Irrelevant Antibody Research Articles

Resistance to Cryptococcus neoformans infection in the absence of CD4 + T cells

Previous studies of Cryptococcus neoformans infection have revealed a role for CD4+ T cells and CD8+ T cells in anticryptococcal resistance in the lungs, but such a role has been revealed only for CD4+ T cells in the brains of experimentally infected mice. In this study, we found that mice genetically engineered to lack CD4+ T cells could be successfully vaccinated to express resistance to a rechallenge with Cryptococcus neoformans, provided the challenge dose was kept to lower than 1000 organisms per mouse. The challenge infection was uniformly lethal for unvaccinated control mice. Depletion of CD8+ T cells weakened this resistance to re-challenge: both naïve and vaccinated mice that were treated with antibody raised against CD8+ T cells died significantly earlier than did mice that received an irrelevant control antibody. In vitro, purified CD8+ T cells taken from draining lymph nodes of antigen-experienced mice were less efficient than were identically prepared CD4+ T cells at stimulating the cells of a transformed microglial cell line to inhibit C. neoformans proliferation, possibly mirroring the inferiority of CD8+ T-cell-mediated protection observed in vivo. RNase protection assays showed similar IFN-gamma mRNA levels in both lymphocyte subsets. Class II major histocompatibility antigen expression was up-regulated strikingly on microglia cultured with IFN-gamma, but class I expression was less dramatically affected. Therefore microglial cell interaction may be more greatly enhanced with CD4+ cells than with CD8+ cells.

Biodistributions of intact monoclonal antibodies and fragments of BLCA-38, a new prostate cancer directed antibody.

Monoclonal antibodies (MAbs) are used for targeting agents to tumours while minimizing normal tissue exposure. A new anti-prostate cancer MAb, BLCA-38, was radioiodinated (I125) and assessed for its ability to target subcutaneous human prostate cancer (DU-145) xenografts after systemic intraperitoneal administration. For comparison, the profile of J591 MAb (now in clinical trial) against LNCaP-LN3 tumours was examined. Biodistribution profiles were obtained at various times, by assessing injected dose/gram (%ID/g) and xenograft to blood (X/B) ratios. Microautoradiography of xenografts was performed. After conjugation with a melittin peptide toxin, the profiles of BLCA-38 and J591 were compared with that of an irrelevant antibody, DS-1. Xenograft localization by 125I-labeled BLCA-38 and J591 MAbs to their relevant antigen-positive tumors was comparable, and there was no unusual localization in nontumour tissues. F(ab')2 and Fab fragments gave improved X/B ratios, but the %ID/g xenograft was decreased and they accumulated in kidneys, bladder and stomach. In contrast, the conjugates of irrelevant antibody showed no tumour targeting. Microautoradiography showed more tumour accumulation of MAbs than F(ab')2s or Fabs. BLCA-38 can target prostate cancer in vivo almost as effectively as J591. Given that J591 is used clinically, BLCA-38, which targets a different antigen, has potential for radioimmunoscintigraphy and for therapeutic targeting of prostate cancer.

Resistance toCryptococcus neoformansinfection in the absence of CD4+T cells

Previous studies of Cryptococcus neoformans infection have revealed a role for CD4+ T cells and CD8+ T cells in anticryptococcal resistance in the lungs, but such a role has been revealed only for CD4+ T cells in the brains of experimentally infected mice. In this study, we found that mice genetically engineered to lack CD4+ T cells could be successfully vaccinated to express resistance to a re-challenge with Cryptococcus neoformans, provided the challenge dose was kept to lower than 1000 organisms per mouse. The challenge infection was uniformly lethal for unvaccinated control mice. Depletion of CD8+ T cells weakened this resistance to re-challenge: both naïve and vaccinated mice that were treated with antibody raised against CD8+ T cells died significantly earlier than did mice that received an irrelevant control antibody. In vitro, purified CD8+ T cells taken from draining lymph nodes of antigen-experienced mice were less efficient than were identically prepared CD4+ T cells at stimulating the cells of a transformed microglial cell line to inhibit C.neoformans proliferation, possibly mirroring the inferiority of CD8+ T-cell-mediated protection observed in vivo. RNase protection assays showed similar IFN-γ mRNA levels in both lymphocyte subsets. Class II major histocompatibility antigen expression was up-regulated strikingly on microglia cultured with IFN-γ, but class I expression was less dramatically affected. Therefore microglial cell interaction may be more greatly enhanced with CD4+ cells than with CD8+ cells.

