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Biomedical subjects

J Novotny

Publications and source records attributed to J Novotny.

At least 91 records · Page 5Linked to original sources
PubMed1994

Low incidence of invasive fungal infections after bone marrow transplantation in patients receiving amphotericin B inhalations during neutropenia.

The incidence of invasive fungal infections after bone marrow transplantation (BMT) was analyzed in 303 consecutive marrow graft recipients (allogeneic n = 271, autologous n = 27, syngeneic n = 5). All patients received inhalations with amphotericin B (10 mg twice daily) during neutropenia. The overall incidence of invasive fungal infections within the first 120 days after transplant was 3.6% (11/303; aspergillosis: 6; yeast infection: 5). Four of the 11 cases occurred early, and seven cases were observed after neutrophil recovery and discontinuation of amphotericin B inhalation treatment. Late infection was significantly associated with the development of acute graft-versus-host disease. Four of the 11 infections (early 2/4; late: 2/7) were observed in patients with a history of previous fungal infection. Other patient and treatment characteristics were not helpful in defining potential risk factors. In particular, the incidence of invasive fungal infections did not differ between patients with more or less strict reverse isolation measures. Occasional side effects such as initial mild cough and bad taste were rare, usually disappeared during continued administration, and were in no case the reason for discontinuation of treatment. These data suggest that aerosolized amphotericin B may be a useful, convenient, and efficient prophylactic antifungal regimen in BMT.

Administration, Inhalation↗
PubMed1994

The Escherichia coli Dga (MurI) protein shares biological activity and structural domains with the Pediococcus pentosaceus glutamate racemase.

The Pediococcus pentosaceus glutamate racemase gene product complemented the D-glutamate auxotrophy of Escherichia coli WM335. Amino acid sequence analysis of the two proteins revealed 28% identity, primarily in six clusters scattered throughout the sequence. Further analyses indicated secondary structure similarities between the two proteins. These data support a recent report that the dga (murI) gene product is a glutamate racemase.

Amino Acid Isomerases↗
PubMed1994

Cardiac toxicity of bone marrow transplantation: predictive value of cardiologic evaluation before transplant.

PURPOSE: This study analyses the risk of cardiac complications and its individual predictability in bone marrow transplantation (BMT). PATIENTS AND METHODS: One hundred seventy patients undergoing allogeneic (n = 150) or autologous (n = 20) BMT were evaluated by physical examination, history, rest and exercise ECG, chest x-ray, two-dimensional echocardiography, and radionuclide ventriculography (RNV) before BMT, and monitored for 3 months thereafter. RESULTS: Following BMT, cardiac toxicity occurred in eight patients (4.7%). Three patients (1.8%) developed life-threatening toxicity (pericardial effusion and left ventricular failure, n = 2; sudden cardiac arrest, n = 1). Thirty-eight patients (22%) had pathologic findings before BMT. In 22 patients, left ventricular ejection fraction (EF) determined by RNV was reduced to less than 55%. This was the only abnormality in 17 patients and was generally mild, with a lowest EF of 42%. There was no correlation between overall results of cardiologic evaluation before BMT and cardiac toxicity. Cardiotoxic events occurred more frequently in patients with a reduced EF (P < .05). However, this was restricted to minor cardiac events. Life-threatening cardiac toxicity was not significantly increased in patients with pathologic results before BMT. Moreover, none of the patients with an EF less than 50% developed cardiac toxicity. CONCLUSION: Life-threatening cardiac toxicity is rare after BMT, occurring in less than 2% of all patients. Although the occurrence of cardiac toxicity is correlated with a reduction of EF before BMT, life-threatening cardiac toxicity cannot be predicted in individual patients.

Adolescent↗
PubMed1994

Adenovirus infection of myocardial cells induces an enhanced sensitivity to beta-adrenergic agonists by increasing the concentration of the stimulatory G-protein.

