A Phase 2b randomized trial of lorecivivint, a novel intra-articular CLK2/DYRK1A inhibitor and Wnt pathway modulator for knee osteoarthritis.

OBJECTIVE: Lorecivivint (LOR; SM04690), an investigational Wnt pathway modulator, previously demonstrated patient-reported and radiographic outcome improvements vs placebo in clinically relevant subjects with moderate to severe knee osteoarthritis (OA). This study's objective was to identify effective LOR doses. DESIGN: Subjects in this 24-week, Phase 2b, multicenter, randomized, double-blind, placebo (PBO)-controlled trial received an intra-articular injection of 2 mL LOR (0.03, 0.07, 0.15, or 0.23 mg), PBO, or dry-needle sham. The primary efficacy endpoints were changes in Pain NRS [0-10], WOMAC Pain [0-100], WOMAC Function [0-100], and radiographic mJSW outcomes, which were measured using baseline-adjusted analysis of covariance at Week 24. Multiple Comparison Procedure-Modeling (MCP-Mod) was performed for dose modeling. RESULTS: In total, 695/700 subjects were treated. Pain NRS showed significant improvements vs PBO after treatment with 0.07 mg and 0.23 mg LOR at Weeks 12 (-0.96, 95% CI [-1.54, -0.37], P = 0.001; -0.78 [-1.39, -0.17], P = 0.012) and 24 (-0.70 [-1.34, -0.06], P = 0.031; -0.82 [-1.51, -0.12], P = 0.022). Additionally, 0.07 mg LOR significantly improved WOMAC Pain and Function subscores vs PBO at Week 12 (P = 0.04, P = 0.021), and 0.23 mg LOR significantly improved both WOMAC subscores at Week 24 (P = 0.031, P = 0.017). No significant differences from PBO were observed for other doses. No radiographic progression was observed in any group at Week 24. MCP-Mod identified 0.07 mg LOR as the lowest effective dose. CONCLUSION: This 24-week Phase 2b trial demonstrated the efficacy of LOR on PROs in knee OA subjects. The optimal dose for future studies was identified as 0.07 mg LOR.

A novel Wnt pathway inhibitor, SM04690, for the treatment of moderate to severe osteoarthritis of the knee: results of a 24-week, randomized, controlled, phase 1 study.

OBJECTIVE: To assess the safety, pharmacokinetics, and exploratory efficacy of SM04690, a novel Wnt pathway inhibitor, as a potential disease modifying treatment for knee osteoarthritis (OA). DESIGN: Subjects with Kellgren-Lawrence grade 2-3 knee OA were randomized in successive dose-escalation cohorts to receive a knee intra-articular (IA) injection with 0.03, 0.07, or 0.23 mg SM04690, or placebo (PBO) (4:1 ratio). Safety, pharmacokinetics, efficacy (WOMAC Total/Function/Pain, Pain VAS, Physician Global Assessment [MDGA], and OMERACT-OARSI Response), OA-related biomarker (P1NP, ß-CTX, and cartilage oligomeric matrix protein [COMP]), and radiographic/imaging data were collected at baseline and during 24-week follow-up. RESULTS: 61 subjects (SM04690 n = 50; PBO n = 11) enrolled. Two dose limiting toxicities (DLTs), increased pain following injection and paroxysmal tachycardia (also the single serious AE), were reported in the 0.07 mg cohort. A total of 72 AEs were reported; Sixteen (occurring in eight subjects) were considered related to study medication. There were three discontinuations; one due to an AE (0.03 mg cohort). Bone marrow edema (BME) remained constant for most subjects. No doses were excluded from further study due to DLT criteria. Plasma levels of SM04690 were below the limit of detection at all time points. At Week 24, improvements from baseline were seen in all cohorts for the exploratory measures WOMAC Total, WOMAC Function, WOMAC Pain, MDGA, Pain VAS, and OMERACT-OARSI response. Joint space width (JSW) improvement was observed in the 0.07 mg cohort (P = 0.02 vs PBO). CONCLUSION: SM04690 appeared safe and well tolerated, with no evidence of systemic exposure. Exploratory efficacy analyses suggested positive trends for measurements of OA pain, function and disease-modifying osteoarthritis drug (DMOAD) properties. CLINICALTRIALS. GOV REGISTRATION: NCT02095548.

