Extrahepatic transcription of human C-reactive protein.

We synthesized and cloned cDNA from human peripheral blood mononuclear cell (PBMC) transcripts that were hybrid selected by pCRP5, a liver C-reactive protein (CRP)-specific cDNA (Woo, P.,J.R. Korenberg, and A.S. Whitehead. 1985. J.Biol. Chem. 260:13384). Three hybrid-selected cDNA clones, HScDNA1, HScDNA3, and HScDNA8, were isolated and characterized. Nucleotide sequence analysis of the 5' end of the smaller clones, HScDNA1 and HScDNA8, demonstrated that these two PBMC clones are homologous to the 3' and 5' ends, respectively, of pCRP5. Our largest clone, HScDNA3, is larger than pCRP5, extending beyond both the 5' and 3' limits of pCRP5. Therefore, HScDNA3 was coded by human PBMC and not by the hybrid selection vehicle, pCRP5. HScDNA3 lacks the intervening sequence verifying that this clone is DNA made from a PBMC mRNA and not genomic DNA. The complete nucleotide sequence revealed that HScDNA3 is greater than 99% homologous to the CRP gene. These results demonstrate that PBMC express the CRP gene. Based on our previous report, which shows that peripheral blood cells synthesize a peptide recognized by anti-CRP (Kuta, A.E., and L.L. Baum. 1986. J. Exp. Med. 164:321), in conjunction with the data presented here, we conclude that human PBMC can synthesize CRP.

Inhibition of vacuolar ATPase subunit in tumor cells delays tumor growth by decreasing the essential macrophage population in the tumor microenvironment.

In cancer cells, vacuolar ATPase (V-ATPase), a multi-subunit enzyme, is expressed on the plasma as well as vesicular membranes and critically influences metastatic behavior. The soluble, cleaved N-terminal domain of V-ATPase a2 isoform is associated with in vitro induction of tumorigenic characteristics in macrophages. This activity led us to further investigate its in vivo role in cancer progression by inhibition of a2 isoform (a2V) in tumor cells and the concomitant effect on tumor microenvironment in the mouse 4T-1 breast cancer model. Results showed that macrophages cocultivated with a2V knockdown (sh-a2) 4T-1 cells produce lower amounts of tumorigenic factors in vitro and have reduced ability to suppress T-cell activation and proliferation compared with control 4T-1 cells. Data analysis showed a delayed mammary tumor growth in Balb/c mice inoculated with sh-a2 4T-1 cells compared with control. The purified CD11b(+) macrophages from sh-a2 tumors showed a reduced expression of mannose receptor-1 (CD206), interleukin-10, transforming growth factor-ß, arginase-1, matrix metalloproteinase and vascular endothelial growth factor. Flow cytometric analysis of tumor-infiltrated macrophages showed a significantly low number of F4/80(+)CD11c(+)CD206(+) macrophages in sh-a2 tumors compared with control. In sh-a2 tumors, most of the macrophages were F4/80(+)CD11c(+) (antitumor M1 macrophages) suggesting it to be the reason behind delayed tumor growth. Additionally, tumor-infiltrating macrophages from sh-a2 tumors showed a reduced expression of CD206 compared with control whereas CD11c expression was unaffected. These findings demonstrate that in the absence of a2V in tumor cells, the resident macrophage population in the tumor microenvironment is altered which affects in vivo tumor growth. We suggest that by involving the host immune system, tumor growth can be controlled through targeting of a2V on tumor cells.

Antigen-binding molecules of T-cells charge heterogeneity and structural lability.

