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Biomedical subjects

R Khan

Publications and source records attributed to R Khan.

At least 19 recordsLinked to original sources
PubMed1992

Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA.

The nucleocapsid protein (NC) of all animal retroviruses, encoded by the gag gene, is the major structural protein of the core ribonucleoprotein complex, bound to genomic RNA in mature virions. NC is also thought to play one or more accessory roles in reverse transcription. Mature NC (p7NC) from human immunodeficiency virus type 1 (HIV-1) is a 71-amino acid, basic protein which contains two Cys3His Zn(II) retroviral-type zinc finger domains. Herein, we describe the subcloning and expression of HIV-1 NC, denoted NC71, from an inducible phage T7 RNA polymerase promoter in Escherichia coli. Purified NC71 can be reversibly reconstituted with 2 g.at Zn(II) determined by atomic absorption. Ultraviolet circulation dichroism spectroscopy has been used to characterize the complexes between highly purified NC71 and the RNA homopolynucleotide poly(A) and E. coli tRNA(mixed). On poly(A), Zn2 NC71 is characterized by an apparent site size n = 15 +/- 3 nucleotides and high affinity (Kapp = 3 x 10(7) M-1) and moderately cooperative (omega approximately 170 +/- 25) binding. A mixture of E. coli tRNA species (tRNA(mixed) was used to probe the conformational changes induced in tRNA upon binding of HIV-1 NC71. Two structural forms of tRNA(mixed), which differ in their degree of tertiary structure, were assayed for their susceptibility to denaturation by NC71. Five molar monomer equivalents of NC71 are required to denature the "inactive" tRNA in the absence of Mg2+. A Zn(II)-free, oxidized form of NC71 was also shown to unwind inactive tRNA with the same efficiency and stoichiometry. The detailed spectral changes which occur on NC-induced denaturation closely mimic temperature-induced denaturation of inactive tRNA(mixed). The prototype helix-destabilizing protein, T4 gene 32 protein, is unable to unwind this form of tRNA under the same conditions. The stoichiometry of unwinding of inactive tRNA by NC71 is consistent with the site size determined with poly(A). An "active" form of tRNA(mixed), prepared by thermal denaturation and refolding of the inactive form with Mg2+, proved less susceptible to both temperature and NC71-induced unwinding. The mechanistic implications of these findings on the reported biochemical activities of RNA:RNA annealing and replication primer tRNA positioning by NC are discussed.

Amino Acid Sequence↗
PubMed1992

Zn(II) coordination domain mutants of T4 gene 32 protein.

Gene 32 protein (g32P), the replication accessory single-stranded nucleic acid binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. Zinc coordination provides structural stability to the DNA-binding core domain of the molecule, termed g32P-(A+B) (residues 22-253). Optical absorption studies with the Co(II)-substituted protein and 113Cd NMR spectroscopy of 113Cd(II)-substituted g32P-(A+B) show that the metal coordination sphere in g32P is characterized by approximately tetrahedral ligand symmetry and ligation by the Cys-S- atoms of Cys77, Cys87, and Cys90. These studies predicted the involvement of a fourth protein-derived non-thiol ligand to complete the tetrahedral complex, postulated to be His81 on the basis of primary structure prediction and modeling [Giedroc, D.P., Johnson, B.A., Armitage, I.M., & Coleman, J.E. (1989) Biochemistry 28, 2410-2418]. To test this model, we have employed site-directed mutagenesis to substitute each of the two histidine residues in g32P (His64 and His81), accompanied by purification and structural characterization of these single-site mutant proteins. We show that g32P's containing any of three substitutions at residue 64 (H64Q, H64N, and H64L) are isolated from Escherichia coli in a Zn(II)-free form [less than or equal to 0.03 g.atom Zn(II)]. All derivatives show extremely weak affinity for the ssDNA homopolymer poly(dT). All are characterized by a far-UV-CD spectrum reduced in negative intensity relative to the wild-type protein. These structural features parallel those found for the known metal ligand mutant Cys87----Ser87 (C87S) g32P. In contrast, g32P-(A+B) containing a substitution of His81 with glutamine (H81Q), alanine (H81A) or cysteine (H81C), contains stoichiometric Zn(II) as isolated and binds to polynucleotides with an affinity comparable to the wild-type g32P-(A+B). Spin-echo 1H NMR spectra recorded for wild-type and H81Q g32P-(A+B) as a function of pH allow the assignment of His81 ring proteins to delta = 6.81 and 6.57 ppm, respectively, at pH 7.8, corresponding to the C and D histidyl protons of 1H-His-g32P-(A+B) [Pan, T., Giedroc, D.P., & Coleman, J.E. (1989) Biochemistry 28, 8828-8832]. These resonances shift downfield as the pH is reduced from 7.8 to 6.6 without metal dissociation, a result incompatible with His81 donating a ligand to the Zn(II) in wild-type g32P. Likewise, Cys81 in Zn(II) H81C g32P is readily reactive with 5,5'-dithiobis(2-nitrobenzoic acid), unlike metal ligands Cys77, Cys87, and Cys90.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Physicochemical action of potassium-magnesium citrate in nephrolithiasis.

