Effect of sterilization method and other modifications on the wear resistance of acetabular cups made of ultra-high molecular weight polyethylene. A hip-simulator study.

BACKGROUND: Wear of ultra-high molecular weight polyethylene acetabular cups in hip prostheses produces billions of submicrometer wear particles annually that can cause osteolysis and loosening of the components. Thus, substantial improvement of the wear resistance of ultra-high molecular weight polyethylene could extend the clinical life span of total hip prostheses. It has become apparent that the conditions under which ultra-high molecular weight polyethylene cups have been sterilized can markedly affect their long-term wear properties, and new sterilization methods and other modifications have been developed to minimize the negative effects. METHODS: In the present study, a hip-joint simulator was used to assess whether it is preferable to sterilize ultra-high molecular weight polyethylene cups without gamma irradiation, to avoid radiation-induced oxidative degradation, or to sterilize with gamma irradiation while the cups are packaged in a suitable low-oxygen atmosphere to minimize oxidation while retaining the increased wear resistance conferred by the radiation-induced cross-linking. Ion-implanted cups and cups made of a highly crystalline polyethylene (Hylamer) also were investigated. Cups made of each material were subjected to wear-testing prior to and after artificial thermal aging to accelerate oxidative degradation. RESULTS: The results of the present study demonstrated that the cross-linking induced by gamma irradiation improves the wear resistance of ultra-high molecular weight polyethylene, while oxidation reduces it. Without thermal aging, the two types of cups that were sterilized with gamma irradiation while in low-oxygen packaging exhibited about a 50 percent lower rate of wear than did either the nonsterilized cups or the nonirradiated cups sterilized with gas plasma. There was a comparable advantage in the rate of wear after fourteen days of thermal aging. However, after thirty days of aging, the cups sterilized with gamma irradiation in low-oxygen packaging wore several times faster than did the nonirradiated cups. Ion-implanting improved the wear resistance without thermal aging, but after extensive thermal aging the oxidation and wear were greater than those of the controls. Hylamer cups (that is, those that were sterilized with gas plasma) exhibited wear properties very close to those of the nonsterilized ultra-high molecular weight polyethylene cups (the controls) with or without aging. CONCLUSIONS: Sterilizing an ultra-high molecular weight polyethylene acetabular cup without radiation (for example, with ethylene oxide or gas plasma) avoids immediate and long-term oxidative degradation of the implant but does not improve the inherent wear resistance of the polyethylene. Sterilizing with use of gamma irradiation with the implant packaged in a low-oxygen atmosphere avoids immediate oxidation and cross-links the polyethylene, thereby increasing its wear resistance, but long-term oxidation of the residual free radicals may markedly reduce the wear resistance. Ideally, cross-linking with gamma irradiation to reduce wear should be done in a manner that avoids both immediate and long-term oxidation.

The immunological effect of 8-methoxypsoralen and UVA treatment on murine T-cell leukemia.

8-Methoxypsoralen (8-MOP) plus long-wavelength UV radiation (UVA, 320-400 nm) have been used to treat various diseases such as cutaneous T-cell lymphoma, systemic scleroderma, rheumatoid arthritis and rejection of heart transplants. However, the immunological mechanism of this treatment remains unknown. In this report, we investigated the effect of 8-MOP/UVA on the modulation of the immunogenicity of a T-cell leukemia cell line (RL male 1 cells). The results demonstrated that the stimulator function of the in vitro 8-MOP/UVA-treated RL male 1 cells was enhanced in both RL male 1-specific allogeneic and syngeneic immune responses. Furthermore, the enhancement of the immunogenicity of the 8-MOP/UVA-treated RL male 1 cells was found to be strongly associated with the increase of intercellular adhesion molecule-1 expression on these 8-MOP/UVA-treated tumor cells. Therefore, our findings suggested that the alteration of the expression of the immune-related cell surface molecules might be an important effect of 8-MOP/UVA treatment on the elevation of the immunogenicity of the 8-MOP/UVA-treated tumor cells.

A function of Ly-5 (CD-45) in the generation of lymphokine-activated killer cells defined by Ly-5 anti-sense oligodeoxyribonucleotides and Ly-5 monoclonal antibody.