Neonatal rat cardiocytes were infected with a recombinant adenovirus type 5 containing the SV40 early promoter-Gsa fusion gene in order to evaluate the presumed role of the stimulatory G-protein (Gs) in hypertrophy of myocardial cells. In vitro infection of myocardial cells with the recombinant adenovirus induced a 79-fold increase in Gs alpha mRNA and a 5-fold increase in Gs alpha protein, which was accompanied by a pronounced cell hypertrophy but not cell proliferation. Interestingly, adenovirus-infected cells displayed features of cell hypertrophy, an increase in sodium fluoride-stimulatible membrane-bound activity of adenylyl cyclase, and an enhanced beta-adrenergic sensitivity irrespective of the presence or absence of the SV40 early promoter-Gs alpha fusion gene in the virus. While the recombinant adenovirus induced a 5-fold versus 3-fold increase for plain adenovirus in cellular Gs alpha, membrane-bound Gs alpha was increased about 2-fold in both instances, which can explain similar increase in the G-protein-modulated adenylyl cyclase activity determined in membranes derived from myocardial cells infected with both types of the virus. It is concluded that adenovirus infection per se can lead to overexpression of Gs alpha and myocardial hypertrophy and thus may be of importance in the pathogenesis of virus-induced cardiomyopathy.

Adenoviruses, Human↗
PubMed1994

Self-renewal of a transplantable murine leukemia induced by co-culture with human stromal cell lines.

The PGM-1 murine leukemic cell line can be serially transplanted in syngeneic C3H/HeJ mice but cannot be maintained in in vitro culture. In response to a wide range of known growth factors, the PGM-1 cells either die or differentiate into mature granulocytes and macrophages with loss of all clonogenic (i.e. agar culture colony-forming) cells within 7 days. In this report we show that coculture with human, but not mouse, bone marrow stromal cell lines allows maintenance of clonogenic cells. One line in particular (197/17) allowed continuous expansion of clonogenic cells with no evidence of differentiation. The maintenance of clonogenic cells correlated with maintenance of tumor stem cells. Even after 9 months continuous passaging on stromal cells, the cultured cells produced tumors on injection into syngeneic mice with the same latency as cells from explanted tumors. We demonstrated that this activity was due to a soluble factor in 197/17 conditioned medium. An extensive survey of known factors, either alone or in combination, failed to reproduce this effect, implying that the effect was due to a novel factor acting on self-renewal of early stem cells.

Animals↗
PubMed1994

[Leukocyte transfusion as therapy of recurrent CML after allogenic bone marrow transplantation].

Eight patients with relapsed chronic myelogenous leukemia (CML) after allogeneic bone marrow transplantation (BMT) were treated with alpha-interferon and leukocyte transfusions of the bone marrow donor. Six patients responded with disappearance of leukemic cells (Ph1, BCR-ABL) and reestablished donor hemopoiesis. All six patients developed bone marrow hypoplasia and graft-versus-host disease (GvHD). Three of the six patients died of cerebral bleeding, infection and GvHD, respectively. The remaining three patients are alive and well at day 418, 677, 818 after leukocyte transfusions. Two patients relapsed with more advanced disease of CML after BMT and failed treatment. Donor leukocyte transfusions provide an effective therapy for patients with relapsed CML after BMT, but are associated with a high mortality due to bone marrow hypoplasia and GvHD.

Antigens, CD↗
PubMed1993

Affinity and specificity of serine endopeptidase-protein inhibitor interactions. Empirical free energy calculations based on X-ray crystallographic structures.