Effect of potassium-magnesium citrate on urinary biochemistry and crystallization of stone-forming salts was compared with that of potassium citrate at same dose of potassium in five normal subjects and five patients with calcium nephrolithiasis. Compared to the placebo phase, urinary pH rose significantly from 6.06 +/- 0.27 to 6.48 +/- 0.36 (mean +/- SD, p less than 0.0167) during treatment with potassium citrate (50 mEq/day for 7 days) and to 6.68 +/- 0.31 during therapy with potassium-magnesium citrate (containing 49 mEq K, 24.5 mEq Mg, and 73.5 mEq citrate per day). Urinary pH was significantly higher during potassium-magnesium citrate than during potassium citrate therapy. Thus, the amount of undissociated uric acid declined from 118 +/- 61 mg/day during the placebo phase to 68 +/- 54 mg/day during potassium citrate treatment and, more prominently, to 41 +/- 46 mg/day during potassium-magnesium citrate therapy. Urinary magnesium rose significantly from 102 +/- 25 to 146 +/- 37 mg/day during potassium-magnesium citrate therapy but not during potassium citrate therapy. Urinary citrate rose more prominently during potassium-magnesium citrate therapy (to 1027 +/- 478 mg/day from 638 +/- 252 mg/day) than during potassium citrate treatment (to 932 +/- 297 mg/day). Consequently, urinary saturation (activity product) of calcium oxalate declined significantly (from 1.49 x 10(-8) to 1.03 x 10(-8) M2) during potassium-magnesium citrate therapy and marginally (to 1.14 x 10(-8) M2) during potassium citrate therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Neuroleptic malignant syndrome associated with metoclopramide antiemetic therapy.

A case of neuroleptic malignant syndrome in a 50-year-old woman with ovarian cancer is reported. The syndrome was associated with metoclopramide antiemetic therapy given for post-operative nausea and vomiting. Dantrolene, bromocriptine, and supportive measures were effective in obtaining a complete resolution of the syndrome. This potentially lethal syndrome is perhaps underreported in the gynecologic literature. Heightened awareness of its signs and symptoms may cause more cases to be recognized and help improve its outcome.

Adenocarcinoma↗
PubMed1992

Gastrointestinal citrate absorption in nephrolithiasis.

Gastrointestinal absorption of citrate was measured in stone patients with idiopathic hypocitraturia to determine if citrate malabsorption could account for low urinary citrate. Citrate absorption was measured directly from recovery of orally administered potassium citrate (40 mEq.) in the intestinal lavage fluid, using an intestinal washout technique. In 7 stone patients citrate absorption, serum citrate levels, peak citrate concentration in serum and area under the curve were not significantly different from those of 7 normal subjects. Citrate absorption was rapid and efficient in both groups, with 96 to 98% absorbed within 3 hours. The absorption of citrate was less efficient from a tablet preparation of potassium citrate than from a liquid preparation, probably due to a delayed release of citrate from wax matrix. However, citrate absorption from solid potassium citrate was still high at 91%, compared to 98% for a liquid preparation. Thus, hypocitraturia is unlikely to be due to an impaired gastrointestinal absorption of citrate in stone patients without overt bowel disease.