Identification and pharmacological characterization of a novel inhibitor of autotaxin in rodent models of joint pain.

OBJECTIVE: Autotaxin is a secreted lysophospholipase that mediates the conversion of lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA), a bioactive lipid mediator. Autotaxin levels in plasma and synovial fluid correlate with disease severity in patients with knee osteoarthritis (OA). The goal of this study was to develop and characterize a novel small molecule inhibitor of autotaxin to inhibit LPA production in vivo and determine its efficacy in animal models of musculoskeletal pain. DESIGN: Compound libraries were screened using an LPC coupled enzyme assay that measures the amount of choline released from LPC by the action of autotaxin. Hits from this assay were tested in a plasma assay to assess inhibition of endogenous plasma autotaxin and subsequently tested for their ability to lower plasma LPA levels upon oral dosing of rats. The best compounds were then tested in animal models of musculoskeletal pain. RESULTS: Compound screening led to the identification of compounds with nanomolar potency for inhibition of autotaxin activity. Studies in rats demonstrated a good correlation between compound exposure levels and a decrease in LPA levels in plasma. The leading molecule (compound-1) resulted in a dose dependent decrease in joint pain in the mono-sodium iodoacetate (MIA) and meniscal tear models and a decrease in bone fracture pain in the osteotomy model in rats. CONCLUSION: We have identified and characterized a novel small molecule inhibitor of autotaxin and demonstrated its efficacy in animal models of musculoskeletal pain. The inhibitor has the potential to serve as an analgesic for human OA and bone fracture.

A short-term pharmacodynamic model for monitoring aggrecanase activity: injection of monosodium iodoacetate (MIA) in rats and assessment of aggrecan neoepitope release in synovial fluid using novel ELISAs.

OBJECTIVE: To develop a short-term in vivo model in rats, with an enzyme-linked immunosorbent assay (ELISA) readout for specific aggrecanase-cleaved aggrecan fragments, to facilitate testing of aggrecanase inhibitors. METHODS: Monosodium iodoacetate (MIA), a metabolic inhibitor, was injected into the right knee joint of male Lewis rats and the release of aggrecanase-cleaved fragments of aggrecan containing the NITEGE or ARGN neoepitope was measured in the synovial fluid at 7 days post MIA injection using novel ELISAs. The ELISAs utilize a commercial antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan, in combination with either an alpha-NITEGE antibody (NITEGE ELISA) or an alpha-ARGS/BC3 antibody (ARGS ELISA), to detect aggrecanase-cleavage of aggrecan within the interglobular domain (IGD). Aggrecan fragments present in in vitro digests, in cytokine-treated cartilage explant culture supernatants and in rat synovial fluid lavage samples were detected and quantified using the two ELISAs. Small molecule inhibitors of aggrecanase activity were dosed orally on days 3-7 to determine their ability to inhibit MIA-induced generation of the NITEGE and ARGN neoepitopes measured in the rat synovial fluid. RESULTS: The NITEGE assay was shown to specifically detect the N-terminal fragment of aggrecan comprising the G1 domain and the NITEGE neoepitope sequence. This assay can readily measure aggrecanase-cleaved bovine, human and rat aggrecan without the need for deglycosylation. The ARGS assay specifically detects C-terminal fragments of aggrecan comprising the ARGS/ARGN neoepitope and the G2 domain. Keratan sulfate (KS) residues of aggrecan interfere with this ELISA, and hence this assay works well with native rat articular cartilage aggrecan (that lacks KS residues) and with deglycosylated bovine and human aggrecan. Injection of MIA into the rat knee joints resulted in a time-dependent increase in the release of aggrecanase-cleaved aggrecan fragments into the synovial fluid and treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of the generation of these neoepitopes. CONCLUSIONS: We have established a short-term in vivo model in rats that involves measurement of synovial fluid biomarkers that are dependent on aggrecanase activity in the joint. The short duration of the model combined with the mechanistic biomarker readout makes it very useful for the initial in vivo screening of aggrecanase inhibitors prior to testing them in time and resource-intensive disease models of osteoarthritis (OA).

Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine.

OBJECTIVE: Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA. METHODS: The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA. RESULTS: The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity. CONCLUSIONS: We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.

