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Synovial T cells from rheumatoid patients, when activated by IL-15, stimulate production of the proinflammatory cytokine, TNF-α (pages 189–195).

The course of chronic lymphocytic leukemia (CLL) is variable. In aggressive disease, the CLL cells usually express an unmutated immunoglobulin heavy-chain variable-region gene (IgVH) and the 70-kD zeta-associated protein (ZAP-70), whereas in indolent disease, the CLL cells usually express mutated IgVH but lack expression of ZAP-70. The reasons for the differences in clinical characteristics are unknown. Examination of microarray data has shown that these two subtypes of CLL share a common gene-expression pattern, suggesting that they constitute a single entity. However, the microarray data have also revealed some important differences between the two types of CLL in the expression of a small number of genes. While mRNA expression profiling by microarray is a useful and convenient way for signature gene recognition, systematic proteomic analysis may be more relevant to understanding the pathophysiology of disease. To our knowledge, few studies have performed systematic proteomic profiling on CLL and none have tried to identify proteins that are differentially expressed among patients with differing outcomes. We have applied multidimensional LC-ESI-tandem mass spectrometry to identify proteins that are differentially expressed between aggressive (5 ZAP-70 pos/IgVH unmutated) and indolent (5 ZAP-70 neg/IgVH mutated) purified B cell samples. A total of more than 3,000 proteins were identified in our proteomic analysis. We observed a positive correlation in expression of protein and mRNA of three genes (ZAP-70, gravin, and dystrophin); these genes were consistently associated with disease progression in CLL as reported by microarray analyses. This indicates that the proteomic data is of high quality. We also compared the proteomic and transcriptomic patterns between these two groups. In general, the correlation between mRNA and protein expression was poor. To identify the genes that appear coordinately regulated at the mRNA and protein level, we examined the mRNA expression pattern of about 200 proteins that were differentially expressed in our proteomic data between aggressive and indolent CLL. We found 37 genes were differentially regulated post-transcriptionally, perhaps through the influence of microRNA or protein stability. In addition, we found 117 genes to be differentially expressed in microarray but not proteomic analysis. This result raises the question of how reliable mRNA expression levels reflect the biological activity of protein function. In conclusion, we have identified a number of candidate proteins that are differentially expressed in CLL of distinctive clinical outcomes by comparing high quality proteomic and transcriptomic data. These proteins might serve as biomarkers or therapeutic targets. We also found genes that might be differentially regulated in CLL post-transcriptionally. Further studies of how these genes are regulated will advance our knowledge of CLL pathogenesis.

The clinical course of chronic lymphocytic leukemia (CLL) is variable. DNA-microarray studies have shown that the gene-expression patterns of CLL cells with unmutated IgVH genes are similar to those of cells with mutated IgVH genes, but that the patterns of both are distinct from those of other leukemias and lymphomas. Nevertheless, the two subtypes of CLL can be distinguished by the differential expression of a small number of genes, one of which encodes ZAP-70, an intracellular tyrosine kinase with a critical role in T-cell receptor signaling. Further analysis of the mutation status of IgVH genes and ZAP-70 expression revealed that CLL patients with B cells expressing ZAP-70 and unmutated IgVH genes had a more aggressive disease. LAG-3 (CD223) is thought to play a role in immune responses mediated by T and NK cells. LAG-3, a CD4 homolog, is a ligand for MHC class II antigens. Similar to ZAP-70, LAG-3 is selectively expressed on activated T and NK cells and has recently been shown to be expressed on T-cell activated B cells. We compared the gene expression profiles of the B cells purified from 15 CLL patients using the Affymetrix HG-U133 plus 2.0. This analysis revealed LAG-3 and parathymosin differentially expressed at higher levels by the CLL cells expressing ZAP-70 and unmutated IgVH genes. LAG-3 and parathymosin are located on chromosome 12p13 with head-to-tail orientation. To examine for surface expression of LAG-3, we performed flow cytometry on these same 15 CLL samples using an anti LAG-3 mAb (CD223). We found LAG-3 expressed at high levels by the CD5/CD19 B cells of 4/8 (50%) cases that expressed ZAP-70 and unmutated IgVH genes. Conversely, the samples with B cells lacking ZAP-70 and with mutated IgVH genes did not express LAG-3 (0/7). All samples (15/15) expressed high level of MHC class II antigens, as assessed by flow cytometry. LAG-3 may interact with MHC class II molecules expressed by CLL cells to form an autocrine loop that may further enhance the activation of ZAP-70-expressing CLL B cells. Further analysis is needed to delineate the role of LAG-3 in the pathogenesis and/or progression of this disease.

PURPOSE. Anastomotic vessels in exudative age-related macular degeneration (AMD) represent a serious clinical feature that reportedly does not respond well to either photocoagulation or photodynamic therapy (PDT). Anastomoses also occur in various animal models of choroidal neovascularization (CNV). In the present study, anastomotic vessels and their patency were evaluated in two primate CNV laser-trauma models after PDT, by using two novel photosensitizers. METHODS. In cynomolgus (Macaca fascicularis) and squirrel (Saimiri sciureus) monkey eyes (n = 20), matrix placement of laser photocoagulation sites elicited CNV as a component of the development of fibrovascular tissue (FVT). FVT sites received PDT according to specific drug infusion and laser light treatment parameters. FVTs and anastomoses were evaluated by fundus photography, fluorescein angiography, and histologic examination. RESULTS. Anastomoses averaged approximately 48% of FVT sites, with greatest occurrence in the macaque. Although PDT with each photosensitizer effectively produced FVT closure, both retinal vessels and anastomoses remained patent. CONCLUSIONS. Although PDT is effective in closing the choroidal neovascularization in FVT, this technique was ineffective in occluding anastomotic vessels and their associated tributaries within the mid- to proximal retina. Various factors (vascular diameter, composition, blood flow, orientation) may contribute to continued anastomotic patency. By convention, such vessels would typically be defined as chorioretinal anastomoses (CRAs); however, continuing studies suggest the possibility that these neovessels constitute dual-origin hybrids. Regardless of origin, viable anastomoses provide one potential mechanism for revascularization to occur after PDT and may help to explain why CRAs are considered a poor prognostic sign in patients with AMD.

