Autoimmune ovarian inflammation triggered by proinflammatory (Th1) T cells is compatible with normal ovarian function in mice.

The detection of noninfectious ovarian inflammation (oophoritis) and serum ovarian autoantibodies in a patient with premature ovarian failure is indicative of an autoimmune etiology. The mechanisms of autoimmune ovarian injury leading to loss of function are currently unknown. In this study we investigated the impact of oophoritis on ovarian function based on two murine autoimmune ovarian disease (AOD) models. AOD can be induced by thymectomy at Day 3 after birth (d3tx). D3tx mice develop ovarian inflammation and atrophy with loss of oocytes. In these mice, ovarian atrophy and not oophoritis correlated with abnormal estrous cyclicity. The second AOD model is induced by active immunization of adult mice with a murine ZP3 peptide (pZP3) in adjuvant. After active immunization, the zona pellucida antibody titer, not oophoritis, correlated with reduced fertility. To investigate the effect of oophoritis in the absence of antibody response or ovarian atrophy, pZP3-specific T cells were passively transferred into naive syngeneic mice. This recruited cytokine-producing cells into the ovaries so that elevated cytokine production and its effect on ovarian function could be examined. Recipients of pZP3-specific T cells developed severe granulomatous oophoritis, and the diseased ovaries had elevated ovarian mRNA levels of interferon-gamma, interleukin-1beta, and tumor necrosis factor alpha. Despite these changes, fertility rates and gonadotropin-induced follicular development remained essentially normal. Therefore, normal ovarian function is compatible with severe ovarian inflammation mediated by autoreactive T cells.

Comparison of three solutions of ropivacaine/fentanyl for postoperative patient-controlled epidural analgesia.

BACKGROUND: Ropivacaine, 0.2%, is a new local anesthetic approved for epidural analgesia. The addition of 4 microg/ml fentanyl improves analgesia from epidural ropivacaine. Use of a lower concentration of ropivacaine-fentanyl may further improve analgesia or decrease side effects. METHODS: Thirty patients undergoing lower abdominal surgery were randomized in a double-blinded manner to receive one of three solutions: 0.2% ropivacaine-4 microg fentanyl 0.1% ropivacaine-2 microg fentanyl, or 0.05% ropivacaine-1 microg fentanyl for patient-controlled epidural analgesia after standardized combined epidural and general anesthesia. Patient-controlled epidural analgesia settings and adjustments for the three solutions were standardized to deliver equivalent drug doses. Pain scores (rest, cough, and ambulation), side effects (nausea, pruritus, sedation, motor block, hypotension, and orthostasis), and patient-controlled epidural analgesia consumption were measured for 48 h. RESULTS: All three solutions produced equivalent analgesia. Motor block was significantly more common (30 vs. 0%) and more intense with the 0.2% ropivacaine-4 microg fentanyl solution. Other side effects were equivalent between solutions and mild in severity. A significantly smaller volume of 0.2% ropivacaine-4 microg fentanyl solution was used, whereas the 0.1% ropivacaine-2 microg fentanyl group used a significantly greater amount of ropivacaine and fentanyl. CONCLUSIONS: Lesser concentrations of ropivacaine and fentanyl provide comparable analgesia with less motor block despite the use of similar amounts of ropivacaine and fentanyl. This finding suggests that concentration of local anesthetic solution at low doses is a primary determinant of motor block with patient-controlled epidural analgesia after lower abdominal surgery.

Goodpasture's epitope in development of experimental autoimmune glomerulonephritis in rats.

