The pain control infusion pump for postoperative pain control in shoulder surgery.

PURPOSE: This study was initiated to evaluate the effect of a pain control infusion catheter in managing postoperative pain. TYPE OF STUDY: In a prospective, randomized trial, 62 consecutive patients undergoing arthroscopic subacromial decompression had an indwelling pain control infusion catheter placed at the operative site. MATERIALS AND METHODS: Thirty-one patients received 0.25% bupivacaine and 31 patients received saline infusions, each at a constant rate of 2 mL per hour. Patients evaluated their pain by visual analog scale, and also tabulated the amount of narcotic and nonnarcotic medication used each day in the first week of surgery. RESULTS: There was a statistically significant difference in pain in all parameters tested in the bupivacaine group as compared with the saline control group (P <.05). CONCLUSIONS: The bupivacaine pain control infusion pump is an effective means of decreasing postoperative pain.

Association of clonally expanded T cells with the syndrome of primary biliary cirrhosis and limited scleroderma.

Clinical features of the CREST (calcinosis cutis, Raynaud's syndrome, esophageal dysmotility, sclerodactyly, and telangiectasias) syndrome are sometimes exhibited in patients with primary biliary cirrhosis (PBC), but the postulated autoimmune mechanisms behind these conditions are poorly understood. Clonally expanded T cells may play an important role in disease pathogenesis. In this study, overrepresentation of one T-cell receptor beta chain variable region, TCRBV3, was documented in patients with PBC and/or CREST. Overrepresentation of the TCRBV3 gene mRNA was demonstrated by semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR). T cells expressing TCRBV3 were analyzed by flow cytometry, were primarily CD8(+), and contained activated cells as assessed by expression of CD69. Clonally expanded T cells within this population were documented by both complementarity determining region 3 (CDR3) length polymorphism analysis and sequencing of T-cell receptor CDR3 cDNA. TCRBV3(+) clonal expansions were stable when followed for up to 5 years. The results of this study demonstrate that the T-cell repertoire of patients with PBC and CREST is characterized by expanded clonal populations of CD8(+) TCRBV3(+) T cells. These clonal expansions provide evidence that stimulation of clonal populations of CD8(+) T cells is associated with the clinical syndrome of PBC with CREST.

Pleurisy in primary Sjögren's syndrome: T cell receptor beta-chain variable region gene bias and local autoantibody production in the pleural effusion.

Pleurisy with or without effusion has not been considered to be associated with primary Sjögren's syndrome (SS), but rather to represent a manifestation of the underlying disorder, usually rheumatoid arthritis in secondary SS. We describe a patient with primary SS who presented with pleural effusions (PE) as an initial manifestation. Serological studies of paired serum and PE specimens demonstrated the occurrence of local immune reactions in the pleura, including the production of rheumatoid factor and anti-SS-A antibody, the formation of immune complexes, and activation of complement. In addition, the analysis of T cell receptor beta-chain variable (V beta) regions in the PE revealed the overexpression of a number of V beta gene products, including V beta 2 and V beta 13 that have previously been shown to be over-represented in the salivary glands of patients with SS. Thus, our report not only calls for an awareness of pleurisy as an extraglandular manifestation of primary SS, but suggests that a common biased T cell response might play a critical role in the pathogenesis of the glandular as well as extraglandular manifestations.

T-cell receptor Vbeta gene utilization in primary biliary cirrhosis.

Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) was used to study the T-cell receptor (TCR) beta-chain variable (Vbeta) region gene families expressed by T cells in 28 patients with primary biliary cirrhosis (PBC), 20 normal controls, and 9 patients with other chronic inflammatory hepatic diseases. We hypothesized that activation and clonal proliferation of T cells would lead to biases in the T-cell repertoire of patients with PBC. Freshly harvested T cells from both peripheral blood and liver tissue were examined for evidence of biased Vbeta utilization. Individuals varied considerably in their pattern of Vbeta expression, but several significant differences were noted in the PBC group. In peripheral blood, the mean level of Vbeta6.1,3 expression was greater in PBC patients than in normal controls. In the liver of PBC patients, the mean level of Vbeta6.1,3 expression was even higher than in the peripheral blood, indicating intrahepatic accumulation of these T cells. The mean level of Vbeta6.1,3 expression was not significantly different in the blood compared with liver in patients with other liver diseases. Expression of Vbeta7 and Vbeta13.1 was also significantly greater in the liver than in the blood of PBC patients, but similar trends were also seen in the control liver group. These data show that specific alterations in the TCR repertoire are present in the blood and liver of patients with PBC.

