Protein kinase C inhibitor sotrastaurin in de novo liver transplant recipients: a randomized phase II trial.

Efficacy and safety of protein kinase C inhibitor sotrastaurin (STN) with tacrolimus (TAC) was assessed in a 24-month, multicenter, phase II study in de novo liver transplant recipients. A total of 204 patients were randomized (1:1:1:1) to STN 200 mg b.i.d. + standard-exposure TAC (n = 50) or reduced-exposure TAC (n = 52), STN 300 mg b.i.d. + reduced-exposure TAC (n = 50), or mycophenolate mofetil (MMF) 1 g b.i.d. + standard-exposure TAC (control, n = 52); all with steroids. Owing to premature study termination, treatment comparisons were only conducted for Month 6. At Month 6, composite efficacy failure rates (treated biopsy-proven acute rejection episodes of Banff grade ≥1, graft loss, or death) were 25.0%, 16.5%, 20.9% and 15.9% for STN 200 mg + standard TAC, STN 200 mg + reduced TAC, STN 300 mg + reduced TAC and control groups, respectively. Median estimated glomerular filtration rates were 84.0, 83.3, 81.1 and 75.3 mL/min/1.73 m(2), respectively. Gastrointestinal events (constipation, diarrhea, and nausea), infection, and tachycardia were more frequent in STN groups. More patients in STN groups experienced serious adverse events compared with the control group (62.3-70.8% vs. 51.9%). STN-based regimens were associated with a higher efficacy failure rate and higher incidence of adverse events with no significant difference in renal function between the groups.

Sotrastaurin in calcineurin inhibitor-free regimen using everolimus in de novo kidney transplant recipients.

Sotrastaurin, a novel selective protein-kinase-C inhibitor, inhibits early T cell activation via a calcineurin-independent pathway. Efficacy and safety of sotrastaurin in a calcineurin inhibitor-free regimen were evaluated in this two-stage Phase II study of de novo kidney transplant recipients. Stage 1 randomized 131 patients (2:1) to sotrastaurin 300 mg or cyclosporine A (CsA). Stage 2 randomized 180 patients (1:1:1) to sotrastaurin 300 or 200 mg or CsA. All patients received basiliximab, everolimus (EVR) and prednisone. Primary endpoint was composite efficacy failure rate of treated biopsy-proven acute rejection, graft loss, death or lost to follow-up. Main safety assessment was estimated glomerular filtration rate (eGFR) by MDRD-4 at Month 12. Composite efficacy failure rates at 12 months were higher in sotrastaurin arms (Stage 1: 16.5% and 10.9% for sotrastaurin 300 mg and CsA; Stage 2: 27.2%, 34.5% and 19.4% for sotrastaurin 200 mg, 300 mg and CsA). eGFR was significantly better in sotrastaurin groups versus CsA at most time points, except at 12 months. Gastrointestinal and cardiac adverse events were more frequent with sotrastaurin. Higher treatment discontinuation, deaths and graft losses occurred with sotrastaurin 300 mg. Sotrastaurin combined with EVR showed higher efficacy failure rates and some improvement in renal allograft function compared to a CsA-based therapy.

Compound odontoma associated with impacted maxillary incisors.

Odontomas are considered to be the most common odontogenic tumors of the oral cavity. Some authors consider it as malformations rather than true neoplasms. The exact etiology of odontomes is still not known. Most odontomes are asymptomatic and are discovered during routine radiographic investigations. Odontomes generally cause disturbances in the eruption of the teeth, most commonly delayed eruption or deflection. The present report describes the surgical management of a case of compound odontoma in a 10-year-old boy who presented with a complaint of swelling in the maxillary right anterior region and retained deciduous incisors. The related literature is also being reviewed in this article.

Generation and monitoring of cell lines producing humanized antibodies.