Thrombin stimulates production of fibronectin by human proximal tubular epithelial cells via a transforming growth factor-beta-dependent mechanism.

Tubulointerstitial fibrosis contributes to the progression of many forms of glomerular disease and to end-stage renal failure. Inflammatory mediators generated during glomerular injury may induce tubulointerstitial lesions by stimulating tubular cells. Thrombin has multiple biological functions in addition to its role in haemostasis and has been detected in the urine of patients with glomerular diseases. The present study investigated whether thrombin can modulate the production of fibronectin (FN) in cultured human proximal tubular epithelial cells (PTEC). Cultured PTEC were incubated with or without thrombin to examine the effect of thrombin on FN production in PTEC. FN and transforming growth factor-beta (TGF-beta) levels were measured in culture supernatants by enzyme-linked immunosorbent assay (ELISA). Expression of FN mRNA was analysed by reverse transcriptase-polymerase chain reaction. Effects of thrombin on matrix metabolism were examined by enzyme immunoassay for the detection of secreted matrix metalloproteinase (MMP) and its inhibitors (TIMPs) as well as by zymography. Thrombin stimulated FN secretion in PTEC. Thrombin also stimulated TGF-beta secretion in PTEC in a dose-dependent manner. Expression of FN mRNA by PTEC was augmented by thrombin. The stimulatory effect of thrombin on FN secretion was inhibited by neutralizing antibodies against TGF-beta but not by an irrelevant antibody. Thrombin-induced FN secretin was also inhibited by thrombin inhibitors, such as antithrombin III, hirudin and argatroban. Although thrombin stimulated TIMP-1 and -2 secretion by PTEC, the stimulatory effect of thrombin on MMP-2 was not statistically significant. Thrombin did not affect the expression of MMP-2 in zymography studies. We found that thrombin stimulates FN production in PTEC without causing matrix degradation, an effect that may contribute to the formation of tubulointerstitial fibrosis associated with glomerular disease. The stimulatory effect of thrombin on FN production in PTEC is, at least in part, mediated by TGF-beta.

Cellular immunity elicited by human immunodeficiency virus type 1/ simian immunodeficiency virus DNA vaccination does not augment the sterile protection afforded by passive infusion of neutralizing antibodies.

High levels of infused anti-human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibodies (MAbs) can completely protect macaque monkeys against mucosal chimeric simian-human immunodeficiency virus (SHIV) infection. Antibody levels below the protective threshold do not prevent infection but can substantially reduce plasma viremia. To assess if HIV-1/SIV-specific cellular immunity could combine with antibodies to produce sterile protection, we studied the effect of a suboptimal infusion of anti-HIV-1 neutralizing antibodies in macaques with active cellular immunity induced by interleukin-2 (IL-2)-adjuvanted DNA immunization. Twenty female macaques were divided into four groups: (i). DNA immunization plus irrelevant antibody, (ii). DNA immunization plus infusion of neutralizing MAbs 2F5 and 2G12, (iii). sham DNA plus 2F5 and 2G12, and (iv). sham DNA plus irrelevant antibody. DNA-immunized monkeys developed CD4 and CD8 T-cell responses as measured by epitope-specific tetramer staining and by pooled peptide ELISPOT assays for gamma interferon-secreting cells. After vaginal challenge, DNA-immunized animals that received irrelevant antibody became SHIV infected but displayed lower plasma viremia than control animals. Complete protection against SHIV challenge occurred in three animals that received sham DNA plus MAbs 2F5 and 2G12 and in two animals that received the DNA vaccine plus MAbs 2F5 and 2G12. Thus, although DNA immunization produced robust HIV-specific T-cell responses, we were unable to demonstrate that these responses contributed to the sterile protection mediated by passive infusion of neutralizing antibodies. These data suggest that although effector T cells can limit viral replication, they are not able to assist humoral immunity to prevent the establishment of initial infection.