T-cell products released by immune cells during culture and which bind specifically the nominal antigens, trinitrophenol (TNP) or oxazalone, were isolated from culture media by hapten-affinity chromatography and compared by isoelectric focusing and 2D-gel analysis. These proteins and an azobenzenearsonate-specific T-cell product synthesized in vitro by translation of mRNA from an azobenzenearsonate-specific T-cell hybrid were also compared for structural lability of the polypeptides. Polyclonal T-cell antigen-binding molecules (TABM) specific for TNP or oxazalone showed marked charge heterogeneity and distinctions in isoelectric focusing in an acidic pH gradient, while the azobenzenearsonate-specific, clonal T-cell product displayed restricted focusing. All TABM studied showed dissociation of Mr 70,000 polypeptides to Mr 45,000 and 25,000 polypeptides after treatment with guanidine. The results provide further evidence for distinctions and similarities between TABM.

Cloning of a cDNA for a T cell produced molecule with a putative immune regulatory role.

An expression cDNA library was constructed from the helper T cell hybridoma, A.1.1, which has been shown to produce constitutively proteins involved in the down regulation of the immune response. From this library we identified and characterized a cDNA clone, J6B7, by screening with a polyclonal antibody specific for secreted immune regulatory proteins. The mRNA for J6B7 is expressed specifically in some T cells, but not in the thymoma BW5147 or liver cells. J6B7 is 2937 nucleotides in length and contains one open reading frame encoding for a peptide of predicted Mr of 98,042. The nucleotide and deduced amino acid sequences of J6B7 did not reveal significant homology to any published sequences. Hybridization and translation experiments reveal that the J6B7 can hybrid select mRNA from total RNA isolated from either A.1.1 cells or thymic tissue which can be translated in vitro to a peptide which is bound by a monoclonal antibody (mAb) specific for antigenic determinant(s) shared by immune regulatory proteins. Furthermore, the in vitro translated proteins obtained from A.1.1 cells and thymus showed significant suppression of a mixed lymphocyte reaction (MLR) in a dose dependent manner, reaching maximum suppression of 71% and 89%, respectively. These results suggest that the cDNA, J6B7, codes for an immune regulatory protein.

Sequence analysis of porcine immunoglobulin light chain cDNAs.

A porcine cDNA library was constructed using poly(A)+ RNA isolated from the spleen of an adult Minnesota miniature swine. Screening the library with antisera specific for porcine immunoglobulin light chains resulted in the selection and isolation of two recombinant clones, PLC18 and PLC3, which encode for kappa and lambda light chains, respectively. These cDNAs contain sequence information for a portion of the variable region and all of the constant region. The lengths of the constant regions are 105 amino acids for lambda and 108 amino acids for kappa. The deduced amino acid sequences of porcine immunoglobulin light chains share a high degree of homology with similar sequences from other species in both the fourth framework region and the constant region.

Characterization of a cDNA clone encoding for a porcine immunoglobulin mu chain.

We present the complete sequence of cDNA clone BLM6, isolated from a porcine spleen cDNA library. BLM6 encodes for the constant region of a secreted mu heavy chain. The cDNA is 1342 bp in length, begins within C mu 1, and terminates in the 3' translated region. The deduced amino acid sequence indicates there are 111 amino acids in C mu 2, 106 in C mu 3, and 113 in C mu 4, along with 19 carboxy-terminal residues that comprise the secreted terminus. The full length transcript is approximately 2.4 kb as determined by Northern blot analysis. Southern blot analysis suggests there is only one mu chain gene within the porcine genome. Comparison of the deduced amino acid sequence of porcine C mu with other mammalian C mu sequences indicates that the overall identity is approximately 60%, with the greatest identity seen in the secreted terminus (approximately 85%) and C mu 4 (approximately 70%); and less identity in C mu 3 (60%) and C mu 2 (50%).

Regeneration and tolerance factor's potential role in T-cell activation and apoptosis.

