Subtractive immunization yields monoclonal antibodies that specifically inhibit metastasis.

Subtractive immunization allowed the isolation and characterization of monoclonal antibodies that specifically inhibit metastasis but not proliferation of highly metastatic human tumor cells. The tolerizing agent cyclophosphamide was used to suppress the immune system in mice to dominant immunodeterminants present on a non-metastatic variant (M-) of the human epidermoid carcinoma cell line (HEp3). Mice were then inoculated with a highly metastatic variant (M+) of HEp3 to enhance an immune response to antigenic determinants present on metastatic cells. Hybridomas were generated and screened by ELISA for differential reactivity to M+ HEp3 over M- HEp3 cells. This experimental approach, termed subtractive immunization (S.I.), was compared to a control immunization protocol, which eliminated the cyclophosphamide treatment. The S.I. protocol resulted in an eight-fold increase in the proportion of mAbs that react with molecules enriched on the surface of the M+ HEp3 cells. Two of the mAbs derived from the S.I. protocol, designated DM12-4 and 1A5, were purified and examined for their effect in a metastasis model system in which chick embryos are transplanted with primary HEp3 tumors. Purified mAbs DM12-4 and 1A5, inoculated i.v. into the embryos, inhibited spontaneous metastasis of HEp3 cells by 86 and 90%, respectively. The mAbs are specifically anti-metastatic in that they have no effect on the growth of HEp3 cells in vitro nor did they inhibit primary tumor growth in vivo. The mAbs recognize M+ HEp3 cell surface molecules of 55 kD and 29 kD, respectively. These data demonstrate that the S.I. protocol can be used for the development of unique mAbs that are reactive with antigenic determinants whose expression is elevated on metastatic human tumor cells and which function mechanistically in the metastatic cascade.

The role of somatic hypermutation in the generation of antibody diversity.

The immune system is capable of establishing an enormous repertoire of antibodies before its first contact with antigen. Most antibodies that express germ-line sequences are of relatively low affinity. Once antigen enters the system, it stimulates a somatic mutational mechanism that generates antibodies of higher affinity and selects for the expression of those antibodies to produce a more effective immune response. The details of the mechanism and regulation of somatic hypermutation remain to be elucidated.

Correlation of the in situ detection of polymerase chain reaction-amplified metalloproteinase complementary DNAs and their inhibitors with prognosis in cervical carcinoma.

The purpose of this study was to correlate the presence of matrix metalloproteinase (MMP)-9 and MMP-2 and tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 mRNAs, detected in serial sections using the reverse transcriptase in situ PCR technique, with prognosis in 23 cases of cervical carcinoma. PCR-amplified MMP and TIMP cDNA were restricted to the invasive cancers cells and the surrounding stromal cells. The ratios of cancer and stromal cells expressing MMP-9 and MMP-2 to those expressing TIMP-1 and TIMP-2 were approximately 1 in those cancers with a good prognosis. This MMP:TIMP ratio in the cancer and stromal cells with a poor prognosis was significantly increased to 5.4 and 3.4 (P < 0.0001), respectively, reflecting a marked reduction in the TIMP detection rate in cancers with a poor prognosis. In cervical cancer cell lines SiHa and HeLa, the MMP:TIMP ratio was also close to 1 and, interestingly, these cell lines are invasive but rarely metastatic in nude mice. These data suggest that the balance of MMP-9 and MMP-2 to TIMP-1 and TIMP-2 expression is an essential factor in the aggressiveness of cervical cancer.

CC chemokine I-309 is the principal monocyte chemoattractant induced by apolipoprotein(a) in human vascular endothelial cells.

BACKGROUND: Lipoprotein(a) [Lp(a)] is a risk factor for atherosclerosis; however, the mechanisms are unclear. We previously reported that Lp(a) stimulated human vascular endothelial cells to produce monocyte chemotactic activity. The apolipoprotein(a) [apo(a)] portion of Lp(a) was the active moiety. METHODS AND RESULTS: We now describe the identification of the chemotactic activity as being due to the CC chemokine I-309. The carboxy-terminal domain of apo(a) containing 6 type-4 kringles (types 5 to 10), kringle V, and the protease domain was demonstrated to contain the I-309-inducing portion. Polyclonal and monoclonal anti-I-309 antibodies as well as an antibody against a portion of the extracellular domain of CCR8, the I-309 receptor, inhibited the increase in monocyte chemotactic activity induced by apo(a). I-309 antisense oligonucleotides also inhibited the induction of endothelial monocyte chemotactic activity by apo(a). I-309 mRNA was identified in human umbilical vein endothelial cells. Apo(a) induced an increase in I-309 protein in the endothelial cytoplasm and in the conditioned medium. Immunohistochemical studies have identified I-309 in endothelium, macrophages, and extracellular areas of human atherosclerotic plaques and have found that I-309 colocalized with apo(a). CONCLUSIONS: These data establish that I-309 is responsible for the monocyte chemotactic activity induced in human umbilical vein endothelial cells by Lp(a). The identification of the endothelial cell as a source for I-309 suggests that this chemokine may participate in vessel wall biology. Our data also suggest that I-309 may play a role in mediating the effects of Lp(a) in atherosclerosis.

