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PubMed1996

[Autologous stem cell transplantation. From bone marrow to selected blood stem cells: 100 consecutive procedures at a single center].

One hundred consecutive autologous stem cell transplants are reported: Non-Hodgkin's lymphoma 51 cases, Hodgkin's disease 27 cases, acute leukaemia 14 cases, multiple myeloma seven cases and chronic myeloid leukaemia one case. Most patients were in their second or later remission. The overall three-year survival for all patients was 60% and the three-year disease-free survival was 50% for lymphoma patients and 30% for acute leukaemia patients. The dominant source of stem cells was bone marrow during 1993, but from 1994 it has been peripheral blood, now totalling 33 cases. There were 12 toxic deaths, all among patients who were heavily treated before bone marrow harvest and transplantation. The patients transplanted with blood stem cells had significantly shorter duration of pancytopenia, and hospital stay, but their disease-free survival was not longer than that of a comparable group of bone marrow transplanted patients. Six patients were transplanted with purified CD34+ cells (selected by avidity column (Ceprate (R)), and had duration of thrombocytopenia and hospital stay similar to the patients transplanted with unmanipulated blood stem cells, but slightly longer duration of neutropenia. We conclude that high-dose therapy with autologous stem cell transplantation in not too heavily pretreated patients is a safe procedure irrespective of the source of stem cells.

Adolescent↗

PubMed2000

Pasteurized, monoclonal antibody factor VIII concentrate: establishing a new standard for purity and viral safety of plasma-derived concentrates.

A factor VIII concentrate (Monoclate-P) manufactured using a combination of pasteurization and immunoaffinity chromatography has been chosen to compare and contrast manufacturing aspects of plasma-derived factor VIII concentrates. Pasteurization is a virucidal method with a long safety record in clinical practice, while immuno-affinity chromatography selectively isolates and purifies the procoagulant protein of factor VIII, and partitions potential viral contaminants and nonessential proteins to the unbound fraction. The complete Monoclate-P production process reduces human immunodeficiency virus by > or = 10.5 log10, Sindbis (a model for hepatitis C virus) by > or = 6.5 log10, and murine encephalomyocarditis virus (a non-enveloped model virus) by 7.1 log10. The viral safety of Monoclate-P has been further demonstrated in clinical studies in patients not previously treated with blood or plasma-derived products. Additionally, the manufacture of Monoclate-P includes careful donor screening and plasma testing for antibodies to syphilis and human immunodeficiency, hepatitis B, and hepatitis C viruses to enhance source plasma safety. Combined with donor selection and plasma testing, multiple viral reduction steps effectively eliminate both lipid-enveloped viruses (e.g. human immunodeficiency, hepatitis B and C) and non-lipid-enveloped viruses (e.g. hepatitis A). In addition, polymerase chain reaction-based nucleic acid detection tests for hepatitis B and C viruses and for human immunodeficiency virus-1 have been introduced as part of an investigational new drug mechanism.

Antibodies, Monoclonal↗

PubMed1992

Identification of human satellite DNA sequences associated with chemically resistant nonhistone polypeptide adducts.

A fraction of DNA fragments of highly purified and completely unfolded eukaryotic DNA inevitably remains associated with chemically resistant nonhistone DNA-polypeptide complexes. This fraction can be isolated by nitrocellulose filtration because the polypeptide-associated DNA fragments are retained on nitrocellulose filters while bulk DNA passes through the filters. The fraction of AluI-fragmented DNA from human placenta retained on filters as a result of the binding factors (R-DNA, approximately 12%) represents a subset of genomic sequences with a sequence complexity different from unfractionated DNA and DNA recovered in the filtrate (F-DNA). DNA sequences prevalent in the retained fraction were detected by differential plaque hybridization of a recombinant lambda gt10 library with radiolabeled F- and R-DNA fractions. Several recombinant phages showing much stronger hybridization signals with the R-DNA probe than with the F-DNA probe were selected, plaque-purified and analyzed. Analysis of the inserts of such clones showed that repetitive DNA sequences of the alphoid dimeric and tetrameric family, satellite III and satellite III-like sequences are highly enriched in the retained fraction, which indicates that these sequences specifically attract the polypeptides involved in the tightly bound and resistant complexes. This property of repetitive sequences is of interest since tandemly repetitive sequences have been suggested to code for locus-specific fixation and stabilization of the chromatin fiber in the cell nucleus.