Costs analysis of successful rotator cuff repair surgery: an outcome study. Comparison of gatekeeper system in surgical patients.

In an effort to determine the cost effectiveness of rotator cuff repair surgery in workers' compensation patients, a financial analysis of 50 consecutive patients with a "successful" result was performed. Treatment costs were analyzed from the date of initial injury through all evaluations, diagnostic studies, surgical reconstruction, physical therapy and work hardening. Additionally, all workers' compensation payments and the cost of settlement was analyzed. The average cost of medical care was $50,302.25 per patient. The average time to return to unrestricted duty from the date of injury was 11 months. However, patients referred to a specialist immediately following the diagnosis of a rotator cuff tear had total costs that averaged $25,870.64 and returned to work an average of 7 months postoperatively. Patients managed via a "gatekeeper" system averaged $100,280.10 in total costs and the average return to work was 18 months. These differences in cost and return to work were both statistically significant, P < .05. In conclusion, immediate referral of rotator cuff tears for specialized care results in decreased cost and earlier return to work.

Inhibition of antigen-specific T cell activation by staphylococcal enterotoxins.

The staphylococcal enterotoxins SEA, SEB, SEC2, and TSST-1 bind to MHC class II molecules and stimulate polyclonal T cell populations on the basis of the expression of responsive TCR V beta domains. CL-1 is a human T cell clone that is specific for a peptide derived from influenza hemagglutinin (HA 307-319) presented in the context of HLA-DR1. CL-1 expresses the TCR V beta 13.1 domain, and does not respond to SEA, SEB, or TSST-1. This T cell was used to test the effect of nonstimulatory staphylococcal enterotoxins on a response to antigenic peptide. These toxins inhibit peptide-specific activation of CL-1 in a concentration-dependent manner. These toxins also inhibit the response of an HLA-DR1-specific alloreactive T cell clone. This inhibition seems to be a result of impaired access of TCR to the MHC/peptide complex rather than negative signaling by toxin via class II interaction or induction of T cell anergy. SEA, but neither SEB nor TSST-1 impedes avidin access to a biotin group attached to the amino terminus of HA 307-319. SEA partially impairs access of avidin to HA peptide biotinylated at residue 313, but is unable to inhibit avidin access to biotin at residue 318. This demonstrates that SEA binds to HLA-DR molecules that have also bound the antigenic peptide and suggests a topology for the interaction of SEA with class II, whereby the toxin interferes with peptide/MHC-TCR contact.

T cell receptor V beta gene bias in rheumatoid arthritis.

Polymerase chain reaction (PCR) technology was employed to examine peripheral blood and synovial T cells in patients with rheumatoid arthritis (RA) for biased utilization of T cell receptor (TCR) variable region (V) genes. Oligonucleotide primers specific for individual TCR V beta gene families were used to amplify TCR gene products in a semiquantitative assay of their relative utilization in unselected T cell populations. Mean V beta expression in 24 RA peripheral blood samples was very similar to that in a panel of 15 normal subjects, except for a slight decrease in V beta 13.2 expression. V beta utilization in 8 RA synovial tissue samples and 13 synovial fluid samples was compared to simultaneously obtained blood samples. Although heterogeneous patterns of skewed V beta utilization were observed, several significant trends emerged. By a number of approaches to data analysis, a statistically significant increase in expression of V beta 6 and V beta 15 in synovial T cells was documented. In addition, increased synovial expression of V beta 14 was found, but only in the synovial fluid samples. Reduced expression of V beta 1, V beta 4, V beta 5.1, V beta 10, V beta 16, and V beta 19 was also observed in synovial T cells. These results indicate that biased V beta gene utilization in different peripheral compartments of RA patients can be observed in unselected T cell populations, and are consistent with the conclusion that populations of T cells expressing these V beta gene products may be involved in the pathogenesis of the disease.

Distinct binding sites on HLA-DR for invariant chain and staphylococcal enterotoxins.