Efficacy of prednisone 1-4 mg/day in patients with rheumatoid arthritis: a randomised, double-blind, placebo controlled withdrawal clinical trial.

OBJECTIVE: A randomised double-blind placebo controlled withdrawal clinical trial of prednisone versus placebo in patients with rheumatoid arthritis (RA), treated in usual clinical care with 1-4 mg/day prednisone, withdrawn to the same dose of 1 mg prednisone or identical placebo tablets. METHODS: All patients were from one academic setting and all trial visits were conducted in usual clinical care. Patients were taking stable doses of 1-4 mg prednisone with stable clinical status, documented quantitatively by patient questionnaire scores. The protocol included three phases: (1) equivalence: 1-4 study prednisone 1 mg tablets taken for 12 weeks to ascertain their efficacy compared with the patient's usual tablets before randomisation; (2) transfer: substitution of a 1 mg prednisone or identical placebo tablet every 4 weeks (over 0-12 weeks) to the same number as baseline prednisone; (3) comparison: observation over 24 subsequent weeks taking the same number of either placebo or prednisone tablets as at baseline. The primary outcome was withdrawal due to patient-reported lack of efficacy versus continuation in the trial for 24 weeks. RESULTS: Thirty-one patients were randomised, 15 to prednisone and 16 to placebo, with three administrative discontinuations. In "intent-to-treat" analyses, 3/15 prednisone and 11/16 placebo participants withdrew (p = 0.03). Among participants eligible for the primary outcome, 3/13 prednisone and 11/15 placebo participants withdrew for lack of efficacy (p = 0.02). No meaningful adverse events were reported, as anticipated. CONCLUSION: Efficacy of 1-4 mg prednisone was documented. Evidence of statistically significant differences with only 31 patients may suggest a robust treatment effect.

Erythrocyte sedimentation rate and C-reactive protein levels are poorly correlated with clinical measures of disease activity in rheumatoid arthritis, systemic lupus erythematosus and osteoarthritis patients.

OBJECTIVE: To determine the patterns and correlation of elevated erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels with outcome measures in rheumatoid arthritis (RA), and compare it to systemic lupus erythematosus (SLE) and osteoarthritis (OA) patients. METHODS: Brooklyn Outcomes Arthritis Registry Database (BOARD) was analyzed to determine both first visit and mean values of ESR and CRP, along with disease activity measures in each patient. Data were analyzed with descriptive statistics and correlations. RESULTS: Among all patients half of all (n=377) ESR results were elevated. In RA patients the proportions of having both ESR and CRP elevated, both within normal levels, and only one elevated and the other normal were similar. For all diagnosis, both ESR and CRP have weak positive correlations with disease activity measures measured at first visits. ESR and CRP have a modest positive correlation with each other across all three disease groups. CONCLUSION: In this cohort of RA, SLE and OA patients, ESR and CRP values were modestly correlated with each other and they were weakly correlated with disease activity measures. These data suggest that another look at the role of ESR and CRP as markers of inflammation in RA patients seen in routine care may be in order.

The effect of statin pretreatment on infarct volume in ischemic stroke.

BACKGROUND: Treatment with statins reduces infarct volume in animal models of ischemic stroke, independently of the effect on cholesterol. This study examined this effect in humans by testing whether patients taking statins at onset of ischemic stroke had smaller infarct volumes than those not taking statins. METHODS: The study design was a retrospective cohort analysis of all verified ischemic stroke patients admitted to the Medical University of South Carolina Hospital, in 2002-2006, with magnetic resonance diffusion-weighted imaging (DWI). Patients were grouped according to statin status at admission and compared relative to infarct volume, calculated (blinded to statin status) from DWI. RESULTS: A smaller than median infarct volume following ischemic stroke was associated with the interaction of statin pretreatment and positive diabetes status. This association remained significant (p = 0.01) in multivariable analysis even after controlling for factors related to demographics, comorbidities and risk factors, clinical features on admission, use of other medications and stroke features. CONCLUSIONS: Among ischemic stroke patients in this study, infarct volume below the median was significantly associated with statin pretreatment among those with diabetes.

A proposed approach to recognise "near-remission" quantitatively without formal joint counts or laboratory tests: a patient self-report questionnaire routine assessment of patient index data (RAPID) score as a guide to a "continuous quality improvement" s.