Bacteriophage lambda↗

PubMed1991

Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells.

The addition of mitogen-prestimulated periferal blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) cultures to enriched populations of natural killer (NK) cells obtained from PBL of normal donors in the presence of rIL-2 resulted in highly significant increases in proliferation, purity, and cytolytic activity of cultured NK cells. Two sources of enriched NK cell preparations were used: (i) Adherent-lymphokine activated killer (A-LAK) cells obtained by adherence to plastic during 24 hr activation with 10(3) Cetus U/ml rIL-2; and (ii) NK cells negatively selected from PBL by removal of high-affinity rosette-forming cells and CD3+ lymphocytes. Coculture of A-LAK cells for 14 days with autologous or allogeneic Con A-activated PBL (10(6) cells/ml) or selected EBV-transformed LCL (2 x 10(5) cells/ml) as feeder cells increased fold expansion by a mean +/- SEM of 629 fold +/- 275 (P less than 0.019) and 267 fold +/- 54 (P less than 0.0001), respectively, compared to 55 +/- 20 in A-LAK cultures without feeder cells. The addition of either activated PBL or EBV lines to A-LAK cultures also led to a significant increase in the percentage of NK cells (CD3- CD56+) (84 +/- 2.4 and 84 +/- 2.6%, respectively, P less than 0.0001 for both), compared to 53 +/- 7.2% in cultures without feeders. The presence of feeder cells in cultures of A-LAK cells also led to significantly higher anti-tumor cytolytic activity compared to control cultures, as measured against NK-sensitive (K562) and NK-resistant (Daudi) target cells. Mitogen-stimulated CD4+ PBL purified by positive selection on antibody-coated flasks were better feeders than CD8+ or unseparated PBL. In the presence of feeder cells, it was possible to generate up to 6 x 10(9) activated NK cells from 2 x 10(8) fresh PBL by Day 13 of culture. Enhanced NK cell proliferation in the presence of feeder cells was not attributable to a detectable soluble factor. The improved method for generating A-LAK or activated-NK cells should facilitate cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with high anti-tumor activity.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1993

Generation and characterization of ex vivo propagated autologous CD8+ cells used for adoptive immunotherapy of patients infected with human immunodeficiency virus.

Cytolytic T lymphocytes play an important role in host defense against viral infections, including human immunodeficiency virus (HIV). In a phase I clinical trial (protocol 080 of the AIDS Clinical Trials Group), generation of CD8+ effector cells from peripheral blood of patients with acquired immunodeficiency syndrome (AIDS)-related complex (ARC) or AIDS and safety of autologous adoptive transfer of these cells were evaluated. For therapeutic infusions, CD8+ T cells were purified by positive selection on anti-CD8 monoclonal antibody-coated flasks from leukapheresed peripheral blood of seven patients. These CD8+ T cells were cultured in the presence of interleukin-2 and phytohemagglutinin for up to 3 weeks to obtain cells sufficient for therapeutic infusions (10(8) to 10(10)). All 31 cell cultures established from the seven patients and used for therapy were highly enriched in CD8+ (mean, 97%), CD8+HLA-DR+ (50%), cytotoxic CD8+CD11b- (82%), and memory CD29+ (78%) T lymphocytes. In vitro expanded CD8+ cells had excellent cytotoxic function at the time they were used for therapy, including HIV-specific activity against autologous targets infected with vaccinia vectors expressing HIV-IIIb antigens, gag, pol, and env. Anti-HIV activity of cultured CD8+ cells was significantly higher than that of autologous fresh peripheral blood lymphocytes. Our results show that CD8+ T lymphocytes obtained from peripheral blood of symptomatic HIV-infected patients can be purified, cultured to obtain large numbers of cells with enhanced anti-HIV activity, and safely infused into patients with AIDS as a form of immunotherapy.

AIDS-Related Complex↗

PubMed1990

Choice and use of tracers.