During biosynthesis, class II molecules of the major histocompatibility complex exist as complexes of the polymorphic alpha and beta chains in association with trimers of the invariant chain (Ii). The nonpolymorphic Ii contains sequences necessary for proper targeting of class II to endosomal compartments, where Ii is degraded. Ii also prevents the premature association of antigenic peptides with class II molecules. It is not known whether the effect of Ii on peptide binding extends to other ligands of class II, specifically exogenous superantigens. Cells expressing a mutant Ii molecule stably associated with HLA-DR at the cell surface were tested for their ability to interact with staphylococcal toxins. Most toxins (staphylococcal enterotoxins A-E and exfoliative toxin) were found to bind to cells expressing this alpha beta Ii complex with levels comparable to cells expressing only alpha beta chains at the cell surface. Cells expressing surface alpha beta Ii complexes stimulated polyclonal populations of peripheral blood T cells in association with these toxins. Binding of toxic shock syndrome toxin (TSST) and subsequent stimulation of T cells were reduced by the presence of the Ii. This reduction was not due to an alteration in the repertoire of T cells responding to TSST in the presence of Ii. Data from experiments with a T-cell clone suggest that interactions between class II molecules and T-cell antigen receptors occur during staphylococcal enterotoxin-mediated stimulation and that surface Ii does not interfere with such interactions.

Structure and chromosomal location of the human gene encoding cartilage matrix protein.

Cartilage matrix protein (CMP) is a major component of the extracellular matrix of nonarticular cartilage. The structure and chromosomal location of the human gene encoding CMP was determined by molecular cloning analysis. We used a partial chicken CMP cDNA probe to isolate three overlapping human genomic clones. From one of these clones, a probe containing 2 human CMP exons was isolated and used to map the gene to chromosome 1p35 and to screen a human retina cDNA library. Two overlapping cDNA clones were isolated. The predicted protein sequence of 496 amino acids includes a 22-residue signal peptide and a 474-residue mature protein of Mr 51,344. The human CMP gene and polypeptide are strikingly similar to the chicken CMP gene and polypeptide. Human CMP is 79% identical to chicken CMP and contains two homologous domains separated by an epidermal growth factor-like domain. One potential N-glycosylation site is conserved between the two species. The human CMP gene spans 12 kilobase pairs with 8 exons and 7 introns which are similar in size to those of the chicken CMP gene. Both RNA splice junctions of intron G in the human and chicken CMP genes are nonconforming to the consensus splice sequences. This suggests that the CMP gene utilizes a new RNA splicing mechanism.

Clonal analysis of the anti-Qa-1 cytotoxic T lymphocyte repertoire: definition of the Qa-1d and Qa-1c alloantigens and cross-reactivity with H-2.

To characterize the four common Qa-1 allelic products, we examined in detail the CTL-defined determinants encoded by Qa-1. In previous studies with anti-Qa-1 CTL and alloantisera, investigators have described antigenic determinants present on Qa-1a and Qa-1b antigens, but they have defined Qa-1c and Qa-1d exclusively by their cross-reactivity with Qa-1a and/or Qa-1b determinants. To delineate further the CTL-defined determinants encoded by Qa-1d, we generated CTL clones with Qa-1d specificity and demonstrated that the Qa-1d molecule expressed determinants that were not detected on Qa-1a, Qa-1b, or Qa-1c target cells. Other CTL clones derived from anti-Qa-1d MLC recognized new antigenic determinants on Qa-1c that cross-reacted with Qa-1d. Each of the four common Qa-1 phenotypes was shown to exhibit unique antigenic determinants. In addition, Qa-1d anti-Qa-1a and Qa-1d anti-Qa-1b CTL confirmed extensive cross-reactivity among these Qa-1 alloantigens. Analysis of CTL from these four immunizations also resulted in the isolation of Qa-1a-specific and Qa-1d-specific CTL clones that cross-reacted with H-2Df and H-2Ks, respectively.

Oligosaccharide-dependent and independent Qa-1 determinants.

A contribution of N-linked oligosaccharides to determinants recognized by alloreactive cytotoxic T lymphocytes has not been demonstrated. Employing cloned CTL and tunicamycin, an inhibitor of protein glycosylation, we found that carbohydrate addition was required for the formation of two of six Qa-1 determinants. The other determinants were detectable on nonglycosylated Qa-1 molecules, similar to observations in most reports that allodeterminants on class I molecules are not dependent on glycosylation for serologic detection. Examination of TM-treated, Con A-activated lymphoblasts revealed a direct correlation between the determinants defined by the reactivity of CTL clones with target cells from four Qa-1 genotypes and their dependence on carbohydrate side chains for expression. Most anti-Qa-1b CTL clones recognized either a glycosylation-dependent determinant found only on Qa-1b cells or glycosylation-independent determinants on both Qa-1b and Qa-1c cells. Similarly, clones that killed only Qa-1a cells recognized a glycosylation-independent determinant. However, clones reactive with both Qa-1a and Qa-1d cells recognized a glycosylation-dependent determinant on Qa-1a molecules and a glycosylation-independent determinant on Qa-1d molecules. This result indicates that such clones recognize cross-reactive conformational determinants, not carbohydrate itself. Thus, N-linked oligosaccharides serve to stabilize the conformation of some Qa-1 determinants, but others remain intact on nonglycosylated molecules. The absence of similar data for H-2K/D/L molecules suggest that a reexamination of other class I antigens with cloned CTL is in order to determine whether Qa-1 molecules are unique.