Activation of proMMP-9 by a plasmin/MMP-3 cascade in a tumor cell model. Regulation by tissue inhibitors of metalloproteinases.

To examine MMP-9 activation in a cellular setting we employed cultures of human tumor cells that were induced to produce MMP-9 over a 200-fold concentration range (0.03 to 8.1 nM). The secreted levels of TIMPs in all the induced cultures remain relatively constant at 1-4 nM. Quantitation of the zymogen/active enzyme status of MMP-9 in the cultures indicates that even in the presence of potential activators, the molar ratio of endogenous MMP-9 to TIMP dictates whether proMMP-9 activation can progress. When the MMP-9/TIMP ratio exceeds 1.0, MMP-9 activation progresses, but only via an interacting protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin, generated by the endogenous plasminogen activator (uPA), is not an efficient activator of proMMP-9. Plasmin, however, is very efficient at generating active MMP-3 from exogenously added proMMP-3. The activated MMP-3, when its concentration exceeds that to TIMP, becomes a potent activator of proMMP-9. Addition to the cultures of already-activated MMP-3 relinquishes the requirement for plasminogen and proMMP-3 additions and results in direct activation of the endogenous proMMP-9. The activated MMP-9 enhances the invasive phenotype of the cultured cells as their ability to transverse basement membrane is significantly increased following zymogen activation. That this enhanced tissue remodeling capability is due to the activation of MMP-9 is demonstrated through the use of a specific anti-MMP-9-blocking monoclonal antibody.

Inhibition of matrix metalloproteinase 9 activation by a specific monoclonal antibody.

Two members of the matrix metalloproteinase (MMP)1 family of enzymes are expressed at elevated levels in highly aggressive human tumor cells and have been implicated in the catalytic functions of extracellular proteolysis. The zymogen forms of these enzymes are designated proMMP-2 and proMMP-9, also known as 72kDa and 92kDa type IV collagenases/gelatinases, respectively. The MMP family of enzymes can be activated in vitro by a number of compounds including the organomercurial 4-aminophenylmercuric acetate (APMA). The natural or in vivo activators of MMP-2 and MMP-9 are at present unknown. A partially purified preparation of MMP-9 was used to immunize mice for the isolation of monoclonal antibodies (mAbs). Three IgG1 mAbs were identified by immunoreactivity with purified MMP-9 and are designated 6-6B, 7-11C, and 8-3H. These mAbs react specifically with MMP-9 by ELISA and Western blot. Additionally, these mAbs react with N-glycanase treated 92kDa protein. These mAbs were tested for their ability to inhibit enzyme activation in a radio-labeled gelatin assay. The 6-6B mAb inhibited the activation of MMP-9, but had no effect on MMP-2. These mAbs are highly specific to human MMP-9 and the 6-6B mAb will be extremely useful for examining the autolytic and catalytic activity of MMP-9 in normal and abnormal biological processes.

The ovariectomized, mature rat model of postmenopausal osteoporosis: an assessment of the bone sparing effects of curcumin.

Identification of natural health products that might benefit skeletal health could reduce the negative impact of osteoporotic bone fractures upon society. The objectives of this study were to evaluate an animal model of postmenopausal osteoporosis and to search for evidence that curcumin reduces bone mineral losses in a dose-dependent manner when endogenous estrogen levels are reduced. Bone mineral density was measured at the spine, femur and whole body before and at 2, 4 and 6 months after ovariectomy in each of 40 mature rats. Serum osteocalcin and C-telopeptide were measured as indicators of bone formation and resorption rates. Femoral compressive strength was measured at 6 months. Ovariectomy alone resulted in loss of mineral from the spine (p<0.005) and an increase in osteocalcin levels (p<0.05). At the same time, there was an increase in energy to fracture (p<0.01) due to an increased bone size. When ovariectomized animals were given etidronate there was no loss of mineral from the spine, the size of the femur increased (p<0.005), C-telopeptide levels were reduced (p<0.001) and femoral compressive strength increased (p<0.025). Administration of curcumin to ovariectomized animals resulted in changes that were intermediate between those produced by etidronate and by ovariectomy alone. The increase in femur size produced by the highest dose of curcumin was statistically significant (p< 0.01) and curcumin administration resulted in a significant, dose dependent, increase in energy to fracture. Curcumin produces beneficial changes in bone turnover and increases in bone strength using the ovariectomized mature rat model of postmenopausal osteoporosis.