An empirical function was used to calculate free energy change (delta G) of complex formation between the following inhibitors and enzymes: Kunitz inhibitor (BPTI) with trypsin, trypsinogen and kallikrein; turkey ovomucoid 3rd domain (OMTKY3) with alpha-chymotrypsin and the Streptomyces griseus protease B; the potato chymotrypsin inhibitor with the protease B; and the barely chymotrypsin inhibitor and eglin-c with subtilisin and thermitase. Using X-ray coordinates of the nine complexes, we estimated the contributions that hydrophobic effect, electrostatic interactions and side-chain conformational entropy make towards the stability of the complexes. The calculated delta G values showed good agreement with the experimentally measured ones, the only exception being the kallikrein/BPTI complex whose X-ray structure was solved at an exceptionally low pH. In complexes with different enzymes, the same inhibitor residues contributed identically towards complex formation (delta G(residue) Spearman rank correlation coefficient 0.7 to 1.0). The most productive enzyme-contacting residues in OMTKY3, eglin-c, and the chymotrypsin inhibitors were found in analogous positions on their respective binding loops; thus, our calculations identified a functional (energetic) motif that parallels the well-known structural similarity of the binding loops. The delta G values calculated for BPTI complexed with trypsin (-21.7 kcal) and trypsinogen (-23.4 kcal) were similar and close to the experimental delta G value of the trypsin/BPTI complex (-18.1 kcal), lending support to the suggestion that the 10(7) difference in the observed stabilities (KA) of these two complexes reflects the energetic cost of conformational changes induced in trypsinogen during the pre-equilibrium stages of complex formation. In almost all of the complexes studied, the stabilization free energy contributed by the inhibitors was larger than that donated by the enzymes. In the trypsin-BPTI complex, the calculated delta G contribution of the amino group from the BPTI residue Lys15 (9.7 kcal) was somewhat higher than that arrived at in experiments with semisynthetic inhibitor analogs (7.5 kcal). In OMTKY3, different binding loop residues are known to affect differently the binding (delta delta G) to alpha-chymotrypsin and protease B; a good qualitative agreement was found between the calculated delta G(residue) estimates and the experimental delta delta G data (correlation coefficient 0.7). Large variations were observed in local surface complementarity and related interfacial volume in the two OMTKY3 complexes (by 20 to 60% for some side-chains).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Effect of heavy chain signal peptide mutations and NH2-terminal chain length on binding of anti-digoxin antibodies.

In certain instances, antibody variable region mutations outside of the antigen-combining site influence antigen binding. We reported previously that a heavy chain mutation (Ser-94-->Arg) decreased binding of the anti-digoxin antibody 40-150, whereas an additional signal peptide mutation at the -2 position (Gln-->Pro) causing NH2-terminal 2-residue truncation partially restored binding. To assess the combined effects on binding of two seemingly distant mutations, we constructed signal peptide mutations and NH2-terminal deletions in the presence of Ser-94 and Arg-94. Deletions of one to three amino acids had little effect on binding for Ser-94 mutants, whereas 2-residue truncations produced directly or by signal peptide mutation increased affinity approximately 40-fold for Arg-94 mutants. These observations are consistent with the reported computer-generated model of antibody 40-150. Introduction of Pro at the signal peptide -3 position in 40-150 resulted in cleavage at alternative sites, with varying effects on affinity. Introduction of Pro at -2 into the anti-digoxin antibody 26-10 resulted, unexpectedly, in expression of heavy chains with 3 extra NH2-terminal residues, causing an approximately 100-fold reduction in affinity. Thus, both extensions and deletions of the heavy chain amino terminus can enhance or reduce antigen binding, depending on the structural context of specific antigen combining sites.

Amino Acid Sequence↗
PubMed1993

Heavy chain position 50 is a determinant of affinity and specificity for the anti-digoxin antibody 26-10.

