Zolpidem use and risk of fractures: a systematic review and meta-analysis.

UNLABELLED: Zolpidem is a representative of non-benzodiazepine hypnotics. Recent epidemiologic studies have reported increased fracture risk in patients taking zolpidem, but the results have been inconsistent. The present meta-analysis shows that the use of zolpidem is associated with an increased risk of fractures. PURPOSE: Previous studies have reported inconsistent findings regarding the association between the use of zolpidem and the risk of fractures. We performed a systematic literature review and meta-analysis to assess the association. METHODS: We identified relevant studies by searching MEDLINE, EMBASE, Cochrane Library, and PsycINFO without language restrictions (until August 2014). Methodological quality was assessed based on the Newcastle-Ottawa Scale (NOS). RESULTS: A total of 1,092,925 participants (129,148 fracture cases) were included from 9 studies (4 cohort, 4 case-control, and 1 case-crossover study). Overall, the use of zolpidem was associated with an increased risk of fracture (relative risk [RR] 1.92, 95 % CI 1.65-2.24; I (2) = 50.9 %). High-quality subgroups (cohort studies, high NOS score, adjusted for any confounder, or adjusted for osteoporosis) had higher RRs than the corresponding low-quality subgroups (high quality, 1.94-2.76; low quality, 1.55-1.79). Of note, the risk for hip fracture was higher than that for fracture at any site (hip fracture, RR 2.80, 95 % CI 2.19-3.58; fracture at any site, RR 1.84, 95 % CI 1.67-2.03; P < 0.001). CONCLUSIONS: The use of zolpidem may increase the risk of fractures. Clinicians should be cautious when prescribing zolpidem for patients at high risk of fracture.

Oxygen-vacancy-induced orbital reconstruction of Ti ions at the interface of LaAlO3/SrTiO3 heterostructures: a resonant soft-X-ray scattering study.

Resonant soft-x-ray scattering measurements have been performed to investigate interface electronic structures of (LaAlO(3)/SrTiO(3)) superlattices. Resonant scattering intensities at superlattice reflections show clear evidence of degeneracy lifting in t(2g) states of interface Ti ions. Polarization dependence of intensities indicates the energy of d(xy) states is lower by ~1 eV than two other t(2g) states. The energy splitting is insensitive to epitaxial strain. The orbital reconstruction is induced by oxygen vacancies and confined to the interface within two unit cells, indicating charge compensation at the polar interfaces.

The murine MHC class I genes, H-2Dq and H-2Lq, are strikingly homologous to each other, H-2Ld, and two genes reported to encode tumor-specific antigens.

Two phenomena appear to distinguish the D region class I genes from those in the K region in the murine MHC: (a) haplotype disparity in the number of expressed D region class I molecules has been observed; and (b) clines of closely related D region class I molecules among and within mice of different H-2 haplotypes can be defined. Both of these observations have been based on serological and peptide mapping analyses of these molecules. Recent reports using molecular biological approaches have corroborated these findings. Since the mouse strain B10.AKM expresses multiple D region class I antigens, all of which are closely related to the prototypic Ld molecule, we investigated the Dq region of B10.AKM using molecular approaches. Three D region class I genes were isolated from genomic B10.AKM bacteriophage and cosmid libraries. Based on alignment of those genes with the BALB/c D region class I genes by analogous restriction endonuclease sites and by hybridization of one of those genes with a D4d gene-derived oligonucleotide probe, we have designated these genes as Dq, Lq, and D4q. As determined by DNA-mediated gene transfer to mouse L cells followed by serological analyses, the Dq and Lq genes encode previously characterized Dq region class I antigens. The nucleic acid sequence comparisons of the Dq and Lq genes demonstrated a higher level of homology with the Ld and Db genes than with other D region class I genes. In addition, CTL stimulated with a Dq, Lq, or Ld gene transfectant showed strong crossreactions with the other transfectants as targets, suggesting that the products of these genes are also functionally related. Thus, these studies suggest that the L molecule represents a prototypic structure shared by several D region gene products, and furthermore, the duplication of an Ld-like progenitor gene resulted in two Dq region class I genes, Dq and Lq. Unexpectedly, the sequences determined for the Dq and Lq genes are nearly identical to the sequences of two genes, A166 and A149, respectively, which were reported to encode the tumor-specific antigens; these novel class I genes were isolated from an H-2k fibrosarcoma, 1591. This raises the distinct possibility that these purported tumor-specific class I genes were introduced into this tumor by contamination.