Cellular requirements for the generation of primary cell-mediated lympholysis responses to Qa-1 antigens.

Mixed leukocyte cultures (MLC) between NZB responder spleen cells and Qa-1-disparate stimulator spleen cells were employed to determine the cellular requirements for the generation of primary anti-Qa-1 cell-mediated lympholysis (CML) responses. Although primary anti-Qa-1 cytotoxic lymphocytes (CTL) were generated during H-2-homologous stimulation, anti-Qa-1 CTL were not detectable from MLC in which the stimulators were H-2 allogeneic. Anti-Qa-1 CTL also were not generated from MLC in which the stimulators were semiallogeneic. Thus, H-2 identity between responder and stimulator cells was not sufficient to permit the generation of primary anti-Qa-1 CTL when H-2 disparity was also present. The capacity for H-2 disparity to prevent anti-Qa-1 CML responses was further demonstrated in MLC containing both H-2-allogeneic and H-2-homologous stimulator cells. Therefore, in subsequent studies we employed NZB responders and H-2-homologous, Qa-1-disparate stimulators. When various subpopulations of stimulator cells were studied for their ability to induce anti-Qa-1 CTL, nylon wool-adherent cells were found to be potent stimulators, but nylon wool-nonadherent cells were not. Furthermore, depletion of macrophages from the stimulator population abrogated the generation of anti-Qa-1 CML responses, despite the presence of responder macrophages in the culture. In contrast, all fractionated subpopulations stimulated anti-H-2 CML responses. When macrophage-enriched cells were used as stimulators, anti-Qa-1 CTL could be generated with approximately 80-fold fewer stimulator cells than when unfractionated splenocytes were used as stimulators. These findings indicated that stimulator macrophages were essential for the generation of primary anti-Qa-1 CTL. Direct evidence for macrophage expression of Qa-1-antigens was obtained by using a Qa-1b-specific CTL clone. These studies provide i) the first evidence for Qa-1 expression on macrophages, ii) a basis for comparison of the cellular interactions necessary to generate CTL against H-2K/D-encoded vs Qa-1-encoded class 1 antigens, and iii) a model for investigating the mechanisms responsible for the immunodominance of H-2K/D alloantigens.

Correlation of Qa-1 determinants defined by antisera and by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) activated in H-2 identical, Qa-1 disparate mixed leukocyte cultures recognize H-2-nonrestricted target antigens indistinguishable by strain or tissue distribution from serologically defined Qa-1 antigens. Cloned Qa-1-specific CTL define determinants encoded by four Qa-1 genotypes; we used anti-Qa-1 sera in antibody blocking experiments to determine if these determinants reside on molecules recognized by Qa-1-specific antibodies. Antisera containing Qa-1.1-specific and TL-specific antibodies blocked recognition of two CTL-defined determinants associated with Qa-1a. Although both Qa-1 and TL molecules are expressed on activated T cells from appropriate strains, our studies indicated that the CTL recognized Qa-1, not TL. In addition, anti-Qa-1.2 serum inhibited CTL recognition of Qa-1b- and Qa-1c-encoded determinants. Qa-1d target cells are unique in that they express determinants recognized by anti-Qa-1a CTL and by anti-Qa-1b CTL. Killing of Qa-1d targets by anti-Qa-1a CTL was not inhibited by anti-Qa-1.1 serum, but was partially inhibited by anti-Qa-1.2 serum. Cytotoxicity of Qa-1d cells by one anti-Qa-1b CTL clone was inhibited by both anti-Qa-1.2 and anti-Qa-1.1 sera, indicating close association of both serological determinants with the determinants recognized by the CTL. Thus, all of the CTL-defined Qa-1 determinants resided on molecules recognized by Qa-1-specific antibodies, but anti-Qa-1a CTL and Qa-1.1-specific antibodies did not have identical specificities.

Mitochondria control expression of a murine cell surface antigen.