Antibody produced by a variant of the murine antidigoxin hybridoma 26-10 has reduced affinity for digoxin but enhanced recognition of the digoxin 12-hydroxyl due to a Tyr to His substitution at heavy chain position 50 (Schildbach, J. F., Panka, D. J., Parks, D. R., Jager, G. C., Novotny, J., Herzenberg, L. A., Mudgett-Hunter, M., Bruccoleri, R. E., Haber, E., and Margolies, M. N. (1991) J. Biol. Chem. 266, 4640-4647). Consistent with these data, the 26-10 Fab-digoxin x-ray crystal structure (Jeffrey, P. D., Strong, R. K., Sieker, L. C., Chang, C. Y., Campbell, R. L., Petsko, G. A., Haber, E., Margolies, M. N., and Sheriff, S. (1993) Proc. Natl. Acad. Sci. U. S. A., in press) reveals that Tyr-50 contacts a region of digoxin that includes the hapten-12 carbon. To determine the effects of other heavy chain position 50 substitutions, mutant antibodies were engineered, and their affinities for digoxin and digoxin analogues were measured. The affinity of the mutant antibodies for digoxin roughly correlates with the size of the position 50 side chain. Substitutions of Trp or Phe have no effect on affinity, whereas substitutions of Asn, His, Leu, Ala, Gly, and Asp confer progressively lower affinities. Although Trp and Phe mutants exhibit wild-type specificity, Asn and Asp mutants have improved affinity for digoxin relative to digitoxin (12-deshydroxydigoxin). Leu, Ala, and Gly mutants have improved affinity for 12-acetyldigoxin relative to digoxin as compared with 26-10. These results indicate that position 50 is a determinant of both antibody affinity and fine specificity for antibody 26-10 and that single-amino acid substitutions can alter antibody fine specificity. Models of the mutants were computationally constructed, and haptens were docked into the modeled binding sites. The results suggest that 12-acetyldigoxigenin occupies different orientations in the 26-10 and in the Ala mutant binding sites, resulting in altered binding.

Amino Acids↗
PubMed1993

A new alpha-thalassemia-2 deletion resulting in microcytosis and hypochromia and in vitro chain imbalance in the heterozygote.

We have detected a novel alpha-thalassemia-2 with a large (18+ kb) deletion involving the alpha 1- and theta 1-globin genes and the 3' hypervariable region sequence. Unexpectedly, the heterozygote had a mild anemia with a marked microcytosis and hypochromia, and an in vitro alpha/beta chain synthesis ratio of 0.62-0.66. It is suggested that the deletion includes a sequence that is involved in the in cis regulation of the alpha 2-globin gene.

Adult↗
PubMed1993

Modulation of antibody affinity by a non-contact residue.

Antibody LB4, produced by a spontaneous variant of the murine anti-digoxin monoclonal antibody 26-10, has an affinity for digoxin two orders of magnitude lower than that of the parent antibody due to replacement of serine with phenylalanine at position 52 of the heavy chain variable region (Schildbach, J.F., Panka, D.J., Parks, D.R., et al., 1991, J. Biol. Chem. 266, 4640-4647). To examine the basis for the decreased affinity, a panel of engineered antibodies with substitutions at position 52 was created, and their affinities for digoxin were measured. The antibody affinities decreased concomitantly with increasing size of the substituted side chains, although the shape of the side chains also influenced affinity. The crystal structure of the 26-10 Fab complexed with digoxin (P.D.J., R.K. Strong, L.C. Sieker, C. Chang, R.L. Campbell, G.A. Petsko, E.H., M.N.M., & S.S., submitted for publication) shows that the serine at heavy chain position 52 is not in contact with hapten, but is adjacent to a tyrosine at heavy chain position 33 that is a contact residue. The mutant antibodies were modeled by applying a conformational search procedure to position side chains, using the 26-10 Fab crystal structure as a starting point. The results suggest that each of the substituted side chains may be accommodated within the antibody without substantial structural rearrangement, and that none of these substituted side chains are able to contact hapten. These modeling results are consistent with the substituents at position 52 having only an indirect influence upon antibody affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Characterization of an anti-digoxin antibody binding site by site-directed in vitro mutagenesis.