1010 Hairy cell leukemia: early immunological diagnosis and quantitative analysis of flow cytometry

Lack of standardization of methods and detection limits contributes to the controversial results regarding microchimerism after organ transplantation and has prompted the development of a standardized, reproducible, “easy-to-use” flow cytometric method for this type of “rare event analysis”.EDTA-blood of healthy, HLA-typed donors was stained simultaneously with FITC- and biotin-labeled HLA-class I antibodies (One Lambda) as well as Cy5-PE-labeled CD45 (Medac, Hamburg) according to a standard protocol and analysed on a Coulter EPICS XL Flow cytometer (FCM).An absolute range of positivity (mean MESF± 1 STD) was determined for 22 HLA-specific antibodies. The range of positivity ranged between 5000 and 20,000 MESF (anti-A23, 24(9) FITC) and 40,000–140,000 (anti-Bw6 FITC). The frequency of nonspecific positive signals using nonstained cells, isotype-controls and irrelevant HLA antibodies was between 0.01% and about 0.5%, in some samples up to 1.4%, with an MESF between 8000 and 150,000, thus interfering clearly with the defined positive range of most antibodies tested. Using an “HLA antibody cocktail”, combining FITC- and PE-labeled antibodies for different HLA specificities and thereby creating an internal control, the identification of donor cells was improved but was only rarely applicable.Due to the lack of highly reactive antibodies, FCM analysis was not suitable for the reliable identification of very low numbers of donor hematopoetic cells despite the theoretical advantages of flow cytometric detection of rare events. The single parameter approach was hampered by a significant frequency of nonspecific positive signals, which were easily mistaken as specific (true) positive signals, whereas the multiparameter approach could only be used in selected cases.

Experimental radioimmunotherapy of small peritoneal metastases of colorectal origin.

Radioimmunotherapy using radiolabeled monoclonal antibodies (MoAbs) directed against tumor-associated antigens might be an effective treatment modality for small volume disease. Our aim was to optimize an experimental model of radioimmunotherapy for small peritoneal metastases of colorectal origin using the anti-CEA MoAb MN-14. In nude mice with intraperitoneal (i.p.) LS174T tumors, a protein dose-escalation study was carried out to determine the maximal dose of radioiodinated MN-14 to be used in radioimmunotherapy. The biodistribution of radioiodinated MN-14 was determined after intravenous (i.v.) and i.p. administration. Finally, the therapeutic efficacy of escalating activity doses of (131)I-labeled MN-14 (62.5-500 microCi) was assessed and compared to that of unlabeled MN-14 or 500 microCi of (131)I-labeled irrelevant control antibody. At protein doses higher than 25 microg, uptake in tumor was reduced, presumably due to saturation of tumor antigen. During the first 24 hours i.p. administration led to higher tumor uptake and higher tumor:blood ratios than i.v. administration. Median survival of the control groups was 38 days (unlabeled MN-14) and 52 days ((131)I-labeled nonspecific antibody). Median survival of the groups treated with increasing activity doses of (131)I-labeled MN-14 was 42 days (62.5 microCi), 49 days (125 microCi), 63 days (250 microCi) and 101 days (500 microCi), respectively (p < 0.0001 compared to unlabeled MN-14). The present study shows that the anti-CEA-antibody MN-14 preferentially accumulates in i.p. LS174T tumor xenografts after both i.p. and i.v. administration. Intraperitoneal radioimmunotherapy using (131)I-labeled MN-14 delays significantly the outgrowth of peritoneal LS174T metastases, even at relatively low activity doses.