Antibody humanization has eliminated or reduced the human antimouse antibody response associated with the administration of murine antibodies. We have successfully humanized three different antibodies: (a) hMN-3 (granulocyte targeting); (b) hMu-9 (colorectal cancer targeting); and (c) hWI2 (anti-idiotype to the anti-carcinoembryonic antigen antibody MN-14). All humanized antibodies demonstrated immunoreactivities comparable to their parent counterparts. Previously, we reported the generation of high productivity cell lines for hMN-14 and hLL2 using the amplifiable vector pdHL2. Through amplification, selection, and cloning procedures, cell lines capable of large scale production were established, and further enhancement of production was achieved by a fed-perfusion bioreactor process. Using a similar and improved approach, we have enhanced the production of the above-mentioned humanized antibodies by gene amplification induced by a stepwise increase in the concentration of methotrexate in the culture media. A reliable IgG determination method is essential to monitor amplification, especially at the final cloning stage, for the selection of the subclones with the highest productivity. We found that measurement of humanized IgG concentration in culture media supplemented with more than 1 microM methotrexate by a standard ELISA assay could be unreliable and misleading. Whereas the determination of antibody by adsorption/elution on protein A from a 100-ml culture is accurate and reproducible, the method is time-consuming, tedious, and labor intensive. We have recently developed a Western blot assay that enables us to monitor the productivity of the cultures. The assay is simple and sensitive, and it makes simultaneous determinations of relative antibody production from individual clones at the 96-well stage feasible. With this method, amplification, cloning, and adaptation to serum-free conditions of multiple cell lines can be monitored in an efficient manner.

Co-secretion of two distinct kappa light chains by the mu-9 hybridoma.

Mu-9 is a monoclonal antibody (MAb) specific for the CSAp antigen (Ag) expressed by colorectal cancers. By using variable (V)-region-specific primers, the respective VH and VL sequences of Mu-9 were polymerase chain reaction (PCR)-amplified. However, chimeric Ab (cMu-9-1) constructed from these PCR-amplified V sequences failed to bind the CSAp Ag. Although the light chain of murine Mu-9 was not glycosylated, that of cMu-9-1 was found to be O-glycosylated, as confirmed by reducing SDS-PAGE analyses, glycoprotein blotting and O-linked specific deglycosylation studies. Removal of O-linked oligosaccharides either by enzymatic digestion or by blocking O-glycosylation with a specific inhibitor did not restore the immunoreactivity of cMu-9-1, indicating that light chain O-glycosylation was not the cause for lack of immunoreactivity. We reported earlier that screening of a Mu-9 cDNA library uncovered the presence of an additional light chain sequence that was later proven to be the authentic light chain of Mu-9. Analyses of the cDNA sequence encoding the nonimmunoreactive light chain, however, revealed no defects that would preclude the sequence from being translated and secreted by the murine hybridoma. By adapting the Mu-9 hybridoma culture to serum-free conditions, we confirmed the secretion of low levels of O-glycosylated light chain. The biological significance of the O-glycosylation as well as the cosecretion of both light chains with respect to allelic exclusion are discussed.

Generation of a high-producing clone of a humanized anti-B-cell lymphoma monoclonal antibody (hLL2).

BACKGROUND: LL2 is a murine immunoglobulin (Ig)G2a-kappa anti-B-cell monoclonal antibody with proven targeting and therapeutic efficacy in the management of non-Hodgkin's lymphoma (NHL). The authors had previously generated a humanized LL2 (hLL2) that demonstrated binding properties identical to those of LL2. Nevertheless, the productivity of the cell line was insufficient for large-scale production of the antibody for clinical studies. Therefore, the authors chose an amplifiable system for the generation of hLL2. METHODS: The hLL2 sequences were ligated into the expression vector pdHL2, which has a dhfr amplifiable gene, and were incorporated into the SP2/0 cells by electroporation. A methotrexate (MTX) resistant clone producing hLL2 was identified. Stepwise increases in MTX concentrations, from 0.1 to 5 microM, and subcloning of the cells by limiting dilution were performed. RESULTS: By amplifying the dhfr and hLL2 genes with stepwise increases in the MTX concentration, the antibody production was enhanced from its original 1.4 to 70 +/- 5 mg per liter of culture media. Subsequent subcloning further improved the productivity. Immunoreactivity of the antibody was conserved, as proven by enzyme-linked immunosorbent assay and cell-binding assays. By isoelectrofocusing, the isoelectric point (pI) of the antibody was measured at approximately 9.6. The productivity of the clone was not affected by culture conditions or storage of the cells in liquid nitrogen. CONCLUSIONS: By means of gene amplification, the authors have generated a high-producing hLL2-IgG clone suitable for production of the quantity of antibody necessary for clinical diagnostic and therapeutic trials of NHL patients.