The Goodpasture's epitope (GP) has recently been localized to the last 36 AA of the non-collagenous (NCl) domain of the alpha 3 chain of type IV collagen [alpha 3(IV)]. Since alpha 3(IV) induces glomerulonephritis (GN) in rats and rabbits, the purpose of the present study was to determine if the GP epitope itself could induce GN. We immunized rats with synthetic peptides of GP epitope, 36-mer, alone or as protein conjugates. Rats immunized with bovine GBM served as positive controls. Peptide immunized rats developed high titer antibodies to peptides, but only unconjugated 36-mer induced antibody against human and bovine GBM, but not to rat GBM. Acidic residues and the full length 36-mer were important in production of GBM reactive antibodies. Positive controls developed antibody to GBM without reactivity against 36-mer, had IgG and fibrin on the basement membrane, GN and proteinuria. Kidney eluted antibody was reactive with rat, bovine, and human GBM but not 36-mer. GN rat lymphocytes underwent blast transformation to GBM but not peptide, and peptide immunized animals responded only to the respective peptides. None of the animals immunized with GP peptide epitope, despite the development of anti-peptide antibodies or anti-GBM antibodies, developed any in vivo fixation of antibody to the GBM, abnormal proteinuria, or GN. The present study shows that the GP epitope is sufficient to induce an immune response to the epitope, but it is not sufficient to induce GN. This demonstrates that other factors or epitopes are important in the pathogenicity of GBM induced GN in this model. These remain to be delineated.

Study of EHS type IV collagen lacking Goodpasture's epitope in glomerulonephritis in rats.

The Goodpasture's epitope has been mapped to the alpha 3 non-collagenous chain (NC1) of type (IV) collagen [alpha 3col(IV)]. We have developed a model of experimental autoimmune glomerulonephritis (EAG) in rats immunized once with collagenase solubilized GBM (csGBM). Engelbreth-Holm-Swarm (EHS) tumor contains abundant col(IV) with little or no alpha 3col(IV). To test the hypothesis that antigens related to Goodpasture epitope are required to produce EAG in our model, we immunized rats once with 40 micrograms csEHS. Positive controls immunized with csGBM developed typical EAG with GBM bound antibody, proteinuria, and glomerulonephritis. EHS rats developed circulating and bound antibody to mesangium and tubular basement membrane with minimal GBM deposits, but did not develop proteinuria or glomerulonephritis. Although circulating antibody in EHS rats bound to csGBM by ELISA, there was no binding in ELISA to M2 antigen containing the Goodpasture epitope while EAG rat's serum did bind. By Western blot with antisera to Goodpasture epitope, EHS antigen was less complex than GBM in the monomer/dimer regions and appeared to lack NC1 corresponding to alpha 3col(IV). Blotting with sera from EHS rats demonstrated reactivity to various components of GBM but not to alpha 3col(IV). EAG sera and renal eluates bound to alpha 3col(IV). EAG rats evidenced cell mediated immunity while EHS rats did not (stimulation index EHS 1.1, EAG rats 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)

Contraceptive vaccine assessment based on a murine ZP3 mini-autoantigen.

A summary is presented of published and some unpublished observations from studies on the immunological response of mice to a 13-mer peptide of the murine ovarian zona pellucida glycoprotein ZP3. The findings have the following implications for the design of immunocontraceptive vaccines. To be reversible, a ZP3 vaccine must not contain pathogenic T cell epitopes of ZP3, but contraception without autoimmune oophoritis may be feasible. The immune response to the ZP3 mini-autoantigen is highly variable among inbred mouse strains, suggesting that a single oophoritogenic peptide would not achieve irreversible contraception in an outbred population. The discovery of antigen mimicry at the level of T cell peptide has thrown doubt on the validity of current strategy in detecting relevant self-antigens that might cross react with vaccine immunogens and on the feasibility of fully predicting the cross-reactive autoimmunogenic potential of a peptide or polypeptide vaccine antigen. Autoantibodies directed against epitopes outside the ZP3 mini-autoantigen, produced by immunization with the pure T cell epitope, react with high affinity, with native zona pellucida, and may be useful in identifying B cell epitopes in ZP3.

Antigen mimicry in autoimmune disease sharing of amino acid residues critical for pathogenic T cell activation.

