The diagnosis of hairy cell leukemia was made in three patients by phase-contrast microscopy and histochemistry of the abnormal peripheral blood cells. Both IgM and IgD surface immunoglobulins were resynthesized after these cells were trypsinized and cultured. Aggregate or Fc receptors were demonstrated on hairy cells. The ability to phagocytose latex was also a property of hairy cells; however, these cells did not demonstrate nonspecific esterase activity. Stimulation by phytohemagglutinin resulted in very low incorporation of tritiated thymidine. Cytofluorographic analysis of the phytohemagglutinin- stimulated cell population revealed less than 9% of the cells in an interploid or tetraploid state. The abnormal mitogen response was largely restored when purified T lymphocytes obtained from the peripheral blood of the patients were cultured with phytohemagglutinin. Hairy cells cultured with normal allogeneic mononuclear cells did not undergo blast transformation. These data strongly suggest that the cells of at least some patients with hairy cell leukemia are B lymphocytes with phagocytic capabilities.

Inhibitors of clotting factors occuring in humans are often antibody molecules synthesized in response to exogeneous proteins used in replacement therapy. Extensive studies of inhibitors to factor VIII indicate such antibodies may be monoclonal or polyclonal in nature. To date, only one factor IX inhibitor has been subjected to detailed immunochemical analysis and it appears to be a monoclonal IgGA lambda antibody. We have discovered a second inhibitor of factor IX in a patient with severe hemophilia B and have subjected it to immunochemical analysis. Studies on this second inhibitor have been carried out before and after an anamnestic response. Column chromatography, preparative zone electrophoresis, and specific inhibitor neutralization assays using monospecific heterologous antisera to human immunoglobulin classes, subclasses, and light-chain types indicate that the antibody is of the IgG class and contains both kappa and lambda light chains and probably all four IgG subclasses. Thus, the inhibitor appears to be polyclonal by immunochemical and structural criteria. In addition, preparative isoelectric focusing of pre- and postanamnestic inhibitor samples indicates that recruitment of new clones of IgG antibody occurs as a result of anamnesis. It is conceivable that an antibody initially restricted in immunoglobulin subclass became polyclonal following an anamnestic response.

Previous studies using immunoneutralization techniques have shown that many factor VIII inhibitors are IgG antibodies of a single light chain type. We have investigated this apparent homogeneity by immunoneutralization assay and liquid isoelectric focusing of inhibitor fractions from five hemophiliacs and two nonhemophiliacs. By immunoneutralization assay, inhibitors from four hemophiliacs and one nonhemophiliac were exclusively k light chain type: the fifth hemophilic inhibitor was predominantly k1 and the second nonhemophilic inhibitor was a mixture of k and gamma. However, heavy chain subtyping of the six predominantly or exclusively k inhibitors showed all to be mixtures of IgG4 and IgG1. By isoelectric focusing, two inhibitors showed multiple peaks of activity between pH 5 and 9. The remaining five showed predominant peaks of activity, which were solely IgGk1 between pH 5.8 and 7, with smaller peaks between pH 7 and 9. The most acidic major peak, focusing at pH 6, was IgG4 in the three cases tested. Two of these acidic peaks neutralized factor VIII more efficiently than other peaks in the same focusing profiles, suggesting greater affinity for factor VIII. These studies demonstrate that factor VIII inhibitors are composed of heterogenous subpopulations of immunoglobulins which can be separated by isoelectric focusing.

Surface immunofluorescence experiments using a human anti-i and two anti-I antisera have been performed on human peripheral blood lymphocytes. These are known to contain cold-reactive monoclonal IgM antibodies against the carbohydrate sequence:Galfl -4GlcNAc#1 -3Galjfl 4GlcNAc/31 -3Gal-(anti-i Den) and the 1 --4, 1 -6 domain (anti-I Ma) and the 1 --4, 1 3 domain (anti-I Step) of the branched I-active structure:Galf1l 4GlcNAcfll \6G lfli -4GIcNAcfl -3Gal-A high proportion of B-and T-type lymphocytes express these I and i determinants. In the presence of anti-human immunoglobulin, the cold-reactive membrane-associated complexes of I-anti-I and i-anti-i become stabilized, and redistribution (with patching and capping) can be elicited at 370C. Dual fluorescence experiments have shown striking concordant staining of I or i (fluorescein) caps and patches with concanavalin A (rhodamine) reactive sites on normal and leukemic cells, suggesting that a proportion of I and i active structures of lymphocyte membranes are structurally associated or physiologically coupled with glycoproteins carrying oligosaccharides with branched mannosyl cores.

Concepts of plasma membrane structure and function have changed markedly during the past decade (1). The cell surface is no longer considered to be a rigid, static barrier between cytosol and extracellular milieu but rather a dynamic matrix in which cell surface constituents can change their relative and absolute positions (2). Lymphocyte immunoglobulin (Ig) capping is an example of this phenomenon (3, 4). Human peripheral blood B lymphocytes have surface Ig evenly dispersed on the plasma membrane which can be induced to aggregate when cross-linked by polyvalent antisera (5). The formation of local Ig patches is followed by capping which brings the Ig patches to one pole of the cell. Why or how such cross-linking of Ig results in the observed translocation is unknown. However, recent experiments have suggested the involvement of the cell's contractile (microfilaments) (3, 6) and cytoskeletal (microtubule) (7, 8) elements. The data contained in this communication indicate that cyclic adenosine 3':5'-monophosphate (cAMP) may act as one of the signals which regulate cell surface modulation. A dual immunofluorescence technique has been used to demonstrate a topographical correlation between patched and capped surface Ig labeled with rhodamine and specific cAMP immunofluorescence. Materials and MethodsSurface Immunoglobulin and cAMP Staining. Mononuclear cells were separated from peripheral blood by Ficoll-Isopaque centrifugation. The cells were washed three times in 10% (fetal calf serum (FCS)-RPMI 1640 at room temperature. Viability was assessed by trypan blue exclusion.The lymphocytes, in a concentration of 1-2 x 10 e in 0.05 ml of 10% FCS-RPMI 1640, were incubated with 0.05-0.1 ml of rhodamine-conjugated purified Ig or F(ab')2 fragments prepared from goat polyvalent anti-human immunoglobulin (9). After a i h incubation at 4°C the cells were washed three times with a total of 10 ml of cold 10% FCS-RPMI 1640. Redistribution of cell-bound