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PubMed1993

The adenovirus E1A 12S product displays functional redundancy in activating the human proliferating cell nuclear antigen promoter.

The adenovirus E1A 243R oncoprotein stimulates expression from the promoter of the human proliferating cell nuclear antigen (PCNA). To gain insight into the mechanism of activation, we analyzed deletion and point mutations of the 243R protein for their abilities to activate PCNA promoter-directed reporter gene expression upon cotransfection into HeLa cells. Large deletions that in combination span the entire protein severely impaired the ability of E1A 243R to induce PCNA expression. Smaller deletions and specific point mutations that target specific E1A-binding proteins were less deleterious to PCNA induction. The data suggest that E1A activates transcription of the PCNA gene by multiple mechanisms and that, of the known 243R-associated proteins, p300 and p107-cyclin A can mediate the response while p105-RB does not appear to participate. Presumably, the functional redundancy ensures that 243R can activate expression of this essential DNA replication protein regardless of cell type and physiological conditions.

Adenovirus E1A Proteins↗
PubMed2004

CP-64131, an aminobenzazepine with cytokine-like properties, stimulates human neutrophil functions through the p38-MAPK pathway.

CP-64131 (CP), an aminobenzazepine with cytokine-like, physiologic effects similar to granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage (GM)-CSF, increases the number of neutrophils and stimulates marrow recovery after doxirubicin ablation. CP can also function as a neutrophil agonist, like formyl-Met-leu-Phe (fMLP). In these studies, we show that CP is unique in that it stimulates the p38-mitogen-activated protein kinase (MAPK) pathway but not extracellular signal-regulated kinase (ERK)1/2 or c-jun N-terminal kinase MAPKs in human neutrophils from peripheral blood. This is in contrast to other neutrophil agonists such as fMLP, interleukin (IL)-8, or GM-CSF, which stimulate multiple MAPK pathways. Like fMLP and IL-8, CP is capable of stimulating superoxide (O2-) production, CD11b expression, and cell polarization in human neutrophils. CP-stimulated O2- production is completely dependent on p38-MAPK activation, as determined by sensitivity to the p38-MAPK inhibitor SB203580. In contrast, SB203580 only partially inhibits expression of CD11b and has no effect on cell polarization stimulated by CP. Therefore, CP treatment of neutrophils activates p38-MAPK but has effects independent of p38-MAPK activation. In human embryonic kidney 293 cells, a human kidney epithelial cell line CP stimulates p38-MAPK and modestly activates ERK1/2. The findings define CP as a novel, small molecule, which has little cellular toxicity in vitro. CP has the ability to activate specific MAPK pathways in different cell types and should prove to be an effective agonist in combination with inhibitors to study biological responses regulated by MAPKs.

Adjuvants, Immunologic↗
PubMed2001

Antigen-specific primary activation of CD8+ T cells within the liver.

It is generally accepted that naive T cells recirculate via the blood and lymph, but do not enter nonlymphoid tissues without prior activation and differentiation. In this study, we demonstrate that the liver is an exception to this rule. Naive Des-TCR transgenic CD8(+) T cells specific for H-2K(b) were selectively retained in the liver within a few minutes of adoptive transfer into transgenic Met-K(b) mice expressing H-2K(b) in the liver. Activated CD8(+) cells were found in the liver, but not the blood, as soon as 2 h after transfer and underwent cell division and started to recirculate within 24 h of transfer. In contrast, CD8(+) cells activated in the lymph nodes remained sequestered at that site for 2 days before entering the blood. Our results therefore suggest that, in addition to its previously described role as a non Ag-specific activated T cell graveyard, the liver is involved in Ag-specific activation of naive recirculating CD8(+) T cells. This particular property of the liver, combined with the previously demonstrated ability of hepatocytes to induce tolerance by means of premature CD8(+) T cell death, may be a major mechanism contributing to the acceptance of liver allografts and the chronicity of viral hepatitis.

Adoptive Transfer↗
PubMed1994

Role of endothelin and tumour necrosis factor in the renal response to sepsis.