Regeneration and tolerance factor (RTF) is a novel membrane protein that has a diverse expression pattern and immunoregulatory properties. RTF is expressed in vivo on the surface of individuals with B cell chronic lymphocytic leukemia and on activated T lymphocytes of HIV infected individuals as determined by their coexpression with CD38 and HLA-DR. The unique expression patterns of this protein in vivo lead us to investigate its expression in vitro. The activation of human PBMCs through the TCR, using anti-CD3 antibody and PMA, upregulated cell surface expression of RTF from 2. 3% to 91.2% (mean channel fluorescence [MCF] increased threefold). The activation of Jurkat T cells through the TCR upregulated surface expression of RTF from 8.3% (MCF-1.3) to 58.7% (MCF-13.1). The Jurkat T-cell line was used as a model system to explore RTF's role in cellular activation. Using the Jurkat T-cell model, we found anti-RTF antibody induces apoptosis. The addition of anti-RTF antibody increased annexin V binding by threefold compared with the IgG1 kappa isotype control antibody (p < 0.00002) and activated caspase 3. These data indicate that RTF is expressed during T-cell activation and may be associated with apoptosis.

Regeneration and tolerance factor is expressed during T-lymphocyte activation and plays a role in apoptosis.

Regeneration and tolerance factor (RTF) is a protein cloned from the thymus and expressed on B lymphocytes in normal pregnancy, B lymphocytic leukemia lines, and T and B lymphocytes in individuals with HIV infection. Findings, using the Jurkat T-cell model, revealed that RTF is upregulated after activation and anti-RTF antibody-induced apoptosis. In this article anti-RTF antibody-induced apoptosis of both unstimulated and activated T lymphocytes. RTF expression was examined in human PBMC or purified T lymphocytes after their in vitro activation. Kinetic studies indicated maximal RTF cell surface expression on activated T lymphocytes occurred between expression of the early activation antigen CD69 and the IL-2alpha receptor (CD25) by multiparameter flow cytometry. RTF receptor expression correlated with Fas (CD95) and CD25 receptor expression (r2 = 0.6 and 0.5, respectively). RTF surface expression was dependent on the stimuli used to activate T lymphocytes. T lymphocytes obtained maximal RTF expression when activated through the TCR signal complex using anti-CD3epsilon antibody alone when compared with T lymphocytes activated with costimulation provided by anti-CD28 antibody alone or with anti-CD28 and anti-CD3epsilon antibody. RTF is expressed under conditions of both activation and anergy. The RTFs increased concentration on the surface of anergic T cells may protect these cells from apoptosis because increased RTF concentrations inhibited anti-RTF induced apoptosis. These data further characterize the expression of RTF on activated T lymphocytes and the role of anti-RTF antibody in T-lymphocyte apoptosis.

Presence of multiple anti-phospholipid antibody specificities in a pediatric population.

Thrombotic related events are thought to be associated with the presence of anti-phospholipid antibodies (APA). However, the association of anti-cardiolipin antibody is much weaker than the association with antibodies to other phospholipids. Much of the literature equates antiphospholipid antibodies and anticardiolipin antibodies because of the relationship of APA and false positive tests for syphilis. However, recently the presence of antibodies to naturally occurring phospholipids other than cardiolipin have been reported. In fact, some investigators report that antibodies to phosphatidylserine appear to correlate more closely to disease processes than anti-cardiolipin antibodies. We describe here the presence of non-anti-cardiolipin antiphospholipid antibodies in a pediatric population that lack anti-cardiolipin antibodies and demonstrate the association of these antibodies with thrombotic disease. Antibodies to phosphatidic acid were the most prevalent and correlated (p < .001) with thrombotic disease and idiopathic thrombocytopenia purpura. The rank order of prevalence of antibodies to phospholipids was phosphatidic acid, phosphatidylglycerol, phosphatidylinosital, phosphatidylserine, cardiolipin and phosphatidylethanolamine. Antiphospholipid antibodies of the three major sera isotypes were present in the positive sera examined. These descriptive findings suggest that the significance of APA other than anti-cardiolipin antibodies in pediatric patients should be further investigated.

T-cell suppressor factors play an integral role in preventing fetal rejection.

