S, G(2), and M phases when T lymphocytes from the hypocomplementemic patients were stimulated on either autologous or normal allogeneic monocyte monolayers. Likewise, normal lymphocytes incorporated similar amounts of [(3)H]TdR and had similar percentages of cells in S, G(2), and M phases whether their T lymphocytes were stimulated on autologous or SLE monocyte monolayers. Highly purified subpopulations of B and T lymphocytes were obtained by density sedimentation or Fenwal Leuko-Pak passage of lymphocyte populations. The response to PHA by lymphocytes from the hypocomplementemic lupus patients could be seen to involve at least two abnormalities. One, in reference to normal lymphocytes, SLE T lymphocytes plus monocytes had an impaired response; two, SLE B lymphocytes plus SLE T lymphocytes plus SLE monocytes had an impaired response. Two patients in the hypocomplementemic group were treated with steroids. 5 days after steroid treatment was initiated, the percentage of cells in S, G(2), and M phases and the [(3)H]TdR response of PHA-stimulated lymphocytes returned to normal. The normalization of the [(3)H]TdR response was explained both by a return of purified T cells plus monocytes, purified B cells plus monocytes, and whole lymphocyte populations to normal responsiveness. These studies suggest that a steroid-correctable defect exists in T and B lymphocytes in SLE.

Factor XI deficiency, juvenile rheumatoid arthritis and systemic lupus erythematosus: Report of the first case☆

Abstract A number of inborn errors have recently been associated with a diathesis for collagen vascular disorders. For example, hereditary deficiencies of C1r or of the second component of complement (C2) have been associated with certain features of systemic lupus erythematosus (SLE). We describe the association of juvenile rheumatoid arthritis and SLE with a familial deficiency of factor XI (plasma thromboplastin antecedent). The propositus, a 26 year old Ashkenazi woman, presented at age four with symmetrical destructive polyarthritis, fever and rash. Progressive renal disease began at age seven, and nine criteria for SLE became manifest beginning at age 15. Three maternal relatives have arthritis, two maternal relatives had mild bleeding after surgery. Physical findings included a deforming arthritis and rash. Low levels of C3 and C4, antinuclear antibodies, high titers of native DNA antibodies, positive lupus erythematosus cell preparations and C1q precipitins were present. A skin biopsy specimen showed deposits of immunoglobulin G, immunoglobulin M, C1q and C3 at the dermal-epidermal junction. Factor XI levels were 5 per cent in the propositus and mother, 25 per cent in the maternal grandfather and within normal limits in her sister, father, and a maternal and paternal aunt, implying a dominant mode of inheritance. An HL-A identical sister whose lymphocytes showed no stimulation with those of the patient in unidirectional mixed lymphocyte culture manifested neither collagen vascular disease nor factor XI deficiency. There was no evidence for a circulating anticoagulant, and other clotting factors were normal. The present case represents a third and new association of an inborn error in the clotting system (factor XI deficiency) and collagen vascular disease with immune complex deposition.