In vitro mutagenesis and immunoglobulin gene transfection were used to investigate the binding site of a monoclonal antibody, 2610, that binds to digoxin, a cardiac glycoside. A computer model was generated in order to select sites in the complementarity determining regions (CDR) that would participate in binding. Residues in the CDR segments were chosen that possess high solvent exposure and were located in a putative cleft. The cloned heavy and light chain variable regions were subjected to in vitro mutagenesis at these sites. The mutated variable regions in M13 were then subcloned into expression vectors and transfected. The affinities and specificity binding properties of the resultant expressed antibodies were measured. Many of the mutants of the putative contact residues showed significant but not major alterations of binding properties. Since most of the residues in the binding site are non-polar and aromatic and since many of the mutations resulted in only modest binding changes, we theorize that much of the high affinity binding (> 10(9)/M) is the cumulation of many weak interactions, arising from dispersion forces and hydrophobic effects in the pocket. Preliminary mutagenesis of two L chain positions proposed to bind to the lactone end of digoxin have larger binding effects. Specificity studies show that the mutants more frequently possess altered binding to the lactone ring of digoxin that altered binding to other digoxin moieties. The data are most suggestive of a model in which lactone is at the bottom of a binding pocket, followed by the steroid nucleus and then by the sugar moiety extruding out of the pocket. The binding information may be useful in understanding the immune response to large, hydrophobic haptens.

Amino Acid Sequence↗
PubMed1993

Interferon-alpha and donor buffy coat transfusions for treatment of relapsed chronic myeloid leukemia after allogeneic bone marrow transplantation.

Eight patients with chronic myeloid leukemia relapse after allogeneic BMT were treated with IFN-alpha and buffy coat transfusions (BC) of the bone marrow donor. The antileukemic effect of this treatment was directly demonstrated in 4 patients by the disappearance of Philadelphia chromosome-positive metaphases or the loss of detectable BCR-ABL transcripts by polymerase chain reaction. In 2 patients in whom cytogenetic or polymerase chain reaction analysis was not performed, a change in hemopoietic chimerism with recurrence of donor-type hemopoiesis was demonstrated. Two patients, both treated in advanced stages of hematological relapse after BMT, did not respond. However, severe side effects of the treatment were observed: graft-versus-host disease (GVHD) occurred in 5 patients. Two of these patients progressed to severe chronic GVHD and 1 patient ultimately died of this complication. GVHD occurred in 5 of the 6 responding patients; one patient responded without developing clinical symptoms of GVHD. Six patients developed bone marrow hypoplasia after IFN/BC treatment, and pancytopenia occurred in 4 patients. None of these 4 patients recovered spontaneously and 2 patients died of complications of pancytopenia (cerebral bleeding, infection). Our results demonstrate that treatment of chronic myeloid leukemia relapse with IFN and BC transfusions is highly effective in patients with relapse in chronic phase. The occurrence of GVHD and pancytopenia, however, resulted in a high treatment-associated morbidity and mortality. Whereas a response to treatment was observed in 1 patient without GVHD, indicating that GVHD and a graft-versus-leukemia effect may be clinically separable, bone marrow hypoplasia occurred in all responding patients.

Adult↗
PubMed1992

Similarity between fluorescein-specific T-cell receptor and antibody in chemical details of antigen recognition.

A computer-generated model of the single-chain variable V alpha V beta fragment of the RFL3.8 T-cell receptor (TCR) specific for fluorescein served as a starting point for mutagenesis aimed at identification of its antigen-contacting residues. Selected backbone segments of the model representing regions of prominent sequence similarity between antibodies and TCRs were least-squares superimposed onto the corresponding segments of the crystallographically resolved 4-4-20 antibody complexed with its antigen, fluorescein. The superimposition placed the antibody-bound fluorescein molecule close to a cavity on the surface of the TCR model formed by the complementarity-determining region (CDR) loops. Some of the TCR cavity forming loops displayed sequence motifs related to canonical CDR loops previously found in antibodies. Six putative amino acid contacts were identified and single-chain TCRs with mutations at each of these positions were expressed in Escherichia coli, purified, refolded, and assayed for fluorescein binding. Five of the six mutations resulted in a loss of detectable binding. These RFL3.8 antigen combining site residues are distributed among the beta 3, alpha 1, and alpha 2 CDR loops and show striking chemical similarity to the known fluorescein contact residues on 4-4-20. Thus, antibodies and TCRs are similar both in their overall architecture and in the chemical details of specific antigen recognition.