The isotope dilution method has been widely employed to measure glucose turnover in man. The validity of this technique depends upon several assumptions. First, it is assumed that the selected model of glucose kinetics is valid. Under nonsteady state conditions this has recently been challenged for the most commonly used single compartment model. Secondly, it is assumed that the isotope is metabolized in the same manner as unlabeled glucose. If the isotope loses its label when subject to a substrate cycle (for example, [2-3H]glucose in glucose to glucose 6-phosphate cycling), an overestimate of glucose disposal will result. In contrast, if isotope that has been cleared (for example [6-3H]glucose in hepatic glucogen) is recycled into the systemic circulation, an underestimation of glucose turnover will result. Thirdly, it is assumed that measurement of specific activity is accurate. It has recently been shown that many commercially available tritiated (but not 14C) isotopes contain radioactive nonglucose contaminants which have a slower clearance rate than glucose under conditions of high glucose turnover. This can result in overestimates of specific activity and underestimates of glucose appearance, leading to calculated negative hepatic glucose release. While this problem may be avoided with purified tritiated glucose tracers, a similar problem with stable isotopes of glucose remains to be determined. In summary, the choice of glucose isotope should reflect the probability of substrate cycling and isotope cycling in the experiment in question. Care should be taken to ensure purified isotopes are selected and that the model of glucose kinetics used is valid during the conditions to be studied.

Animals↗

PubMed1976

Isolation and characterization of purified rat casein messenger ribonucleic acids.

Purification of casein messenger ribonucleic acids (mRNAs) from lactating rat mammary gland RNA has been accomplished by a combination of sizing techniques, including Sepharose 4B chromatography and preparative agarose-urea gel electrophoresis, and affinity chromatography of poly(adenylic acid)-containing mRNA on oligo(dT)-cellulose. The separation of the individual casein mRNAs into discrete molecular species free of apparent ribosomal RNA contaminants was facilitated by the use of denaturing conditions either prior to or during each of the fractionation procedures. Two casein mRNA fractions were isolated: (1) a 15S mRNA doublet which directed the synthesis of the two largest rat caseins in the wheat-germ, cell-free, translation assay, and (2) a 12S mRNA which migrated as a single species during agarose-urea gel electrophoresis and directed the in vitro synthesis of the smallest of three rat caseins. These mRNAs had apparent molecular weights of 450 000 +/- 30 000 and 320 000 +/- 25 000 and contained poly(adenylic acid) sequences at their 3' termini ranging from 15 to 150 residues with number average lengths of 42 and 38 adenosines, respectively. The purity of the isolated casein mRNA'S was determined both by agarose-urea gel electrophoresis and by a careful comparison of the total products synthesized in the wheat-germ translation assay with those recognized by a specific casein antibody using an indirect immunoprecipitation technique. The specificity of the indirect immunoassay procedure was demonstrated by the selective displacement by purified rat casein of greater than 95% of the radioactive product synthesized in the cell-free system. Under optimal translation conditions for casein mRNA, at least 90% of the released protein synthesized in response to the 15S casein mRNA was specifically immunoprecipitable, representing a 178-fold purification compared with the initial RNA extract. Using these techniques a comparable purification was also obtained for a 15S mouse casein mRNA fraction. Finally, an analysis by fluorography on 5-20% (w/v) polyacrylamide gradient slab gels of the total proteins synthesized in response to both the 15S and 12S casein mRNAs revealed a close correspondence with those proteins which were specifically immunoprecipitated.

Animals↗

PubMed1979

Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA.

Transcription of Ad2 DNA templates in the presence of crude cellular extracts supplemented with exogenous (purified) RNA polymerase II is selectively and accurately initiated at the major late viral promoter at map position 16.45. Specific initiation has been demonstrated by a combination of hybridization, nuclease S1 mapping, size and partial sequence (fingerprint) analyses of the transcripts generated with various templates. With intact Ad2 DNA, transcription is terminated ell before the end of the 28 kb transcription unit is reached. With truncated templates (which contain intact promoter regions and several hundred base pair segments of the transcribed region) the expected run-off products are observed, along with a low level of prematurely terminated transcripts. The 560 nucleotide run-off product of the Sma l-f template (coordinates 11.6-18.2) was shown to contain all the large RNAase T1 oligonuc eotides that are characteristic of the corresponding in vivo transcript from this region; in addition, the 5 terminal undecanucleotide appears to be both capped and methylated. We have investigated various parameters (salt, metal ion and template concentrations) that affect the level of specific transcription in the crude system and have found that, under optimal conditions, specific transcription of Ad2 DNA continues for several hours. In addition, specific transcription initiation at the late promoter is observed with extracts derived from either virus-infected or uninfected KB cells and with class II RNA polymerases isolated from either human calf, murine or amphibian cells. RNA polymerase II from wheat germ does not function in this system.