Molecular evidence that the H-2D and H-2L genes arose by duplication. Differences between the evolution of the class I genes in mice and humans.

To resolve issues regarding the evolution of D region class I MHC genes and their relationship to other class I-encoding regions of the mouse, as well as man, we characterized the class I genes from the Dq region of the B10.AKM mouse strain. The Dq region was selected because it was known to express multiple gene products, yet two of the products previously characterized have structural features in common with the Ld molecule. Since DNA hybridization data defined similarities between the Dd and Dq regions, we used low-copy genomic or oligonucleotide probes derived from the Dd region of BALB/c (H-2d) to screen a B10.AKM cosmid library. Cosmid clones containing Dq, D2q, D3q, D4q, Lq, and Q1q genes have been isolated and aligned with the corresponding genes of the BALB/c MHC, thus demonstrating a similar gene organization. The two classical transplantation genes, Dq and Lq were found to be strikingly similar to each other such that exons 1-3 of Dq and Lq, are approximately 97% homologous, and exons 4-8 are identical. Furthermore, the implied amino acid sequences of both Lq and Dq molecules show considerable homology to Ld, particularly in regions presumed to be involved in ligand binding. These comparisons suggest not only that the Dq and Lq genes arose from the duplication of an Ld-like progenitor, but also that there is a selective advantage for the maintenance of an Ld-like structure. In addition, the 5' portion of the D4q gene was sequenced and found to have a 13-bp deletion and a 4-bp insertion within the alpha 2 exon. These result in a frame shift that creates a premature termination codon and potential polyadenylation site, respectively. Thus, D4q does not encode a typical class I molecule. Sequence comparisons suggest that the D4q gene did not arise from a duplication event involving an Ld-like gene such as Dq and Lq. Interestingly, the D4q molecule, if produced, would have amino acid residues in common with K and/or Q molecules that differ from those observed in D/L molecules. These findings, in conjunction with hybridization data, provide evidence that the D2, D3, and D4 genes were derived from Q genes by an unequal crossover event. Additional hybridization data using low-copy D region probes suggest that several different D region gene organizations exist among mice of different haplotypes. These and other recent molecular studies provide multiple examples of expansion and contraction of the class I genes in the D region.(ABSTRACT TRUNCATED AT 400 WORDS)

The specific binding of peptide ligand to Ld class I major histocompatibility complex molecules determines their antigenic structure.

To better understand the biological implications of the association of ligand with major histocompatibility complex class I molecules, we have studied the Ld molecule of the mouse. The culturing of various nonselected cell lines with three different known Ld peptide ligands resulted in a two- to fourfold specific increase in surface Ld expression as detected by 10 of 11 different monoclonal antibodies (mAbs) recognizing Ld epitopes. These findings suggest that Ld molecules are not saturated with endogenous peptide ligands and thus have accessible binding sites. Exploiting this feature of Ld we demonstrate that the physical association of Ld with ligand is exquisitely specific, indicating that they function in determinant selection. In addition, a non-peptide-bound antigenic variant of Ld was specifically detected with an exceptional mAb designated 64-3-7. In comparison with other Ld molecules, 64-3-7+ Ld molecules are not peptide ligand inducible, are more susceptible to proteolysis, lack beta 2 microglobulin association, and display a slower rate of oligosaccharide maturation. In spite of their deficiencies, the non-ligand-associated 64-3-7 Ld molecules were detected on the surface of all cell types tested; however, they appear not to be recognized by alloreactive cytotoxic T lymphocytes.

Reduced viscosity of the free surface in entangled polymer melt films.

By embedding "dilute" gold nanoparticles in single polystyrene thin films as "markers", we probe the local viscosity of the free surface at temperatures far above the glass transition temperature (T(g)). The technique used was x-ray photon correlation spectroscopy with resonance-enhanced x-ray scattering. The results clearly showed the surface viscosity is about 30% lower than the rest of the film. We found that this reduction is strongly associated with chain entanglements at the free surface rather than the reduction in T(g).