Chronic Lymphocytic Leukemia (CLL) is the most frequent leukemia afflicting the Western world. Unlike other more aggressive leukemias and lymphomas, CLL B-cells do not divide at a high rate and expansion of the malignant clone appears to be due to underlying defect in apoptosis. Elevated levels of anti-apoptotic Bcl-2 are thought to be involved in the pathogenesis of CLL. The activity of Bcl-2 can be modulated my interactions with other cellular proteins. Recently, orphan nuclear receptor TR3 (nur77) was shown to exit the nucleus, bind Bcl-2, and convert Bcl-2 into a killer protein (Lin, et al., CELL, 116:527, 2004). We evaluated the expression of TR3 by immunoblotting in CLL B-cells specimens. High levels of TR3 protein were present in 8 of 8 CLL patient specimens. Subcellular fractionation studies showed predominately nuclear localization of TR3 in 2 of 2 CLL specimens examined. Because Akt is known to regulate TR3 activity in the nucleus (Pekarsky, et al., Proc. Natl. Acad. Sci.98:3690, 2001), we also examined the expression of the Akt regulator, Tcl-1. The Tcl-1 protein was expressed in the majority of CLL samples and at high levels in 8 of 16 CLL specimens, correlating with ZAP70-positivity. In gene transfection experiments using tumor cell lines, over-expression of Tcl-1 inhibited nuclear export of TR3, as determined by immunofluorescence confocal microscopy. We propose a model in which expression of the oncogene Tcl-1 in CLL B-cells (perhaps related to ZAP70 expression) modulates nuclear Akt activity, in turn suppressing TR3 function, and thus reducing activity of the TR3-mediated pathway for conversion of Bcl-2 from protector to killer. This hypothesis requires further experimental testing.

Gene expression analyses of isolated chronic lymphocytic leukemia (CLL) cells have identified gene expression profiles for CLL that are distinct from those of other B lymphoid malignancies or normal B cells. These signature genes may play a role in disease pathogenesis or progression and/or may encode proteins that can be targeted by immune therapy for patients with this disease. One such gene encodes ROR1, a surface receptor tyrosine kinase that ordinarily is expressed by a subset of developing neurons during embryogenesis but not by cells of normal adults, including blood and tissue lymphocytes. The extracellular domain of ROR1 contains a frizzled (FRZ) module that was first defined in G-protein-coupled receptors of the frizzled and smoothened families and that has been found in several frizzled-related proteins involved in Wnt signaling. Because our recent studies found that Wnt signaling genes are over-expressed and active in CLL (PNAS10:3118, 2004), we examined CLL B cells for expression of ROR1 protein and studied the capacity of this orphan receptor tyrosine kinase to mediate signaling in response to stimulation by any one of the multiple members of the Wnt factor family. Immunoblot analyses with antibodies raised against ROR1-peptides revealed expression of full-length ROR1 of appropriate size in each of the CLL samples examined (n = 12). However, we did not detect expression of ROR1 protein by other tissues, including blood or splenic lymphocytes of non-leukemic patients or normal adults donors. Moreover, we generated mouse antisera against full-length human ROR1 via DNA immunization with ROR1 cDNA expression vectors. These antisera reacted specifically with CHO cells transfected to express ROR1, but not with non-transfected CHO cells. Anti-ROR1 antisera, but not control sera, reacted specifically with intact CLL B cells, but not with lymphocytes of healthy adult donors, revealing that expression of surface-membrane ROR1 may be unique to leukemia B lymphocytes. We studied the functional significance of ROR1 by transfecting HEK293 cells with expression constructs encoding ROR1, one of several Wnt factors, low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6), and/or reporter constructs. We observed that expression of ROR1 with any Wnt factor did not activate T-cell transcription factor (TCF), nuclear factors of activated T cells (NFAT), or AP-1 dependent gene expression, suggesting that ROR1 does not signal via the canonical Wnt-signaling pathway. However, we observed that co-expression of ROR1 in HEK293 cells with Wnt5a, but not with any other Wnt factor, induced activation of NF-κB. Induction of NF-κB was dose dependent on expression of ROR1 and Wnt5a, but independent of expression of LPR5/6 that ordinarily serve as co-receptors for the frizzled family of Wnt receptors. We conclude that CLL cells have distinctive expression of a surface tyrosine kinase that ordinarily is expressed during early development. Moreover, this tyrosine kinase may serve as a receptor for Wnt5a, a factor expressed in stromal microenvironments that can trigger activation of NF-κB in CLL cells via a non-canonical Wnt-signaling pathway. Because constitutive activation of NF-κB is implicated in oncogenesis of many types of cancers, the capacity of ROR1 to induce activation of NF-κB suggests that the unusual expression of this developmentally-regulated tyrosine kinase may play an important role in the pathogenesis of this disease.

