ABSTRACT
During the development of indazolylpyrimidines as novel and potent inhibitors of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) tyrosine kinase, we observed that some human tumour xenografts are more sensitive to VEGFR2 kinase inhibitors than others. A better understanding of the basis for this differential response may help to identify a predictive marker that would greatly aid in the identification of a suitable patient population for treatment. One representative compound from the indazolylpyrimidine series is GW654652 that inhibited all three VEGFRs with similar potency. The inhibition of VEGFR2 kinase by GW654652 was about 150 to >8800 more potent than the inhibition of eight other kinases tested. GW654652 inhibited VEGF- and bFGF-induced proliferation in endothelial cells with an IC(50) of 110 and 1980 nM, respectively, and has good pharmacokinetic profile in mouse and dog. We investigated the association between VEGF and VEGFR2 expression and the antitumour efficacy of GW654652, in various xenograft models. Statistically significant associations were observed between the antitumour efficacy of GW654652 in xenografts and VEGF protein (P=0.005) and VEGFR2 expression (P=0.041). The oral dose of GW654652 producing 50% inhibition of tumour growth (ED(50)) decreased with increasing levels of VEGF (r=-0.94); and, in contrast, the ED(50) increased with the increased expression of VEGFR2 (r=0.82). These results are consistent with the observed inverse correlation between VEGF and VEGFR2 expression in tumours. These findings support the hypothesis that VEGF and VEGFR2 expression by tumours may predict the therapeutic outcome of VEGFR kinase inhibitors.
Subject(s)
Imidazoles/pharmacology , Pyrimidines/pharmacology , Vascular Endothelial Growth Factor Receptor-2/physiology , Animals , Biomarkers, Tumor , Cell Division , Disease Models, Animal , Endothelial Cells/physiology , Enzyme Inhibitors/pharmacology , Humans , Mice , Mice, Nude , Prognosis , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Tumor Cells, Cultured , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitorsABSTRACT
A series of 2'-deoxy analogues of the antiviral agent 5,6-dichloro-2-isopropylamino-1-(beta-L-ribofuranosyl)-1H-benzimidazole (1263W94) were synthesized and evaluated for activity against human cytomegalovirus (HCMV) and for cytotoxicity. The 2-substituents in the benzimidazole moiety correspond to those that were used in the 1263W94 series. In general, as was found in the 1263W94 series, cyclic and branched alkylamino groups were needed for potent activity against HCMV. Three analogues 3a, 3b and 3d were as potent as 1263W94. Further evaluation of two analogues, 3a and 3b, suggested that these 2'-deoxy analogues may act via a novel mechanism of action similar to that of 1263W94. These 2'-deoxy analogues generally lacked cytotoxicity in vitro. Pharmacokinetic parameters in mice and protein binding properties of 3a were quite similar to 1263W94. However, the oral bioavailability of 3a was only half of that observed for 1263W94.
Subject(s)
Antiviral Agents/chemical synthesis , Benzimidazoles/chemical synthesis , Cytomegalovirus/drug effects , Ribonucleosides/chemical synthesis , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Benzimidazoles/chemistry , Benzimidazoles/pharmacokinetics , Benzimidazoles/pharmacology , Biological Availability , Cells, Cultured , Humans , Magnetic Resonance Spectroscopy , Male , Mice , Mice, Inbred Strains , Ribonucleosides/chemistry , Ribonucleosides/pharmacokinetics , Ribonucleosides/pharmacologyABSTRACT
1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.
Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , Anti-HIV Agents/pharmacokinetics , Dideoxynucleosides/pharmacokinetics , Acquired Immunodeficiency Syndrome/metabolism , Adenosine Deaminase/metabolism , Administration, Oral , Animals , Anti-HIV Agents/blood , Anti-HIV Agents/chemistry , Anti-HIV Agents/urine , Antiviral Agents/blood , Antiviral Agents/pharmacokinetics , Area Under Curve , Biotransformation , Cells, Cultured , Dideoxynucleosides/blood , Dideoxynucleosides/chemistry , Dideoxynucleosides/urine , Drug Resistance, Microbial , Female , HIV-1/drug effects , Half-Life , Humans , Injections, Intravenous , Macaca fascicularis , Male , Rats , Structure-Activity RelationshipABSTRACT
Patients infected with the human immunodeficiency virus experienced severe hematopoietic toxicity after treatment with the deoxynucleoside analog 3'-fluorothymidine (FLT). Using several methods for the analysis of genome integrity, including histochemical staining of the 3' ends of DNA and both conventional and pulsed-field agarose gel electrophoresis, we demonstrated that FLT caused extensive DNA fragmentation in CEM cells that was not observed when these cells were treated with other, less toxic thymidine analogs. In addition, a distinctive pattern of small DNA fragments that is characteristic of cells in the process of programmed cell death was observed in the genomic DNA of CEM cells treated with FLT. We conclude that FLT induces DNA fragmentation and apoptosis in a human cell line of hematopoietic origin, and we offer this observation as a possible explanation for the severe toxicity of FLT observed in vivo.