Chimerization of Mu-9: a colon-specific antigen-p antibody reactive with gastrointestinal carcinomas.

BACKGROUND: Mu-9 is a murine monoclonal antibody that is directed against affinity-purified colon-specific antigen-p (CSAp). CSAp is a tumor-associated antigen that is present in 60% of colorectal carcinomas. Preclinical and clinical studies have shown Mu-9 to have excellent targeting abilities. However, as administration of the murine immunoglobulin G (IgG) provoked a human anti-mouse antibody response, chimerization of Mu-9 is warranted for decreasing immunogenicity. METHODS: Polymerase chain reaction and cDNA library screening methods were used for the cloning of Mu-9 heavy and light chain variable regions for the construction of chimeric Mu-9. RESULTS: The functional chimeric Mu-9 antibody binds to the CSAp antigen in the GW-39 extracts. It has immunochemical properties similar to that of murine Mu-9. CONCLUSIONS: The V-region sequence information will be used for design of humanized Mu-9, which will be evaluated for targeting gastrointestinal carcinomas.

Isolation and characterization of mutants of human mitogen-activated protein kinase (ERK2).

Site directed mutagenesis/charged-to-alanine scanning mutagenesis of the amino terminal portion of human ERK2 (from amino acids 1 to 150) purified as a glutathione-S-transferase fusion protein (GST-ERK2) from E. coli has been done to determine regions/amino acids important for activation by rabbit skeletal muscle MAP kinase kinase (rMEK) and kinase activity towards myelin basic protein (MBP). Five classes of mutants have been isolated. The first class of mutants comprises of G30A/G32A, A50D and R65A/R68A/E69A, that can be phosphorylated by rMEK and have no kinase activity towards MBP, the second class includes mutants D122A/H123A and N142A which have lower kinase activities but no change in their activation by rMEK; third class being Y34A, E58A/H59A, which have neutral effect towards either activity, the fourth class that includes completely inactive mutants D42A/K46A/R48A, the deletion mutant in the same region (-9aa[40-48]) and D104A/E107A/D109A and finally the fifth class that include K53A, E94A/K97A/D99A, K112A/K115A and R133A/K136A that are phosphorylated 140-240% but with kinase activity toward MBP ranging from 50-100% of the wild type.

Stabilization, purification, and characterization of glutamate synthase from Clostridium pasteurianum.

Clostridium pasteurianum possesses a high level of glutamate synthase (EC 1.4.1.14) activity and cell yield when grown on 4 mM ammonium chloride and molasses as the sole nitrogen and carbon sources, respectively. The enzyme activity is stabilized by addition of alpha-ketoglutarate, EDTA, and 2-mercaptoethanol. Ammonium sulfate precipitation and single-step combined gel and ion-exchange chromatography followed by fractional dialysis yield a homogeneous protein with 40% recovery of the glutamate synthase activity. The native enzyme (Mr congruent to 590,000) gives five different subunits (as dimers) upon SDS gel electrophoresis. The enzyme has been characterized for pH and temperature optimum, substrate specificity, Kmapp values, energy of activation, half-life, and thermal stabilization. Metal ions and citric acid cycle metabolites do not affect the enzyme activity. Glutamate synthase shows fluorescence maximum at 370 nm when excited at 280 nm. The fluorescence is quenched upon the addition of NADH. Spectroscopic examination of the enzyme gave absorption maximum at 280 and none at 380 and 440 nm, indicating the absence of iron and flavin. The absence of iron and flavin was also confirmed by atomic absorption, chemical analysis, and fluoroscopy, respectively. The C. pasteurianum enzyme differs from that of other aerobic bacterial sources.

Cloning of the gene coding for human class 1 heparin-binding growth factor and its expression in fetal tissues.