Randomized, Open-Label, Phase IV, Korean Study of Kidney Transplant Patients Converting From Cyclosporine to Prolonged-Release Tacrolimus Plus Standard- or Reduced-Dose Corticosteroids.

BACKGROUND: This 24-week, multicenter, randomized, exploratory, comparative, open-label, phase-IV study assessed the safety and efficacy of prolonged-release tacrolimus (PR-T) with reduced-dose versus standard-dose corticosteroids in stable kidney transplant recipients in Korea after converting from cyclosporine-based therapy. METHODS: At baseline, patients were converted from cyclosporine-based to PR-T-based immunosuppression and randomized (1:1) to receive either corticosteroids maintained at prestudy dose (standard-dose group) or tapered from week 4 to 50% of the prestudy dose by week 12 (reduced-dose group). Patients were seen at baseline and weeks 1, 4, 12, and 24. The primary endpoint was change in estimated glomerular filtration rate (Modification-of-Diet-in-Renal-Disease-4) between baseline and week 24. Secondary endpoints included either acute rejection or patient-reported satisfaction with PR-T. Adverse events (AEs) were recorded. RESULTS: Overall, 150 patients were randomized into a reduced-dose group (n = 73) and a standard-dose group (n = 77). At week 24, mean ± standard deviation for corticosteroid dose was 2.5 ± 0.9 mg and 5.0 ± 1.3 mg, respectively. Mean change in estimated glomerular filtration rate from baseline to week 24 was +1.5 ± 9.1 mL/min/1.73 m2 (P = .1567) and +3.4 ± 10.6 mL/min/1.73 m2 (P = .0065), respectively, and not significantly different between groups. There were no acute rejection episodes. Most respondents (>70%) considered PR-T more convenient than cyclosporine. AE incidence was similar between groups. The most common AEs experienced by ≥3% of patients in either treatment group were gastrointestinal events (20.8% and 28.6% of patients receiving reduced- and standard-dose corticosteroids, respectively). Most AEs in both treatment groups were mild or moderate in severity. CONCLUSION: Renal function was maintained following conversion from cyclosporine to PR-T, irrespective of corticosteroid regimen; PR-T enables reduced corticosteroid dosage.

C3d-binding Donor-specific HLA Antibody Is Associated With a High Risk of Antibody-mediated Rejection and Graft Loss in Stable Kidney Transplant Recipients: A Single-center Cohort Study.

BACKGROUND: One risk factor for antibody-mediated rejection (ABMR) and poor outcome after kidney transplantation is donor-specific anti‒human leukocyte antigen (anti-HLA) antibodies (DSAs). In this study we sought to determine whether the presence of DSAs that bind complement component C3d could better predict ABMR and graft loss in stable kidney transplant recipients (KTRs). METHODS: We included 220 stable KTRs in this study and screened them for DSAs from July 2013 to July 2016. RESULTS: Of the 220 KTRs, DSAs were detected in 24 (10.9%). The incidence of ABMR was 3.6% (8 of 220) overall, and C3d-DSA‒positive KTRs had a significantly higher incidence than SA-DSA‒positive KTRs (63.3% vs 38.9%, P = .03). Most C3d-binding DSAs were anti-HLA class II antibodies (11 of 13, 84.6%). Class II C3d-binding DSA was also significantly associated with graft failure on multivariate analysis, as were ABMR, chronic ABMR, and high serum creatinine. Class II C3d-binding DSA was also significantly associated with lower graft survival after ABMR. CONCLUSION: C3d-binding DSA, especially class II, was significantly associated with the risk of ABMR and graft loss in stable KTRs. We suggest that monitoring of stable KTRs for C3d-binding DSA, followed by biopsy, could aid in early recognition of ABMR and prevention of graft loss.

Disulfide structure of murine Ia alloantigens.