Purpose To evaluate and characterize the New-World squirrel monkey as a primate model for experimental choroidal neovascularization (CNV) studies and to compare it with the current Old-World macaque monkey model. Methods Fibrovascular tissues (FVT) were elicited in 12 maculae of seven squirrel monkeys by laser photocoagulation using optimized laser parameters (532 nm, 0.05 second, 75 micro m, 650 mW). Follow-up fundus and fluorescein angiography (FA) examinations were conducted on postlaser days 30 and 35, followed by euthanasia and histologic analysis of tissues. For comparative evaluations, FVT development also was induced and analyzed in eight maculae of four macaque monkeys with laser parameters previously used in this species (514 nm, 0.1 second, 50 micro m, 390 and 455 mW). Results FVT developed in both primate species, consisting of fibrous tissue that contained vessels that ranged from sparse but identifiable capillaries to well-established neovascular networks. Overall, 65% of the photocoagulation sites in the squirrel monkey and 37% of sites in macaque monkey elicited development of FVT. Localized FVT ranged from modest to extensive thickenings of the choriocapillaris layer. Unexpectedly, 76% of the FVT sites in squirrel monkey eyes and 27% of the sites in macaque eyes showed diffuse FVT that expanded beyond the original photocoagulation sites, accompanied by neovascular infiltration of the retina. Conclusions Like the macaque, the squirrel monkey can be considered a useful primate model for experimental CNV investigations, while additionally offering certain species-specific advantages. Diffuse FVT permit studies of antiangiogenic therapies in areas distant from laser photocoagulative trauma sites.

B lymphocytes play several critical roles in the pathogenesis of rheumatoid arthritis. They are the source of the rheumatoid factors and anticitrullinated protein antibodies, which contribute to immune complex formation and complement activation in the joints. B cells are also very efficient antigen-presenting cells, and can contribute to T cell activation through expression of costimulatory molecules. B cells both respond to and produce the chemokines and cytokines that promote leukocyte infiltration into the joints, formation of ectopic lymphoid structures, angiogenesis, and synovial hyperplasia. The success of B cell depletion therapy in rheumatoid arthritis may depend on disruption of all these diverse functions.

Human malignant T cell lines have high levels of deoxyribonucleoside phosphorylating activity and low levels of deoxyribonucleotide dephosphorylating activity. When incubated with deoxyadenosine or thymidine, the malignant T cell lines rapidly accumulate toxic concentrations of dATP and dTTP, respectively. This unusual pattern of deoxyribonucleotide metabolism renders the malignant T cells especially vulnerable to the toxic effects of deoxyribonucleosides and related analogues.

Antibody against a cross-reactive idiotype (CRI) on human IgM-rheumatoid factor (RF) antibodies was induced by immunization of rabbits with a synthetic peptide ( PSL2 ) corresponding to the second complementarity-determining region (CDR), and adjacent amino acid residues of the kappa light chain of the IgM-RF Sie . The anti-peptide antibody bound efficiently to IgM-RF proteins known to share a cross-reactive idiotype, and to their isolated kappa chains. The anti-CRI was absorbed by, and eluted from, a peptide-Sepharose affinity column. The antibody activity was inhibited by the free peptide in solution. The anti-peptide antibody thus identifies a public idiotype on human IgM-RF, that is largely dependent on the primary sequence of the second CDR of the light chain. Such peptide-induced antiidiotypes of predefined specificity may facilitate studies of the molecular basis of idiotypic cross-reactions, the inheritance and somatic diversification of antibody molecules, and the regulation of the idiotype network.

Rat leukemic basophils with surface-bound IgE were reacted with fluoresceinated anti-IgE. The results were qualitatively similar to those obtained in analogous studies with lymphocytes, normal human basophils, and other cells: local aggregation (patch formation) was only moderately temperature-dependent and was uninhibited by NaN3. Polar cap formation was significantly more sensitive to temperature and was completely inhibited by 0.01 to 0.1 M NaN3 and cytochalasin B. The amount and rate of capping could be altered by varying either the number of bound IgE molecules or the anti-IgE concentration indicating that lattice formation is required for redistribution. Monovalent Fab′ fragments of the anti-IgE caused no redistribution. No endocytosis of the antibody-surface determinant complexes was observed. Under conditions adequate to inhibit cap formation on mouse lymphocytes, concanavalin A did not inhibit cap formation on the leukemic basophils.

The exact route of metabolism of 5′-isobutylthioadenosine is controversial. Using human cell lines deficient in methylthioadenosine phosphorylase, purine-nucleoside phosphorylase, or adenosine deaminase, we have ascertained the relative roles of the three enzymes in isobutylthioadenosine metabolism. The results showed that viable human cells progressively converted isobutylthioadenosine to 5′-isobutylthioinosine via sequential metabolism by methylthioadenosine phosphorylase and purine nucleoside phosphorylase acting in opposite directions, rather than through direct deamination. An identical pathway converted 5′-methylthioadenosine to 5′-methylthioinosine.