Maternally transmitted antigen (Mta) is a murine cell-surface molecule defined by the reactivity of specific H-2 nonrestricted cytotoxic T lymphocytes (CTL-s). Maternal transmission appears to be under control of a stable genetic factor in the cytoplasm of the ovum. In view of the known maternal inheritance of mitochondria we have assessed their involvement in Mta expression using the mitochondria specific poison Rhodamine 6G (R6G). We report here that Mta expression in somatic cell hybrids requires functional mitochondria from the Mta+ parent cell line. Mta expression was dominant in hybrids from the fusion of Mta+ and Mta- cells. However, pretreatment of the Mta+ parent with R6G resulted in hybrids which were Mta-, or diminished in Mta expression. These data strongly implicate mitochondrial DNA (mtDNA) in the expression of a cell-surface molecule, and define a system for studying a previously unrecognized mitochondrial function. To our knowledge, this is the first evidence for mitochondrial control of the expression of a cell membrane molecule in eukaryotes.

Characterization of determinants encoded by four Qa-1 genotypes and their recognition by cloned cytotoxic T lymphocytes.

The alloantigens encoded by the four defined Qa-1 genotypes were characterized by cloned cytotoxic T lymphocyte (CTL) recognition. CTL clones specific for Qa-1a- and for Qa-1b-encoded antigens were generated. Examination of the reactivity of these clones with target cells from H-2r and H-2f strains provided the strongest evidence to date for the designation of the Qa-1c and Qa-1d genotypes, respectively, for these strains. Qa-1c-encoded antigens were recognized by most, but not all CTL clones that specifically lysed Qa-1b target cells, thus demonstrating that these antigens lack a Qa-1b-associated determinant. Similarly, Qa-1d encoded antigens were recognized by only half of the CTL clones that lysed Qa-1a target cells. In addition, one CTL clone that was cytotoxic for Qa-1b and Qa-1c target cells demonstrated low affinity, cross-reactive recognition of a Qa-1d encoded antigen. The reactivity patterns of the monoclonal CTL defined five Qa-1 determinants. Qa-1a, Qa-1b, and Qa-1d each encode multiple determinants. Two Qa-1d encoded determinants probably reside on different molecular species. Finally, large numbers of CTL clones tested on panels of target cells indicated that the Qa-1a strains expressed indistinguishable Qa-1.1 antigens and the Qa-1b strains expressed indistinguishable Qa-1.2 antigens. Therefore, additional polymorphism among these strains is improbable.

The Qa-1 alloantigens. II. Evidence for the expression of two Qa-1 molecules by the Qa-1d genotype and for cross-reactivity between Qa-1 and H-2K.

The nature of cell surface determinants detected by Qa-1-specific alloantisera and cytotoxic T lymphocytes (CTL) in mice of the H-2f, Qa-1d genotype was investigated. The H-2f, Qa-1d strains A.CA and B10.M express both Qa-1a and Qa-1b encoded alloantigenic determinants (Qa-1.1 and Qa-1.2, respectively), as defined in the prototypic A (or B6-Tlaa) and C57BL/6 (or A-Tlab) strains, respectively. Both anti-Qa-1.1 and -Qa-1.2 sera immunoprecipitate 46K m.w. glycoproteins from H-2f, Qa-1d strains. In addition to 46K m.w. proteins, anti-Qa-1.1 sera, but not anti-Qa-1.2 sera, precipitate 55 to 75K m.w. proteins; the nature of these proteins and their relationship to Qa-1 is unclear at present. Sequential immunoprecipitation experiments and the analysis of several recombinant strains revealed that anti-Qa-1.1 sera also cross-react with a 46K m.w. H-2f-encoded alloantigen, probably H-2Kf. Both Qa-1.1 and the non-Qa-1.1 cross-reacting determinants were detected by polyclonal anti-Qa-1a CTL on the Qa-1d strains. The Qa-1a encoded but not the cross-reacting determinants were detected by a cloned anti-Qa-1a CTL line. Sequential immunoprecipitation experiments on the recombinant strains B6.AC2 and B10.M(17R), which are Qa-1d but not H-2Kf, revealed that the Qa-1.1 and Qa-1.2 determinants do not reside on the same molecule. Furthermore, although Qa-1b-encoded determinants were detected on these strains with anti-sera and with bulk CTL cultures, cloned anti-Qa-1b CTL lines thus far analyzed have failed to react with Qa-1d targets, indicating that some but not all of the prototypic Qa-1b-encoded determinants are expressed by the Qa-1d strains.