The unique feature of the Ly-5 system is that it is a major cell surface glycoprotein, representing up to 10% of the total cell surface complement, confined to the hematopoietic cells as a family of isoforms generated by alternative splicing of a single Ly-5 gene. The cytoplasmic domain of Ly-5 has protein tyrosine phosphatase activity suggesting that Ly-5 is involved in signal transduction. We used Ly-5 anti-sense oligodeoxyribonucleotides (oligo) and Ly-5 monoclonal antibody (mAb) to study the functional role of Ly-5 in the concanavalin A mitogenesis response by spleen cells, as well as in the generation of lymphokine-activated killer cells and the proliferative response by spleen cells induced by recombinant human interleukin-2 (rhIL-2). Our results indicate that the Ly-5 mAb could enhance these activities whereas the anti-sense oligo was inhibitory. These data clearly suggest that Ly-5 is involved in the IL-2 and IL-2 receptor responsive circuit.

CD45 cell surface antigens are linked to stimulation of early human myeloid progenitor cells by interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (a c-kit ligand).

CD45 antigens are protein tyrosine phosphatases. A possible link was evaluated between expression of CD45 antigens on human myeloid progenitor cells (MPC) (colony-forming unit-granulocyte/macrophage [CFU-GM], burst-forming unit-erythroid [BFU-E], and colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte [CFU-GEMM]) and regulation of MPC by colony-stimulating factors (CSF) (interleukin 3 [IL-3], GM-CSF, G-CSF, M-CSF, and erythropoietin [Epo]), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (MGF; a c-kit ligand). Treatment of cells with antisense oligodeoxynucleotides (oligos) to exons 1 and 2, but not 4, 5, or 6, of the CD45 gene, or with monoclonal anti-CD45, significantly decreased CFU-GM colony formation stimulated with GM-CSF, IL-3, fusion protein, and GM-CSF + MGF, but not with G-CSF or M-CSF. It also decreased GM-CSF, IL-3, fusion protein, and MGF-enhanced Epo-dependent BFU-E and CFU-GEMM colony formation, but had little or no effect on BFU-E or CFU-GEMM colony formation stimulated by Epo alone. Similar results were obtained with unseparated or purified (greater than or equal to one of two cells being a MPC) bone marrow cells. Sorted populations of CD343+ HLA-DR+ marrow cells composed of 90% MPC were used to demonstrate capping of CD45 after crosslinking protocols. Also, a decreased percent of CD45+ cells and CD45 antigen density was noted after treatment of column-separated CD34+ cells with antisense oligos to exon 1 of the CD45 gene. These results demonstrate that CD45 cell surface antigens are linked to stimulation of early human MPC by IL-3, GM-CSF, a GM-CSF/IL-3 fusion protein, and MGF.

Properties of the promoter region of the T18d (T13c) Tla gene.

The T18d (formerly T13c) gene of BALB/c mice belongs to the category of Tla genes which is expressed by both thymocytes and TL+ T-cell leukemias. To elucidate the regulation of T18d, different restriction fragments of the 5' flanking region between -457 and +146 were linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into TL+ and TL- cells. By comparison of transiently expressed CAT activity among cells transfected with different CAT constructs, the results suggest that determination of TL+ vs TL- phenotypes is located within the region -105 to -33, and that an element essential to T18d expression resides within the region -33 to +54. Putative DNA-binding factors characterizing particular cell types and displaying selective affinity for particular T18d restriction fragments were identified by electrophoretic mobility shift assays with nuclear extracts (NEs). Two factors (or complexes) which bound specifically to the T18d fragment -105 to -33 were expressed preferentially in TL+ cells and thus may be involved in determining the tissue-selective expression of T18d. The close proximity of negative and positive cis-acting elements within the promoter region is consistent with regulation of T18d gene expression by a variety of trans-acting factors whose production is attuned to development and differentiation. The data provided may serve as a guide to study the regulation of other categories of Tla genes that are normally silent in thymocytes but may become expressed by leukemia cells.

Organization of the Ly-5 gene.

