We have examined whether the toxic effects of homocysteine on cultured endothelial cells could result from the formation and action of hydrogen peroxide. In initial experiments with a cellfree system, micromolar amounts ofcopper were found to catalyze an oxygen-dependent oxidation of homocysteine. The molar ratio of homocysteine oxidized to oxygen consumed was 4.0, which suggests that oxygen was reduced to water. The addition of catalase, however, decreased oxygen consumption by nearly onehalf, which suggests that H202 was formed during the reaction. Confirming this hypothesis, H202 formation was detected using the horseradish peroxidase-dependent oxidation of fluorescent scopoletin. Ceruloplasmin was also found to catalyze oxidation of homocysteine and generation of H202 in molar amounts equivalent to copper sulfate. Finally, homocysteine oxidation was catalyzed by normal human serum in a concentration-dependent manner.

To gain insight into the mechanisms of autoantibody induction, sera from 40 patients with systemic lupus erythematosus (SLE) were tested by ELISAs for antibody binding to denatured individual histones, native histone-histone complexes, histone-DNA subnucleosome complexes, three forms of chromatin, and DNA. Whole chromatin was the most reactive substrate, with 88% of the patients positive. By chi-square analysis, only the presence of anti-(H2A-H2B), anti-[(H2A-H2B) -DNA], and antichromatin were correlated with kidney disease measured by proteinuria > 0.5 g/d. SLE patients could be divided into two groups based on their antibody-binding pattern to the above substrates. Antibodies from about half of the patients reacted with chromatin and the (H2A-H2B) -DNA subnucleosome complex but displayed very low or no reactivity with native DNA or the (H3-H4)2-DNA subnucleosome complex. An additional third of the patients had antibody reactivity to chromatin, as well as to both subnucleosome structures and DNA. Strikingly, all sera that bound to any of the components of chromatin also bound to whole chromatin, and adsorption with chromatin removed 85-100% of reactivity to (H2A-H2B)-DNA, (H3-H4)2-DNA, and native DNA. Individual sera often bound to several different epitopes on chromatin, with some epitopes requiring quaternary protein-DNA interactions. These results are consistent with chromatin being a potent immunogenic stimulus in SLE. Taken together with previous studies, we suggest that antibody activity to the (H2A-H2B) -DNA component signals the initial breakdown of immune tolerance whereas responses to (H3-H4)2-DNA and native DNA reflect subsequent global loss of tolerance to chromatin. (J. Clin. Invest. 1994. 94:184-192.)

The lymphoproliferative disease of granular lymphocytes (LDGL), also referred to as LGL leukemia, is a heterogeneous disorder, but is clinically, morphologically, and immunologically distinct. Although LDGL has recently been included in the revised classification of lymphomas as an independent clinical entity, no consensus exists on the criteria to establish the diagnosis. The aim of this report was to refine the parameters needed to make the diagnosis of LDGL. We studied 11 patients with chronic granular lymphocytosis selected from among 195 cases observed by our institutions from three different geographic areas (North America, Europe, and Asia). These cases did not meet the current criteria for inclusion in LDGL, since all patients had less than 2,000 GL/μL. However, in each of these patients, we found evidence for expansion of a discrete GL population. Clonal rearrangement of the T-cell receptor (TCR) β gene was found in peripheral blood mononuclear cells (PBMC) of all nine patients with CD3+ LDGL. Using recently generated monoclonal antibodies (MoAbs) against the TCR Vβ gene regions, we identified a unique TCR Vβ on GL from each of three patients studied. In two patients with CD3− LDGL, we also identified a restricted pattern of reactivity, by staining with MoAbs against p58 antigen found on normal natural killer (NK) cells. The clinical features of these 11 patients with relatively low absolute number of GL were similar to those reported previously for patients with greater than 2,000 GL/μL. These data demonstrate that newer techniques such as MoAbs against Vβ gene regions and p58 molecules and molecular analyses are useful to identify expansions of discrete GL proliferations. Demonstration of an expansion of a restricted GL subset is evidence for the diagnosis of LDGL, even in patients with a relatively low GL count. Our results also contribute to distinguish between the end of normality and the beginning of pathology in the broad spectrum of GL lymphocytoses.

Three patients had leukocytosis of large granular lymphocytes and chronic neutropenia. Clonal chromosomal abnormalities (trisomy 8 and trisomy 14) and lymphocytic infiltration of splenic red pulp, hepatic sinusoids, and bone marrow indicated the neoplastic nature of the large granular lymphocytes. Demonstration of a T3+, T8+, HNK-1 + phenotype and low natural killer cell activity that was augmented by interferon treatment showed the leukemic cells to be immature natural killer cells. Multiple autoantibodies were present and included rheumatoid factor and antinuclear, antineutrophil, antiplatelet, and antierythrocyte antibodies, suggesting a defect of B-cell immunoregulation. In addition, in-vitro studies showed impaired suppression of immunoglobulin biosynthesis by abnormal cells from one patient. Antineutrophil antibodies and absence of direct cell-mediated inhibition of granulocyte-macrophage colony formation supported a humoral immune mechanism for the neutropenia. In these patients the syndrome of splenomegaly, multiple autoantibodies with neutropenia, and lymphocytosis of large granular lymphocytes is due to a neoplastic proliferation of immature natural killer cells.

Lymphoproliferative disease of granular lymphocytes (LDGL) is a recently recognized, relatively rare atypical lymphocytosis characterized by the presence of over 2000 lymphocytes with cytoplasmic azurophilic granules/mm3 in the peripheral blood. The clinical course is heterogeneous, varying from spontaneous regression to progressive, malignant disease. As a consequence, clinical intervention is not standardized. In a worldwide multicenter study, the authors observed 151 patients with LDGL for a mean follow-up time of 29 months. Forty-three patients were asymptomatic at the time of diagnosis. In the remaining cases, clinical symptoms included fever (41 cases), infections (58), neutropenia (47), anemia (17), and thrombocytopenia (12). In 69 cases, LDGL coexisted with an associated disease. Most patients had a nonprogressive clinical course despite the presence of severe symptoms. In 19 patients, death related to LDGL occurred within 48 months. The authors investigated which features at diagnosis were significantly associated with increased mortality. In the univariate analysis, lymph node and liver enlargement, fever at presentation, skin infiltration, a low (less than or equal to 5000/mm3) or high (greater than 20,000/mm3) peripheral leukocyte count, relatively low (less than or equal to 3000) or high (greater than 7000/mm3) absolute peripheral granular lymphocyte (GL) count, and a low (less than or equal to 15%) percentage of HNK-1-positive cells were found to be predictors of increased mortality. In the multivariate analysis, significant independent predictors were fever at diagnosis, a low (less than or equal to 15%) percentage of HNK-1-positive peripheral blood mononuclear cells (PBMC) and a relatively low (less than or equal to 3000) GL count. These results showed that about 25% of the patients with LDGL were diagnosed after a routine blood count and had no clinical symptoms. The remaining patients were symptomatic, with some experiencing a fatal clinical course. The author's analysis of the significant prognostic features of LDGL may help in understanding the heterogeneous nature of this syndrome.