Subject(s)
Antiviral Agents/toxicity , Apoptosis , CD4-Positive T-Lymphocytes/drug effects , DNA Damage , Dideoxynucleosides/toxicity , Antineoplastic Agents, Phytogenic/toxicity , Antiviral Agents/metabolism , CD4-Positive T-Lymphocytes/metabolism , Camptothecin/toxicity , Cell Line , DNA/drug effects , DNA/metabolism , Dideoxynucleosides/metabolism , Electrophoresis, Agar Gel , Humans , Zidovudine/metabolismABSTRACT
Inhibition of in vitro colony formation of human hematopoietic progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid) by the antiviral nucleoside drugs alovudine, zalcitabine, zidovudine, ganciclovir, stavudine, didanosine, lamivudine, and acyclovir was measured. Significant correlations between in vitro 50% inhibitory concentrations and the daily human exposures (area under the concentration-time curve from 0 to 24 h; in micromolar.hour) of these chronically administered drugs in human immunodeficiency virus-positive patients that induced neutropenia or anemia were demonstrated by both linear regression and Spearman rank-order analyses. These quantitative correlations allow estimation of the exposure at which bone marrow toxicity may occur with candidate compounds.
Subject(s)
Antiviral Agents/adverse effects , Antiviral Agents/pharmacokinetics , Antiviral Agents/toxicity , Hematopoietic Stem Cells/drug effects , Cells, Cultured , Colony-Forming Units Assay , Dideoxynucleosides/adverse effects , Dideoxynucleosides/pharmacokinetics , Dideoxynucleosides/toxicity , Humans , Linear Models , Zalcitabine/adverse effects , Zalcitabine/pharmacokinetics , Zalcitabine/toxicity , Zidovudine/adverse effects , Zidovudine/pharmacokinetics , Zidovudine/toxicityABSTRACT
4(S)-(6-Amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol (IsoddA) is the most antivirally active member of a novel class of optically active isomeric dideoxynucleosides in which the base has been transposed from the natural 1' position to the 2' position and the absolute configuration is (S,S). IsoddA was active against human immunodeficiency virus type 1 (HIV-1) (strain IIIB), HIV-2 (strain ZY), and HIV-1 clinical isolates. Combinations of the compound with zidovudine (3'-azido-3'-deoxythymidine), 2',3'-dideoxyinosine, or 5-fluoro-2'-deoxy-3'-thiacytidine showed synergistic inhibition of HIV. A moderate reduction of activity was observed with clinical isolates resistant to zidovudine. An IsoddA-resistant virus (eightfold-increased 50% inhibitory concentration) was selected in vitro by repeated passage of HIV-1 (HXB2) in the presence of increasing concentrations of IsoddA. The reverse transcriptase-coding region of the mutant virus contained a single base change resulting in a change at codon 184 from Met to Val. IsoddA was also active against hepatitis B virus (HBV) in vitro; however, it lacked substantial selective activity in an in vivo HBV model. IsoddA was inefficiently phosphorylated in CEM cells; however, the half-life of the triphosphate was 9.4 h, and IsoddATP was a potent inhibitor of HIV-1 reverse transcriptase, with a Ki of 16 nM. The cytotoxicity 50% inhibitory concentrations of IsoddA were greater than 100 microM for CEM, MOLT-4, IM9, and the HepG2-derived HBV-infected 2.2.15 (subclone P5A) cell lines but were 12 and 11 microM for human granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Antiviral Agents/pharmacology , Dideoxyadenosine/analogs & derivatives , Adenosine Deaminase/metabolism , Animals , Antibody-Dependent Cell Cytotoxicity , Antiviral Agents/metabolism , Antiviral Agents/pharmacokinetics , Cells, Cultured , DNA, Viral/analysis , Dideoxyadenosine/metabolism , Dideoxyadenosine/pharmacokinetics , Dideoxyadenosine/pharmacology , Drug Resistance, Microbial , Erythroid Precursor Cells/physiology , HIV-1/drug effects , HIV-2/drug effects , Hepatitis B virus/drug effects , Humans , Nucleic Acid Synthesis Inhibitors , Phosphorylation , Polymerase Chain Reaction , Rats , Viral Plaque AssayABSTRACT