Adult↗
PubMed1992

Characterization of the fetal inflammatory response to cytomegalovirus placentitis. An immunohistochemical study.

The histopathologic features of cytomegalovirus placentitis, an established cause of chronic villitis, are well documented. However, the immunologic features of the fetal inflammatory response to placental cytomegalovirus infection are largely unknown. The characterization of the fetal-derived inflammatory cell reaction may be important in our understanding of both the intrauterine as well as the antenatal immunological response of the neonate to this important viral infection. We examined formalin-fixed placentas from four cases of confirmed congenital cytomegalovirus infection using an in situ DNA probe to cytomegalovirus, and a variety of antibodies to leukocyte antigens, including anti-CD68, CD45RO, CDw75, CD74, IgG, IgM, and IgA. All four placentas showed marked hyperplasia of fetal-derived placental macrophages, termed Hofbauer cells. A lymphocytic villitis was present in all placentas, which was characterized by positive staining in all cases with T-cell antibodies. There was no evidence of positive staining of lymphocytes using B-cell antibodies in any of the cases. Two placentas showed plasmacellular villitis, which stained positively for both IgG- and IgM-secreting cells, that was present as early as the second trimester of gestation. No IgA positivity of plasma cells was observed. These data are presented in light of current theories of fetal viral immunity.

Adolescent↗
PubMed1991

Site-specific 1,N6-ethenoadenylated single-stranded oligonucleotides as structural probes for the T4 gene 32 protein-ssDNA complex.

Bacteriophage T4 gene 32 protein (g32P) is a DNA replication accessory protein that binds single-stranded (ss) nucleic acids nonspecifically, independent of nucleotide sequence. G32P contains 1 mol of Zn(II)/mol of protein monomer, which can be substituted with Co(II), with maintenance of the structure and activity of the molecule. The Co(II) is coordinated via approximately tetrahedral ligand symmetry by three Cys sulfur atoms and therefore exhibits intense S(-)----Co(II) ligand to metal charge-transfer (LMCT) transitions in the near ultraviolet [Giedroc, D. P., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8452-8456]. A series of fluorescent 1,N6-ethenoadenosine (epsilon A)-containing oligonucleotides conforming to the structure (5'----3') d[(Tp)m epsilon A(pT)l-m-1] where 0 less than or equal to m less than or equal to l - 1 and length (l) six or eight nucleotides have been evaluated as dynamics probes and potential fluorescence energy transfer donors to Co(II) in mapping the spatial proximity of the (fixed) intrinsic metal ion and a variably positioned epsilon A-base in a series of protein-nucleic acid complexes. We provide spectroscopic evidence that the epsilon A-oligonucleotides bind to g32P-(A + B) with a fixed polarity of the phosphodiester chain. A Trp side chain(s) makes close approach to a epsilon A base positioned toward the 3' end of a bound l = 8 oligonucleotide. Six oligonucleotides of l = 8 and m = 0, 1, 3, 5, 6, or 7 were investigated as energy transfer donors to Co(II) at 0.1 M NaCl, pH 8.1, 25 degrees C upon binding to Co(II)-substituted or Zn(II) g32P-(A + B), i.e., in the presence and absence of an energy acceptor, respectively. Detectable quenching of the epsilon A-fluorescence by the Co(II)-LMCT acceptors was found to occur in all epsilon A-oligonucleotide-protein complexes, yielding energy transfer efficiencies (E) of 0.43, 0.31, 0.26, 0.26, 0.28, and 0.41 for l = 8 and m = 0, 1, 3, 5, 6, and 7 epsilon A-oligonucleotides, respectively. The two-dimensional distances R (in A) were found to vary as follows: d[epsilon A(pT)7] (m = 0), 16.0 (15.5-16.9); d[Tp epsilon A(pT)6] (m = 1), 17.7 (16.9-19.1); d[(Tp)3 epsilon A(pT)4] (m = 3), 20.7 (19.5-22.1); d[(Tp)5 epsilon A(pT)2] (m = 5), 20.5 (19.5-21.9); d[(Tp)6 epsilon ApT] (m = 6), 19.0 (18.0-20.4); and d[(Tp)7 epsilon A] (m = 7), 18.6 (17.8-19.8).(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine↗
PubMed1991