We have identified four overlapping genomic DNA clones coding for human class 1 heparin-binding growth factor (HBGF-1), also known as acidic fibroblast growth factor, by screening genomic DNA libraries with an HBGF-1 cDNA probe. The exon-intron structure of the HBGF-1 gene was determined by Southern hybridization and nucleotide sequence analysis. The complete amino acid sequence of human HBGF-1 was deduced from the nucleotide sequence of these genomic DNA clones. The predicted amino acid sequence is identical to the published amino acid sequence determined by protein sequencing. Southern blot analysis of human DNA suggested that there is a single-copy gene coding for HBGF-1. A 4.5-kilobase mRNA and two minor species (3.4 and 2.0 kilobases) homologous to the HBGF-1 gene were detected in cellular RNA isolated from human adult brain and kidney. The HBGF-1 mRNAs from brain and kidney had slightly different sizes. The mechanism for the synthesis of different sizes of mRNA was not determined. We also detected HBGF-1 transcript from glioblastoma cells, fetal brain, and kidney but not from placenta or fetal liver. Since HBGF-1 is an angiogenic factor, these data suggest that it may play a role in embryonic angiogenesis during fetal development.

Purification and characterization of glutamine synthetase from Clostridium pasteurianum.

Glutamine synthetase from Clostridium pasteurianum grown on molasses as the sole carbon source and ammonium chloride as the nitrogen source has been purified to homogeneity (45-fold) with 32% recovery. The procedure involves ammonium sulfate precipitation and chromatography on a combined Sepharose 4B/DEAE-Sephadex A-50 column. The purified enzyme being very unstable was stabilized by the addition of 25% (v/v) glycerol. The enzyme has an unusually high molecular weight of 1 X 10(6) and 20 subunits of Mr 50 000 each, as determined by gel filtration and sodium dodecyl sulfate gel electrophoresis, respectively. It has an absorption maximum at 280 nm and a fluorescence emission maximum at 380 nm when excited at 280 nm. Its substrate binding pattern as studied by fluorescence quenching studies is different from that of the Escherichia coli enzyme. Both the gamma-glutamyltransferase and synthetase activities reside in the same protein as the ratio of the two activities at each step of purification remains constant and the enzyme exhibits optimal transferase and synthetase activities at the same pH (7.2) and temperature (50 degrees C). The thermal stabilities of both activities were also similar, and decay of both the activities at 50 degrees C ran parallel. The enzyme shows stabilization by substrates, as L-glutamate, Mg2+, and ATP + Mg2+ protected both the synthetase and gamma-glutamyltransferase activities against thermal inactivation. Storage in 25% (v/v) glycerol enhanced the thermal stability of glutamine synthetase. Metal ion requirement and substrate specificity of the enzyme have been examined. Maximum synthetase activity occurs when [Mg2+]: [ATP] = 2. The Km app values are as follows (in parentheses): ATP (0.34 mM), NH2OH (0.4 mM in the synthetase reaction and 4.1 mM in the transferase reaction), glutamine (14.7 mM), ADP (3.8 X 10(-4) mM), arsenate (2.5 mM), and L-glutamate (3.4 mM, 22.2 mM). The enzyme exhibits negative cooperativity in the binding of glutamate. Amino acids such as L-serine, glycine, L-alanine, and L-aspartic acid inhibit the enzyme.

Clostridium pasteurianum glutamine synthetase mechanism. Evidence for active site tyrosine residues.

Preliminary chemical modification studies indicated the presence of tyrosine, carboxyl, arginine, histidine and the absence of serine and sulfhydryl residues at or near the active site of Clostridium pasteurianum glutamine synthetase. The conditions for tyrosine modification with tetranitromethane were optimized. The inactivation kinetics follow pseudo-first-order kinetics with respect to enzyme and second order with respect to modifier per active site. There was no inactivation at pH 6.5 suggesting the absence of thiol oxidation. The synthetase and transferase reactions followed the same pattern of inactivation on enzyme modification and both were equally protected by glutamate plus ATP. Thus tyrosine residues are present at the active site of the enzyme and are essential for both transferase and synthetase activities.

Elevated levels of (2'-5')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.