A rapidly increasing body of evidence is implicating endothelin and TNF in the pathogenesis of septic acute renal failure. TNF causes renal damage by recruiting leukocytes, accelerating fibrin accumulation, promoting cell lysis, stimulating the release of vasoconstrictor substances, and other mechanisms. ET-1 causes renal dysfunction in sepsis and endotoxaemia primarily by evoking severe reductions in RBF and GFR. While these are only two of the many agents that mediate renal dysfunction during sepsis, they stand out by virtue of their combined ability to modulate numerous inflammatory pathways and to elicit marked alterations in renal function. Clearly the development of specific TNF and endothelin antagonists holds out promise for the treatment and prevention of septic acute renal failure.

Acute Kidney Injury↗
PubMed1994

[Diagnostic and prognostic contribution of somatosensory evoked potentials by truncular and dermatomal stimulation in lumbosacral radiculopathy. Apropos of 120 cases surgically-treated].

Lower-limb somatosensory evoked potentials (SEPs) of nerve-trunk (peroneal nerve, posterior tibial nerve) and dermatomal (L5 and S1) stimulation were recorded in 120 patients suffering from lumbosacral disk disease. Recordings were performed before surgery and on Days 4 and 50 after surgery. Electromyography was also performed before surgery. Dermatomal SEPs were shown to be sensitive (70%) and specific in both terms of their lateralizing value and their ability to identify the L5 or S1 level. Moreover, combining SEPs and EMG significantly increased the sensitivity of the electrophysiological testing when compared with each method alone. There was no relationship between SEPs performed before surgery and surgical outcome. Inversely, we demonstrated the prognostic value of SEPs performed on Day 4, SEP normalization at Day 4 was associated with a good outcome at Day 50 in 91.8% of examined cases, while the persistence of pathological SEPs at Day 4 was associated with poor outcome at Day 50 in 56% of the cases.

Adult↗
PubMed1986

Specific, limited tryptic modification of wheat-germ fructose-bisphosphate aldolase subunits: destruction of catalytic activity but not of ability to establish precise subunit-subunit recognition.

We have been using the glycolytic enzyme fructose-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) as a model system to investigate the assembly of oligomeric enzymes. In the present work, we investigate the effect of specific, limited tryptic modification on the properties of aldolase isolated from wheat germ. The wheat-germ enzyme was selected, since several aldolases isolated from animal sources were not readily susceptible to the specific tryptic modification seen with this plant enzyme. We will show that: Low levels of trypsin cause a first-order inactivation of wheat-germ aldolase activity which is associated with a fairly specific cleavage of the enzyme which reduces its subunit molecular weight from 41000 to 39000. The proteolytic modification is greatly inhibited in the presence of the aldolase substrate, fructose bisphosphate. The intact and modified enzymes appear to have similar surface changes, as judged by their behavior during electrophoresis in polyacrylamide gels under non-denaturing conditions. The modified aldolase is not specifically eluted from phosphocellulose columns by fructose bisphosphate under the conditions used in the affinity chromatographic isolation of the intact enzyme, suggesting that the modified enzyme may no longer be able to bind substrate. Although enzymatically inactive, the modified aldolase subunits are able to refold and reassociate into tetrameric combinations following unfolding of the subunits by treatment at low pH; thus, this specific proteolytic modification does not interfere with the ability of wheat-germ aldolase subunits to refold and to establish precise subunit-subunit recognition in vitro.

Carboxypeptidases↗
PubMed1998

Immunology in diabetes: an update.