Carcinoma (malignant mixed müllerian [mesodermal] tumor) of the uterus and ovary. Correlation of clinical, pathologic, and immunohistochemical features in 29 cases.

We examined the histologic, immunohistochemical, and clinical features of a series of 23 endometrial, five cervical, and one ovarian carcinosarcomas (malignant mixed müllerian [mesodermal] tumors) and nine associated distant peritoneal metastases. The primary tumors all showed epithelial differentiation (cytokeratin and/or epithelial membrane antigen expression) of the carcinomatous component, while sarcomatous areas showed epithelial differentiation in all but one case. The metastases showed uniform staining for cytokeratin (eight of eight cases) and epithelial membrane antigen (eight of eight cases), including the spindle cell component that was present in four of nine cases. Desmin significantly changed the interpretation of rhabdomyosarcoma differentiation by refuting putative rhabdomyoblasts in two cases and identifying rhabdomyoblasts in two other cases where they were unrecognized on hematoxylineosin staining. S100 protein was positive in all five cases with chondrosarcoma differentiation. Muscle-specific actin and vimentin were positive in the sarcomatous component of all cases and in the carcinomatous component of seven and 10 cases, respectively. After immunostaining, heterologous elements were present in 18 of 29 cases (11 cases of rhabdomyosarcoma, three cases of chondrosarcoma, three cases of mixed rhabdomyosarcoma and chondrosarcoma, and one case of liposarcoma). Only six of 27 patients with follow-up were disease free for 12 months or longer (associated with stage I or II disease, smaller size, no lymphatic invasion in the resection specimen, and no invasion of the outer two thirds of myometrium). Presence and type of heterologous elements, grade of sarcomatous or carcinomatous components, histologic type of carcinomatous component, gross appearance, presence of necrosis, or use of chemotherapy or radiotherapy did not affect outcome. Carcinosarcomas are clinically aggressive distinctive mixed epithelial-stromal neoplasms with histologic and immunohistochemical features that overlap with metaplastic carcinoma in many cases.

Aged↗
PubMed1990

Binding sites in mammalian genes and viral gene regulatory regions recognized by methylated DNA-binding protein.

Methylated DNA-binding protein (MDBP), a ubiquitous mammalian protein, recognizes a variety of related DNA sequences. Some of these sequences require methylation of their CpG dinucleotides for binding and others do not. We report that MDBP binds, in a DNA methylation-independent fashion, to two sites in the mouse polyomavirus enhancer, one in the enhancer of the human hepatitis B virus, and to one in the long terminal repeat of equine infectious anemia proviral DNA. We have also found a number of MDBP sites in human and rodent DNAs which bind much better to MDBP when they are methylated at CpG dinucleotides within the recognition site. These include sites at the beginning of the human genes for hypoxanthine phosphoribosyl transferase, HLA-A2, -A3, and -A25 antigens, and alpha-galactosidase A. In the case of methylation-responsive MDBP sites, changes in their methylation status during differentiation or DNA replication could help drive development by modulating transcription.

Animals↗
PubMed1990

Halogenation reactions of derivatives of D-glucose and sucrose.