A proposed approach is presented to recognise a status of "near-remission" in a patient with rheumatoid arthritis (RA) on the basis of patient self-report questionnaire data without formal joint counts or laboratory tests. Indices of patient-reported outcome (PRO) measures distinguish active from control treatments in RA clinical trials at levels similar to American College of Rheumatology (ACR) or disease activity score (DAS) 28 improvement levels. PRO measures on a multidimensional health assessment questionnaire (MDHAQ) can be compiled into a routine assessment of patient index data (RAPID) score. RAPID 3 includes the three PRO measures from the ACR Core Data Set - physical function, pain, and global estimate. RAPID 4 adds a self-report joint count from a rheumatoid arthritis disease activity index (RADAI). RAPID 5 adds a physician estimate of global status. RAPID cores may be classified into four preliminary proposed categories, as "near-remission" (0-1), "low severity" (1.01-2), "moderate severity" (2.01-4), and "high severity" (> 4), analogous to the four categories of the DAS28 of "remission" (< 2.6), as well as "low" (2.6-3.19), "moderate" (3.2-5.1), and "high" (> 5.1) disease activity. RAPID scores are correlated significantly with DAS28 (rho = 0.64-0.67, p < 0.001), and about 75% of patients with DAS < 2.6 have RAPID scores < 2, while about 75% of patients with DAS > 5.1 have RAPID scores > 4. RAPID data are available on one side of one page, and are feasible to collect in standard clinical care. RAPID 3 scores may be calculated in about 10 seconds, and RAPID 4 and RAPID 5 scores in 20 to 30 seconds. RAPID scores every 3 months or more on simple flowsheets can be a basis for a "continuous quality improvement" strategy in standard clinical care to recognise a need for aggressive therapy, an inadequate response to a therapy, and "near- remission" status.

Long term safety of methotrexate in routine clinical care: discontinuation is unusual and rarely the result of laboratory abnormalities.

OBJECTIVE: To analyse patients with rheumatoid arthritis, treated with methotrexate in a weekly academic rheumatology clinic over 13 years, for continuation of courses and reasons for discontinuation. METHODS: All 248 patients with an analysable longitudinal course who took methotrexate in standard care between 1990 and 2003 were studied. Continuation of courses was analysed using life tables. All abnormal and severely abnormal values for aspartate aminotransferase (AST) >40 U/l, >80 U/l, albumin <35 g/l, <30 g/l, white blood cell (WBC) count <4.0 x 10(9)/l, <3.0 x 10(9)/l, and platelet count <150 x 10(9)/l, <100 x 10(9)/l, were identified. Responses of the clinician and subsequent laboratory values were reviewed. RESULTS: Over 1007 person-years, the probability of continuing methotrexate over five years was 79% (95% confidence interval, 72% to 84%). Severe laboratory abnormalities occurred in 2.9 per 100 person-years, specifically 0.9 for AST >80 U/l, 1.1 for albumin <30 g/l, 0.7 for WBC <3.0 x 10(9)/l, and 0.3 for platelets <100 x 10(9)/l. No severe laboratory abnormality progressed to further severity or clinical disease. Permanent discontinuations of methotrexate occurred in 46 patients (19%), 26 (10% of all patients) for adverse effects, 15 (32.6%) for inefficacy; only two discontinuations resulted from laboratory abnormalities, both of WBC, possibly from other sources. CONCLUSIONS: Methotrexate was associated with a high rate of continuation, and few clinically significant laboratory abnormalities. Discontinuation primarily reflected clinical rather than laboratory findings. Vigilance for methotrexate toxicity is required but methotrexate appears among the safest treatments for rheumatoid arthritis.

alpha-Amino-beta-sulphone hydroxamates as potent MMP-13 inhibitors that spare MMP-1.

A series of alpha-amino-beta-sulphone hydroxamates was prepared and evaluated for potency versus MMP-13 and selectivity versus MMP-1. Various substituents were employed on the alpha-amino group (P(1) position), as well as different groups attached to the sulphone group extending into P(1)'. Low nanomolar potency was obtained for MMP-13 with selectivity versus MMP-1 of >1000x for a number of analogues.

alpha-Alkyl-alpha-amino-beta-sulphone hydroxamates as potent MMP inhibitors that spare MMP-1.