Intraperitoneal radioimmunotherapy in an ovarian carcinoma mouse model: Effect of the radionuclide

Despite debulking surgery and multidrug chemotherapy, advanced stage ovarian cancer has a high mortality rate. Radioimmunotherapy (RIT) is a treatment modality using specific, radiolabeled antibodies that guide cytotoxic radionuclides to cancer cells. In the present study, the therapeutic efficacy of RIT with murine monoclonal antibody HMFG1 labeled with three different beta-radiation emitting radionuclides (90Yttrium, 186Rhenium, and 131Iodine) was assessed in athymic BALB/c mice with intraperitoneally growing NIH:OVCAR-3 ovarian carcinoma xenografts. Each of the three intraperitoneally administered radiolabeled antibody preparations (90Y-HMFG1, 186Re-HMFG1, and 131I-HMFG1) caused a significant delay in ascites formation and mortality as compared to the control groups treated with 90Y-labeled irrelevant antibody, nonradiolabeled HMFG1, or phosphate buffered saline. Intraperitoneally (ip) administered 90Y-HMFG1 was shown to have a significantly higher abdominal retention as compared to the intraperitoneally administered irrelevant antibody 90Y-G250. Furthermore, intraperitoneally administered 90Y-HMFG1 more effectively inhibited tumor growth than intravenously administered 90Y-HMFG1. It was concluded that in intraperitoneally located malignant disease with ascitic cell clusters and tumor deposits, intraperitoneal administration of RIT seemed preferable as compared to intravenous administration. The choice of the most optimal radionuclide in intraperitoneally located malignancies needs further research, but could well depend on tumor characteristics such as the size of the tumor lesions.

Intraperitoneal radioimmunotherapy in an ovarian carcinoma mouse model: Effect of the radionuclide

Despite debulking surgery and multidrug chemotherapy, advanced stage ovarian cancer has a high mortality rate. Radioimmunotherapy (RIT) is a treatment modality using specific, radiolabeled antibodies that guide cytotoxic radionuclides to cancer cells. In the present study, the therapeutic efficacy of RIT with murine monoclonal antibody HMFG1 labeled with three different beta-radiation emitting radionuclides (90Yttrium,186Rhenium, and131Iodine) was assessed in athymic BALB/c mice with intraperitoneally growing NIH:OVCAR-3 ovarian carcinoma xenografts. Each of the three intraperitoneally administered radiolabeled antibody preparations (90Y-HMFG1,186Re-HMFG1, and131I-HMFG1) caused a significant delay in ascites formation and mortality as compared to the control groups treated with90Y-labeled irrelevant antibody, nonradiolabeled HMFG1, or phosphate buffered saline. Intraperitoneally (ip) administered90Y-HMFG1 was shown to have a significantly higher abdominal retention as compared to the intraperitoneally administered irrelevant antibody90Y-G250. Furthermore, intraperitoneally administered90Y-HMFG1 more effectively inhibited tumor growth than intravenously administered 90Y-HMFG1. It was concluded that in intraperitoneally located malignant disease with ascitic cell clusters and tumor deposits, intraperitoneal administration of RIT seemed preferable as compared to intravenous administration. The choice of the most optimal radionuclide in intraperitoneally located malignancies needs further research, but could well depend on tumor characteristics such as the size of the tumor lesions.