A role for helper cell subpopulations in a genetically controlled cytolytic T lymphocyte response to the H-2Db antigen.

The primary cytolytic T lymphocyte (CTL) response to the H-2Db antigen in several strain combinations appears to be under genetic control. Our studies were undertaken to determine the mechanisms involved in responders that were absent from the nonresponders and that led to CTL generation. In these studies, H-2Dk-anti-H-2Db combinations served as CTL responders, and H-2Dd-anti-H-2Db combinations served as CTL nonresponders. The nonresponsiveness of the H-2Dd-anti-H-2Db strain combinations appeared to be due to the inability of the Db antigen to activate a helper cell subpopulation. The activation of this helper cell subpopulation, as demonstrated in the H-2Dk-anti-H-2Db response, resulted in the production of factors that led to the induction of CTL differentiation. Antigen-specific pre-CTL for Db were present in the nonresponders as well as in the responders. Interleukin 2 (IL 2)-producing cells were also present in both nonresponders and responders, because all strain combinations tested in this system produced detectable levels of IL 2. Additional analysis of these data suggested that different determinants on the H-2Db antigen were recognized by distinct populations of cells. The activation of pre-CTL and IL 2-producing cells by the recognition of determinants on the H-2Db antigen was not sufficient for the generation of effector CTL, as demonstrated in the H-2Dd-anti-H-2Db response. We suggest that determinants on the H-2Db antigen that are distinct from those recognized by pre-CTL and IL 2-producing cells are recognized by a helper cell subpopulation in the H-2Dk strains, and that activation of this subpopulation is required for the production of factors that mediate CTL differentiation. This is the first description of the role that CTL differentiation factors play in a genetically controlled response.

Evaluation of the physicochemical properties and dissolution characteristics of mesalamine: relevance to controlled intestinal drug delivery.

The physicochemical properties of mesalamine and the effect of pH and buffer concentration on the dissolution rate of pure mesalamine and mesalamine with Carbopol 974P were investigated. The aqueous solubilities at 25 and 37 degrees C were 0.844 and 1.41 mg/mL, respectively. Consistent with the observed pKa1 (2.30) and pKa2 (5.69) or mesalamine, the solubility-pH profile is increased at pH < 2.0 and pH > 5.5 and is minimized from pH 2.0 to pH 5.5. The flux data were consistent with the solubility data from pH 1.0 to pH 5.5. The flux increased and plateaued at pH values 5.5 to 7.0 and was dependent on the bulk buffer concentration. At low bulk buffer concentrations, mesalamine reduces the pH in the diffusion layer, which results in a decrease in flux. The medium with the highest buffer capacity has a greater ability to increase the surface pH and dissolution rate. The addition of Carbopol reduces the flux and the sensitivity of the dissolution rate of mesalamine to increasing bulk buffer concentration. This reduction is postulated to be due to neutralization of the basic dissolution media, gel formation, and possible drug-polymer interactions.

Cell-mediated suppression of the fifth component of complement in mice.

Suppression of levels of circulating C5 in (C5- C5+)F1 hybrids by administration of (C5- C5-) parental lymphoid cells in the neonatal period has been accomplished with the three strain combinations tested ((SWR X RIII)F1, (A/He x RIII)F1, and (SWR X DBA/1)F1). Suppression was shown to be specific for C5 and not accompanied by reductions of C1, C2, C6, or other major groups of blood proteins. This demonstrated that the C5 reduction was not due to activation of complement (C) with resultant hypercatabolism of C components. When there was a concurrent chronic GVH reaction induced by lymphoid cells administered to offspring of H-2 incompatible parents, there was usually a resultant hypergammaglobulinemia that was also unrelated to the presence or absence of C5 suppression. Effective suppression required preimmunization of either the cell donor, the mother of the F1 hybrids, or both. This suggests that either two cell types or a single cell plus a humoral factor are required for suppression in this system.