Type 1 (insulin-dependent) diabetes mellitus is strongly associated with autoimmune phenomena connected to the loss of beta-cells in the pancreatic islets. Despite considerable progress in our understanding of genetic susceptibility factors and islet autoimmunity preceding the clinical onset of Type 1 diabetes there are considerable gaps in our knowledge. First, the etiology is unclear. It is speculated that multiple etiological factors may initiate a common pathogenic pathway which results in immune-mediated beta-cell destruction. In 1998 we will need to learn more about the possible importance of gestational infections, as well as isolation of viral DNA or RNA from the blood of new-onset patients or marker-positive individuals. The scan of the whole genome has provided a smorgasbord of genetic regions which confer diabetes risk either alone or in combination. HLA remains the major genetic risk factor, and while HLA peptide binding information is considerable, we understand less of autoantigen processing and presentation. Cloned autoantigens and their use in standardized autoantibody assays have improved our ability to identify individuals at risk for diabetes. The diagnostic sensitivity and specificity of autoantibody markers for Type 1 diabetes are high as are their predictive values. We need methods to combine autoantibodies with genetic risk factors. The identification of individuals in different stages of their pathogenesis, including patients with so-called slowly progressive Type 1 diabetes (SPIDDM, LADA etc.), allow approaches to novel therapeutic interventions. Insulin is currently the therapeutic agent of choice and although spontaneous insulin-dependent diabetes in the NOD mouse and the BB rat can be prevented by immune suppression or modulation, this has not yet been possible in humans. The 1998 research on the interaction between environmental factors and susceptibility genes to initiate beta-cell specific autoreactivity should allow the development of therapies for prevention, and perhaps a cure, of insulin-dependent (Type 1) diabetes.

Allergy and Immunology↗
PubMed1998

Suppression of human prostate carcinoma metastases in severe combined immunodeficient mice by interleukin 2 immunocytokine therapy.

Immunocytokines are antibody-cytokine fusion proteins that combine the unique targeting ability of antibodies with the multifunctional activities of cytokines to activate effector cells in the tumor microenvironment. Here, we demonstrate the therapeutic efficacy of a tumor-specific immunocytokine, huKS1/4-IL2, which effectively inhibited growth and dissemination of lung and bone marrow metastases of human prostate carcinoma in severe combined immunodeficient mice. This antitumor effect was specific and highly effective, irrespective of reconstitution of these mice with human lymphokine-activated killer cells. Survival times of mice treated with huKS1/4-IL2 were increased 4-fold as compared with animals treated with a mixture of the corresponding antibody and recombinant human interleukin-2 (rhIL2). A persistent antitumor response after treatment with the huKS1/4-IL2 immunocytokine in B, T, and natural killer cell-deficient severe combined immuodeficient-BEIGE mice, depleted of granulocytes, implies a major role for macrophages in this treatment effect. Our data demonstrate that immunocytokine-directed interleukin-2 therapy to tumor sites is an immunotherapeutic approach with potent effects against disseminated metastases of human prostate carcinoma and suggest that this treatment could be effective in an adjuvant setting for patients with minimal residual disease.

Animals↗
PubMed2003

Inclusion of a non-immunoglobulin binding protein in two-site ELISA for quantification of human serum proteins without interference by heterophilic serum antibodies.

Measurement of human serum molecules with two-site ELISA can be biased by the presence of human heterophilic anti-animal immunoglobulin antibodies (HAIA) that cause false-positive signals by cross-linking the monoclonal (mAb) and/or polyclonal antibodies (pAb) used for the pre- (capture) and post-analyte steps (detection). To evaluate a novel ELISA format designed to avoid interference by HAIA, a target-specific non-immunoglobulin (Ig) affinity protein (affibody) was used to replace one of the antibodies. First, a human IgA-binding affibody (Z(IgA)) selected by phage display technology from a combinatorial library of a single Staphylococcus aureus protein A domain was used. The detection range of IgA standard using an ELISA based on Z(IgA) for capture and goat pAb against IgA (pAb(IgA)) for detection was comparable with that of using pAb(IgA) for both capture and detection. Secondly, another affibody (Z(Apo)) was combined with mAb and used to detect recombinant human apolipoprotein A-1. The affibody/antibody ELISAs were also used to quantify human serum levels of IgA and apolipoprotein A1. To verify that human serum did not cause false-positive signals in the affibody/antibody ELISA format, the ability of human serum to cross-link affibodies, mAb (mouse or rat) and/or pAb (goat) displaying non-matched specificities was assessed; affibodies and antibodies were not cross-linked whereas all combinations of mAb and/or pAb were cross-linked. The combination of affibodies and antibodies for analysis of human serum molecules represents a novel two-site ELISA format which precludes false-positive signals caused by HAIA.

Animals↗
PubMed2005

IL-7 is a potent and proviral strain-specific inducer of latent HIV-1 cellular reservoirs of infected individuals on virally suppressive HAART.

The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4(+) T lymphocytes from HIV-1-infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4(+) T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.