β1-integrin—ligand disengagement induces in vitro capillary tube disruption mediated by p38 MAPK activity

Background. Disrupting cell-matrix interactions may lead to capillary injury as seen in sepsis and transplant rejection. Previously, we demonstrated capillary disruption mediated by β1-integrin-ligand disengagement. We now determine whether p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways are involved in this capillary injury. Methods. Endothelial capillaries on Matrigel were preincubated with a p38 MAPK inhibitor (SB203580), ERK pathway inhibitor (PD98059), or dimethyl sulfoxide. Subsequently, a β1-integrin blocking (P5D2) or an irrelevant antibody was added. After 24 hours, capillary integrity was quantified as capillary intersections/well. Antibody-treated cell lysates then were immunoprecipitated with either a phospho-p38 MAPK or phospho-ERK1/2 antibody. Kinase activity was measured with ATF-2 and Elk-1 fusion proteins as substrates for p38 MAPK and ERK, respectively, followed by Western blotting. Results. P5D2 disrupted capillary tubes. Increased p38 MAPK activity at 8 hours and ERK activity at 2 and 8 hours were seen in P5D2-treated lysates. Preincubation with SB203580, but not with PD98059 or DSMO, significantly reduced capillary tube disruption. Conclusions. The β1-integrin-ligand disengagement resulted in capillary disruption and stimulated p38 MAPK and ERK activity. In spite of activation of both pathways, the p38 MAPK but not the ERK pathway inhibitor prevented β1-integrin antibody effects. Inhibiting p38 MAPK may mitigate capillary injury associated with sepsis and transplant rejection. (Surgery 2003;134:164-8.)

Add-on anti-TGF-beta antibody to ACE inhibitor arrests progressive diabetic nephropathy in the rat.

Renin-angiotensin system (RAS) inhibitors are effective in reducing renal disease progression in early diabetic nephropathy, but they provide imperfect protection at a later stage. Due to the pivotal role of transforming growth factor-beta (TGF-beta) in the pathogenesis of diabetic kidney disease, this study tested the effect of simultaneously interrupting TGF-beta and angiotensin II on disease progression in diabetic rats with overt nephropathy. Diabetes was induced by streptozotocin injection in uninephrectomized rats. Diabetic rats received murine (1D11) or human (CAT-192) anti-TGF-beta monoclonal antibodies alone or in combination with lisinopril, 13C4 irrelevant murine antibody, saline or lisinopril from month 4 (when animals had proteinuria) to month 8. Normal animals served as controls. Systolic BP increase was controlled by single treatments and even more by the combined therapies. 1D11 and lisinopril kept proteinuria at levels numerically lower than irrelevant antibody and saline, while CAT-192 was ineffective. The addition of either TGF-beta antibody to lisinopril normalized proteinuria. Consistent results were obtained for glomerulosclerosis and tubular damage, which were abrogated by the combined therapy. Interstitial volume expansion and infiltration of lymphocytes/macrophages were limited by 1D11 and lisinopril and further reduced by their combination. The increase of type III collagen in the renal interstitium was partially attenuated by 1D11 and lisinopril while normalized by their combination. It is concluded that anti-TGF-beta antibody when added to a background of chronic angiotensin-converting enzyme (ACE) inhibition fully arrests proteinuria and renal injury of overt diabetic nephropathy, providing a novel route to therapy and remission of disease for diabetic patients who do not respond to RAS inhibition.

Inhibition of gamma interferon decreases bacterial load in peritonitis by accelerating peritoneal fibrin deposition and tissue repair.

