H. G. Kunkel

Complement Fixation with Cell Nuclei and DNA in Lupus Erythematosus

Summary 1. Sera from patients with active lupus erythematosus fixed complement with a wide variety of nuclei from different organs and species, with calf thymus nucleoprotein, and in two instances with histone. Isolated calf thymus, salmon sperm, human leukocyte and pneumococcal DNA also fixed complement with many of these sera. Similar reactions were not encountered in a limited control series including normal individuals and other pathological states. 2. Most active L.E. sera fixed complement with both nuclei and DNA in roughly parallel titer. However, exceptions were encountered and one serum reacted strongly with nuclei but failed to react with DNA. Cross-absorption experiments with nuclei and DNA suggested the presence of 2 distinct serum factors. 3. The L.E. factor appeared to be related to the factor responsible for complement fixation with nuclei but distinct from that responsible for DNA fixation. 4. The significance of these findings with respect to antibodies against nuclear constituents is discussed.

The demonstration of acid α-naphthyl acetate esterase activity in human lymphocytes: Usefulness as a T-cell marker

Abstract The histochemical demonstration of nonspecific acid α-naphthyl acetate esterase (ANAE) activity was evaluated as a T lymphocyte marker primarily with the sheep erythrocyte (E) assay. A distinctive staining pattern characterized T lymphocytes which could be readily distinguished from monocyte staining. The percentage of E + and ANAE + lymphocytes was nearly always comparable in the peripheral blood and lymphoid tissue from normal and selected patients, including those with acute and chronic lymphocytic leukemia. Divergences were noted in certain other tissues including spleen and thymus. Certain mitogen-stimulated cells lost their ANAE activity while retaining their ability to form e n rosettes. Atypical and variable staining patterns were observed in established lymphoid cell lines. The histochemical demonstration of ANAE is simple and reproducible; preparations may be counterstained for cytomorphologic detail and mounted as a permanent record. Certain disadvantages are discussed. The method represents a practical alternative to E rosette assays. It is particularly well suited for certain routine laboratories.

Special characteristics of cellular immune function in normal individuals of the HLA-DR3 type

Abstract Studies with a number of tests of in vitro cellular immune function have shown differences between DR3-positive normal individuals and those of other DR types. Increased Ig synthesis and decreased Con A-induced suppression were the most striking changes observed in this preliminary investigation. Further studies especially with additional suppressor cell assays appear waranted. The possibilities that such changes play a significant role in the development of diseases associated with the DR3 phenotype is discussed.

Stimulation of human B lymphocytes by antibodies to IgM and IgG: Functional evidence for the expression of IgG on B-lymphocyte surface membranes

Abstract Isolated human tonsillar and peripheral blood B lymphocytes were induced to proliferate in vitro after exposure to F(ab′) 2 fragments of affinity purified antibodies to IgM, IgG, Fab, κ, and λ chain determinants. Low levels of DNA synthesis were observed in cultures containing anti-IgA antibodies, whereas DNA synthesis was not detected in cultures stimulated with anti-IgD. Divalent antibodies were essential for the generation of blastogenesis. These proliferative responses were T-cell independent and susceptible to suppression by excess numbers of monocytes. In addition, they were elicitable in cultures not containing FCS or 2-mercaptoethanol. Highly specific antibodies to IgG induced marked proliferation and this was similar in degree to that induced by anti-IgM. Subfractionation studies demonstrated that the anti-IgG responsive cells were contained to a major extent within the surface IgM + B-cell population. None of the antibody preparations elicited B-cell differentiation to antibody producing cells. Moreover, anti-μ antibodies completely abrogated Ig synthesis by pokeweed mitogen-stimulated cultures of unseparated tonsillar mononuclear cells. Anti-IgG antibodies similarly suppressed PWM-induced antibody production, although this was most apparent on the IgG response. In contrast anti-IgD antibodies both failed to suppress Ig production and in certain instances resulted in an increased level of Ig synthesis. These functional studies suggest that IgG molecules are intimately associated with the surface membrane of some B cells and that coexpression of IgG with IgM occurs. In addition, the observations emphasize further the divergent functional roles which surface IgM and IgG vs surface IgD have in B-cell proliferation and differentiation.

Two Types of γ-Globulin Differing in Carbohydrate Content

Summary 1. The heavy components (19 S) have been highly concentrated from normal human serum by repeated cycling in the preparative ultracentrifuge. Electrophoresis of such preparations has permitted isolation of a high molecular weight γ-globulin fraction. 2. Analyses of this material indicate a high carbohydrate content with hexose sugars and sialic acid present at approximately 5 times the concentration found for the main 7 S γ-globulin. The hexosamine-hexose ratio is approximately 0.5. 3. Markedly reduced levels were found in the sera of 4 patients with agammaglobulinemia. The possibility that certain antibodies fall into the 19 S fraction and are similarly rich in carbohydrate is discussed.