A series of alpha-alkyl-alpha-amino-beta-sulphone hydroxamates was prepared and evaluated for potency versus MMP-2 and MMP-13, and for selectivity versus MMP-1. Low nanomolar potency was obtained with selectivity versus MMP-1 ranging from >10 to >1000. Selected compounds were orally bioavailable.

Preference for nonsteroidal antiinflammatory drugs versus acetaminophen and concomitant use of both types of drugs in patients with osteoarthritis.

OBJECTIVE: To analyze results of treatment of osteoarthritis (OA) with acetaminophen and the nonsteroidal antiinflammatory drugs (NSAID) through a patient survey. METHODS: A 15 minute telephone survey was conducted with 300 patients, including 172 with confirmed OA. RESULTS: Twenty-four percent of patients who took acetaminophen rated it as "very helpful," compared to 31% for ibuprofen, 30% for naproxen, and 56% for diclofenac. Drug continuation beyond 24 months was reported by 33% of patients for acetaminophen, 21% for ibuprofen, 17% for naproxen, and 19% for diclofenac. Acetaminophen was significantly less likely to be discontinued because of toxicity than NSAID. Patients who indicated that they would not take a drug again, and therefore be unlikely to participate in a clinical trial involving this drug, were 26% for acetaminophen, 40% for ibuprofen, 38% for naproxen, and 28% for diclofenac. About 30% of patients who took acetaminophen reported concurrent use of ibuprofen, naproxen, or diclofenac. Among the 67% of patients who identified a drug as "most helpful," 80% named an NSAID, compared to 20% who named acetaminophen or another analgesic as the "most helpful" drug. CONCLUSION: Patients take many different drugs for OA, most of which are not continued beyond 2 years. Many patients take both acetaminophen and an NSAID. Most patients who identified a drug as "most helpful" named an NSAID rather than acetaminophen or an analgesic drug. These findings may be of value in further development of management strategies and guidelines for OA.

Toward a multidimensional Health Assessment Questionnaire (MDHAQ): assessment of advanced activities of daily living and psychological status in the patient-friendly health assessment questionnaire format.

OBJECTIVE: To develop components of a multidimensional Health Assessment Questionnaire (MDHAQ) through the addition of new items in the "patient-friendly" HAQ format, including advanced activities of daily living (ADL), designed to overcome "floor effects" of the HAQ and modified HAQ (MHAQ) in which patients may report normal scores although they experience meaningful functional limitations, and psychological items, designed to screen efficiently for psychological distress in routine care. METHODS: The new MDHAQ items, as well as scales for pain, fatigue, helplessness, and global health status on a 2-page questionnaire, were completed by 688 consecutive patients with various rheumatic diseases, including 162 with rheumatoid arthritis (RA), 114 with fibromyalgia, 63 with osteoarthritis, 34 with systemic lupus erythematosus, 20 with vasculitis, 18 with psoriatic arthritis, 16 with scleroderma, and 261 with various other rheumatic diseases, over 2 years at a weekly academic rheumatology clinic. RESULTS: The new MDHAQ items have good test-retest reliability and face validity. MHAQ scores were highest in patients with RA, and scores for other scales were highest in patients with fibromyalgia. On the advanced ADL, 58% of patients reported difficulty with errands, 68% with climbing stairs, 79% with walking two miles, 87% with participating in sports and games, and 94% with running or jogging two miles. On the psychological items, 75% of patients reported difficulty with sleep, 63% with stress, 61% with anxiety, and 57% with depression. Normal MHAQ scores were reported by 23% of patients and normal HAQ scores by 16% of patients who completed these questionnaires, while fewer than 5% had normal scores on the MDHAQ. CONCLUSION: The MDHAQ items overcome in large part the "floor effects" seen on the HAQ and MHAQ, and are useful to screen for problems with sleep, stress, anxiety, and depression in the "patient-friendly" HAQ format. These data support the value of completion of a simple 2-page patient questionnaire by each patient at each visit to a rheumatologist.

Discovery of a novel series of selective MMP inhibitors: identification of the gamma-sulfone-thiols.

We have discovered a new series of potent MMP Inhibitors that are selective for MMP-13 over MMP-1 incorporating a gamma-sulfone thiol.