beta-L-2',3'-Dideoxycytidine (beta-L-ddC) and beta-L-5-fluoro-2',3'-dideoxycytidine (5-F-beta-L-ddC) were prepared and shown to have potent activity against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). These compounds were compared with beta-D-2',3'-dideoxycytidine (beta-D-ddC) and two beta-L-oxathiolane nucleosides (beta-L-3'-thio-2',3'-dideoxycytidine and beta-L-5-fluoro-3'-thio-2',3'-dideoxycytidine) in terms of anti-HIV and anti-HBV activity, cytotoxicity, and development of HIV-1 resistance. Compared with beta-D-ddC, the beta-L-dideoxycytidine nucleosides had similar anti-HIV-1 activities, significantly greater anti-HBV activities, and decreased toxicities to a B-cell line, T-cell lines, and human bone marrow progenitor cells. HIV-1 strains resistant to beta-D-ddC were susceptible to the beta-L-ddC analogs. Compared with the beta-L-oxathiolane nucleosides, beta-L-ddC and 5-F-beta-L-ddC had similar anti-HIV-1 activities, decreased anti-HBV activities, and greater toxicities to B- and T-cell lines and bone marrow progenitor cells. There were similarities between the beta-L-ddC and beta-L-oxathiolane nucleosides in the rate of development and pattern of resistant HIV-1 selection. While the in vitro activity and cytotoxicity profiles of the beta-L-ddC nucleosides differed from those of the beta-D-ddC and beta-L-oxathiolane nucleosides, the data presented herein suggest that the sugar configuration of a dideoxynucleoside analog may play a major role in the rate of development and the pattern of HIV-1 resistance.
Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Viruses/drug effects , Zalcitabine/analogs & derivatives , Animals , Cattle , Cell Line , Cell Survival/drug effects , Colony-Forming Units Assay , Deoxycytidine Kinase/metabolism , Drug Resistance, Microbial , HIV Reverse Transcriptase , HIV-1/drug effects , Hepatitis B virus/drug effects , Humans , Reverse Transcriptase Inhibitors , Structure-Activity Relationship , Thymus Gland/enzymology , Zalcitabine/chemistry , Zalcitabine/pharmacology , Zidovudine/pharmacologyABSTRACT
The racemic isosteric phosphonate of ganciclovir monophosphate (BW2482U89, SR3745, [3-((2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-4- hydroxybutyl]phosphonic acid, 1) has potent and selective in vitro activity against human cytomegalovirus. An enantiospecific synthesis of the R-enantiomer of compound 1 starting from L-arabinose was developed. The synthesis involved (1) the preparation of a chiral acyclic moiety, (2) the coupling of the chiral acyclic moiety to diacetylguanine, (3) the introduction of phosphorus, and (4) the final deprotection. The R-enantiomer, which has stereochemistry analogous to the natural compound GMP, was tested against human cytomegalovirus and had an IC50 of 1.7 microM, which was approximately 2-fold more active than the racemic material. Both racemic and chiral compounds were less toxic than ganciclovir to bone marrow progenitor cells in an in vitro assay.
Subject(s)
Antiviral Agents/chemical synthesis , Cytomegalovirus/drug effects , Ganciclovir/analogs & derivatives , Guanine/analogs & derivatives , Antiviral Agents/pharmacology , Bone Marrow Cells , Ganciclovir/chemical synthesis , Ganciclovir/pharmacology , Ganciclovir/toxicity , Guanine/chemical synthesis , Guanine/pharmacology , Guanine/toxicity , Guanosine Monophosphate/chemistry , Hematopoietic Stem Cells/drug effects , Humans , Magnetic Resonance Spectroscopy , Molecular Structure , Phosphorus/chemistry , StereoisomerismABSTRACT
Four subgroup feline leukemia viruses (FeLV) were tested for their ability to infect primary cultures of human bone marrow in vitro. Using three sequential exposures to both concentrated and unconcentrated virus, FeLV infection of human bone marrow was documented by indirect immunofluorescence, ELISA, and polymerase chain reaction (PCR) assays. Based on comparison to known standards, it was estimated that between 5 and 10% of the bulk human marrow culture could be infected by FeLV. Further evidence for FeLV reverse transcription, integration, and expression was obtained using specific PCR assays.