A single Ly-5 gene is known to generate a variety of transmembrane glycoprotein isoforms that distinguish various cell lineages and stages of differentiation within the hematopoietic developmental compartment of the mouse. Systems homologous to Ly-5 are known in rats and in humans. The complete exon-intron organization of the Ly-5 gene is described in this report. The Ly-5 gene occupies about 120 kilobases of chromosome 1 and comprises 34 exons, of which 32 (Ex-3 to Ex-34) are protein coding. Ex-1, Ex-2, and parts of Ex-3 and Ex-34 are untranslated. In all cDNA clones examined, either Ex-1 or Ex-2 was represented, but not both, implying that Ex-1 and Ex-2 in Ly-5 mRNA may be mutually exclusive. Primer extension and S1 nuclease protection mapping were used to identify initiation (cap) sites for transcription. The finding of putative cap sites for Ex-1 and Ex-2, and of corresponding TATA-like sequences, suggests the presence of two promoters. In both Ex-1+ and Ex-2+ cDNA clones the next exon is Ex-3, which has a translation-initiating codon. The intron between Ex-3 and Ex-4 is unusually long, about 50 kilobases. Evidence is given that Ex-5, like Ex-6 and Ex-7 (studied previously), is another alternative exon that is selectively programmed, alone or together with Ex-6 or Ex-7 or both, to generate actual or potential Ly-5 isoforms by alternative splicing.

Differential induction of class II gene expression in murine pre-B-cell lines by B-cell stimulatory factor-1 and by antibodies to B-cell surface antigens.

We have previously reported that BSF-1 and an alloantibody to the B-cell differentiation antigen Lyb2 induce class II gene expression in two Ia negative pre-B-cell lines. Two questions were asked in these studies. The first question is whether the different stimuli which we and others have shown to induce class II expression in B-cells act via the same signal transduction mechanisms. The second question is whether the traditionally accepted pathway of B-cell differentiation, as defined by immunoglobulin (Ig) gene rearrangement, is applicable to other events that occur during B-cell differentiation. In this report, we have therefore examined a large panel of pre-B-cell lines at different stages of Ig gene rearrangement in an attempt to 1) identify the stage in B-cell development where class II gene expression occurs and where it becomes inducible by BSF-1 or anti-Lyb2, and 2) compare the signal transduction mechanisms used by these ligands. The majority of pre-B-cell lines tested did not express BSF-1 receptors and were consequently noninducible for class II by BSF-1; such cell lines were, however, inducible for class II expression by anti-Lyb2 and, in addition, by antibodies to the B220 membrane glycoprotein. The induction of class II molecules by BSF-1 and by anti-Lyb2 and anti-B220 differed in several respects: 1) Induction by anti-Lyb2 and anti-B220 did not require the presence of BSF-1 receptors; 2) BSF-1 selectively induced class II antigen expression while anti-Lyb2 and anti-B220 induced the expression of other surface markers as well; and 3) PGE2 inhibited BSF-1 but not antibody-mediated class II induction. Finally, the presence of receptors for BSF-1 and the baseline expression of cell surface Ia was shown to be unlinked to Ig gene rearrangement and expression in this series of pre-B-cell lines. The independent regulation of Ia and Ig genes observed here may reflect a branching rather than a linear pathway for B-cell differentiation. The differentiation of pre-B-cells to mature Ig-secreting cells should probably not be defined solely by rearrangement of Ig genes, since this is likely to represent an oversimplified view of B-cell differentiation.

Alternative use of 5' exons in the specification of Ly-5 isoforms distinguishing hematopoietic cell lineages.

Previous inferences that Ly-5 glycoprotein isoforms of murine hematopoietic cells are generated by alternative splicing of primary transcripts of a single Ly-5 gene are supported by the present study. A cDNA library was prepared from B cells by extension from primer representing a known T-cell cDNA sequence. Three different Ly-5 clones from this library included sequences missing in T-cell cDNA clones. From the constitution of cDNA clones and of the Ly-5 gene, and from S1 nuclease mapping, it is concluded that at least two exons, provisionally numbered Ex-6(B) and Ex-7(B), in the 5'-proximal region are mainly represented in mRNA of the B-cell lines examined but not of the T-cell lines examined. Also, exons 1 and 2 appear to be used alternatively in different species of B-cell mRNA and probably also in different species of T-cell mRNA.