In previous studies we showed that the blocking of T-cell suppressor factors (TsF) with monoclonal antibody could completely ablate pregnancy, and demonstrated the presence of TsF in fetal and maternal tissues. In our current study we used a monoclonal antibody specific for TsF to determine the time during gestation when TsF is most integral in the maintenance of pregnancy. Significant decreases in the number of viable pregnancies when monoclonal antibody was administered on days 3, 4 and 5 were demonstrated. In addition, ELISAs were used to measure the levels of TsF in tissues from pregnant and non-pregnant mice. Increased levels of TsF in the uterus and the lymph nodes draining the uterus were observed when compared to the same tissues of non-pregnant animals. These data strengthen the hypothesis that TsF is in part responsible for the fetal specific immune supression during pregnancy in allogeneic mice and more clearly define the importance of TsF in the implantation of the embryo.

Inhibition of binding of anti-CD3 antibodies to paternal lymphocytes correlates with failure of immunotherapy for treatment of recurrent spontaneous abortions.

A two color flow cytometry crossmatch (FCXM) was used to evaluate the induction of anti-lymphocyte antibodies in 34 women undergoing immunotherapy for recurrent spontaneous abortions. All women had anti-lymphocyte antibodies that reacted with T-cells when analyzed by FCXM. However, inhibition of the binding of anti-CD3 to paternal CD3 lymphocytes in the presence of maternal antipaternal lymphocyte antiserum was found for some couples following lymphocyte immunotherapy for spontaneous recurrent abortions. Ten couples who had another spontaneous abortion following immunotherapy showed inhibition. In contrast, eight couples who did not show inhibition of the binding of anti-CD3 T lymphocytes to paternal lymphocytes by maternal anti-lymphocyte antiserum had live births. Women of the remaining 16 couples were either pregnant and awaiting birth or not pregnant. Thus, by FCXM it may be possible to predict those couples who will have successful pregnancies following this treatment.

Autoantibodies in women with primary recurrent spontaneous abortion of unknown etiology.

A group of 153 women with 3 or more recurrent spontaneous abortions (RSAs) with unknown etiology and 90 normal multigravida controls were evaluated for antibodies to phospholipids and nuclear antigens. We demonstrate that women with recurrent spontaneous abortions showed significantly higher incidence of antibodies to phospholipids than normal multigravida controls. In contrast, the incidence of antibodies to polynucleotides and histones was not different between these two groups. These findings suggest that antiphospholipid antibodies are either epiphenomena or causally related to recurrent spontaneous abortions.

Expression of membrane form of the pregnancy associated protein TJ6 on decidual lymphocytes in the first trimester of pregnancy.

TJ6, a newly described protein produced locally in the uterine decidua during pregnancy, may be involved in maintaining a unique immunological environment at the maternal-fetal interface. The aim of this study was to determine whether TJ6 is expressed as membrane form on decidual lymphocytes (DL), to define the phenotypes of TJ6m (membrane form TJ6) expressing cells and to analyze the fluorescence intensity of TJ6m expression. Peripheral blood lymphocytes (PBL) and DL were obtained from first trimester pregnancies undergoing elective termination and immunophenotyped for TJ6m and other cell surface antigens (CD3, CD8, CD19, CD56, CD16) by flow cytometry. This is the first study showing that TJ6 molecules are present on decidual lymphocytes in human pregnancy. TJ6m expression on PBL was not different from that of DL. However, a significantly higher percentage of double positive (TJ6m+CD3+, TJ6m+,CD8+,TJ6m+CD19+) cells were found in PBL when compared to DL. The average fluorescence intensity (AFI) for the TJ6m marker among cells with CD8+, CD19+ and CD56+ double positive was significantly higher in DL as compared with those of PBL. The AFI for granularity of double positive DL was significantly higher than observed in PBL.

Intravenous immunoglobulin infusion therapy in women with recurrent spontaneous abortions of immune etiologies.