Subject(s)
Bone Marrow/microbiology , Leukemia Virus, Feline/physiology , Bone Marrow Cells , Cells, Cultured , DNA, Viral/analysis , HumansABSTRACT
The anti-hepatitis B (anti-HBV) activities of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (2'-deoxy-3'-thia-5-fluorocytosine [FTC]) were studied by using an HBV-transfected cell line (HepG2 derivative 2.2.15, subclone P5A). The (-) isomer was found to be a potent inhibitor of viral replication, with an apparent 50% inhibitory concentration of 10 nM, while the (+) isomer was found to be considerably less active. Both isomers showed minimal toxicity to HepG2 cells (50% inhibitory concentration, > 200 microM) and showed minimal toxicity in the human bone marrow progenitor cell assay. In accord with the cellular antiviral activity data, the 5'-triphosphate of (-)-FTC inhibited viral DNA synthesis in an endogenous HBV DNA polymerase assay, while the 5'-triphosphate of the (+) isomer was inactive. Unphosphorylated (-)-FTC did not inhibit product formation in the endogenous assay, suggesting that the antiviral activity of the compound is dependent on anabolism to the 5'-triphosphate. Both (-)- and (+)-FTC were anabolized to the corresponding 5'-triphosphates in chronically HBV-infected HepG2 cells. The rate of accumulation and the steady-state concentration of the 5'-triphosphate of (-)-FTC were greater. Also, (-)-FTC was not a substrate for cytidine deaminase and, therefore, is not subject to deamination and conversion to an inactive uridine analog. The (+) isomer is, however, a good substrate for cytidine deaminase.
Subject(s)
Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Zalcitabine/analogs & derivatives , Animals , Antiviral Agents/toxicity , Cell Survival/drug effects , Cytidine Deaminase/metabolism , DNA, Viral/biosynthesis , DNA, Viral/drug effects , Emtricitabine/analogs & derivatives , Growth Inhibitors/toxicity , Hepatitis B virus/genetics , Humans , Macaca fascicularis , Phosphorylation/drug effects , Stereoisomerism , Substrate Specificity , Zalcitabine/pharmacology , Zalcitabine/toxicityABSTRACT
The objective of this study was to determine how recombinant human granulocyte colony-stimulating factor (rhG-CSF) affects hematopoiesis in normal cats. Recombinant human G-CSF was given at 3.0, 5.0, and 10.0 micrograms/kg to two cats each s.c. twice daily for 21 days. This resulted in significant (p less than 0.01) elevations of peripheral blood neutrophils from 3.0- to 9.2-fold above pretreatment levels and significantly (p less than 0.02) above levels of nontreated control cats (n = 4). A statistically significant dose-related response was not seen at these dosages in any parameter evaluated. The period of maximum neutrophilia occurred between days 10 and 14 of rhG-CSF treatment, with maximum neutrophil counts ranging from 20,370 cells/microliters to 61,400 cells/microliters (normal is less than 12,500). Lymphocytosis (greater than 7000 lymphocytes/microliters) and monocytosis (greater than 850 monocytes/microliters) were observed in 50% of the cats receiving rhG-CSF during the period of maximal neutrophil stimulation. Monocyte counts in treated cats were significantly (p less than 0.01) elevated over those of treatment controls on days 12-17. Lymphocyte numbers in rhG-CSF-treated cats were significantly elevated (p less than 0.05) over pretreatment controls on days 12 and 14 of rhG-CSF treatment. No significant changes were observed in reticulocyte counts, platelet counts, or hematocrit levels. By day 19, neutrophil levels had dropped significantly (p less than 0.01) from the maximum neutrophil levels, with one cat attaining a normal blood neutrophil count by day 21 of rhG-CSF treatment. Marrow aspirates revealed an overall increase in marrow cellularity through day 14 of treatment in rhG-CSF-treated cats, with increased myeloid:erythroid ratios (two- to ninefold) over those of nontreated