The enzyme methylthioadenosine phosphorylase functions in both purine and polyamine metabolism is dividing mammalian cells. To determine the effects of the loss of this enzyme on cell growth and metabolism, we selected two methylthioadenosine phosphorylase-deficient mutant clones of the transplantable murine T lymphoma cell line R1.1. The first had 3.5% of wild type methylthioadenosine phosphorylase activity. The second was completely enzyme-deficient. The loss of the enzyme did not alter the growth rate, cloning efficiency, or tumor-forming ability of the T lymphoma cells. The methylthioadenosine phosphorylase-deficient clones excreted substantial amounts of methylthioadenosine into the culture medium (0.13 and 0.32 nmol/h/mg of protein, respectively) and were unable to utilize the methylthioadenosine phosphorylase substrate 2',5'-dideoxyadenosine as a purine source when de novo purine synthesis was blocked. Spermine levels were 10-20% lower in the enzyme-deficient clones than in wild type cells. The loss of methylthioadenosine phosphorylase rendered the mutants exquisitely sensitive to the antiproliferative effects of methylthioadenosine. Methylthioadenosine at 3-6 microM inhibited their growth by 50%. The toxic effects of methylthioadenosine were not attributable to inhibition of purine, pyrimidine, or polyamine synthesis.

Naturally developing human antibodies to the Epstein-Barr nuclear antigen recognize synthetic peptides containing sequences from the unusual glycine-alanine region of this protein. We tested antibody binding to a series of peptides of from five to 20 amino acids in length. Peptides as small as seven amino acids could bind but optimal results required chain lengths of 15. Binding was extremely sensitive to small changes in the length and sequence of the peptide, and also to the temp of the reaction. The changes can be ascribed to two factors: (1) deletion of the site of antigen binding and (2) loss of peptide secondary structure.

The presence of receptors for IgG Fc and fragments of C3 on primary cultures and cryostat sections of normal and rheumatoid synovial tissues was assessed. Significant proportions of large rounded cells with asteroid projections found in such cultures had receptors for both IgG Fc and fragments of C3. Moreover, Gram negative bacteria that had fixed complement, but not EAC, bound in a linear fashion on the superficial layers of synovial cryostat sections. On the basis of morphologic and histochemical criteria, the cultured cells bearing these receptors were tentatively determined to represent a subset of synovial lining cells. The possible role of such receptors on synovial lining cells in the pathogenesis of rheumatoid arthritis is discussed.

Interferons (IFN) are antiviral proteins that may be important in mediating cellular defenses against Epstein-Barr virus (EBV) infection. However, the means by which IFN-alpha, -beta and -gamma modify EBV infectivity are not clear. We have evaluated the effects of purified recombinant preparations of all three classes of IFN on EBV-induced B lymphocyte proliferation and Ig secretion. When added early after EBV infection, all three recombinant IFN reduced B cell outgrowth and Ig secretion. IFN-gamma exerted a 7-10-fold more potent antiviral effect than IFN-alpha or -beta. All three types of IFN act directly on B cells. Monocytes and natural killer cells are not necessary for the anti-EBV activity. Of the three recombinant IFN, only IFN-gamma reduced EBV-induced proliferation and Ig secretion when added 3-4 days after virus infection; IFN-alpha/beta were only effective up to 24 h. B lymphoblastoid lines already transformed by EBV are insensitive to the anti-proliferative actions of all three types of IFN. On the basis of these findings, we propose three phases of regulation during EBV infection. In the early phase, EBV-infected cells can be regulated by all IFN. Subsequently, there is an intermediate period where only IFN-gamma is capable of directly affecting EBV-induced B cell responses. In the third phase, B lymphocytes become insensitive to direct actions of all IFN and are now subject to regulation only by cytotoxic cells.

The natures of the IgM rheumatoid factor (RF)-, IgM-, and IgG-secreting cells in the human bone marrow as compared to the peripheral blood, have been investigated by (1) response to the polyclonal B-cell activator, the Epstein-Barr virus (EBV), (2) sensitivity to the S-phase specific antimetabolite hydroxyurea, (3) presence of the BA-1 and Ia antigens on the cell surface, and (4) cell size, as determined by counter flow elutriation. The EBV-inducible bone marrow IgM-RF precursors derived from medium to large B cells that were inhibited by hydroxyurea pretreatment. The marrow total IgM response derived from small to medium size cells, and was only partially inhibited by hydroxyurea. Hydroxyurea had no effect on IgM-RF or IgM synthesis by peripheral blood cells. These results indicate thet the marrow EBV-induced IgM-RF response is not representative of the response by peripheral blood cells, moreover; the marrow RF secreting response arises from a dividing cell pool that may represent newly generated autoreactive B cells.

Summary Forty-two of 115 patients with Type I (insulin dependent) diabetes were found to have limited joint mobility affecting mainly the small joints of the hands. The presence of joint abnormalities was related to duration of diabetes. Patients with limited joint mobility had a significantly higher incidence of proliferative retinopathy than patients with normal joint mobility and a similar duration of diabetes (P

We have determined the relationship of antibody titers to the rheumatoid arthritis nuclear antigen (RANA) and of antibody titers to the Epstein-Barr nuclear antigen (EBNA) to each other and to the DRw4 B cell alloantigen in the sera of 34 normal white adults. By a sensitive indirect immunofluorescence (IF) assay, 76% had RANA antibody, compared to 23% by a micro-immunodiffusion assay. The correlation coefficient for the tube dilution titers of anti-RANA and anti-EBNA was 0.61 (P < 10(-4)). The 14 DRw4-positive subjects and the 20 DRw4-negative subjects did not differ with respect to anti-RANA titers (P = 0.51) or anti-EBNA titers (P = 0.89). We conclude that: 1) most normal adults have RANA antibody by IF; 2) anti-RANA and anti-EBNA titers are closely related; 3) the titers of these antibodies cannot be related to the presence of the DRw4 determinant in normal persons.