Negatively charged residues interacting with the p4 pocket confer binding specificity to DRB1*0401.

OBJECTIVE: To identify critical residues involved in the binding of a selective peptide to DRB1*0401. METHODS: The binding of peptides to native or site-directed mutant DR molecules was evaluated using enzyme-linked immunosorbent assay and flow cytometry. RESULTS: Amino acid substitutions at DR and peptide residues, which were predicted to contribute to interactions within the DR p4 pocket, had the greatest effects on the specificity of binding. CONCLUSION: Differences in the peptide-binding repertoires of DR molecules may contribute to associations with autoimmune diseases.

Pocket 4 of the HLA-DR(alpha,beta 1*0401) molecule is a major determinant of T cells recognition of peptide.

To investigate the functional roles of individual HLA-DR residues in T cell recognition, transfectants expressing wild-type or mutant DR(alpha,beta 1*0401) molecules with single amino acid substitutions at 14 polymorphic positions of the DR beta 1*0401 chain or 19 positions of the DR alpha chain were used as antigen-presenting cells for five T cell clones specific for the influenza hemagglutinin peptide, HA307-19. Of the six polymorphic positions in the DR beta floor that were examined, mutations at only two positions eliminated T cell recognition: positions 13 (four clones) and 28 (one clone). In contrast, individual mutations at DR beta positions 70, 71, 78, and 86 on the alpha helix eliminated recognition by each of the clones, and mutations at positions 74 and 67 eliminated recognition by four and two clones, respectively. Most of the DR alpha mutations had minimal or no effect on most of the clones, although one clone was very sensitive to changes in the DR alpha chain, with loss of recognition in response to 10 mutants. Mutants that abrogated recognition by all of the clones were assessed for peptide binding, and only the beta 86 mutation drastically decreased peptide binding. Single amino acid substitutions at polymorphic positions in the central part of the DR beta alpha helix disrupted T cell recognition much more frequently than substitutions in the floor, suggesting that DR beta residues on the alpha helix make relatively greater contributions than those in the floor to the ability of the DR(alpha,beta 1*0401) molecule to present HA307-19. The data indicate that DR beta residues 13, 70, 71, 74, and 78, which are located in pocket 4 of the peptide binding site in the crystal structure of the DR1 molecule, exert a major and disproportionate influence on the outcome of T cell recognition, compared with other polymorphic residues.

Individual effects of the DR11-variable beta-chain residues 67, 71, and 86 upon DR(alpha,beta 1*1101)-restricted, peptide-specific T cell proliferation.

The four members of the HLA-DR11 family of class II molecules vary only by three or fewer amino acids via dimorphisms among DR beta-chain residues 67, 71, and 86. However, they differ markedly in their abilities to induce proliferation of DR(alpha,beta 1*1101)-restricted, peptide-specific T cell clones. To dissect which DR11-variable residues, individually and in combination, mediate these functional differences, we used as APC transfectants expressing DR molecules with one of all possible permutations of DR11-variable sequences, including the four DR11 family members, and four additional DR11 variant mutants. The abilities of the wild-type or mutant molecules to present two distinct influenza peptide Ags, HA307-19 and HA128-45, to T cells was assessed in in vitro T cell proliferation assays. Of the naturally dimorphic DR11 positions, residue beta 71 variation significantly influenced the ability of DR11 molecules to present both peptides to DR(alpha,beta 1*1101)-restricted T cells. Residue beta 86 variation had relatively less influence than reported in several other DR and peptide systems. Residue beta 67 variation usually appeared irrelevant to T cell proliferation, but in two mutants led to unexpected T cell proliferation independent of nominal peptide Ag. Peptide binding, assessed by flow cytometry, was not found to be altered by any mutations that disrupted DR(alpha,beta 1*1101)-like presentation. These data indicate that residue beta 71 exerts a central role in influencing the functional differences among DR11 molecules, whereas the widely studied dimorphism of residue beta 86 is not as generally influential in DR11 as in other alleles.

Sex- and strain-related differences in megakaryocytopoiesis and platelet production in C3H and BALB/c mice.