Treatment of methyl 4,6-O-benzylidene-alpha-D-glucopyranoside (1) with triphenylphophine-carbon tetrachloride-pyridine (reagent A) gave methyl 4,6-O-benzylidene-2-chloro-2-deoxy-alpha-D-mannopyranoside (2). When reagent A was used in excess, a further elimination reaction occurred to give methyl 4,6-O-benzylidene-2-chloro- (6, 60%) and -3-chloro-2,3-dideoxy-alpha-D-erythro-hex-2-enopyranoside (7, 16%). Treatment of 1 with triphenylphosphine-carbon tetrabromide-pyridine (reagent B) caused little or no elimination, and 47% of methyl 4,6-O-benzylidene-2-bromo-2-deoxy-alpha-D-mannopyranoside (14) was obtained. On treatment with reagent A, methyl alpha-D-glucopyranoside (16) gave exclusively methyl 2,4,6-trichloro- 2,3,4,6-tetradeoxy-alpha-D-erythro-hex-2-enopyranoside (17), and methyl 4,6-O-benzylidene-beta-D-glucopyranoside (19) gave methyl 4,6-O-benzylidene-3-chloro-3-deoxy-beta-D-allopyranoside (20, 70%). However, with reagent B, 19 gave methyl 4,6-O-benzylidene-3-bromo-3-deoxy-beta-D-glucopyranoside (23, 66%), probably by way of double inversion of configuration at C-3. Likewise, with reagent A, methyl beta-D-glucopyranoside (25) gave methyl 2,4,6-trichloro- (26) and3,4,6-trichloro-2,3,4,-6-tetradeoxy- beta-D-threo-hex-2-enopyranoside (27), and 4,6-O-isopropylidenesucrose (28) gave mainly 3-chloro-3-deoxy-4,6-O-isopropylidene-alpha-D-allopyranosyl 1,4,6-trichloro-1,4,6-trideoxy-beta-D-lyxo-hexulofuranoside (29) together with 3-chloro-3-deoxy-4,6-O-isopropylidene-alpha-D-allopyranosyl 1,4,6-trichloro-1,4,6-trideoxy-beta-D-fructofuranoside (30). The assignment of structure to 29 is tentative.

Carbohydrate Sequence↗
PubMed1990

Overexpression, purification, and characterization of recombinant T4 gene 32 protein22-301 (g32P-B).

Gene 32 protein (g32P), the replication accessory protein from bacteriophage T4, is a zinc metalloprotein which binds with high cooperativity to single-stranded (ss) nucleic acids. The basic N-terminal 21 amino acids (termed the "B" domain) is required for highly cooperative (omega approximately 500) binding of g32P monomers to ss nucleic acids. As part of our studies to systematically evaluate the structural features of the B domain important for cooperative binding, a homogeneous source of g32P which binds noncooperatively to nucleic acids (omega = 1) and is devoid of contamination by native g32P is needed. Herein, we describe large-scale overexpression and purification of recombinant g32P lacking the tryptic N-terminal B domain (residues 1-21), designated g32P-B, as well as its physiochemical and nucleic acid binding properties. G32P-B is readily purified from the soluble fraction of Escherichia coli BL21 (DE3) transformed with the plasmid pT7g32-B.wt which contains the g32P-B coding sequences under inducible transcriptional control of T7 RNA polymerase. Anion exchange, ssDNA-cellulose and phenyl-Sepharose chromatographies give rise to highly homogeneous g32P-B, free of contaminating nucleic acid. Recombinant g32P-B has the expected N-terminal primary structure and contains stoichiometric Zn(II). It also has the expected globular structure as shown by 1H NMR spectroscopy, hydrodynamic measurements, and the ability to selectively remove the carboxyl-terminal "A" domain to form the trypsin-resistant g32P-(A + B) DNA-binding core fragment. Quantitative ss nucleic acid binding experiments of g32P-B to poly(dT) (0.05 M NaCl, pH 8.1, 20 degrees C) show that all equilibrium binding isotherms can be fit with omega = 1 and Kobs = 5.2 (+/- 1.6) x 10(5) M-1, with a moderate electrostatic component to the binding free energy, delta log Kobs/delta log[NaCl] = -3.0 +/- 0.2. Under identical solution conditions, g32P-(A + B) derived from g32P-B binds to poly(dT) noncooperatively as expected, but with an approximately 80-fold higher apparent affinity, Kobs = 4.0 (+/- 2.0) x 10(7) M-1, and detectable enhanced salt sensitivity, delta log Kobs/delta log[NaCl] = -3.9 +/- 0.3. As the salt concentration is raised, the relative difference in Kobs between the g32P-(A + B) and g32P-B is gradually reduced such that extrapolation of the log-log plots to 1 M Na+ standard state gives similar Kobs within experimental error. Qualitatively similar observations are also found upon binding to the ribohomopolymer, poly(U).(ABSTRACT TRUNCATED AT 400 WORDS)