Anti-HIV Agents↗
PubMed1996

The use of intracellular single-chain antibody fragments to inhibit specifically the expression of cell surface molecules.

One possible method to inhibit specifically the function of a protein inside a cell is to express an intracellular antibody combining site that can block function or prevent expression of the targeted molecule. In this report the parameters involved in the production and expression of functional, endoplasmic reticulum-retained, single chain Fv antibody fragments (scFv) were investigated. These intracellular scFv constructs were tested for their ability to inhibit specifically the expression of a CHO cell line pretransfected with the relevant cell surface antigen CD2. No scFv was detected in the cell supernatant although functional scFv, as assayed by ELISA, was detected in an NP-40 soluble fraction if an N-linked glycosylation site had been introduced into the antibody construct. This demonstrates that functional antibody combining sites can be produced even though they are not secreted. Inhibition of CD2 was obtained but was not complete and differed between clones. Levels of scFv could be increased by gene amplification but the level of functional binding activity remained constant and no further inhibition of CD2 expression was obtained. Immunofluorescence analysis at the single-cell level of the permeabilised transfected cell lines showed that less than 8% of cells expressed detectable levels of intracellular scFv, indicating selection against cells producing high levels of single-chain antibody. This selection was not seen when comparable single-chain TCR constructs, known to be retained intracellularly, were used. Thus, production of scFv with binding activity is not sufficient for good inhibition of gene expression although introduction of an N-linked glycosylation site is beneficial. The best strategy is probably to screen a panel of scFv constructs and use those that are secreted rather than those that are retained intracellularly.

Amino Acid Sequence↗
PubMed1995

Prediction of prostate cancer volume using prostate-specific antigen levels, transrectal ultrasound, and systematic sextant biopsies.

OBJECTIVES: Prostate-specific antigen (PSA) levels, transrectal ultrasound, and systematic sextant biopsies have each shown limited ability to predict prostate cancer volume. In combination, these studies may allow more accurate estimation of volume and prognosis. METHODS: One hundred twenty-four patients were evaluated prior to radical prostatectomy. Interactive stepwise multiple regression and separate logistic regression analysis were performed for prediction of prostate cancer volume and volume range. RESULTS: The cancer volumes calculated correlated with the volumes in the radical prostatectomy specimens with R2 of 0.76. Cancers were predicted to be in the volume range associated with poor prognosis (more than 12 cc) or clinically insignificant cancer (less than 1.0 cc) with bias corrected error rates of 5.3% and 10%, respectively. CONCLUSIONS: The formula for prediction of cancer volume correlates well with actual cancer volume in 92 patients but is not adequate to predict volume for an individual patient. The formulas for prediction of volume range show promising predictive ability and may be useful if the extent of disease is unclear.

Aged↗
PubMed1995

T-cell mediated responses in a murine model of orthotopic corneal transplantation.

PURPOSE: To evaluate the role that delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte responses play in a murine model of orthotopic corneal allograft transplantation. METHODS: Corneal transplantation was performed by grafting C57BL/6 donor corneas into BALB/c corneal beds. After transplantation, the mice were observed by slit lamp biomicroscopy on a weekly basis and graded for signs of graft rejection and delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) responses to donor alloantigens assessed at selected times after grafting. RESULTS: It was determined that between 40% and 65% of BALB/c mice rejected C57BL/6 corneas by 8 weeks after engraftment. Mice with opacity scores > 2 demonstrated significantly greater DTH responses than did mice with opacity scores < 2 at 2, 3, and 4 weeks after engraftment. After 4 weeks, the DTH responses for all groups were essentially the same as for naive BALB/c mice. The DTH responses were specific for C57BL/6 alloantigens and are primarily directed against non-major histocompatibility complex (MHC) C57BL/6 alloantigens and are primarily directed against non-major histocompatibility complex C57BL/6 alloantigens, as evidenced by the ability of B10.D2 cells to elicit DTH responses whereas C.B10-H-2b cells did not. However, although BALB/c mice engrafted with C57BL/6 tail skin demonstrated significantly greater CTL activity than naive BALB/c mice, there was no significant difference in CTL activity between BALB/c mice whose C57BL/6 corneal allografts displayed opacity scores greater than (rejected) or less than (accepted) 2. CONCLUSIONS: The mechanism whereby corneal allografts in this strain combination are rejected is best associated with the ability to generate strong DTH responses and not CTL activity. This DTH response also demonstrates alloantigen specificity and appears to be primarily directed against the non-MHC component of the corneal transplant.