We have investigated clinical effectiveness of intravenous immunoglobulin G infusion (IVIg) on antiphospholipid antibody titers in five women with evidence of antiphospholipid antibody-associated recurrent spontaneous abortions and one with antinuclear antibody who became refractory to conventional autoimmune treatment during pregnancy and experienced pregnancy complications. Three women developed intrauterine growth retardation and three had complicated twin pregnancies with rising autoantibody titers. Antiphospholipid antibody and antinuclear antibody titers were tested pre and 2 weeks after each IVIg infusion. We report that: (i) IgG antiphospholipid antibody titers were significantly suppressed after each IVIg infusion (P < 0.05); (ii) IgM antiphospholipid antibody titers were also significantly suppressed after each IVIg infusion (P < 0.0001); (iii) decreased titers of autoantibodies paralleled increased levels of maternal IgG which lasted for at least 30 days; the autoantibodies showed a definite rise again prior to the next infusion; (iv) antinuclear antibody titers were effectively suppressed; and (v) rising autoantibody titers combined clinical manifestation of intrauterine growth retardation and women with complicated twin pregnancies. We conclude that IVIg infusion effectively suppresses IgM and IgG autoantibodies to phospholipids and antinuclear antibody in autoimmune women with a history of recurrent spontaneous abortions and refractory to conventional anticoagulation or immunosuppressive treatment.

Effect of intravenous immunoglobulin G on natural killer cell cytotoxicity in vitro in women with recurrent spontaneous abortion.

Intravenous immunoglobulin (IVIg) has been used to treat women with recurrent spontaneous abortion (RSA), particularly for women with elevated natural killer (NK) cells. We investigated the effect of IVIg on peripheral blood NK cell activity in vitro in women with RSA. 51Cr-release assays using K562 in the presence of varying concentrations of IVIg were performed using PBL from 16 women with RSA. Antibody dependent cellular cytotoxicity (ADCC) was evaluated using Daudi cells. Effectors and targets were preincubated with IVIg. Binding of IVIg to K562 and Daudi was evaluated by flow cytometry. The effect of K562 absorbed IVIg on NK activity was compared to that of non-absorbed IVIg. NK cytotoxicity and ADCC in the presence of F(ab')2 fragments were compared with those in the presence of intact IVIg. IVIg produced a significant, dose dependent inhibition of NK activity in vitro. Inhibition of NK activity occurred when effectors but not targets were preincubated with IVIg. IVIg binds to K562 and Daudi. IVIg increased ADCC when targets but not effectors were incubated with IVIg. K562 absorbed IVIg produced more inhibition of NK cytotoxicity than non-absorbed IVIg. Suppression of NK cytotoxicity by F(ab')2 was as effective as that of IVIg. However, F(ab')2 did not increase ADCC. IVIg effectively reduces peripheral blood NK cytotoxicity in vitro. Inhibition of NK cytotoxicity is mediated at the effector cell level through the antigen binding portion of the immunoglobulins. Women with RSA and elevated NK cells may benefit from IVIg treatment.

Characterization of antibodies induced by paternal lymphocyte immunization in couples with recurrent spontaneous abortion.

This study was designed to identify and characterize the allo- and autoantibodies induced following successful paternal lymphocyte immunization to prevent recurrent spontaneous abortion. Firstly the titers of maternal anti-paternal antibodies in women with successful pregnancies as determined by the flow cytometry crossmatch (FCXM) were highly variable; however, in all cases, the initial pre-immunization titers were negative and the post-immunization titers were positive by the FCXM in successfully treated women. Secondly, the specificities of maternal alloantibodies to paternal HLA antigens (immunogen) were evaluated. No all predicted antibodies to mismatched paternal HLA antigens were found by microlymphocytotoxicity (MCX) assays and the specificities varied. Thirdly, antibodies in post- but not preimmunization sera reacted with two lymphoid cell lines, SupT1 and SB; in addition, the rise and fall of the titers of these sera with paternal cells seemed to be reflected with the cell lines by the FCXM. Fourthly, autoantibodies to activated lymphocytes were detected and seemed to correlate with successful immunization since women who had another abortion following immunotherapy lacked these autoantibodies. These findings suggest that the antibody response following successful immunotherapy is complex and needs to be studied further to understand the mechanism of this treatment.