Relation between Certain Myeloma Proteins and Normal Gamma Globulin

Summary 1. Antisera against two preparations of normal gamma globulin reacted quantitatively with the special myeloma protein found in the serum of 4 patients with multiple myeloma. 2. The myeloma protein from the serum of a fifth patient showing the most rapid electrophoretic mobility failed to react with gamma globulin antisera. 3. Absorption of gamma globulin antiserum with purified myeloma proteins removed only a portion of the total antiserum, thus dividing the antibody into fractions. 4. Antiserum prepared against a highly purified preparation of a gamma myeloma protein gave a precipitin reaction with normal gamma globulin.

Phospholipid studies of different serum lipoproteins employing P32.

Summary 1. Administration of P32 to humans by mouth resulted in labelling of the α1 α2 and β lipoproteins separated by zone electrophoresis in a starch supporting medium. 2. Both phospholipid phosphorus and radioactivity of α2 lipoprotein fraction increased after ordinary meals. 3. Specific activities of the different lipoproteins were very similar even at various time intervals following the oral radioactive phosphate. The only exception was a slightly higher specific activity for the α1 lipoprotein in certain sera taken at early times. 4. In vitro experiments demonstrated transfer of phospholipid radioactivity from labelled β lipoproteins to unlabelled α1 lipoproteins; also from labelled α1 lipoproteins to unlabelled β lipoproteins.

Regulatory role of circulating monocytes in the differentiative and proliferative responses of human B lymphocytes

Abstract Depletion of monocytes from peripheral blood mononuclear cells had a profound influence on mitogen- and antigen-induced B-cell proliferation and differentiation to antibody-secreting cells. Such depletion enhanced the generation of plasma cells identifiable by immunofluorescence and specific plaque-forming cells (PFC) against sheep erythrocytes in cultures containing pokeweed mitogen, staphylococcal protein A (SPA), concanavalin A (Con A), and phytohemagglutinin (PHA). This enhancement was especially marked in the cases of SPA and Con A. Without monocyte depletion only 0.1 to 0.4% of the cultured cells were shown to be plasma cells and as much as a 100-fold increase was seen with monocyte removal. Similar results were also obtained in the PFC assay. These studies suggest that Con A, SPA, and PHA may be considered as inducers of B-cell differentiation to plasma cells under appropriate conditions. In two B-cell differentiation systems initiated by antigen involving allogeneic helper cells and autologous helper factors, the monocyte-dependent inhibition was also demonstrated. In addition, monocyte depletion enhanced proliferation of B cells in the presence of irradiated autologous T cells and pokeweed mitogen. This enhancement was also seen when B cells were stimulated to divide by purified anti-μ antibodies. The addition of adherent cells to monocyte-depleted cultures confirmed the suppressive effect of excessive monocytes but also demonstrated that the presence of a certain number of monocytes was necessary for optimal responses in at least some of the systems studied. The striking effect of monocytes in these different systems emphasizes the importance of their consideration in B-cell stimulation studies, especially those involving human peripheral blood.

A comparison of peroxidase- and fluorochrome-conjugated antisera for the demonstration of surface and intracellular antigens

Abstract The preparation of highly effective peroxidase-labeled F(ab′) 2 antibody fragments allowed the development of direct immunoperoxidase techniques analogous to existing immunofluorescent methodology. Peroxidase-labeled and rhodamine-labeled antisera were used in parallel experiments to demonstrate lymphocyte surface immunoglobulin, B-lymphocyte alloantigens and the intracellular immunoglobulin of cultured lymphoblastoid cells and mitogen-stimulated peripheral blood lymphocytes. Immunoperoxidase and immunofluorescence were comparable in specificity and sensitivity. Intracellular immunoglobuin was demonstrated by direct immunoperoxidase in formalin-fixed, paraffinembedded bone marrow biopsies of patients with multiple myeloma. Current immunoperoxidase methodology was modified to avoid nonspecific interference by endogenous peroxidase activity and the tissue affinity of peroxidase. Immunoperoxidase was found to be a reasonable alternative to immunofluorescence for lymphocyte analysis, offering the advantages of permanent slide preparations, improved cytomorphologic detail, routine light microscopic examination, and application to fixed tissue sections, where immunofluorescence is unsuccessful.

Portal blood in collateral veins of patients with cirrhosis; acetylation by the intestine.

Summary 1. Portal origin of blood in abdominal collateral veins of patients with portal hypertension may be demonstrated simply and rapidly by feeding fluorescein or thiocyanate and finding higher concentrations in the abdominal veins than in the antecubital veins. 2. Seven out of 12 patients with cirrhosis gave positive tests for the portal origin of abdominal collateral vein blood. Patients with acites are more likely to give positive results than those without ascites. 3. Evidence is presented for the acetylation of PABA by the human intestinal tract.