controls. The erythroid and lymphoid component of the marrow decreased from day 0 to day 14, whereas the early myeloid progenitors (myeloblasts, progranulocytes, and myelocytes) increased significantly (p less than 0.05). No significant differences in the percentage of later myeloid forms in the marrow were observed over the treatment period. In vitro colony-forming assays of marrow obtained from treated cats revealed increases in granulocyte-macrophage colony-forming units (CFU-GM) through day 14, with subsequent decreases by day 21 of rhG-CSF treatment. Recombinant human G-CSF was also effective at in vitro stimulation of feline marrow cells from untreated cats in a dilution study, with maximal CFU-GM formation at 0.1 microgram rhG-CSF/ml assay.(ABSTRACT TRUNCATED AT 400 WORDS)
Subject(s)
Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoiesis/drug effects , Animals , Bone Marrow Cells , Cats , Female , Granulocytes/cytology , Leukocyte Count/drug effects , Lymphocytes/cytology , Male , Monocytes/cytology , Recombinant Proteins , Species Specificity , Time FactorsABSTRACT
The anti-human immunodeficiency virus (HIV) activity and hemopoietic toxicity of zidovudine (AZT) and didanosine (dideoxyinosine;ddI), alone and in combination, were assessed in a variety of cell types. AZT was more potent than ddI as an inhibitor of HIV in vitro. Synergistic inhibition of HIV by the combination of these agents was observed in MT4 cells, peripheral blood lymphocytes, and macrophages. Toxicity assessment in vitro by using progenitor (erythroid and granulocyte-macrophage) colony-forming assays with normal human bone marrow showed ddI to be less toxic than AZT. Addition of inhibitory concentrations of ddI to AZT resulted in additive inhibition of progenitor CFUs. These in vitro findings suggest that combinations of ddI and AZT at appropriately modified doses may provide an enhanced degree of selectivity in anti-HIV chemotherapy.
Subject(s)
Bone Marrow Cells , Didanosine/pharmacology , HIV/drug effects , Stem Cells/drug effects , Zidovudine/pharmacology , Bone Marrow/drug effects , Bone Marrow Diseases/chemically induced , Bone Marrow Diseases/physiopathology , Cell Division/drug effects , Colony-Forming Units Assay , Drug Synergism , Erythroid Precursor Cells/drug effects , Humans , In Vitro Techniques , RNA-Directed DNA Polymerase/metabolismSubject(s)
Anemia, Aplastic/veterinary , Cat Diseases/microbiology , Feline Acquired Immunodeficiency Syndrome/microbiology , Leukemia Virus, Feline/genetics , Anemia, Aplastic/microbiology , Animals , Cats , Cytopathogenic Effect, Viral , Leukemia Virus, Feline/physiology , Repetitive Sequences, Nucleic Acid , Virus ReplicationABSTRACT
Although feline leukemia viruses (FeLV) cause a spectrum of proliferative and anti-proliferative diseases in vivo, in vitro studies demonstrating cell lineage-specific pathogenic properties of feline retroviruses have been rare. We describe here an efficient in vitro system that demonstrates the selective cytopathic effect of a molecularly cloned anemogenic FeLV (FeLV-Sarma-subgroup C; FSC) on erythroid progenitor cells. Forty-eight-hour coculture of normal feline bone marrow mononuclear cells with an underlayer of FSC-infected feline fibroblasts (FeF) resulted in infection of 60% to 90% of marrow mononuclear cells and pronounced depletion of early erythroid progenitor cells (BFUe). The dramatic depletion of BFUe was specific for FSC and did not occur in marrow cells infected with a molecularly cloned nonanemogenic subgroup A FeLV (FeLV 1161E; F6A). The ablation of BFUe by FSC in vitro paralleled both the decrease in BFUe and the induction of aplastic anemia in vivo. This combination of marrow cell infection by coculture and colony-forming unit (CFU) assessment by methylcellulose assay provides a reliable in vitro technique for studies of mechanisms involved in retrovirus-induced marrow aplasias.