Sequences of Ly-5 cDNA: isoform-related diversity of Ly-5 mRNA.

The Ly-5 system of the mouse is expressed exclusively by hematopoietic cells and comprises a series of glycoprotein isoforms that typify different hematopoietic cell lineages. The 200-kDa isoform of T cells and the 220-kDa isoform of B cells are known to differ in peptide composition. The complete 1152 amino acid sequence of the 200-kDa isoform protein deduced from cDNA sequence appears to comprise a leader sequence of some 30 residues, an external N-terminal domain of 370 residues, a probably single transmembrane domain of 22 residues, and an unusually large cytoplasmic domain of 730 residues. Both the external and cytoplasmic domains include regions of internal homology suggestive of evolution from a smaller ancestral gene. RNA transfer blotting has previously shown that B-cell mRNA for Ly-5 is larger than T-cell mRNA. S1 nuclease protection mapping with Ly-5 cDNA probes suggests that this difference can be ascribed to interpolation of an extra B-cell sequence located at the 5' end of B-cell mRNA, probably immediately following the leader sequence. From restriction mapping of overlapping Ly-5 genomic clones spanning 60 kilobases it is concluded that Ly-5 isoforms are generated by differential processing of transcripts of a single gene, rather than from a family of linked Ly-5 genes.

Selective inhibition of lipopolysaccharide-induced polyclonal IgG response by monoclonal Ly-5 antibody.

We have identified two important molecules involved in the regulation of B cell differentiation, namely Lyb-2 and Ly-5. To gain further insight into the function of these two molecules, we examined the effect of monoclonal Lyb-2 and Ly-5 antibodies on lipopolysaccharide (LPS)-induced B cell growth and maturation. We found that Lyb-2 antibody does not have any effect on LPS-induced proliferation and on polyclonal IgM or total IgG responses. On the other hand, although Ly-5 antibody did not affect proliferation and polyclonal IgM responses, it strongly inhibited polyclonal IgG responses, presumably by direct action on B cells. This inhibition was not caused by direct suppressive effect of Ly-5 antibody or Fc receptor-mediated negative signaling. To exert maximal inhibitory effect, Ly-5 antibody had to be added to the culture during the initial 48 hr. However, the presence of Ly-5 antibody during the first 2 days did not cause a significant inhibition. It is thus likely that Ly-5 plays a critical role in the regulation of LPS-induced B cell maturation into IgG-secreting cells at a phase starting within 48 hr after LPS stimulation and continuing thereafter.

Biochemical genetics of TL antigens.

TL antigens are class I glycoproteins which are expressed on thymocytes and which are coded by the Tla region of the major histocompatibility complex of the mouse. Biochemical analysis of TL molecules from different strains of mice revealed structural variation determined by the Tla region which is detectable by peptide mapping, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional gels, and by differential reactivity of allelic forms of TL molecules with a panel of anti-TL reagents. The quantity of TL expressed on thymocytes is also influenced by the Tla region; three quantitative phenotypes were identified: high (Tlaa, Tlad, Tlae), intermediate (Tlac, Tlaf), and low (Tlab). (Relative amounts: 1000: 100: 1.) Some thymic leukemias arising in (Tlab, Tlac) mice with genetically determined reduced levels of thymic TL were found to express TL molecules which were structurally indistinguishable from TL isolated from thymocytes but were present in larger amounts. This suggests that TL structural genes are intrinsically capable of full expression in all mice but that the Tla region of mice expressing an intermediate or low quantity of TL is marked by some feature which causes the thymocyte to express less than the full amount of TL possible.

Further definition of the Ly-5 system.