In an effort to explain the different platelet production capabilities of male and female mice, megakaryocyte and platelet indices were measured on castrated male and oophorectomized female C3H and BALB/c mice, along with suitable intact controls. In agreement with our previous work, intact male BALB/c mice had higher platelet counts and percent incorporation of sulfur 35 into platelet values than did intact female BALB/c mice. Also, both intact BALB/c and C3H male mice had higher platelet counts than their castrated counterparts. Fewer femoral megakaryocytes were found in intact BALB/c and C3H male mice than in their female counterparts (p less than 0.05), but only BALB/c male mice had larger megakaryocytes than BALB/c female mice (p less than 0.0005). Castration caused increased numbers and decreased sizes of megakaryocytes (p less than 0.05) in both strains of mice, but oophorectomy did not change the characteristics of megakaryocytes in these mice. In all treatment groups, C3H mice had megakaryocytes with higher average deoxyribonucleic acid content than did BALB/c mice (p less than 0.0005), that is, BALB/c mice had greater percentages of 8N and 16N megakaryocytes than did C3H mice, but C3H mice had higher proportions of 32N and 64N megakaryocytes than did BALB/c mice (p less than 0.05 to p less than 0.0005). Although a difference in megakaryocyte ploidy was not detected between intact male and intact female C3H mice, BALB/c female mice had elevated percentages of low ploidy classes (8N) when compared with BALB/c male mice (p less than 0.005). Intact male C3H mice had higher percentages of 16N megakaryocytes (p less than 0.05) than did their neutered counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of platelet production in two strains of mice with different modal megakaryocyte DNA ploidies after exposure to hypoxia.

Thrombocytopenia develops with prolonged exposure to hypoxia. Although decreases in megakaryocyte numbers due to hypoxia have been well documented, the effects of hypoxia on megakaryocyte DNA content have not been reported. In this study, megakaryocytopoiesis and platelet production were compared in both C3H mice (whose megakaryocyte modal ploidy class is 32N) and C57/BL mice (whose modal ploidy class is 16N), by enclosure in cages covered with silicone-rubber membranes. After equilibration, O2 levels inside the cages were 6%-7%. Hematocrits, platelet counts, platelet sizes, percent 35S incorporation into platelets, megakaryocyte size and number, and megakaryocyte DNA content of mice were measured before and at various days after hypoxia. Although hematocritis increased and platelet counts decreased in both strains of mice with time in hypoxic chambers, megakaryocyte and platelet responses of C3H mice differed from those of C57/BL mice in several respects; hematocrits of C3H mice were higher and platelet counts were lower than those in C57/BL mice. C3H mice produced larger platelets than C57/BL mice in response to hypoxia. Total circulating platelet counts (TCPC) and total circulating platelet masses (TCPM) of both mouse strains showed similar biphasic responses, that is, elevated TCPC and TCPM on days 2-4 and decreased values after 6-14 days of hypoxia. However, hypoxic C3H mice had lower TCPC on days 4-14 and lower TCPM on days 10-14 of hypoxia than C57/BL mice. Both C3H and C57/BL mice had decreased megakaryocyte numbers at 6-10 days of hypoxia, but only C3H mice had decreased numbers of megakaryocytes at day 14. Elevated megakaryocyte size was observed in both mouse strains at day 14 of hypoxia. However, after hypoxia, C3H mice showed a greater depression in megakaryocyte number and a larger increase in megakaryocyte sizes than did C57/BL mice. C3H mice maintained 32N as the modal megakaryocyte DNA content through day 10 of hypoxia, but 64N was the modal megakaryocyte DNA content at day 14; 16N remained the modal megakaryocyte DNA content in hypoxic C57/BL mice. Hypoxic C3H mice had an increase in 16N megakaryocytes after 6 days of hypoxia, followed by an increase in the proportion of 64N cells at 14 days compared to values of untreated C3H control mice. Hypoxic C57/BL mice had an increased proportion of 16N cells at 6 days but a decreased proportion of 32N cells at 14 days. These studies demonstrate that the decreased platelet production resulting from prolonged exposure to hypoxia is primarily the result of decreased differentiation of hematopoietic precursors into the megakaryocyte lineage rather than decreased megakaryocyte DNA content, because higher ploidy classes actually increase as thrombocytopenia becomes more severe. Stem cell competition could explain the findings of reduced platelet production and increased red blood cell production in both strains of mice after exposure to hypoxia.