Amino Acid Sequence↗
PubMed1992

Support of human cord blood progenitor cells on human stromal cell lines transformed by SV40 large T antigen under the influence of an inducible (metallothionein) promoter.

We describe the development of a human bone marrow (BM) culture system which allows study of the interaction of stromal cell lines (SCL) and highly purified hematopoietic progenitor cells. Normal BM stromal cells were electroporated with a plasmid containing the simian virus 40 (SV40) large T antigen (SV40 T Ag) under the control of a synthetic metallothionein promoter (MT4); this construct is designated MT4 SV40 T Ag. SCL in which the rate of proliferation could be controlled by altering the zinc (Zn) concentration were characterized, demonstrating that the SCL were heterogeneous with respect to G-CSF and GM-CSF production. Suppression of SCL proliferation on removal of Zn made it possible to use these lines in coculture with purified CD34+ progenitor cells from umbilical cord blood. The ability to control proliferation of SCL has allowed us to maintain the survival and expansion of colony-forming cells in culture for up to 2 months. These lines have enabled us to test for stromal cell characteristics at a clonal level and provided us with a tool to analyze the events leading to lineage commitment and hematopoietic differentiation, as demonstrated by suppression of hematopoiesis by an antibody directed against the c-kit molecule.

Antigens, CD↗
PubMed1992

Distances between the antigen-binding sites of three murine antibody subclasses measured using neutron and X-ray scattering.

For three different murine immunoglobulins (IgG subclasses 1, 2a, and 2b), the distances between their antigen-binding sites have been measured using neutron scattering from deuterated antigens complexed with proteated IgG. Neutron-scattering data were measured for each antibody-antigen complex in a 41% D2O solvent. Unlike the proteated antibody molecule, the deuterated antigens are strongly contrasted against the 41% D2O solvent and give rise to a scattering profile that contains an interference term related to the distance between the deuterated antigens. For all three subclasses, the damping of this interference term, which gives information on the relative flexibility of the antigen-binding sites, indicates that a single distance is inadequate to describe the observed scattering and a distribution of distances is needed. The scattering profile has been modeled for each subclass to give the mean distance between the antigens and the variance of this distance. For all three IgG subclasses, the mean distance is between 117 and 134 A, and the variance is large (approximately 40 A), indicating a high degree of flexibility of the Fab arms. Small-angle X-ray scattering measurements on the same samples are consistent with the neutron-scattering results.

Animals↗
PubMed1991

A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties.

A strategy for the production of small, soluble, single-chain T-cell receptor (scTCR) fragments that carry an intact TCR antigen-combining site is presented. The rationale is based on structural similarity between TCR and antibody molecules and use of computer modeling methods to derive a model structure of a human scTCR variable (V)-domain dimer. A gene encoding the RFL3.8 TCR protein, specific for the hapten fluorescein in the context of major histocompatibility complex class II and composed of one V alpha and one V beta domain joined via a flexible peptide linker, was assembled in an Escherichia coli plasmid. Subsequently, the protein was produced in a bacterial expression system, purified, refolded, and found to be poorly soluble at neutral pH in aqueous buffers. An inspection of the computer-generated V alpha-V beta domain model showed several surface exposed hydrophobic residues. When these were replaced by water-soluble side chains via site-directed mutagenesis of the corresponding gene, a soluble protein resulted and was shown to have antigen-binding properties equivalent to those of the intact TCR of the RFL3.8 T-cell clone. These results demonstrate the feasibility of obtaining TCR fragments endowed with antigen-combining properties by protein engineering in E. coli.

Amino Acid Sequence↗