Animals↗
PubMed1988

Acute scrotal abnormalities in children: diagnosis by combined sonography and scintigraphy.

Both scintigraphy and real-time sonography have been used to assess acute symptoms involving the scrotum. However, because of its high sensitivity and ability to document physiologic abnormalities, scintigraphy has been the procedure of choice. Scintigraphy, however, lacks specificity; its value lies mainly in serving to distinguish torsion from nontorsion. The purpose of this study was to supplement scrotal scintigraphy with sonography to determine if the combination improves diagnosis and management compared with scintigraphy alone. Forty-three scrotal scintigrams and sonograms were performed on 40 consecutive patients with acute scrotal symptoms. The interpretation of the scintigram was altered by sonography in six (14%) of the combined scans, directly affecting clinical management. In three patients with acute hydroceles diagnosed by sonography, exploratory surgery was avoided despite scintigraphic findings suggesting testicular torsion. Scintigraphy was normal in two patients with spontaneous testicular detorsion, whereas sonography showed recent spermatic-cord torsion that required subsequent orchiopexy. In a patient with epididymitis and orchitis, sonography showed a complicating scrotal abscess, which was not apparent on scintigraphy and which required antibiotic treatment. The addition of sonography to the scintigraphic evaluations of children with acute scrotal abnormalities changed the diagnosis and clinical management in 14% of the patients studied.

Acute Disease↗
PubMed2000

Characterization of N-linked oligosaccharides bearing sialyl lewis x moieties on an alternatively glycosylated form of soluble complement receptor type 1 (sCR1).

We sought to produce a complement inhibitory protein possessing oligosaccharides specifically modified to contain the sialyl Lewis x (sLe(x)) moiety. This modified glycoprotein could combine anti-complement activity with the ability to inhibit selectin-mediated interactions and concentrate this activity to sites of activated endothelium where selectins are upregulated. Soluble complement receptor type 1 (sCR1), previously shown to be effective in inhibiting the complement cascade, was produced in a cell line capable of adding fucose to N-linked oligosaccharides in the alpha1-3 linkage, which is necessary for sLe(x) glycosylation. The glycoprotein purified from these cells was designated sCR1sLe(x), and may prove to be more effective than sCR1 in some clinical applications. Detailed analysis and characterization of sCR1sLe(x) was performed to confirm that the N-linked oligosaccharides possessed sLe(x) moieties and also to determine the extent of sLe(x) glycosylation. The glycoproteins were characterized by oligosaccharide profiling, sequencing, linkage analysis and quantified by differential enzymic digestion, using fluorophore-assisted carbohydrate electrophoresis. The major glycans were identified as biantennary oligosaccharides (including sialylated and non-core fucosylated glycans). The linkages of sialic acid and the branched fucose were analysed by digestion with linkage-specific enzymes and subsequent separation by electrophoresis. All data were consistent with the presence of sLe(x) moieties on the N-linked oligosaccharides of sCR1sLe(x). sCR1sLe(x) is a prime example of a recombinant protein expressed with oligosaccharides engineered for a specific biological function, and produced using a commercially viable method.

Carbohydrate Conformation↗
PubMed2000

Characterization of osteoblast-like behavior of cultured bone marrow stromal cells on various polymer surfaces.

The creation of novel bone substitutes requires a detailed understanding of the interaction between cells and materials. This study was designed to test certain polymers, specifically poly(caprolactone) (PCL), poly(D,L-lactic-CO-glycolic acid) (PLGA), and combinations of these polymers for their ability to support bone marrow stromal cell proliferation and differentiation. Bone marrow stromal cells were cultured from New Zealand White rabbits and were seeded onto glass slides coated with a thin layer of PCL, PLGA, and combinations of these two polymers in both a 40:60 and a 10:90 ratio. Growth curves were compared. At the end of 2 weeks, the cells were stained for both matrix mineralization and alkaline phosphatase activity. There was no statistically significant difference in growth rate of the cells on any polymer or polymer combination. However, there was a striking difference in Von Kossa staining and alkaline phosphatase staining. Cells on PCL did not show Von Kossa staining or alkaline phosphatase staining. However, in the 40:60 and 10:90 blends, there was both positive Von Kossa and alkaline phosphatase staining. These data indicate that PCL alone may not be a satisfactory material for the creation of a bone substitute. However, it may be used in combination with PLGA for the creation of a bone substitute material.