Cloning of a 72 kDa matrix metalloproteinase (gelatinase) from chicken embryo fibroblasts using gene family PCR: expression of the gelatinase increases upon malignant transformation.

Chicken embryo fibroblasts secrete a 72 kDa progelatinase that displays all of the characteristics of a matrix metalloproteinase. Employing reverse-transcription PCR and degenerate oligonucleotide primers that are specific for two highly conserved sequences found in all matrix metalloproteinases, a DNA fragment specific for the chicken gelatinase was generated. Using this PCR product as a probe, cDNA clones were isolated from a chicken embryo cDNA library and the entire protein coding sequence was determined. The chicken progelatinase is 84% identical, at the amino acid level, with human and mouse 72 kDa progelatinase/type-IV procollagenase, with the greatest degree of similarity occurring in the propeptide and catalytic domains. The avian and mammalian proteinases diverge significantly in the C-terminal, hemopexin-like domain. The last 100 residues of the chicken gelatinase are only 66% identical with mammalian gelatinases. Mouse 72 kDa progelatinase, however, does not diverge significantly (> 98% identity) from human progelatinase in the hemopexin-like domain. The divergence in this domain of the chicken progelatinase may explain some of the distinct catalytic and inhibitory properties of the 72 kDa chicken progelatinase. Northern-blot analysis reveals that steady-state levels of the chicken progelatinase mRNA are increased 5-fold upon malignant transformation of chicken embryo fibroblasts with Rous sarcoma virus (RSV) and 3-fold by treatment with the tumour-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA). This represents the first reported cloning of an avian matrix metalloproteinase. The increased expression of the chicken progelatinase by RSV transformation and the tumour promoter PMA suggests that the progelatinase is regulated differently in chicken cells.

Identification of mutant monoclonal antibodies with increased antigen binding.

Sib selection and an ELISA have been used to isolate hybridoma subclones producing mutant antibodies that bind antigen better than the parental monoclonal antibody. Such mutants arise spontaneously in culture at frequencies of 2.5-5 X 10(-5). The sequences of the heavy and light chain variable regions of the mutant antibodies are identical to that of the parent and the Ka values of the mutants and the parent are the same. The increase in binding is associated with abnormalities of the constant region polypeptide and probably reflect changes in avidity of these antibodies.

The molecular and biochemical characterization of mutant monoclonal antibodies with increased antigen binding.

Mutant mAb with increased Ag binding were generated from a hybridoma cell line, 36-65, that secretes an IgG1,kappa anti-p-azophenylarsonate-(Ars) specific antibody. The mutant antibodies were identified using an Ars-specific ELISA and sib selection so that approximately 10(6) cells could be analyzed. The ELISA used as Ag a low ratio of Ars coupled to BSA and was set up so that only those antibodies that had higher binding than the parent would be detected. Seven mutant producing cell lines were isolated from five independent clones of 36-65. The mutant antibodies bind Ag 20 to more than 200-fold better than the parent and have wild type V region sequences. All have C region mutations that result in an increased avidity. At least five different genetic events are responsible for the C region mutations.

Controlled release of substituted benzoic and naphthoic acids using Carbopol gels: measurement of drug concentration profiles and correlation to release rate kinetics.

PURPOSE: The objective of this study is to correlate drug release mechanism with measured drug concentration profiles in gel layers of Carbopol matrices containing mesalamine or benzoic acid. METHODS: Release rate experiments with Carbopol matrices were performed using a rotating disk apparatus. Matrices were frozen and the gel layer in the matrices was sliced using a microtome in a cryostat. Drug concentration profiles were determined by direct measurement of the concentration of the drug in the gel slices. The pH of the slices was measured using microelectrodes, and water content was measured by Karl Fisher titration. RESULTS: The concentration gradient in mesalamine matrices decreased over time and correlated with square root of time release rate kinetics. The concentration profiles of benzoic acid were unchanged over time and correlated with zero order release rate kinetics. Carbopol gel layers were highly hydrated (93-95% water). Gel layers in matrices with mesalamine had a more alkaline microenvironmental pH. This higher pH resulted in increased growth of the thickness of the gel layer and a reduction drug diffusivity in comparison to benzoic acid matrices. CONCLUSIONS: The release rate kinetics of mesalamine and benzoic acid correlated to the measured concentration profiles. The shape of the concentration profiles is determined by the rate of growth of the Carbopol gel layer and drug diffusivity.

Distinct STAT structure promotes interaction of STAT2 with the p48 subunit of the interferon-alpha-stimulated transcription factor ISGF3.