The tryptic (T) and T insoluble chymotryptic (TIC) peptide maps from 35S-cysteine (Cys) labeled, disulfide-linked I-Ak dimer were compared to those from 35S-Cys labeled I-Ak alpha and beta chains which were not covalently linked. These comparisons indicated that the alpha and beta chains found in the covalent I-Ak dimer were not a specialized subset of I-A alpha and beta chains. Furthermore, these data, along with the knowledge that alkylation of spleen cells prior to and during detergent solubilization prevents the formation of disulfide-linked I-Ak dimer, indicate that covalent dimer formation is an inefficient and artifactual process. Comparison of the T and TIC peptide maps of reduced and nonreduced 35S-Cys labeled I-Ak alpha and beta chains suggests that the I-Ak alpha chain contains one intrachain disulfide bond, whereas the I-Ak beta chain contains two intrachain disulfide bonds. Examination of the T and TIC peptide maps of the reduced and nonreduced 35S-Cys labeled I-Ak dimer identifies the Cys-containing peptides which are involved in the formation of the artifactual I-Ak dimer interchain (alpha-beta) disulfide bond. Comparison of 35S-Cys labeled I-Ak and I-Ek alpha and beta chains by T and TIC peptide mapping reveals considerably more homology between the two alpha-chains and between the two beta-chains than is observed using other 3H-amino acid precursors, thus indicating that the I-Ak and I-Ek alloantigens are homologous in their amino acid sequences adjacent to the Cys resides. The reasons for the inability to induce formation of interchain (alpha-beta) disulfide bonds in I-Ek molecules are discussed.

Slow egress of a mouse MHC class I molecule to the cell surface despite its strong association with beta 2-microglobulin.

Two H-2D region class I genes from the wild-derived mouse strain B10.GAA37 provisionally encoding the Dw16 and Lw16 molecules, respectively, were transfected into mouse L cells, and the expressed gene products were analyzed serologically by flow cytometry. As expected from nucleotide sequence comparisons, these analyses revealed that several Ld-reactive monoclonal antibodies (mAbs) recognize Lw16 and not Dw16. As detected by flow cytometry of intact L.Lw16 cells and B10.GAA37 splenocytes, and by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) of immunoprecipitates from splenocyte lysates, the alpha 2 domain-reactive mAb 30-5-7 detected less Lw16 than did the alpha 3 domain-reactive mAb 28-14-8, suggesting the existence of two populations of Lw16 molecules: 30-5-7+ 28-14-8+ and 30-5-7- 28-14-8+. Sequential immunoprecipitation studies provided further evidence for these two Lw16 subsets; furthermore, the 30-5-7- 28-14-8+ subset was found predominantly on the cell surface and in association with beta 2-microglobulin (beta 2-m). Pulse-chase studies of B10.GAA37 splenocytes revealed that Lw16, like Ld, is trafficked slowly to the cell surface, whereas Dw16 is trafficked quickly, like most other mouse K and D region class I molecules. Despite these similarities, Lw16 and Ld differ in their association with beta 2-m, in that the immunoprecipitates of Lw16 contained much higher levels of radiolabeled beta 2-m per heavy chain. Together, these studies indicate that the slower trafficking of Lw16 to the surface does not result from a weaker association with beta 2-m, suggesting that other factors, such as peptide ligand-induced assembly, and/or retention by ER-resident proteins play an important role in the trafficking of major histocompatibility (MHC) class I molecules to the cell surface.

Association of Ld-restricted peptides with the wild-derived mouse Lw16 MHC class I molecule.

Previous serological analysis of untreated splenocytes and L cell transfectants expressing the wild-derived mouse major histocompatibility complex (MHC) class I molecule, Lw16, demonstrated the presence of two forms of this molecule in the cell lysates, one reactive with both the alpha 2 domain-reactive monoclonal antibody (mAb) 30-5-7 and the alpha 3 domain-reactive mAb 28-14-8 (30-5-7+ 28-14-8+), and the other reactive with only the latter of the two (30-5-7- 28-14-8+). Furthermore, the analysis suggested the presence of both forms on the cell surface. Due to the similarity of Lw16 to the inbred mouse-derived Ld molecule, we tested a panel of Ld-restricted and control peptides for their ability to bind to Lw16 molecules. Here, we report that two Ld-restricted viral peptides, lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) 118-126 and murine cytomegalovirus (MCMV) pp89 168-176, significantly increase the number of Lw16 molecules on the cell surface as measured by the mAb 28-14-8, and the proportion of those molecules that are recognized by the mAb 30-5-7. This was further supported by an increase in mAb 30-5-7-reactive molecules in L.Lw16 cell lysates following treatment with either of these peptides. Examination of the stability of the different forms on the cell surface suggested that the 30-5-7+ Lw16 molecules induced with these peptides were unstable and probably lost their Ld-restricted peptides to generate 30-5-7- 28-14-8+ molecules; these latter molecules were also unstable. In contrast, putative 30-5-7+ and 30-5-7- 28-14-8+ Lw16 molecules on untreated cells were stable. Together, these results suggest that two Ld-restricted, viral peptides can induce assembly of or stabilize 30-5-7+ 28-14-8+ Lw16 molecules, mimicking endogenous self peptides. However, the association of the Ld-restricted peptides with Lw16 is apparently not optimal, since it results in unstable Lw16 molecules.