Animals↗
PubMed2005

Functional groups based on leaf physiology: are they spatially and temporally robust?

The functional grouping concept, which suggests that complexity in ecosystem function can be simplified by grouping species with similar responses, was tested in the Florida scrub habitat. Functional groups were identified based on how species regulate exchange of carbon and water with the atmosphere as indicated by both instantaneous gas exchange measurements and integrated measures of function (%N, delta13C, delta15N, C:N ratio) in fire-maintained Florida scrub, which was considered the natural state for scrub habitat. Using cluster analysis, five distinct physiologically based functional groups were identified in the fire-maintained scrub and were determined to be distinct clusters and not just arbitrary divisions in a continuous distribution by the non-parametric multivariate analysis of similarities (ANOSIM; R=0.649, P=0.005). These functional groups were tested for robustness spatially, temporally, and with management regime using ANOSIM. The physiological functional groups remained distinct clusters in this broader array of sites (R=0.794, P=0.001) and were not altered by plot differences, primarily, water table depth (R=-0.115, P=0.893) or by the three different management regimes: prescribed burn, mechanically treated and burned, and fire-suppressed (R=0.018, P=0.349). The physiological groupings also remained robust between the two climatically different years, with 1999 being a much wetter year than 2000 (R=-0.027, P=0.725). Easy-to-measure morphological characteristics, if they indicate the same functional groups, would be more practical for scaling and modeling ecosystem processes than detailed gas exchange measurements; therefore, we tested a variety of morphological characteristics as functional indicators. A combination of non-parametric multivariate techniques were used to compare the ability of life form, leaf thickness (LT), and specific leaf area (SLA) classifications to identify the physiologically based functional groups. Life form classifications (ANOSIM; R=0.629, P=0.001) were able to depict the physiological groupings more adequately than either SLA (ANOSIM; R=0.426, P=0.001) or LT (ANOSIM; R=0.344, P=0.001). The ability of life forms to depict the physiological groupings was improved by separating the parasitic Ximenia americana from the shrub category (ANOSIM; R=0.794, P=0.001). Therefore, a life form classification including parasites was determined to be a good indicator of the physiological processes of scrub species and would be a useful method of grouping species for scaling physiological processes to the ecosystem level.

Carbon↗
PubMed2003

The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS.

It is well established that the humoral immune response can generate antibodies to many different antigens. The antibody diversity required to achieve this is believed to be substantial. However, the extent to which the immune repertoire can generate structural diversity against a single target antigen has never been addressed. Here, we have used phage display to demonstrate the extraordinary capacity of the human antibody repertoire. Over 1000 antibodies, all different in amino acid sequence, were generated to a single protein, B-lymphocyte stimulator (BLyS protein). This is a highly diverse panel of antibodies as exemplified by the extensive heavy and light chain germline usage: 42/49 functional heavy chain germlines and 19/33 V(lambda) and 13/35 V(kappa) light chain germlines were all represented in the panel of antibodies. Moreover, a high level of sequence diversity was observed in the V(H) CDR3 domains of these antibodies, with 568 different amino acid sequences identified. Thus we have demonstrated that specific recognition of a single antigen can be achieved from many different VDJ combinations, illustrating the remarkable problem-solving ability of the human immune repertoire. When studied in a biochemical assay, around 500 (40%) of these antibodies inhibited the binding of BLyS to its receptors on B-cell lines. The most potent antibodies inhibited BLyS binding with sub-nanomolar IC(50) values and with sub-nanomolar affinities. Such antibodies provide excellent choices as candidates for the treatment of BLyS-associated autoimmune diseases.

Amino Acid Sequence↗