Bowel perforation can lead to significant bacterial spillage, which may then cause septic peritonitis, characterized by a systemic inflammatory response and organ dysfunction. There are several reports that have shown that the development of peritoneal adhesions is dependent on inflammatory cytokine levels and that these adhesions can reduce bacterial spread, possibly by sealing off the cecum in the cecal ligation and puncture (CLP) model of septic peritonitis. There have not, however, been any studies that have utilized a strategy to accelerate tissue repair in order to seal off the injured cecum and reduce bacterial spread as well as ameliorate systemic inflammation. In the present study, we demonstrate that the administration of anti-gamma interferon (IFN-gamma) antibody (1.2 mg/kg of body weight, intravenously) accelerated tissue repair via increased fibrin deposition 12 and 24 h after CLP in rats. This increase in fibrin deposition was associated with peritoneal adhesion 24 h after CLP and a reduction in bacterial load compared to the bacterial load of rats given irrelevant antibody. Plasma fibrin levels, however, were not altered after IFN-gamma antibody administration, suggesting that the inhibition of IFN-gamma activity specifically increased fibrin deposition to the site of injury. Furthermore, plasma interleukin-6, used as a marker of systemic inflammatory response, was reduced in CLP rats given IFN-gamma antibody compared to that found in those given irrelevant antibody. These results suggest that the early inhibition of IFN-gamma activity in the CLP model is beneficial by accelerating fibrin deposition in cecal tissue to prevent bacterial spread and reduce the systemic inflammatory response. Importantly, increased fibrin deposition in the ceca was not associated with increased plasma fibrin whereas the latter may have detrimental effects associated with coagulation disorders.

Development of a quantitative antigen-specific cell-based ELISA for the 7G7/B6 monoclonal antibody directed toward IL-2Rα

Interleukin-2 receptor α (IL-2Rα, CD25) has been identified as a valuable target for immunotherapy. The 7G7/B6 monoclonal antibody, a mouse IgG2a kappa, recognizes an epitope of the IL-2Rα peptide, other than that identified by anti-Tac. This antibody is currently being explored for potential therapeutic and diagnostic applications. Here, we show a cell-based enzyme-linked immunosorbent assay (CbELISA) method for quantitative measurement of the binding activity of the 7G7/B6 antibody to the Kit-225-iG3 cell line expressing IL-2Rα antigen on the cell surface. The cell- and antigen-specificity of the assay was established using specific cell lines and irrelevant control antibodies. Satisfactory binding curves were demonstrated with Kit-225-iG3 cells grown between 3 and 25 passages in culture and at seed densities of 2×10 5–4×10 6 cells/well. The assay shows reproducible dose–response curves in the concentration range of 10–1000 ng/ml. The assay validation data presented here indicate that this CbELISA assay is quantitative, reproducible, robust, precise, and can be used to test the biological activity, lot to lot comparison, and stability of 7G7/B6 monoclonal antibody.

F(ab)′2-mediated neutralization of C3a and C5a anaphylatoxins: a novel effector function of immunoglobulins

High-dose intravenous immunoglobulin (IVIG) prevents immune damage by scavenging complement fragments C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylatoxins C3a and C5a, thereby neutralizing their pro-inflammatory effects. Single-cell calcium measurements in HMC-1 human mast cells showed that a rise in intracellular calcium caused by C3a and C5a was inhibited in a concentration-dependent manner by IVIG, F(ab)2-IVIG and irrelevant human monoclonal antibody. C3a- and C5a-induced thromboxane (TXB2) generation and histamine release from HMC-1 cells and whole-blood basophils were also suppressed by exogenous immunoglobulins. In a mouse model of asthma, immunoglobulin treatment reduced cellular migration to the lung. Lethal C5a-mediated circulatory collapse in pigs was prevented by pretreatment with F(ab)2-IVIG. Molecular modeling, surface plasmon resonance (SPR) and western blot analyses suggested a physical association between anaphylatoxins and the constant region of F(ab)2. This binding could interfere with the role of C3a and C5a in inflammation.