(2'-5')Oligoadenylic acid [(2'-5')An] polymerase activity was measured in extracts of human lymphoblastoid cells of the Namalva line cultured under different conditions. Exponentially growing cells had a relatively low polymerase activity level, whereas cells grown to limit density showed elevated levels. When fresh medium was added to growth-arrested cells, (2'-5')An polymerase activity decreased concomitantly with the initiation of active deoxyribonucleic acid synthesis. An increase in polymerase activity level was also observed after exponentially growing cells were transferred from medium containing 20% serum to fresh medium containing 0.2% serum. These cells diminished deoxyribonucleic acid synthesis and remained quiescent until 20% serum was again added. Polymerase activity level decreased as the cells entered into S phase. The addition of the inhibitor of deoxyribonucleic acid synthesis, hydroxyurea, to exponentially growing cells did not increase polymerase level, indicating that cells blocked in S phase and at the G1-S boundary maintained the basal level of this enzyme. Degradation of labeled (2'-5')An was measured in extracts of Namalva cells cultured under different conditions, but no significant differences among degradative activities were observed. Since (2'-5')An polymerase activity is one of the enzymatic activities induced by interferon, we measured interferon titers in Namalva cell medium. Less than 1 reference unit per ml was detected in cells grown under different conditions. Moreover, the increase in (2'-5')An polymerase activity level in cells transferred from 20 to 0.2% serum was not prevented by including anti-lymphoblastoid interferon antibody in the medium. These results suggest that the activity level of (2'-5')An polymerase is regulated in Namalva cells on the basis of the growth status of the cells and that this regulatory mechanism is apparently not activated by interferon.

Increased levels of (2'-5')oligo(A) polymerase activity in human lymphoblastoid cells treated with glucocorticoids.

An enzymatic activity that synthesizes (2'-5')-oligo(A) from ATP is induced in animal cells treated with interferon. This activity, designated (2'-5')A polymerase, is also elevated in human lymphoblastoid Daudi and Raji cells treated with hydrocortisone. The polymerase activity increases significantly after 24 hr of treatment and declines when hydrocortisone is removed from the culture medium. The product of the enzyme prepared from hydrocortisone-treated cells is indistinguishable from (2'-5')oligo(A) synthesized with polymerase of interferon-treated cells either by an endonuclease activation assay or by chromatographic analysis. The increase in (2'-5')A polymerase is not mediated by secretion of interferon by hydrocortisone-treated cells; less than 1 unit of interferon per ml is present in the culture medium during treatment with this glucocorticoid hormone. Moreover, this increase is related to the concentration of hydrocortisone in the culture medium and is inhibited by the addition of cortexolone. This steroid interferes with the interaction between glucocorticoid hormones and their receptor. Cortexolone has no effect, however, on the induction of (2'-5')A polymerase by interferon. The synthetic glucocorticoid dexamethasone also increases the polymerase activity. Experiments with inhibitors show that such an increase requires RNA and protein synthesis.

2'5' oligo(A) polymerase activity in serum of mice infected with EMC virus or treated with interferon.

Interferon-treated cells show an increase in two double-stranded RNA (dsRNA)-dependent enzymatic activities involving an oligoadenylate polymerase and a protein kinase (ref. 1 and refs therein). The polymerase converts ATP into a series of oligonucleotides characterized by 2'5'-phosphodiester bonds, designated 2'5'-oligo(A) or 2-5A (ref. 1). These oligonucleotides activate an endoribonuclease that degrades RNA in extracts of control and interferon-treated cells. These observations have been made in tissue culture cells and no informatin is yet available on these enzymatic activities in animals with elevated interferon levels. We report here on 2-5A synthesis in tissue homogenates and serum of mice infected with encephalomyocarditis virus (EMCV); this virus induces interferon synthesis when injected intraperitoneally into mice. Significant synthesis of 2-5A was detected in extracts of spleen and lungs, but also, surprisingly, in the serum of these mice. Subsequent experiments showed synthesis of 2-5A in serum of mice treated with the interferon inducer poly(I) x poly(C) (ref. 3) or with mouse fibroblast interferon.