Adenoviruses, Human↗

PubMed1985

Transcription of vaccinia virus early genes by a template-dependent soluble extract of purified virions.

An extract capable of selectively transcribing early vaccinia virus genes was prepared by disrupting purified vaccinia virions and passing the soluble material through a DEAE-cellulose column to remove endogenous DNA. Runoff transcripts of predicted size were synthesized by using double-stranded DNA templates that contained truncated early vaccinia virus genes, whereas several late vaccinia virus genes were not transcribed under these conditions. Proper dilution of the enzyme extract was critical, and a threshold concentration of DNA was required. At 30 degrees C, runoff transcripts were detected after 5 min and synthesis slowed appreciably after 30 min. Mg2+ was the preferred divalent cation, and KCl concentrations above 20 mM were inhibitory. Correct initiation of transcription was demonstrated by high-resolution analysis of S1 nuclease-digested hybrids formed by annealing in vitro-synthesized RNA with 5'-end-labeled DNA. A requirement for a 31-base-pair transcriptional regulatory sequence was found by using templates with deletions in an early promoter region. This in vitro system may be useful for mapping early transcriptional initiation sites, measuring the effects of additional promoter mutations, and isolating transcription factors.

Cell-Free System↗

PubMed1992

Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP.

8-(p-Chlorophenylthio)-cGMP (8-pCPT-cGMP) and 8-bromo-cGMP were compared with respect to their chemical and biological properties in order to evaluate their potential as selective activators of cGMP-dependent protein kinase (cGMP-PK; EC 2.7.1.37) in intact human platelets. 8-pCPT-cGMP, 8-Br-cGMP and cGMP were shown to be potent and selective activators of purified bovine lung cGMP-PK and of cGMP-PK present in human platelet membranes when compared with the activation of cAMP-dependent protein kinase (cAMP-PK; EC 2.7.1.37). 8-pCPT-cGMP was not hydrolysed by the purified cGMP-stimulated phosphodiesterase (cGS-PDE), cGMP-inhibited phosphodiesterase (cGI-PDE) and Ca(2+)-calmodulin-dependent phosphodiesterase (CaM-PDE), whereas cGMP and, to a lesser extent, 8-Br-cGMP were hydrolysed by all three types of 3',5' cyclic nucleotide phosphodiesterases (EC 3.1.4.17) examined. Also, 8-pCPT-cGMP was not hydrolysed by a human platelet homogenate which contains a high level of the cGMP-specific cGMP-binding phosphodiesterase (cGB-PDE). Additionally, 8-pCPT-cGMP did not activate the cGS-PDE or inhibit the cGI-PDE, whereas half-maximal inhibition of cGI-PDE occurred at 8 microM 8-Br-cGMP. The apparent lipophilicity of 8-pCPT-cGMP was higher than that of 8-Br-cGMP. Extracellular application of 8-pCPT-cGMP to intact human platelets reproduced the pattern of protein phosphorylation induced by sodium nitroprusside (SNP), a cGMP-elevating inhibitor of platelet activation. Quantitatively, 8-pCPT-cGMP was more effective than 8-Br-cGMP in inducing phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein, a major substrate of cGMP-PK in intact platelets. As observed with SNP, pretreatment of human platelets with 8-pCPT-cGMP prevented the aggregation induced by thrombin. The results suggest that 8-pCPT-cGMP is a very potent and selective activator of cGMP-PK in cell extracts and in intact human platelets and, in this respect, is superior to 8-Br-cGMP and other cGMP analogs used for intact cell studies. The data also suggest that inhibition of platelet activation in intact human platelets by nitrovasodilators is mediated by cGMP-PK.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1988

Factor D is a selective single-stranded oligodeoxythymidine binding protein.