Chromatography, Gel↗
PubMed1990

Cyclic acetals of 4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose and their conversion into methyl ether derivatives.

The acid-catalysed reaction of 4,1',6'-trichloro-4,1',6'-trideoxy-galacto- sucrose (1) with 5.5 equiv. of 2-methoxypropene in N,N-dimethylformamide followed by acetylation gave 3',4'-di-O-acetyl-4,1',6'-trichloro-4,1',6'-trideoxy-2,3-O- isopropylidene-6-O-(1-methoxy-1-methylethyl)-galacto-sucrose (2, 2%), 6,3',4'- tri- O-acetyl-4,1',6'-trichloro-4,1',6'-trideoxy-2,3-O-isopropylidene-galacto -sucrose (3, 31%), 3',4'-di-O-acetyl-4,1',6'-trichloro-4,1',6'-trideoxy-2,3-O- isopropylidene- galacto-sucrose (4, 38%), 3'-O-acetyl-4,1',6'-trichloro-4,1',6'-trideoxy-2,3-O- isopropylidene- galacto-sucrose (5, 13%), and 2,3',4'-tri-O-acetyl-4,1',6'-trichloro- 4,1',6'-trideoxy-galacto-sucrose (6, 13%). Methylation of 4 followed by removal of the protecting groups gave 4,1',6'-trichloro-4,1',6'-trideoxy-6-O-methyl- galacto- sucrose (8). 4,1',6'-Trichloro-4,1',6'-trideoxy-3-O-methyl-galacto-sucrose (11) was synthesised from 6 by preferential tert-butyldiphenylsilylation of HO-6 followed by methylation and removal of the protecting groups. Likewise, 4,1',6'-trichloro- 4,1',6'-trideoxy-4'-O-methyl-galacto-sucrose (14) was synthesised from 5. Treatment of 3 with aqueous acetic acid followed by methylation and removal of the protecting groups afforded 4,1',6'-trichloro-4,1'6'-trideoxy-2,3-di-O-methyl- galacto-sucrose (17).

Acetals↗
PubMed1990

Synthesis and ring-opening reactions of 4-chloro-4-deoxy-alpha-D-galactopyranosyl 3,4-anhydro-1,6-dichloro-1,6-dideoxy-beta-D-lyxo-hexulofuranoside.