A polymorphic residue in the amino terminal alpha 1 hemi-domain of the mouse Ld class I molecule affects its assembly and surface expression.

In comparison to Dd and most other mouse major histocompatibility complex class I molecules, the Ld molecule is poorly expressed on the cell surface, has a lower affinity for beta 2-microglobulin and is trafficked more slowly to the cell surface. Previous studies using Ld-Dd exon-shuffled constructs and the chimeric Ddm1 molecules suggested that the Ld alpha 1 domain was responsible for this phenotype. Two constructs, one containing an Ld-Dd hemi-exon-shuffled alpha 1 exon and the other containing a Dd-Ld hemi-exon-shuffled alpha 1 exon, were inserted into either Ld or Dd to replace the intact alpha 1 exon. These constructs were transfected into mouse L cells. Flow cytometric analyses of the resulting transfectants indicate that the Dd-Ld alpha 1/Ld molecules, similar to the Dd alpha 1/Dd alpha 2/Ld molecules, were expressed at a higher level on the cell surface than either the Ld-Dd alpha 1/Ld molecules or intact Ld molecules. Analyses of the molecules in lysates suggested that a higher proportion of the Dd-Ld alpha 1/Ld molecules, like the Dd alpha 1/Dd alpha 2/Ld molecules, as compared to the Ld-Dd alpha 1/Ld and intact Ld molecules were assembled as detected by alpha 2 domain-reactive monoclonal antibodies. Pulse-chase and lysate stability studies suggested that the lower steady state levels of assembled Ld-Dd alpha 1 molecules resulted from a slower assembly rate rather than instability. Collectively, these studies suggest that residues in the amino terminal half of the Ld alpha 1 domain are responsible for its inefficient assembly, probably leading to its low cell surface expression. To determine which polymorphic residues in the amino terminal alpha 1 hemi-domain might influence this phenotype, several Ld point mutants, in which a Dd amino terminal alpha 1 hemi-domain residue was substituted into the corresponding position of Ld, were analysed. These analyses suggested that, while the residue at position 9 has only a slight effect on beta 2-microglobulin association, it has a striking effect on assembly and cell surface expression.

Sequence comparisons of non-human primate HIV-1 coreceptor homologues.

Infection of non-human primate peripheral blood mononuclear cells (PBMCs) in vitro with primary human immunodeficiency virus type 1 (HIV-1) isolates is extremely inefficient and often unattainable. The mechanism of resistance to infection by primary HIV-1 isolates in chimpanzee and baboon PBMCs is unknown. In this study, two HIV-1 coreceptors, CCR5 and CXCR4, were sequenced from chimpanzee and baboon PBMCs to determine if any sequence variations or mutations in these genes could be responsible for resistance to HIV infection. Primers were designed from the human coreceptor sequences and were able to amplify the CCR5 and CXCR4 genes from these non-human primate cells. No 32 base pair deletion (delta32) mutations were found in any of the non-human primate samples tested. CXCR4 sequence analysis showed chimpanzee and baboon share 99.7 and 98% nucleotide sequence homology and 100 and 98.9% amino acid sequence homology, respectively, compared to the human sequence. CCR5 sequence analysis demonstrated that chimpanzee and baboon share 99.6 and 98% nucleotide homology and 100 and 98% amino acid homology, respectively, with the human sequence. These data indicate that no variations in these coreceptor gene sequences exist that can explain the lack of susceptibility to infection with primary HIV-1 isolates in non-human primate PBMCs.

Structural analyses define an additional H-2D region class I antigen that is expressed by a variant mouse strain.