Factor D, a protein purified from rabbit liver that selectively enhances traversal of template oligodeoxythymidine tracts by diverse DNA polymerases, was examined for the sequence specificity of its binding to DNA. Terminally [32P]-labeled oligomers with the sequence 5'-d[AATTC(N)16G]-3', N being dT, dA, dG, or dC, were interacted with purified factor D and examined for the formation of protein-DNA complexes that exhibit retarded electrophoretic mobility under nondenaturing conditions. Whereas significant binding of factor D to 5'-d[AATTC(T)16G]-3' is detected, there is no discernable association between this protein and oligomers that contain 16 contiguous moieties of dG, dA, or dC. Furthermore, factor D does not form detectable complexes with the duplexes oligo(dA).oligo(dT) or poly(dA).poly(dT). The preferential interaction of factor D with single-stranded poly(dT) is confirmed by experiments in which the polymerase-enhancing activity of this protein is protected by poly(dT) against heat inactivation two- and four-fold more efficiently than by poly(dA) or poly(dA).poly(dT), respectively.

Animals↗

PubMed2007

Binding of antiphospholipid antibodies to discontinuous epitopes on domain I of human beta(2)-glycoprotein I: mutation studies including residues R39 to R43.

OBJECTIVE: Pathogenic antiphospholipid antibodies (aPL) bind the self antigen N-terminal domain (domain I) of beta(2)-glycoprotein I (beta(2)GPI), with residues G40-R43 being important. However, peptides homologous to other regions of domain I have also been shown to bind aPL. Furthermore, there are no published reports of the effects of altering R39, which has greater surface exposure than the G40-R43 residues. METHODS: We used a novel, efficient method of production and purification of human domain I by Escherichia coli to create multiple mutants of domain I. These domain I mutants were then screened for binding to a range of polyclonal IgG purified from patients with antiphospholipid syndrome, using both solid-phase and fluid-phase assays. RESULTS: E coli-expressed purified domain I selectively bound IgG derived from patients with antiphospholipid syndrome. In region R39-R43, the R39S mutation had the greatest effect in terms of reducing binding to a panel of aPL in the fluid phase (mean +/- SD inhibition 14 +/- 18.5% versus 44.1 +/- 31.7% for G40E and 62.9 +/- 25.7% for wild-type domain I). Conversely, altering both D8 and D9 to S8 and G9, respectively, had the effect of enhancing binding to aPL in the fluid phase. Adding the remainder of the domain I-II interlinker resulted in enhanced binding over wild-type in the solid phase but not the fluid phase. CONCLUSION: The binding of aPL to beta(2)GPI domain I is complex and likely to involve discontinuous epitopes that include R39 in addition to G40-R43, the domain I-II interlinker, and possibly D8 and D9. Domain I variants with enhanced binding to aPL compared with wild-type domain I may aid in the development of novel therapies.

Antibodies, Antiphospholipid↗

PubMed1995

Altered T lymphocyte signaling in rheumatoid arthritis.

Synovial and peripheral blood T cells from patients with rheumatoid arthritis are functionally deficient. This may be secondary to their reduced cytokine (e.g. interleukin-2) synthesis. We have investigated the possibility of an alteration in pathways common to interleukin-2 production and proliferation in peripheral blood T cells from patients with active rheumatoid arthritis. Intracellular calcium levels ([Ca2+]i) were analyzed by flow cytometric methods in Indo1-loaded T cells. These were purified by negative selection from patients or age/sex-matched controls, and stimulated with phytohemagglutinin-P or anti-CD3. Rheumatoid [Ca2+]i responses to both stimuli were reduced (p < 0.005). Patient cell samples included a larger proportion of non-responding cells, but even in the responsive population the magnitude of the response in rheumatoid cells was impaired compared with those in normal cell samples (p < 0.0001) for both stimuli. Proliferation responses were also impaired (p < 0.005), and there was a positive correlation between the paired [Ca2+]i elevation and proliferative responses for both stimuli. CD2 and CD3 expression were normal, and the proportions of CD4, CD8 and CD45RO and CD45RA subsets were also unaffected by disease. Thus a signaling defect downstream of CD2 or CD3 surface molecules may contribute to functional deficiencies in rheumatoid T lymphocytes. This effect is not due to non-steroidal anti-inflammatory drugs which some patients were taking. We have demonstrated similar alterations in [Ca2+]i responses and proliferation in a smaller study of patients with inflammatory bowel disease, indicating that such changes might be present in other chronic inflammatory states.