Treatment of 4-chloro-4-deoxy-alpha-D-galactopyranosyl 1,6-dichloro-1,6-dideoxy-beta-D-fructofuranoside (1) with 2.3 mol. equiv. of diethyl azodicarboxylate (DEAD) and 1.3 mol. equiv. of triphenylphosphine (TPP) in toluene gave a mixture of 3,6-anhydro-4-chloro-4-deoxy-alpha-D-galactopyranosyl 3,4-anhydro-1,6-dichloro-1,6-dideoxy-beta-D-lyxo-hexulofuranosi de (2, 55%) and 4-chloro-4-deoxy-alpha-D-galactopyranosyl 3,4-anhydro-1,6-dichloro-1,6-dideoxy-beta-D-lyxo-hexulofuranosi de (3, 35%). Compound 3 was also synthesised from 6-O-tert-butyldiphenylsilyl-4-chloro-4-deoxy-alpha-D-galactopyrano syl 1,6-dichloro-1,6-dideoxy-beta-D-fructofuranoside by epoxidation with DEAD-TPP and removal of the silyl ether group with tetrabutylammonium fluoride. The SN2 reactions of 2,3,6-tri-O-acetyl-4-chloro-4-deoxy-alpha-D-galactopyranosyl 3,4-anhydro-1,6-dichloro-1,6-dideoxy-beta-D-lyxo-hexulofuranosi de (5) with fluoride, chloride, bromide, iodide, and azide ions gave the corresponding 4'-derivatives 10, 12, 14, 18, and 20, respectively. Reduction of 4-chloro-4-deoxy-alpha-D-galactopyranosyl 4-bromo-1,6-dichloro-1,4,6-trideoxy-beta-D-fructofuranoside (15) gave 4-chloro-4-deoxy-alpha-D-galactopyranosyl 1,6-dichloro-1,4,6-trideoxy-beta-D-fructofuranoside (16). A similar reduction of 4-chloro-4-deoxy-alpha-D-galactopyranosyl 4-azido-1,6-dichloro-1,4,6-trideoxy-beta-D-fructofuranoside (21) gave 4-chloro-4-deoxy-alpha-D-galactopyranosyl 4-amino-1,6-dichloro-1,4,6-trideoxy-beta-D-fructofuranoside (22).

Carbohydrate Sequence↗
PubMed1990

HLA antibody responses in HLA class I transgenic mice.

In a previous report we described how cross-immunizations of pairs of transgenic mice expressing different HLA class I antigens led to the production of antibodies directed exclusively at polymorphic epitopes. This was ascribed to self-tolerance of HLA that prevents immune responses to monomorphic epitopes and focuses responses on polymorphic ones. In the present report we extend our findings and demonstrate that immunizations of class I transgenic mice with HLA transfected mouse fibrosarcoma as well as with human lymphoblastoid cells also preferentially yield antibodies to polymorphic epitopes. This was the case whether or not immunizations were carried out across locus barriers [e.g., Tg(HLA-A *0201) or Tg(HLA-Cw*0301) transgenic mice immunized with HLA-B27 transfectants] or within the same locus [e.g., Tg(HLA-B*1302) transgenic mice immunized with HLA-B27 transfectants or B27-expressing lymphoblastoid cells]. Use of an extended immunization protocol with four or more booster injections favored antibodies of IgG isotype with affinities high enough to lyse normal peripheral blood lymphocytes (PBLs) in complement-dependent cytotoxicity assays and to immunoprecipitate HLA antigens. The specificities covered by the monoclonal antibodies (mAbs) could be either broad or narrow, depending on the genetic distance of the HLA antigens or alleles involved. For instance, a Tg(HLA-B*1302) transgenic mouse immunized with B27 produced both broad B7/B27-specific antibodies, Bw4-specific antibodies, and one antibody reacting with all B alleles except B13 and with some C alleles. On the other hand, a Tg(HLA-B*1302) transgenic mouse immunized with Bw47 transfectants responded narrowly with an antibody to Bw60 and Bw47. Thus it appears that by choosing appropriate recipient mice and closely related or more distant HLA antigens, antibodies of a programmed specificity can be generated.

Alleles↗
PubMed1990

Self-tolerance to HLA focuses the response of immunized HLA-transgenic mice on production of antibody to precise polymorphic HLA alloantigens.

HLA class I-transgenic mice express HLA antigen on their tissues and establish self-tolerance to the expressed monomorphic and polymorphic determinants. When challenged with skin grafts and lymphoid cells of a second HLA-transgenic mouse expressing a different HLA molecule, a specific immune response is elicited that is focused on the determinants specified by the allelic HLA differences between donor and recipient transgenic mice. In the studies described, this has led to the production of a number of monoclonal antibodies with specificity for the HLA-Cw3 and HLA-B7 crossreactive groups of class I antigens. These results indicate that immunization of appropriate transgenic strains of mice with murine cells expressing a different HLA allelic transgene should permit the generation of monoclonal antibodies of narrow specificity against virtually any polymorphic epitope on HLA antigens.

Animals↗