The relative frequencies of IgM antiIgG autoantibody (rheumatoid factor) producing cells induced by the polyclonal B cell activator Epstein-Barr virus were measured in peripheral blood lymphocyte cultures of normal children and patients with juvenile rheumatoid arthritis. The frequencies of rheumatoid factor precursor B cells in normal children were lower than adults, but higher than neonates. The frequency increased with the age of the donor. In seronegative children with the systemic-onset or pauciarticular-onset types of juvenile rheumatoid arthritis, the number of IgM antiIgG inducible B cells was not significantly different (P greater than 0.05) from age-matched controls. Patients with seropositive juvenile rheumatoid arthritis or seropositive adult rheumatoid arthritis had significantly higher IgM antiIgG precursor cell frequencies than age-matched normal subjects (P less than 0.01 and P less than 0.02, respectively). In contrast, the patients with seronegative polyarticular-onset juvenile rheumatoid arthritis had an average precursor frequency significantly lower than normal age-matched controls (P less than 0.05), analogous to results previously noted in adult seronegative rheumatoid arthritis. Thus, both children and adults with seronegative polyarticular rheumatoid arthritis had a deficiency in B cells that produce IgM antiIgG and that are induced by Epstein-Barr virus. This distinguished them from seropositive juvenile rheumatoid arthritis and rheumatoid arthritis patients, normal subjects, and patients with the pauciarticular-onset and systemic-onset types of seronegative juvenile rheumatoid arthritis.

Most patients with seropositive rheumatoid arthritis, and a variable but lesser percentage of normal subjects, have precipitating antibodies to a nuclear antigen, rheumatoid arthritis nuclear antigen, present in Epstein-Barr virus-infected human B lymphoblastoid cells. We have used a sensitive indirect immunofluorescence assay for antibody to rheumatoid arthritis nuclear antigen in a study of patients with infectious mononucleosis and healthy control subjects. Of 110 sera from normal, college-age cadets, 58 were from individuals without prior Epstein-Barr virus infection, as indicated by the lack of antibody to viral capsid antigen. All of these also lacked activity to rheumatoid arthritis nuclear antigen. 52 sera were positive for antibody to viral capsid antigen, and antibody to rheumatoid arthritis nuclear antigen was present in 26 (50%) of these. In 67 sequential sera from 11 college-age students with infectious mononucleosis who became positive for antibody to rheumatoid arthritis nuclear antigen, only 2 were positive during the 1 mo. Thereafter the incidence and titers increased progressively through the 1st yr after infection. This time-course resembled that for the development of antibody to Epstein-Barr nuclear antigen, another transformation antigen in Epstein-Barr virus-infected B lymphocytes. The development of positivity for both was much later than that of antibody to the structural viral capsid antigen, which in the current study was always positive by 1 wk. Thus, antibody to rheumatoid arthritis nuclear antigen is present in a large proportion of normal individuals and can now be clearly ascribed, from both in vivo and in vitro studies, to prior infection with Epstein-Barr virus.

Current findings have suggested that the T3 molecular complex is an essential antigenic signal transducer during T cell activation. Lectins, such as phytohemagglutinin (PHA), activate T cells nonspecifically. Concesivably, lectins may mediate their stimulatory action by affecting the T3 complex. In the present investigation we have studied the involvement of the T3 molecular complex in the PHA-mediated activation of T cells. We selectively modulated the surface expression of T3 molecules by anti-T3 antibody and subsequently tested the ability of the modulated cells to respond to PHA. Reduction of T3 expression by 70% resulted in 80% inhibition of the PHA response. This effect was specific for T3 since modulation of other T cell surface molecules (T4, T8) did not affect the PHA-induced mitogenesis. To determine if PHA could interact directly with the T3 complex, immunoblotting (Western blot) analyses of anti-T3 immunoprecipitates were performed. A 20-kDa member of the T3 complex reacted not only with the anti-T3 antibody, but also with PHA itself. These results provide the first evidence for direct binding of PHA to one of the molecules of the T3 complex. The combined data suggest that a major pathway of PHA-induced T cell activation involves the T3 complex. Possible activation mechanisms are discussed.

Adenosine phosphorylase (adenosine: orthophosphate ribosyltransferase) activity is low in mammalian cells, but is abundant in certain prokaryotes. It is an appropriate target enzyme for the development of diagnostic and chemotherapeutic agents. Adenosine phosphorylase from Bacillus subtilis and mycoplasmas that commonly infect cell cultures converted the non-toxic deoxyadenosine analog, 6-methylpurine deoxyriboside (6MPDR) into the potent anti-metabolites 6-methylpurine and 6-methylpurine riboside. Consequently 6MPDR selectively killed mammalian cell cultures infected with mycoplasmas. 6MPDR was cytotoxic to 87 of 90 mycoplasma-infected cultures (96.6%). No toxicity was observed in nine different types of mycoplasma-free cell cultures. Use of a 3T6 mouse embryo fibroblast indicator cell culture improved standardization. Cytotoxicity was apparent 3–4 days after inoculation of 10 μM of 6MPDR to 3T6 cultures infected with the following mycoplasmas: M. hyorhinis, M. orale, M. arginini, M. salivarium, A, laidlawii, M. hominis, M. fermentons, M. sp . 70–159 and M. buccale . Cytotoxicity was produced in 28/28 3T6 indicator cultures inoculated with mycoplasma-infected cell cultures in the presence of 10 μM 6MPDR. The analog is apparently non-toxic to the mycoplasmas.