Antigens, CD↗

PubMed2005

Separation, identification, and interaction of heparin oligosaccharides with granulocyte-colony stimulating factor using capillary electrophoresis and mass spectrometry.

A capillary electrophoresis (CE) method was developed for the separation of heparin oligosaccharides compatible to study the interactions between the oligosaccharides and granulocyte-colony stimulating factor (G-CSF). Unfractionated heparin was eliminitively degraded to heparin oligosaccharides by an endolytic heparinase. The degraded smaller oligosaccharides (M(r) < 1000) were baseline-separated by CE under a 50 mM phosphate buffer (pH 9.0) in 10 min. Standard heparin disaccharides and larger oligosaccharides (1000 < M(r) < 8000) were all separated under optimized separation conditions. Compared with standard heparin disaccharides, smaller oligosaccharides contained one nonsulfated, two monosulfated, and two disulfated disaccharides, but trisulfated disaccharides were not found. The smaller oligosaccharides were also identified and molecular mass was deduced by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, interactions between G-CSF and the oligosaccharides were studied by using capillary zone electrophoresis (CZE) under the above separation conditions. It was found that larger oligosaccharides could interact with G-CSF while smaller oligosaccharides were not observed to bind to G-CSF under the experimental conditions. In conclusion, the purified heparinase could selectively degrade heparin into oligosaccharides and the interaction between G-CSF and heparin was correlated with the chain length of heparin.

Chromatography, Gel↗

PubMed1976

Interactions of murine leukemia virus (MuLV) with isolated lymphocytes. III. Alterations of splenic B and T cells in Friend virus-infected mice.

Lymphoid tissues of mice infected with murine leukemia virus (Friend) (MuLV-F) were examined for the presence of cellular markers of MuLV-F infection. The Friend virus-associated cell membrane antigen (FVMA) and the virus group-specific antigen (GSA) were detectable on cells from the spleen and, to a lesser degree, on cells from the bone-marrow. In contrast, neither FVMA nor GSA was found in cells from the thymus. Alterations in the B-cell and T-cell spleen populations of MuLV-F-infected mice were then studied. The proportion of Ig-positive cells declined from the initial 45% (in non-infected controls) to about 10% after 2 weeks of infection. A similar decline of theta-positive cells was noted. However, complement-bearing cells (EAC rosettes) declined even more rapidly and became undetectable in the second week after infection. The treatment of spleen cells from MuLV-F-infected mice with anti-FVMA serum plus complement in vitro reduced the number of detectable Ig-positive cells, specifically, whereas the number of theta-positive cells remained unchanged. Furthermore, B and T cells from spleens of infected mice were separated on an affinity column with anti-Ig antibody-coated beads. The initial cell suspension contained about 45% FVMA-positive cells, about 40% Ig-positive cells and about 40% theta-positive cells. Ig+ cells were retained on the column. The theta-positive cell fraction was collected in the eluate and contained very few FVMA-positive cells with some "null" cells. Most of the FVMA-positive cells were retained on the column, which strongly suggested that they were B cells. These results confirm the previous experiments which showed the selective infections of purified splenic B cells by MuLV-F in cultures.

Animals↗

PubMed1995

Natural killer (NK) cells as effectors of antibody-dependent cytotoxicity with chimeric antibodies reactive with human squamous-cell carcinomas of the head and neck.