Ly-5 is expressed by cells of the hematopoietic branch of development. Further serological analysis of the Ly-5 system, aided by Ly-5 monoclonal antibodies and by two Ly-5 congenic mouse strains, reveals two new Ly-5 alloantigens, Ly-5.3 and Ly-5.4. The data define three thymocyte phenotypes, Ly-5.1,3, Ly-5.2,4, and Ly-5.2,3, and three corresponding genotypes, Ly-5a, Ly-5b, and Ly-5c, respectively. Ly-5a is by far the most common allele. The Ly-5c allele is found only in the ST/bJ strain, a finding that accords with the presently unique pattern of restriction fragments previously observed in Southern blotting of ST/bJ DNA with an Ly-5 cDNA probe. Present serological and biochemical data favor the interpretation that the compound Ly-5 phenotype of thymocytes is attributable to two separate Ly-5 molecular isoforms that exhibit a discrete difference in protein composition, bear different Ly-5 antigens, and are produced jointly by thymocytes, unlike other Ly-5 isoforms previously shown to distinguish different hematopoietic cell lineages.

Cloning of Ly-5 cDNA.

A notable feature of Ly-5, among immunogenetic systems that identify glycoproteins of the cell surface and define the surface phenotype of cells according to their lineage, is that the Ly-5 locus specifies a range of molecular isoforms that distinguish cells of different stages and branches of hematopoietic development. The composition of the Ly-5 locus is of much interest in regard to how these isoforms are constructed and differentially regulated according to cell lineage. We describe here a cDNA clone, pLy-5-68, that identifies Ly-5. The Ly-5 specificity of the pLy-5-68 clone was first indicated by a restriction fragment length polymorphism (RFLP), which in Southern blotting distinguishes genomic DNA of C57BL/6 (B6) mice (Ly-5a) from that of B6-Ly-5b congeneic mice whose genome is the same as B6 except for the segment of chromosome 1 that bears Ly-5b. For the following reasons it is unlikely that pLy-5-68 represents a gene linked to Ly-5 that was carried over with Ly-5b during serial backcrossing to make the B6-Ly-5b congeneic strain. In all mouse strains tested, the serological Ly-5 allotype (Ly-5.1 vs. Ly-5.2) accorded with the RFLP pattern. Cells of the ST/bJ mouse strain have unique Ly-5 serological reactions and ST/bJ DNA gives a unique (third) RFLP pattern (Ly-5c) with pLy-5-68. All Ly-5+ cell types reacted positively with pLy-5-68 in RNA transfer blotting, and all Ly-5- cell types tested did not. The difference in size of mRNA reactive with pLy-5-68 in cells expressing the 200-kDa Ly-5 isoform as compared with cells expressing the 220-kDa Ly-5 isoform corresponded with the difference in size of the protein components of those isoforms.

Additional products of the Tla locus of the mouse.

Thymocytes and leukemia cells of some mouse strains yield TL proteins, precipitable by anti-TL antiserum and by anti-TL monoclonal antibodies, that include not only the familiar heavy (H) chain of 45-50 kDa but also products of higher molecular mass. Production of a 53-kDa TL form by Tlad thymocytes was studied in detail. A cross was made between B10.M (Tlad) mice, which produce the 53-kDa TL, and mice of the A strain (Tlaa), which make only the usual H chain. Hemi-expression of apparently unaltered 53-kDa TL was observed in thymocytes of the Tlad/Tlaa heterozygous F1 progeny. Thus, there was no indication of positive or negative trans interaction with respect to production of the 53-kDa TL form associated with Tlad. We conclude that production of 53-kDa TL is governed intrachromosomally. Two-dimensional chymotryptic peptide maps of the TL H chain and the 53-kDa TL of Tlad thymocytes differed only by added features found in the map of the 53-kDa TL. With the exception of Tlaa, all Tla alleles (Tlab-f) yielded TL products of higher molecular weight than the accompanying H chain, although in the case of Tlab this was evident only in TL+ leukemia cells because Tlab thymocytes are TL-. For H-2, representing other class I genes, no products other than the familiar H chain were demonstrable under similar conditions.

Structural characteristics of Tla products.