The genetic complexity of the H-2D region includes haplotype disparities in apparent gene and product number. To probe the genetic basis of this complexity, the products of two independently derived mouse strains (STU and B10.SAA48) that express Dw3 antigens were compared. Serologic, fluorometric and peptide map comparisons were made using monoclonal antibodies. Although both STU and B10.SAA48 mice were found to express indistinguishable Dw3 molecules, only B10.SAA48 mice were found to express an additional antigen designated Lw3. Several lines of evidence are presented that suggest the gene encoding Lw3 maps to the D region. Furthermore peptide map comparisons of Dw3 with Lw3 molecules implied that they are products of separate genes; but Dw3 and Lw3 molecules were found to be more homologous to each other than Dd and Ld molecules are to each other. Inter-haplotype comparisons of Dw3 and Lw3 molecules with other D region molecules showed no striking homologies to Dd, Ld or eight other molecules compared. However, both Dw3 and Lw3 molecules were found to be unexpectedly homologous to the Ddx and Dw25 molecules, thus defining another family of structurally related D region antigens. This so called Dw3-family was found to be quite distinct from the previously defined Ld-family of molecules, since no joint members were found. The results of these studies of Dw3 encoded antigens are discussed as evidence for intra-D region recombination or mutation.

Structural characterization of murine Ia antigen N-linked oligosaccharides and localization of specific structures to two unique alpha-chain glycosylation sites.

The sequence of N-linked oligosaccharides of differentially glycosylated murine I-Ak alpha-(alpha 2- and alpha 3-) and beta-chains was determined. I-Ak beta-chains predominantly bear a biantennary complex oligosaccharide with a core fucose, and with the peripheral sequence SA----Gal----GlcNAc----Man. The I-Ak alpha-chain has two N-linked glycosylation sites at Asn-82 and Asn-122. When Lubrol-insoluble alpha 3-chains are examined they are found to bear high-mannose oligosaccharides of either the Man9GlcNAc2 or Man8GlcNAc2 type at both sites. When Lubrol-soluble alpha 2-chains are examined, in about 85% of the molecules the Asn-82 site bears a biantennary complex oligosaccharide with core fucose, and with the peripheral sequence SA----Gal----GlcNAc----Man. Interestingly, the Asn-122 site bears a variety of structures. In about 50% of the molecules, the structure at Asn-122 is a biantennary complex oligosaccharide without core fucose and with the peripheral sequence SA----Gal----GlcNAc----Man. In addition, it can bear other complex structures which we did not define further. The apparently restricted addition of fucose to the oligosaccharide at the alpha-Asn-82 site, even when both alpha-sites bear biantennary complex structures with the same peripheral sequence, is a feature unique to this system. The unusual variety of structures present at the alpha-Asn-122 site may indicate differential processing in different cell types.

Initial biocompatibility studies of a novel degradable polymeric bone substitute that hardens in situ.

Clinical management of osseous defects often requires bone grafts. The standard for treatment is autogenous bone harvested from sites such as either the iliac crest or the outer table of the calvaria. In addition to the problem of donor site morbidity and the limited supply of graft material, there is the additional operating time associated with harvesting procedures. A synthetic, bone graft substitute that can match the clinical performance of autogenous bone could alleviate these deficiencies. Therefore, a polymeric bone substitute was developed that consists of a four-armed star polymer of poly(dioxanone-co-glycolide) endcapped at each termini with a biocompatible lysine-based diisocyanate crosslinker. The polymer can be mixed with inorganic fillers such as either hydroxyapatite or tricalcium phosphate to form either injectable or moldable putty. The addition of a catalyst (for example, diethylaminoethanol and water) to the polymer produces a crosslinking reaction causing the combination to harden. This reaction is nontoxic, normo-thermic and can be performed in situ. During the course of the polymerization, carbon dioxide is liberated, producing an interconnected porous network within the implant, suitable for bone ingrowth. This paper will describe a preliminary biocompatibility assay of the bone substitute.

Biochemical and functional characterization of soluble multivalent MHC L(d)/Fc gamma 1 and L(d)/Fc mu chimeric proteins loaded with specific peptides.