We investigated anti-rheumatoid arthritis-associated nuclear antigens (RANA) and other anti-Epstein-Barr virus (EBV) antibodies in a uniquely controlled study in female Pima Indians of Arizona, an RA prone population. Four groups of age and sex-matched individuals were formulated: (1) individuals positive for rheumatoid factor (RF) who had clinical evidence of rheumatoid arthritis (RA); (2) individuals seropositive for RF, but without arthritis; (3) individuals seronegative for RF, but with various kinds of arthritis; (4) those seronegative without arthritis. The mean anti-RANA in the seropositive RA group was significantly above those of the other groups but the anti-VCA and anti-EBNA titers did not differ. The anti-RANA was shown to be independent of RF. Comparing the Pima Indians to Caucasians in La Jolla, we found the mean anti-RANA titers of the Pimas to be significantly higher than those of the Caucasians. This study thus establishes clearly that elevated anti-RANA titers are characteristics of this American Indian group, just as they are of Caucasian groups. The elevated anti-RANA titers in RA patients may represent a unique hyperresponsiveness to this antigen, since there is no consistency in the reported levels of antibodies to other EBV-related antigens.

Efforts to determine the role of specific Ig variable region (V) genes in human autoimmune responses have been hampered by the lack of suitably polymorphic probes. Recently we isolated a heavy chain V (Vh) gene, designated Humhv3005, that is 99% homologous to the 1.9III Vh gene and can encode an anti-DNA antibody. To study the relation between these two genes, different DNA fragments from the isolated Humhv3005 clone were used to probe Southern blots of human genomic DNA. A 1.6-kb Eco RI fragment (designated hv3005/E1.6) was found to hybridize with only one band in Eco RI-digested DNA, and with two major bands in Bam HI-digested DNA. Importantly, the sizes of the latter two bands were indistinguishable from the corresponding Bam HI fragment sizes of the isolated hv3005 clone and the isolated 1.9III clone, respectively. Population and family studies with the hv3005/E1.6 probe revealed five different hybridization patterns of these two characteristic bands, which defined nine possible genotypes for two human Ig Vh gene loci. Together the data demonstrate that hv3005/E1.6 is a highly informative probe for an autoantibody-associated Vh gene(s), and should prove useful in elucidating the role of Ig Vh genes in autoimmune diseases.

The adenosine deaminase-resistant purine deoxynucleoside 2-chloro-2'-deoxyadenosine (CdA) is markedly toxic in vitro to nondividing and proliferating normal human lymphocytes and to many leukemia cell specimens. The CdA is also effective against mouse L1210 leukemia in vivo. The present investigations have examined the pharmacology, chemotherapeutic activity, and toxicity of CdA in nine patients with advanced hematologic malignancies refractory to conventional therapy. When administered by continuous intravenous infusion, the deoxyadenosine analog was well tolerated. As monitored by radioimmunoassay, plasma CdA levels rose gradually during the infusions. The CdA was not deaminated significantly. In all patients with leukemia, the CdA lowered the blast count by at least 50%. In one patient with a T-cell leukemia-lymphoma, and in another patient with chronic myelogenous leukemia in blast crisis, the CdA infusion eliminated all detectable blasts from the blood and bone marrow. In a patient with a diffuse lymphoma complicated by severe autoimmune hemolytic anemia, CdA treatment quickly terminated the hemolytic process. Bone marrow suppression represented the dose-limiting toxicity, and was related to plasma CdA levels, cumulative drug dosage, and the rapid release of CdA that accompanied tumor cell lysis.

We have developed a quantitative immunoassay for the measurement of human adenosine deaminase that requires neither purified enzyme nor monospecific antibody. With this technique, cultured fibroblasts from three immunodeficient patients were shown to contain adenosine deaminase as detected both catalytically and immunochemically. In one patient the level of immunoreactive enzyme exceeded the amount of catalytically active adenosine deaminase, suggesting a structural alteration in the enzyme molecule.

Although available literature does not unequivocally establish the presence of open ether bonds (ROR) in low-rank bituminous coals, formation of low-molecular-weight products on reacting such coals with hydrogen donors is usually interpreted in terms of scission of ether bridges. This lack of consistency could be justified by supposing that ethereal and hydroxyl functions in low-rank coals exist in phenoxyphenol-type structures. Ethers of this type have never been cleaved on treatment with sodium in liquid ammonia but, contrary to expectations based on extant literature, can be split by certain hydrogen donors. The results presented here indicate, however, that phenoxyphenol-type ethers do not exist in low-rank vitrinite and/or their cleavage does not result in lowering the molecular weight of the vitrinite. It is concluded that, unless non-reactive oxygen exists in low-rank vitrinite in dialkyl ether structures, formation of low-molecular-weight products on hydrogen donor treatment should be attributed mainly to CC bond cleavage.