Biochemical study of thymus leukemia antigen (TL) from thymocytes of various Tla genotypes and from leukemia cells revealed features that, given present evidence, are peculiar to TL among class I products of the H-2:Qa:Tla region of chromosome 17. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of TL from thymocytes of all TL+ mouse strains, precipitated by anti-TL antiserum or monoclonal antibodies, showed two closely migrating bands of equal intensity in the heavy (H) chain position (45-50,000 mol wt). Comparison of these two bands by two-dimensional isoelectric focusing (2D IEF)-SDS-PAGE and 2D chymotryptic peptide mapping showed no differences indicative of protein dissimilarity. Thus, the two components of the H chain doublet may differ only in a feature of glycosylation that does not affect charge. The two leukemias studied gave only a single band in the H chain position. On 2D peptide mapping and 2D IEF-SDS-PAGE, the patterns for TL of Tlaa and Tlae thymocytes, which are closely related serologically, were broadly similar, but clearly different from the pattern typical of Tlac and Tlad thymocytes. 2D peptide maps of TL from Tlaa thymocytes and Tlaa leukemia cells did not differ. Leukemia cells of Tlab origin (thymocytes TL-) gave 2D peptide and 2D IEF-SDS-PAGE patterns of a third type. With the exception of Tlaa, thymocytes of TL+ mice yielded additional TL products of higher molecular weight than the TL H chain.

Characterization and chromosomal distribution of endogenous mouse mammary tumor viruses of European mouse strains STS/A and GR/A.

The endogenous mouse mammary tumor virus (MMTV) proviral copies in two genetically dissimilar mouse strains, STS/A, a European mouse strain, and BALB/c, were characterized. STS/A carries the same four MMTV proviral copies as GR.Mtv-2-; these strains share also most of the isoenzyme markers and are therefore highly related. Cellular DNA of GR.Mtv-2- contains a partial MMTV provirus that is not present in STS/A. GR.Mtv-2- is derived from GR; they differ in the locus Mtv-2 that contains one MMTV provirus. Expression of this Mtv-2 endogenous MMTV provirus is directly linked to mammary tumorigenesis in GR. MMTV proviral loci were studied using restriction enzyme analysis and the Southern transfer procedure using liver DNAs from recombinant inbred strains between BALB/c and STS/A. All segregating MMTV-specific EcoRI fragments were identified to MMTV proviral loci and most of these were localized by studying the cosegregation of the Mtv units and known chromosomal markers. Since STS/A, GR.Mtv-2-, and GR are highly related, the five complete endogenous MMTV proviruses of GR were located on the following chromosomes: Mtv-2 on chromosome 18, Mtv-3 on 11, Mtv-19 on 1, Mtv-20 on 4, whereas Mtv-8 has tentatively been located on chromosome 18 by Callahan et al. (R. Callahan, D. Gallahan, and Ch. Kozak (1984), J. Virol. 49, 1005-1008). GR and GR.Mtv-2 furthermore contain two incomplete MMTV proviral elements, one of which is also present in STS/A.

Genomic constitution of an H-2:Tla variant leukemia.

A TL+ leukemia of a (B6 X A)F1 hybrid mouse (H-2b/H-2a) was previously subjected to immunoselection against H-2a by passage in (B6 X A.SW)F1 mice (H-2b/H-2s). A variant leukemia line was obtained that serologically lacked not only the H-2a phenotype but also the TL phenotype determined by the linked cis Tlaa allele of strain A. The H-2b phenotype and the TL phenotype of the Tlab allele of the B6 strain, which is expressed only by leukemia cells, were retained by the variant. Southern blotting with an H-2 cDNA probe that identifies restriction fragment polymorphisms distinguishing alleles of the H-2 and Tla regions of the B6 and A strains indicates that both the H-2a and Tlaa alleles are missing from the genome of this H-2a:Tlaa negative variant. Since the variant has two apparently unaltered chromosomes 17, where the H-2:Tla complex is situated, and since the intensity of bands in Southern blotting is suggestive of H-2b homozygosity, it is considered that loss of the H-2a:Tlaa haplotype by the variant was accompanied by duplication of the H-2b:Tlab haplotype. The implied change from heterozygosity to homozygosity that the variant has undergone with respect to H-2:Tla was not paralleled by a similar change at the three other loci tested, since the variant retained heterozygosity for Pep-3 (chromosome 1), Gpi-1 (chromosome 7), and Es-1 (chromosome 8).