Expression of neuropilin-1 in choroidal neovascular membranes

Background: Neovascularization is a serious consequence of several eye diseases, including age-related macular degeneration. Neovascularization is under the control of proangiogenic factors, such as vascular endothelial growth factor and fibroblast growth factor. Recent work in our laboratory has focused on other, novel angiogenic factors, such as neuropilin-I, and their potential role in neovascularization. The purpose of this study was to investigate the role of neuropilin-1 in choroidal neovascularization (CNV).Methods: We examined the localization of neuropilin-1 by immunohistochemistry in nine choroidal neovascular membranes (CNVMs) surgically excised from four patients with age-related macular degeneration who had not undergone laser photocoagulation, four with idiopathic CNV and one with ocular histoplasmosis. We also stained the membranes for markers of endothelial and retinal pigment epithelial cells. Controls included omission of primary antibody, use of an irrelevant primary antibody, and neuropilin-1 staining of the posterior sclera, choroid and retina of four healthy donor eyes.Results: Neuropilin-1 was present in eight of the nine CNVMs. It was localized mainly to the plasma membrane. The more vascular membranes and those consisting of a larger number of retinal pigment epithelial cells were associated with greater neuropilin-1 staining. Neuropilin- I was not seen in the posterior segment of the four healthy eyes.Interpretation: Neuropilin-1 appears to play an active role in CNV. Further study is needed to establish a causal relation.

Microinjection of actin antibodies impaired gap junctional intercellular communication in lens epithelial cells in vitro

Purpose. The aim of this study was to check the importance of cytoskeletal actin for gap junction mediated intercellular communication (GJIC) in cultured lens epithelial cells (LEC). Methods. Bovine LEC were cultured until confluency on cover-slides of a collocate-system. In order to study the cytoskeletal influence on cell communication microcinjection of gap junction permeable neurobiotin ® into a single cell was preceded by microinjection of actin antibodies. Confocal laser scanning microscopy of specimens treated with actin antibodies and/or subsequent phalloidin labelling, and electron microscopy, were applied to check for cytoskeleton cell membrane links. Specificity of actin antibodies was proved by immoblotting techniques. Results. Immunohistochemistry and phalloidin-rhodamine staining displayed bundles of actin-filaments extending through the entire LEC. Quantitative analysis of GJIC showed intensive dye-spreading of neurobiotin between adjacent LEC. Injection of actin antibodies thirty minutes prior to microinjection of neurobiotin significantly reduced GJIC. Microinjection of irrelevant antibodies had no effect on GJIC. Conclusion. Integrity of the actin-cytoskeleton is fundamental for unimpaired GJIC in LEC.

Control of CBP co-activating activity by arginine methylation

The histone acetyltransferases CREB binding protein (CBP) and the related p300 protein function as key transcriptional co-activators in multiple pathways. In the case of transcriptional activation by nuclear receptors, ligand promotes the recruitment of co-activators of the p160 family, such as GRIP-1. Subsequently, the p160 co-activators recruit other co-activators via two activation domains, AD1 and AD2. AD1 binds CBP or p300, whereas AD2 has been shown to activate transcription through the recruitment of the arginine methyltransferase CARM1. Recently, the KIX domain of CBP has been shown to be methylated by CARM1 in vitro. Here, we report that another domain of CBP is specifically methylated by CARM1 on conserved arginine residues in vitro and in vivo. We also provide functional evidence that arginine residues methylated by CARM1 play a critical role in GRIP-1-dependent transcriptional activation and in hormone-induced gene activation. Altogether, our data provide strong evidence that arginine methylation represents an important mechanism for modulating co-activator transcriptional activity.

Synthesis of a group-selective antibody library against haptens

A generic strategy is described for the generation of libraries comprising hapten-selective antibody genes against a group of structurally related low molecular weight target molecules. Hapten antibody libraries are frequently suffering from high background levels of irrelevant antibody genes as a consequence of the immunization, where the small non-immunogenic target molecule is coupled to a large immunogenic carrier protein. In order to elevate the percentage of hapten-specific genes in the library, B cells harboring antibody genes against the group of triazine herbicides were enriched from 21 individual splenocyte populations by means of immunomagnetic separation (IMS). IMS utilizes the specific binding of membrane-associated immunoglobulin receptors on the B cell surface to hapten-coated paramagnetic beads. The variable genes of the specifically enriched subpopulation were cloned into a phagemid vector. The corresponding library yielded up to 75% triazine binding antibody clones after three rounds of phage selection. At least half of these antibodies (abs) were displaceable by triazines resulting in quantitative assays with nanomolar sensitivities. In contrast, no displaceable clone was obtained at the same selection level in a control library, where IMS was omitted. Due to the elevated percentage of relevant antibody genes, the library can be utilized either for the direct isolation of functional antibodies against various triazine herbicides or as group-specific gene source for evolutionary antibody optimization.