The addition of poly(ADP-ribose) chains to nuclear proteins has been reported to affect DNA repair and DNA synthesis in mammalian cells. The enzyme that mediates this reaction, poly(ADP-ribose) polymerase, requires DNA for catalytic activity and is activated by DNA with strand breaks. Because the catalytic activity of poly(ADP-ribose) polymerase does not necessarily reflect enzyme quantity, little is known about the total cellular poly(ADP-ribose) polymerase content and the rate of its synthesis and degradation. In the present experiments, specific human autoantibodies to poly(ADP-ribose) polymerase and a sensitive immunoblotting technique were used to determine the cellular content of poly(ADP-ribose) polymerase in human lymphocytes. Resting peripheral blood lymphocytes contained 0.5 X 10(6) enzyme copies per cell. After stimulation of the cells by phytohemagglutinin, the poly(ADP-ribose) polymerase content increased before DNA synthesis. During balanced growth, the T lymphoblastoid cell line CEM contained approximately 2 X 10(6) poly(ADP-ribose) polymerase molecules per cell. This value did not vary by more than 2-fold during the cell growth cycle. Similarly, mRNA encoding poly(ADP-ribose) polymerase was detectable throughout S phase. Poly(ADP-ribose) polymerase turned over at a rate equivalent to the average of total cellular proteins. Neither the cellular content nor the turnover rate of poly(ADP-ribose) polymerase changed after the introduction of DNA strand breaks by gamma irradiation. These results show that in lymphoblasts poly(ADP-ribose) polymerase is an abundant nuclear protein that turns over relatively slowly and suggest that most of the enzyme may exist in a catalytically inactive state.

Two groups of mediators, the neuropeptides substance P and K and the monocyte-derived cytokines, interact in the neural regulation of immunological and inflammatory responses. Substance P, substance K, and the carboxyl-terminal peptide SP(4-11) induce the release of interleukin-1, tumor necrosis factor-alpha, and interleukin-6 from human blood monocytes. The neuropeptide effects occur at low doses, are specific as shown by inhibition studies with a substance P antagonist, and require de novo protein synthesis. Since monocyte-derived cytokines regulate multiple cellular functions in inflammation and immunity and since neuropeptides can be released from peripheral nerve endings into surrounding tissues, these findings identify a potent mechanism for nervous system regulation of host defense responses.

Using murine monoclonal antibodies (MoAbs) specific for immunoglobulin (Ig) cross-reactive idiotypes (CRI), we performed immunohistochemical analyses on frozen tissue sections and cytocentrifuge preparations of Ig-expressing malignant cells from patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin's lymphomas (NHL) of follicular center cell origin. Twenty percent (4/20) of the Ig kappa light chain- expressing CLL cells reacted with 17.109, a MoAb against a major CRI on human IgM autoantibodies that is encoded by a conserved Ig variable- region gene (V gene) of the V kappa IIIb sub-subgroup. Another MoAb specific for V kappa IIIb framework determinant(s) reacted exclusively with all the 17.109-reactive CLL cells. Only one of 20 kappa light- chain-expressing CLL cells reacted with 6B6.6, a monoclonal antibody specific for a CRI commonly found on rheumatoid factor (RF) paraproteins with light-chain variable regions of the V kappa IIIa sub- subgroup. Finally, greater than 20% (8/34) of all CLL reacted with G6, a MoAb specific for an Ig heavy chain-associated CRI present on several RF paraproteins. In contrast, these CRIs were expressed at significantly lower frequencies in NHL of follicular center cell origin. Only one of 30 NHL expressing kappa light chains reacted with the 17.109 MoAb. Also, in contrast to the concordance between the 17.109-CRI and V kappa IIIb framework determinant(s) in CLL, two lymphomas in addition to the 17.109-reactive lymphoma were recognized by the anti-V kappa IIIb framework MoAb. None of the NHL reacted with either the 6B6.6 or the G6 MoAbs. These results are the first to demonstrate that CLL and NHL differ with respect to the expression of autoantibody-associated CRIs. The data support the notion that NHL of follicular center cell origin differs from CLL in its utilization and/or somatic mutation of Ig variable-region genes. The physiological and immunotherapeutic implications of these findings are discussed.

The idiotypic determinants on IgM rheumatoid factor (RF) from a single family have been analyzed. Rabbit Fab'2 antiidiotypic antibody was prepared against purified IgM-RF from a patient with rheumatoid arthritis. As measured by radioimmunoassay, the antiidiotype reacted with at least 90% of the patient's RF, but not with non-RF immunoglobulins from the same serum, nor with 10 of 11 polyclonal and monoclonal RF from unrelated individuals. Cross-reacting idiotypes were detected on RF in four of the patients' first degree relatives, spanning three generations, without apparent relation of HLA type or clinical rheumatoid arthritis. These results suggest that IgM-RF associated idiotypes were inherited in this family.

The mouse monoclonal antibody 17.109 recognizes a cross-reactive idiotype (CRI) associated with kappa IIIb light chains of human IgM-rheumatoid factor (RF) paraproteins. The 17.109 idiotypic determinant is encoded by one or a group of closely related V kappa genes. The association of the idiotype with IgM- and IgA-rheumatoid factors in certain autoimmune diseases necessitates an understanding of how human B lymphocytes can be induced to express the idiotype. To investigate the cellular expression of the 17.109 CRI, peripheral blood lymphocytes from normal donors were stimulated in vitro with Epstein-Barr virus (EBV) and pokeweed mitogen (PWM). EBV induced greater expression of IgM-associated 17.109 CRI than did PWM. The 17.109 CRI was preferentially associated with IgM rather than with IgG. In vivo EBV infection was studied in college students with infectious mononucleosis and displayed similar elevation of IgM-associated 17.109 CRI in sera obtained at presentation of clinical illness. Later, IgM levels declined while IgG-associated 17.109 CRI rose. The 17.109 idiotype was unrelated to antibodies against the Epstein-Barr virus nuclear antigen and the viral capsid antigen and was probably due to generalized activation of early B cells. These observations support the hypothesis that the 17.109 CRI is expressed by in vitro and in vivo EBV-infected cells. The 17.109 idiotype identifies a highly conserved V kappa gene product, which is expressed preferentially after EBV infection, but not exclusively with RF autoantibodies.