A nonamer peptide from murine nicotinic acetylcholine receptor delta chain (ACR delta), which shared four amino acid residues with a nonamer peptide of murine ovarian zona pellucida glycoprotein ZP3, induced murine autoimmune oophoritis and IgG autoantibody to the zona pellucida. Crossreaction between the ACR delta and ZP3 peptides was established by the response of a ZP3 peptide-specific, oophoritogenic T cell clone to both peptides in association with IA (alpha k beta b). By substituting the ZP3 peptides with a single alanine, four amino acids within the ZP3 peptide were found to be important for ovarian autoimmune disease, autoantibody response, and stimulation of the ZP3-specific T cell clone. Substitution with conservative amino acids of three residues also ablated activity, whereas the fourth, a phenylalanine, was replaceable by tyrosine without loss of activity. Of the four critical amino acids, three were shared between the ZP3 peptide and the ACR delta peptide. Moreover, polyalanine peptides with the four critical ZP3 amino acids or the four amino acids common to the ZP3 and ACR delta peptides induced immune response to ZP3 and elicited severe ovarian autoimmune disease. Thus, organ-specific autoimmune disease can occur through immune response against unrelated self (or foreign) peptides that share with a self-peptide sufficient common amino acid residues critical for activation of pathogenic, autoreactive T cells.

Autoimmune disease of the ovary induced by a ZP3 peptide from the mouse zona pellucida.

We describe a novel experimental system in mice for the study of ovarian autoimmune disease, a condition encountered in women with premature ovarian failure. The ovarian autoimmune disease is induced in B6AF1 mice by a 15-amino acid peptide (Cys-Ser-Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln-Ile-His-Gly-Pro-Arg) from mouse ZP3, the sperm-binding component of the zona pellucida that surrounds growing and mature oocytes. Whereas the peptide induces both T cell and antibody responses, adoptive transfer of CD4+ T cell lines derived from affected animals causes oophoritis without observable antibodies to the zona pellucida peptide. The primacy of the T cell response in the pathogenesis of disease is further substantiated by defining oophoritogenic peptides as small as eight amino acids (Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln) that do not elicit an antibody response to the full-length ZP3 peptide. The identification of a well characterized peptide as a causative agent of autoimmune oophoritis should facilitate understanding of the pathogenesis of this T cell-mediated autoimmune disease. Because the proteins of the zona pellucida are conserved among mammals (the mouse and human ZP3 proteins are 67% identical), this murine model may lead to better understanding of the pathogenesis of human autoimmune oophoritis.

Pharmacokinetic basis for nonadditivity of intraocular pressure lowering in timolol combinations.

The authors determined whether the ocular absorption of topically applied timolol in the pigmented rabbit was affected significantly by coadministration with either pilocarpine or epinephrine in the same drop to explain the nonadditivity in intraocular pressure lowering (IOP) seen clinically. They instilled 25 microliters of 0.65% timolol maleate solution (equivalent to 0.5% timolol), both in the presence and absence of 2.6% pilocarpine nitrate or 1% epinephrine bitartrate, into pigmented rabbit eyes. The time course of timolol concentration in the conjunctiva, anterior sclera, corneal epithelium, corneal stroma, aqueous humor, iris-ciliary body, and lens was monitored for 360 min by using reversed-phase high-performance liquid chromatography. The area under the timolol concentration-time curve in all but one of the anterior segment tissues was reduced by 20-50% (mean, 40%) when timolol was coadministered with pilocarpine and by 20-70% (mean, 42%) when timolol was coadministered with epinephrine. Such an effect was not a result of alterations in corneal permeability or aqueous humor turnover rate, nor was it related to the extent of systemic absorption caused by pilocarpine and epinephrine. Rather, the reduction in ocular timolol absorption may have been caused by the accelerated washout of timolol by tears stimulated by the coadministered drugs and, to a lesser extent, by the loss of timolol through binding to the increased amount of tear proteins induced by the coadministered drugs. Thus, the nonadditivity in IOP lowering from timolol-pilocarpine and timolol-epinephrine combinations is probably caused by changes in precorneal timolol clearance.