Subject(s)
Anemia, Aplastic/etiology , Erythrocytes/pathology , Leukemia Virus, Feline , Anemia, Aplastic/microbiology , Anemia, Aplastic/pathology , Animals , Bone Marrow , Cats , Cells, Cultured , Colony-Forming Units Assay , Erythrocytes/microbiology , Erythropoiesis , Hematopoietic Stem Cells/microbiology , Hematopoietic Stem Cells/pathology , Leukemia, Experimental/blood , Leukemia, Experimental/microbiology , Leukemia, Experimental/pathology , Species Specificity , Time FactorsABSTRACT
Intra-bone marrow inoculation of cells infected with molecularly cloned feline retrovirus (FeLV-C-Sarma [FSC]) associated with aplastic anemia was examined to test the hypothesis that cell-to-cell transmission of virus might facilitate marrow cell infection and anemogenesis, a possibility suggested by in-vitro co-culture experiments. IBM inoculation of either FSC-infected feline marrow cells or fibroblasts of weanling cats bypassed age-related restriction of FSC replication, initiated viremia, caused irreversible depletion of erythroid burst forming units, and induced rapid fatal aplastic anemia. A second significant finding observed with FSC infection was pronounced systemic lymphoid depletion. The direct bone marrow inoculation system described facilitates experimental study of retrovirus-target cell interactions involved in erythroid aplasia.
Subject(s)
Anemia, Aplastic/veterinary , Cat Diseases/microbiology , Leukemia Virus, Feline/pathogenicity , Anemia, Aplastic/microbiology , Animals , Bone Marrow/microbiology , Cats , Cloning, Molecular , Colony-Forming Units Assay , Hematopoiesis , Humans , Leukocyte Count , Lymph Nodes/pathology , Methylprednisolone/pharmacologyABSTRACT
Feline leukemia virus (FeLV) C-Sarma (or FSC) is a prototype of subgroup C FeLVs, which induce fatal aplastic anemia in outbred specific-pathogen-free (SPF) cats. FeLV C isolates also possess an extended host range in vitro, including an ability, unique among FeLVs, to replicate in guinea pig cells. To identify the viral determinants responsible for the pathogenicity and host range of FSC we constructed a series of proviral DNAs by exchanging gene fragments between FSC and FeLV-61E (or F6A), the latter of which is minimally pathogenic and whose host range in vitro is restricted to feline cells. Transfer of an 886-base-pair (bp) fragment of FSC, encompassing the codons for 73 amino acids at the 3' end of pol (the integrase/endonuclease gene) and the codons for 241 amino acids of the N-terminal portion of env [the extracellular glycoprotein (gp70) gene], into the F6A genome was sufficient to confer onto chimeric viruses the ability to induce fatal aplastic anemia in SPF cats. In contrast, no chimera lacking this sequence induced disease. When assayed in vitro, all chimeric viruses containing the 886-bp fragment of FSC acquired the ability to replicate in heterologous cells, including dog and guinea pig cells. Thus, the pathogenic and the host range determinants of the feline aplastic anemia retrovirus colocalize to a 3' pol-5' env region of the FSC genome and likely reside within a region encoding 241 amino acid residues of the N terminus of the extracellular glycoprotein.
Subject(s)
Anemia, Aplastic/veterinary , Leukemia Virus, Feline/pathogenicity , Viral Envelope Proteins/genetics , Anemia, Aplastic/microbiology , Animals , Base Sequence , Cattle , Guinea Pigs , Leukemia Virus, Feline/growth & development , Molecular Sequence Data , Species SpecificityABSTRACT
Feline and human mucopolysaccharidosis VI (MPS VI or Maroteaux-Lamy syndrome) are inherited autosomal recessive deficiencies of lysosomal enzyme arylsulphatase B. Affected cats and children exhibit lesions caused by incompetent degradation, retinal atrophy and excessive urinary excretion of dermatan facial dysmorphia, corneal stromal opacities, leukocyte granulation, retinal atrophy and excessive urinary excretion of dermatan sulphate--and usually die before adulthood. Most attempts to treat humans affected with MPS VI or other mucopolysaccharidoses have been ineffective or logistically prohibitive, but allogeneic bone marrow transplantation (BMT) offers promise for cure of certain inborn errors of metabolism. Engraftment of normal donor marrow may endow the enzyme-deficient recipient with a continuous source of enzyme-competent blood cells and tissue macrophages to facilitate degradation of stored substrate and to prevent genesis of further malformations. To test this hypothesis, we performed allogeneic BMT in a 2-year-old male Siamese cat with advanced MPS VI. Here we describe BMT-induced correction of this hereditary enzyme deficiency.