Recently, an antiidiotype to human monoclonal IgM anti-IgG autoantibodies (rheumatoid factors) was found to react also with human IgG. This peculiar antiidiotype was called an 'epibody'. We describe the induction of a similar epibody by immunization with a synthetic peptide (corresponding to one hypervariable region of the IgM-RF Glo). The results confirm the existence of epibodies, and provide the possible molecular basis of the epibody phenomenon.

Viable human and murine lymphoblasts, and normal human tissue extracts, converted the thioether nucleosides 5′-methylthioadenosine (MeSAdo) and 5′-methylthioinosine (MeSIno) to methionine. Both MeSAdo and MeSIno, but not homocysteine, supported the short-term growth of human or murine lymphoblasts in methionine deficient medium. However, MeSAdo at concentrations greater than 25 μM inhibited cell growth. MeSIno was non-toxic at concentrations up to 200 μM, and supported the long-term growth of lymphoblasts in methionine-free medium.

Using a panel of antibodies specific for H and L chain variable region subgroups, a panel of human monoclonal cold agglutinin (CA) and rheumatoid factor (RF) autoantibodies were analyzed. The vast majority of the two types of autoantibodies utilized VkIII L chains, many of which probably derive from the Humkv325 gene. However, while most RFs (77%) utilized VHI H chains, all the CAs used VHII subgroup H chains. These results are consistent with a model of autoantibody generation, wherein binding specificity is H chain defined in a set of antibodies that use a multipotential L chain.

Investigations of rheumatoid factors by a hemolytic plaque forming cell assay of blood lymphocytes with sensitized sheep cells have suggested that the rheumatoid factors released from the cells have higher affinities for human IgG than do the rheumatoid factors measured in the patient's serum. This study reaffirms this observation and provides evidence that the reported differences are not artifacts of technique. The findings imply that rheumatoid factors of higher affinity for IgG than those of the serum are being released into the tissue fluids by lymphocytes and locally precipitated. Such rheumatoid factors may never reach the peripheral blood serum in detectable quantity or may do so only infrequently.

2-Chlorodeoxyadenosine (CdA), an adenosine-deaminase-resistant purine deoxynucleoside, is markedly toxic toward human T-lymphoblastoid cell lines in vitro and is an effective agent against L1210 leukemia in vivo. The present studies have examined the toxicity, and in some cases, metabolism, of CdA in (1) multiple established human cell lines of varying phenotype, (2) leukemia and lymphoma cells taken directly from patients, (3) normal bone marrow cells, and (4) normal peripheral blood lymphocytes. Nanomolar concentrations of CdA blocked the proliferation of lymphoblastoid cell lines with a high ratio of deoxycytidine kinase to deoxynucleotidase. The drug had virtually no effect on the growth of cell lines derived from solid tissues. The CdA inhibited the spontaneous uptake of tritiated thymidine by many T and non-T, non-B acute lymphoblastic leukemia cell specimens at concentrations less than or equal to 5 nM. The same concentrations did not impair either thymidine uptake or granulocyte-monocyte colony formation by normal bone marrow cells. In common with deoxyadenosine, but unlike several other agents affecting purine and purine metabolism, CdA was lethal to resting normal T lymphocytes and to slowly dividing malignant T cells. In both resting and proliferating lymphocytes, the CdA was phosphorylated by deoxycytidine kinase and entered a rapidly turning over nucleotide pool. Dividing lymphocytes also incorporated abundant CdA into DNA. The selective toxicity of CdA toward both dividing and resting lymphocytes may render the drug useful as an immunosuppressive or antileukemic agent.

An intimate relationship exists between DNA single-strand breaks, NAD metabolism, and cell viability in quiescent human lymphocytes. Under steady-state conditions, resting lymphocytes continually break and rejoin DNA. The balanced DNA excision-repair process is accompanied by a proportional consumption of NAD for poly(ADP-ribose) synthesis. However, lymphocytes have a limited capacity to resynthesize NAD from nicotinamide. An increase in DNA strand break formation in lymphocytes, or a block in DNA repair, accelerates poly(ADP-ribose) formation and may induce lethal NAD and ATP depletion. In this way, the level of DNA single-strand breaks in the lymphocyte nucleus is linked to the metabolic activity of the cytoplasm. The programmed removal of lymphocytes (and perhaps of other cells) with damaged DNA, may represent a novel physiologic function for poly(ADP-ribose)-dependent NAD cycling.

We isolated a VH1 germline gene (Humhv 1263) that is closely related to the heavy chains of 2 human rheumatoid factors (RF), Bor and Kas. We also found that the 783 rearranged VH1 gene is actually 91–93% homologous to Bor and Kas, and it differs from a VH1 complementary DNA (51P1) by only a single base. Thus, 783 is likely to be the unmutated form of a germline VH gene that may encode the heavy chains of RF Bor and Kas and several other human monoclonal RF.

Two US lignites (from Texas and North Dakota) were fractionated by density with appropriately prepared mixtures of liquid halogenated and non-halogenated hydrocarbons. Petrographic analyses confirmed that the procedure resulted in density fractions enriched in specific macerals. Liquid yields obtained upon solubilization at 430 °C and 5.5 MPa hydrogen pressure decreased with increase in the density fraction for both lignites. On plotting the total solubilities of lignites and their density fractions versus their huminite contents a reasonably good linear correlation was obtained (particularly for North Dakota lignite). It is tentatively concluded that in North Dakota lignite, as opposed to Texas lignite, macerals other than huminite did not significantly contribute to generation of liquid products.