Ocular drug interactions involving topically applied timolol in the pigmented rabbit.

The objective of this study was to determine whether the ocular and systemic absorption of topically applied timolol in the pigmented rabbit was significantly affected by the coadministration with other eye medications, including pilocarpine, epinephrine, their prodrugs, phenylephrine, tetracaine, and proparacaine. Twenty-five microliters of 0.65% timolol maleate solutions, both in the presence and absence of a second drug, were instilled to the pigmented rabbit eye. Timolol concentrations in anterior segment tissues were monitored at 30 and 90 min, and the time course of timolol concentration in plasma was monitored over 120 min. All coadministered drugs except proparacaine reduced intraocular timolol concentrations by varying extents depending on the sampling time, while increasing the timolol concentrations in the conjunctiva and sclera. In addition, all coadministered drugs, except pilocarpine, tetracaine, and proparacaine, reduced the systemic absorption of timolol by an average of about 50%. A plausible explanation for the simultaneous reduction in ocular and systemic timolol absorption is changes in tear turnover rate, protein secretion, and binding of timolol to tear proteins, rather than changes in corneal and perhaps conjunctival and nasal permeability, elicited by the second drug. Vasoconstriction on the conjunctival and nasal mucosae is an additional factor possibly contributing to the reduction in systemic timolol absorption by epinephrine, its prodrug, phenylephrine, and perhaps the pilocarpine prodrug. The clinical implication of the above findings is that before instituting combination or multiple drug therapy the possibility of changes in ocular and systemic absorption of the first drug by the second drug must be considered.

Preparation and characterization of monoclonal antibodies against sperm-specific lactate dehydrogenase C4.

LDH-C4, the testis-specific isozymes of lactate dehydrogenase (LDH), is the predominant LDH isozyme in mammalian spermatozoa. Nine monoclonal antibodies against mouse LDH-C4 have been developed. These antibodies were tested for cross reactivity with LDH-C4 from human testis and with LDH-1-5 from mouse and human testes by immunoelectrophoresis, bio-dot, and western blot assays. The results showed that all monoclonal antibodies were specific to LDH-C4 only: they did not react with LDH-1-5 from mice, nor from humans. The immunologic localization of the monoclonal antibodies on capacitated sperm was observed by indirect immunofluorescent assay. On mouse sperm the antibodies were bound to the tail only, but on human sperm eight antibodies were bound to the postacrosome, some to the neck and to the mid-piece. Most of the antibodies belonged to the Ig G class.

The ocular route for systemic insulin delivery in the albino rabbit.

The objective of this study was to determine the extent, pathways and effects of absorption promoters on the systemic absorption of insulin after topical solution instillation to the albino rabbit eye. The absorption promoters were polyoxyethylene-9-lauryl ether, Na glycocholate (GC), Na taurocholate and Na deoxycholate, all at a concentration of 1%. Plasma glucose concentration was measured in a glucose analyzer whereas plasma insulin concentration was measured using radioimmunoassay. The systemic bioavailability of insulin calculated from its area under concentration-time curve was in good agreement with that derived from the glucose concentration-time curve. The bioavailability was 5.7 to 12.6% with polyoxyethylene-9-lauryl ether, 4.9 to 7.9% with GC, 3.6 to 7.8% with Na taurocholate and 8.2 to 8.3% with Na deoxycholate, as compared to 0.7 to 1.3% in the absence of absorption promoters. The absorption promoting effect of GC was dependent on its concentration over the range of 0.1 to 2%. At 1%, the absorption promoting effect of GC persisted for about 5 min but disappeared by 15 to 30 min. The nasal mucosa contributed about 4 times more than the conjunctival mucosa to the systemic absorption of ocularly applied insulin. The conjunctival mucosa was, however, more discriminant in its sensitivity to the nature of the bile salts than the nasal mucosa. The former was most sensitive to Na deoxycholate and least to Na taurocholate. Collectively, our findings indicate that it is feasible to obtain hypoglycemia from ocularly administered insulin.