In patients with cancer, antibody-dependent cellular cytotoxicity (ADCC) may be used as a laboratory test or for enhancing immunotherapy with murine monoclonal or chimeric mouse/human anti-tumor antibodies (mMAbs or cMAbs, respectively). We have established an ADCC assay with IgG1 cMAb SF-25, using human squamous-cell carcinoma of the head and neck (SCCHN) cell lines as targets. By flow cytometry, all SCCHN cell lines tested expressed the antigen recognized by cMAb SF-25. Trypsinization of the cell monolayers facilitated binding of cMAb SF-25 to the antigen on the cell surface of SCCHN targets. Using the PCI-50 SCCHN cell line as a target coated with this cMAb at the optimal concentration of 1.0 micrograms/ml, normal human peripheral blood mononuclear cells (PBMC, n = 28) were found to mediate ADCC at a mean level of 283 +/- 42 (SEM) lytic units (LU20/10(7) effector cells). Non-adherent monocyte-depleted PBMC and natural killer (NK) cells purified by negative selection mediated significantly higher levels of ADCC than unseparated PBMC against SCCHN targets. NK cells, defined as CD3-CD56+ cells, could be effectively armed by cMAb SF-25, as confirmed by flow cytometry and ADCC assays. IL2-activated armed NK cells mediated higher levels of ADCC than non-armed NK cells. Binding of cMAb SF-25 to NK cells and their ADCC were enhanced by pre-incubation with polyethylene glycol. Arming of NK cells with chimeric antibodies should be considered in developing novel strategies for treatment of human SCCHN, especially in the adjuvant setting.

Antibodies, Monoclonal↗

PubMed1990

N-linked glycoprotein synthesis and transport during G1 are necessary for astrocytic proliferation.

The proliferation of astrocytes, purified by a selective detachment technique from mixed glial primary cultures derived from newborn rat cerebrum, was studied. The cells were synchronized by first inducing a quiescent state by removing fetal calf serum (FCS) from the culture medium for 2 days; reversal of the quiescent state by return of serum to the culture medium caused a marked increase in DNA synthesis 12-24 hr later. 2-Deoxyglucose, an inhibitor of dolichol-linked oligosaccharide and thereby N-linked glycoprotein biosynthesis, prevented not only an increase in glycoprotein biosynthesis in G1 phase of the cell cycle but also the burst of DNA synthesis that followed during S phase. Addition of mannose to the culture medium prevented the inhibitions by deoxyglucose of both glycoprotein and DNA syntheses. These data indicated an obligatory relationship in astrocytes between dolichol-linked glycoprotein synthesis and DNA synthesis. To determine whether transport of the newly synthesized glycoproteins to the plasma membrane for incorporation therein or for secretion were necessary for DNA synthesis and astrocytic proliferation, we studied cells treated with monensin, an ionophore for monovalent cations, and an inhibitor of intracellular transport of glycoproteins. The presence of monensin in the first 12 hr after repletion of serum to synchronized astrocytes prevented progression to the S phase and cell proliferation; addition of monensin after the first 12 hr, at the onset of the S phase, had no effect on progression through S phase. Lectin-staining methods combined with fluorescence microscopy demonstrated in monensin-treated cells failure of intracellular glycoproteins to be transported to the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗

PubMedDate not supplied

A nonribosomal peptide synthetase involved in the biosynthesis of ampullosporins in Sepedonium ampullosporum.

Recently, the saprophytic ascomycete Sepedonium ampullosporum strain HKI-0053 was isolated from a basidiomycete on account of its premature induction of pigment formation in Phoma destructiva, a process often related to the neuroleptic activity of the inducing compound. The active substance was identified as the 15-membered peptaibol type peptide Ampullosporin. Although to date more than 300 peptaibols have been discovered, their biosynthetic machinery has not been characterized yet. By improving the culture conditions it was possible to grow S. ampullosporum in a submerged culture and to increase Ampullosporin production by more than three times to 33 mg/l at reduced fermentation times. The appearance of two high molecular weight proteins, HMWP1 (1.5 MDa) and HMWP2 (350 kDa) was closely related to the production of Ampullosporin during the course of fermentation. Both proteins showed a cross-reaction with antibodies against a core fragment of nonribosomal peptide synthetases (NRPSs). Biochemical characterization of the partially purified enzymes exhibited selectivity for the substrate amino acid alpha-aminoisobutyric acid (Aib). substantiating their involvement in Ampullosporin biosynthesis. Our data suggest that Ampullosporin synthetase has been isolated, and provides the basis for the characterization of the entire biosynthetic gene cluster. Furthermore, this knowledge will enable the manipulation of its NRPS template, in order to engineer mutant strains of Sepedonium ampullosporum which could produce more potent analogues of Ampullosporin.

Amino Acid Sequence↗