Cells express a variety of STAT (signal transducer and activator of transcription) transcription factors that are structurally homologous and yet function specifically in response to particular cytokines. The functions of the individual STATs are dependent on distinct protein-protein interactions. STAT1 and STAT2 are activated by tyrosine phosphorylation in response to type I interferons-alpha/beta (IFN-alpha/beta) and subsequently form a multimeric transcription factor designated the IFN-alpha-stimulated gene factor 3 (ISGF3). ISGF3 is a unique STAT complex because it also contains a non-STAT molecule, p48, which is a critical DNA-binding component. We provide evidence that STAT2 specifically interacts with p48 in vivo before and after IFN-alpha stimulation. The specificity of ISGF3 formation is therefore a result of the distinct nature of the STAT2 molecule. Coimmunoprecipitation assays demonstrate p48 association with STAT2 but not STAT1. Hybrid STAT2. STAT1 molecules were used to identify a region of STAT2 which specifically associates with p48. The region of STAT2 interaction spans an amino-terminal region of two predicted coiled coils. The studies demonstrate the in vivo existence of a STAT2.p48 complex and a distinct STAT2.STAT1 complex after IFN-alpha stimulation. Data suggest that distinct bipartite complexes STAT2.p48 and STAT2.STAT1 translocate to the nucleus and associate on the DNA target site as ISGF3.

A Cys374Tyr homozygous mutation of platelet glycoprotein IIIa (beta 3) in a Chinese patient with Glanzmann's thrombasthenia.

A 20-year-old woman from a consanguineous family in the Hunan Province of the People's Republic of China was diagnosed as having Glanzmann's thrombasthenia based on (1) nearly a lifelong history of epistaxis, gum bleeding, petechiae, and purpura; (2) severe menorrhagia resulting in anemia and need for whole-blood transfusion; (3) normal coagulation assays; (4) prolonged bleeding time; (5) absent clot retraction; (6) decreased glass bead retention; (7) absent platelet aggregation in response to adenine diphosphate, epinephrine, and collagen; and (8) normal initial slope of platelet aggregation in response to ristocetin, but with a diminished maximal extent. The patient's platelets had a decreased level of platelet fibrinogen, but the deficiency was not as severe as in other Glanzmann's thrombasthenia patients. As judged by monoclonal antibody binding studies, surface glycoprotein (GP) IIb/IIIa (alpha IIb beta 3) expression was less than 15% of normal and alpha v beta 3 vitronectin receptor expression was 15% to 19% of normal, suggesting that the defect was in GPIIIa (beta 3). Immunoblotting of platelet lysates demonstrated decreased levels of GPIIb (approximately 30% to 35% of normal) and GPIIIa (approximately 10% of normal), and the GPIIb had undergone normal maturational processing into GPIIb heavy and light chains. Sequence analysis of the patient's GPIIIa RNA identified a G to A mutation at nucleotide 1219, predicting a Cys to Tyr substitution at residue 374. The patient's parents, who are first cousins, are asymptomatic and have only minor reductions in platelet aggregation. Direct sequencing of polymerase chain reaction-amplified cDNA and GPIIIa exon VIII indicated that the patient is homozygous and her parents are heterozygous for the mutation. Transient transfection studies in Chinese hamster ovary cells indicated that the mutation results in an 85% to 90% reduction in GPIIb/IIIa surface expression, but these cells retain the ability to mediate adhesion to immobilized fibrinogen. The relative preservation of platelet fibrinogen despite the very low level of platelet surface GPIIb/IIIa expression in this patient raises some interesting questions regarding the mechanism of fibrinogen uptake and the pathophysiology of Glanzmann's thrombasthenia.

Intravascular metabolism of normal and mutant mouse immunoglobulin molecules.

The metabolism of IgG immunoglobulins in the body is tightly regulated in order to maintain their intravascular concentration. Different subclasses may have different intravascular half-lives, and in the mouse, passively administered IgG2b disappears from the circulation more rapidly than IgG2a. We have attempted to localize the sequences in the constant region which are responsible for this difference by examining the intravascular metabolism of mutant immunoglobulins that were generated in tissue culture and have undergone deletions of individual constant region domains or contain different combinations of gamma 2b and gamma 2a CH2 and CH3 domains. Our results suggest that the regulation of intravascular metabolism is complex but indicate that sequences in the CH3 domain are important in determining the different intravascular half-lives of IgG2b and IgG2a antibodies in the mouse.