Enhancement of the antitumor properties of interleukin-2 by its targeted delivery to the tumor blood vessel extracellular matrix

Angiogenic processes depend on the precise coordination of different cell types and a complex exchange of signals, many of which derive from new specific components of the provisional, angiogenesis-related, extracellular matrix (ECM). Angiogenesis-associated ECM components thus represent appealing targets for the selective delivery of therapeutic molecules to newly forming tumor vessels. Results of a previous study indicated that a high affinity recombinant antibody (L19) to ED-B, a domain contained in the angiogenesis-associated isoform of fibronectin (B-FN), selectively and efficiently targets tumor vessels. The present study shows that a fusion protein between L19 and interleukin 2 (L19-IL-2) mediates the selective delivery and concentration of IL-2 to tumor vasculature, thereby leading to a dramatic enhancement of the therapeutic properties of the cytokine. By contrast, IL-2 fused to an irrelevant recombinant antibody used as a control fusion protein showed neither accumulation in tumors nor therapeutic efficacy. Tumors in mice treated with L19-IL-2 were significantly smaller compared to those in animals treated with saline, the control fusion protein, or IL-2 alone (P =.003,.003, and.002, respectively). Moreover, no significant differences in size were observed among the tumors from the different control groups (using the control fusion protein, a mixture of IL-2 and L19, or saline alone). Immunohistochemical analysis of tumor infiltrates demonstrated a significantly higher number of T lymphocytes, natural killer cells, and macrophages, as well as increased interferon-gamma (IFN-gamma) accumulation, in tumors from animals treated with L19-IL-2 compared to tumors from control groups. The fact that ED-B is 100% homologous in human and mouse, thus ensuring that L19 reacts equally well with human and murine antigen, should ultimately expedite transfer of this reagent to clinical trials.

Flow cytometric “rare event analysis”: a standardized approach to the analysis of donor cell chimerism

Lack of standardization of methods and detection limits contributes to the controversial results regarding microchimerism after organ transplantation and has prompted the development of a standardized, reproducible, “easy-to-use” flow cytometric method for this type of “rare event analysis”. EDTA-blood of healthy, HLA-typed donors was stained simultaneously with FITC- and biotin-labeled HLA-class I antibodies (One Lambda) as well as Cy5-PE-labeled CD45 (Medac, Hamburg) according to a standard protocol and analysed on a Coulter EPICS XL Flow cytometer (FCM). An absolute range of positivity (mean MESF± 1 STD) was determined for 22 HLA-specific antibodies. The range of positivity ranged between 5000 and 20,000 MESF (anti-A23, 24(9) FITC) and 40,000–140,000 (anti-Bw6 FITC). The frequency of nonspecific positive signals using nonstained cells, isotype-controls and irrelevant HLA antibodies was between 0.01% and about 0.5%, in some samples up to 1.4%, with an MESF between 8000 and 150,000, thus interfering clearly with the defined positive range of most antibodies tested. Using an “HLA antibody cocktail”, combining FITC- and PE-labeled antibodies for different HLA specificities and thereby creating an internal control, the identification of donor cells was improved but was only rarely applicable. Due to the lack of highly reactive antibodies, FCM analysis was not suitable for the reliable identification of very low numbers of donor hematopoetic cells despite the theoretical advantages of flow cytometric detection of rare events. The single parameter approach was hampered by a significant frequency of nonspecific positive signals, which were easily mistaken as specific (true) positive signals, whereas the multiparameter approach could only be used in selected cases.