Antibodies to phospholipids and nuclear antigens in non-pregnant women with unexplained spontaneous recurrent abortions.

In a collaborative study of 73 non-pregnant Kuwaiti women with unexplained spontaneous recurrent abortion (RSA), 30 control healthy non-pregnant multiparous Kuwaiti women and 20 North American women who received elective abortion(s), autoantibodies to 6 phospholipids and 9 nuclear antigens were measured. Women with recurrent spontaneous abortions demonstrated 3 times higher incidence of antibodies to phospholipids (30.1%) than controls (10% each) (P = 0.029). The incidence of both IgM and IgA class antiphospholipid antibodies were significantly higher than those of controls. The incidence of antibodies to cardiolipin in women with recurrent spontaneous abortions (12.3%) was significantly higher than those of controls (P = 0.035) and incidence of IgM but not IgG anticardiolipin antibody was significantly higher in women with RSAs than in controls (P = 0.053). The incidences of anti-polyinosinic acid (P = 0.035) and anti-histone 1 antibody (P = 0.052) were significantly higher in women with recurrent spontaneous abortions than controls. There was no significant difference in the incidence of autoantibodies between primary and secondary aborters. However, women with a history of second trimester abortions showed a higher incidence of antiphospholipid antibodies than women with first trimester abortions only. Recurrent spontaneous abortion is associated with autoantibodies to phospholipid epitopes including IgA antiphospholipid antibodies.

Antioxidants reduce the mutagenic effect of malonaldehyde and beta-propiolactone. Part IX. Antioxidants and cancer.

Increasing concentrations of malonaldehyde and beta-propiolactone were increasingly mutagenic with 7 mutants of Salmonella typhimurium, 5 of which mutated bya frameshift mechanism and 2 of which mutated through base-pair substitution. The antioxidants vitamin C, vitamin E, selenium and butylated hydroxytoluene (BHT) at 3 logarithmic concentrations markedly reduced mutagenesis in those strains which mutated by frameshift mechanism.

Tumor-associated vacuolar ATPase subunit promotes tumorigenic characteristics in macrophages.

Macrophage polarization contributes to distinct human pathologies. In tumors, a polarized M2 phenotype called tumor-associated macrophages (TAMs) are associated with promotion of invasion and angiogenesis. In cancer cells, vacuolar ATPase (V-ATPase), a multi-subunit enzyme, is expressed on the plasma/vesicular membranes and critically influences the metastatic behavior. In addition, the soluble, cleaved N-terminal domain of a2 isoform of V-ATPase (a2NTD) is associated with in vitro induction of pro-tumorigenic properties in monocytes. This activity of a2 isoform of V-ATPase (a2V) caused us to investigate its role in cancer progression through the evaluation of the immunomodulatory properties of a2NTD. Here, we present direct evidence that surface expression of V-ATPase is associated with macrophage polarization in tumor tissue. Macrophages from BALB/c mice (peritoneal/bone marrow derived) were stimulated with recombinant a2NTD in both ex vivo and in vivo systems and evaluated for TAM characteristics. a2V was highly expressed in tumor tissues (breast and skin) as well as on the surface of tumor cell lines. The a2NTD-stimulated macrophages (a2MΦ) acquired TAM phenotype, which was characterized by elevated expression of mannose receptor-1, Arginase-1, interleukin-10 and transforming growth factor-ß. a2MΦ also exhibited increased production of other tumorigenic factors including matrix metalloproteinase-9 and vascular endothelial growth factor. Further, a2MΦ were cocultured with mouse B-16F0 melanoma cells for their functional characterization. The coculture of these a2MΦ subsequently increased the invasion and angiogenesis of less invasive B-16F0 cells. When cocultured with naive T cells, a2MΦ significantly inhibited T-cell activation. The present data establish the role of V-ATPase in modulating a macrophage phenotype towards TAMs through the action of a2NTD, suggesting it to be a potential therapeutic target in cancer.