Central to the specificity of the immune system is the interaction between the T cell receptor and the major histocompatibility complex (MHC)-peptide ligand complex. To better understand the nature of this interaction, and to investigate possible avenues for specific therapeutic intervention, we have produced soluble recombinant molecules that can modulate antigen-specific T cells. Our approach involved the construction of recombinant murine genes composed of the MHC class I gene H-2L(d) and the Fc portion of immunoglobulin (Ig) heavy chain genes mu or gamma1. Stable transfectants of these L(d)/Fc gamma1 and L(d)/Fc mu genes generated correctly spliced transcripts and were capable of secreting chimeric protein. Immunoprecipitation analyses demonstrated the presence of chimeric L(d)/ Fc gamma1 and L(d)/Fc mu monomers of approximately 69 kDa and 90 kDa, respectively, as well as chimeric dimers under nonreducing conditions. The capacity of L(d)/Ig molecules to bind specific peptide ligands was demonstrated using radiolabeled peptides or with monoclonal reagents that specifically identify peptide-induced conformational changes in the L(d) ligand binding site. Soluble divalent L(d)/Fc gamma1 molecules were loaded with the murine cytomegalovirus-derived peptide and other L(d)-specific peptide ligands and subsequently isolated and purified. Peptide-loaded L(d)/Fc gamma1 molecules were capable of inhibiting the response of class I-restricted T cells in vitro in a peptide-specific fashion. The development of soluble multivalent chimeric proteins that possess unique properties of both the MHC class I and Ig molecules provides a valuable reagent for the study of potential mechanisms of in vitro and in vivo immune modulation.

Solid-phase direct DNA sequencing of allele-specific polymerase chain reaction-amplified HLA-DR genes.

We describe in this report a new strategy to directly sequence polymerase chain reaction-amplified human leucocyte antigen DRB genes using biotinylated allele-specific synthetic oligonucleotide primers coupled to streptavidin-coated magnetic beads. The use of allele-specific primers in the polymerase chain reaction allows for selective amplification of DNA from one haplotype, which when combined with the direct sequencing technique circumvents the need for DNA cloning prior to sequencing. We demonstrate here that this method can be used to characterize human leucocyte antigen DRB genes among heterozygous individuals. This method can be used for the rapid analysis of highly polymorphic genes among individuals heterozygous at the gene of interest.

Detection of human papillomavirus in archival tissues. Comparison of in situ hybridization and polymerase chain reaction.

Formalin-fixed, paraffin-embedded tissues in pathology archives are an important resource for molecular epidemiology studies. Use of these tissues requires that assays be optimized to account for inevitable variations in tissue fixation and processing that occur in the performance of routine histology. We compared results of colorimetric in situ hybridization (ISH) to L1 consensus polymerase chain reaction (PCR) for detection and typing of human papillomavirus (HPV) in 180 blocks of archival tissues (up to 9 years in storage) from cervical cancer patients. Fifteen samples could not be amplified by PCR, but assays were concordant in 75.1% (124/165) of samples that could be analyzed by both methods. Similar numbers of ISH+/PCR- (23) and ISH-/PCR+ (18) cases were found. Eight of the 18 ISH-/PCR+ cases were attributable to PCR detection of HPV types not included in the ISH assay. This degree of concordance required individual optimization of assay conditions for each block. ISH and PCR assays for HPV yield complementary results, and both can be successfully applied to archival tissues.

Conversion of a human immunodeficiency virus cytotoxic T lymphocyte epitope into a high affinity HLA-Cw3 ligand.

To elucidate the residues important for the binding of peptides to HLA-Cw3, a substitutional analysis of two HLA-Cw*0304-binding peptides was performed. The optimal registry and length for a Cw3-restricted epitope from HIV-1 p24gag was determined to be a nonamer, p24gag 144-152. Substituted analogs of this nonamer peptide revealed that substitutions at position 3 (P3) and the carboxyl-terminal P9 were inhibitory to binding, while certain substitutions at the amino-terminal P1 or P2 increased binding significantly. Substituted analogs of another Cw3-restricted peptide, the Cw3 consensus peptide, which binds to HLA-Cw*0304 with a 1,000-fold higher affinity and with a greater stability than the HIV p24gag nonamer revealed that the P1, P2, P6, and P9 residues play important roles in the ligand's binding to Cw*0304. The incorporation of the amino-terminal P1 and P2 residues from the Cw3 consensus peptide into the HIV p24gag 144-152 peptide created a hybrid peptide with profoundly enhanced affinity for and stability with Cw*0304. Collectively, these findings provide a clear insight into how peptides interact with HLA-Cw3 and how high affinity Cw3 ligands can be constructed.