Adherence and persistence with teriparatide among patients with commercial, Medicare, and Medicaid insurance.

UNLABELLED: Adherence to, and persistence with, treatments for osteoporosis are low. Adherence with teriparatide decreases over time. Higher copayments in the commercial/Medicare population were associated with worse persistence. Understanding factors such as prior screening, prior treatment history, and out of pocket costs that influence persistence with teriparatide may help clinicians make informed decisions. INTRODUCTION: The purpose of this study was to evaluate adherence and persistence with teriparatide. METHODS: Beneficiaries with at least one claim for teriparatide in 2003 or 2004 and continuous enrollment in the previous 12 months and subsequent 6 months were identified in a national commercial/Medicare and Medicaid administrative claims database (MarketScan®). Adherence was assessed through calculation of the medication possession ratio (MPR). Persistence was measured by time until discontinuation and time until first 60-day gap in treatment. Factors associated with persistence were assessed using Cox proportional hazards models. RESULTS: The average MPR at 6 months was 0.74 (N=2,218) and at 12 months, was 0.66 (N=1,303). At 6 months, 64.6% of patients remained on therapy and at 12 months, 56.7% remained. Bone mineral density screening and use of antiresorptive therapy within the 12 months pre-period, and lower patient copayments were associated with increased persistence. CONCLUSION: Patients appear to have good adherence with teriparatide over the first 6 months which declines over time. Prior screening and treatment of osteoporosis and out of pocket costs appear to impact persistence. To optimize patient outcomes, clinicians should consider clinical factors that impact persistence, while healthcare decision makers should consider the negative effect of higher patient copayments on persistence.

Relationship between duration of teriparatide therapy and clinical outcomes in postmenopausal women with osteoporosis.

SUMMARY: The extent to which fracture protection and safety varies with increasing time on teriparatide [rhPTH(1-34)] therapy is a clinically relevant unanswered question. In postmenopausal women with osteoporosis, increased duration of teriparatide versus placebo treatment was associated with a progressive decrease in the rates of nonvertebral fragility fractures and back pain. INTRODUCTION: The impact of duration of teriparatide [rhPTH(1-34)] therapy on patient outcomes is a relevant unanswered question. METHODS: Postmenopausal women with osteoporosis were randomized to once-daily subcutaneous injection with placebo (N = 544), teriparatide 20 microg (TPTD20; N = 541), or teriparatide 40 microg (TPTD40; N = 552) plus calcium and vitamin D supplementation. The time to first nonvertebral fragility fracture and new or worsening back pain following treatment initiation was analyzed using Cox partial likelihood regression treating time on therapy as a linear, time-dependent covariate. RESULTS: Compared with placebo, the relative hazard for nonvertebral fragility fractures decreased by 7.3% for each additional month of TPTD20 [hazard ratio = 0.927, 95% CI (0.876 to 0.982), p = 0.009] and by 7.6% for each additional month of TPTD40 [hazard ratio = 0.924, 95% CI (0.871 to 0.981), p = 0.009]. Clinical vertebral fractures appeared to increase over time in the placebo group and occurred primarily in the first time interval in the teriparatide treatment groups. Compared with placebo, the relative hazard of back pain was decreased by 8.3% for each additional month of TPTD20 [hazard ratio = 0.920, 95% CI (0.902 to 0.939), p < 0.001] and 8.7% for each additional month of TPTD40 [hazard ratio = 0.917, 95% CI (0.898 to 0.935), p < 0.001]. CONCLUSIONS: These findings suggest increased nonvertebral fracture protection, reduced back pain, and reduced occurrence of side effects with longer duration of teriparatide therapy.

Characteristics of patients initiating teriparatide for the treatment of osteoporosis.

UNLABELLED: The demographic and clinical characteristics of patients initiating teriparatide were compared with those of patients initiating bisphosphonates for the treatment of osteoporosis. In these samples of commercially insured, Medicare, and Medicaid patients, patients initiating teriparatide were older, in poorer health, and appeared to have more severe osteoporosis than patients initiating bisphosphonates. INTRODUCTION: The demographic and clinical characteristics of patients initiating teriparatide are compared with those of patients initiating bisphosphonates. METHODS: Beneficiaries (45 years and older) with at least one claim for teriparatide or a bisphosphonate from 2003 to 2005 and continuous enrollment in the previous 12 months and subsequent 6 months were identified from commercial, Medicare, and Medicaid administrative claims databases. Patients initiating teriparatide (commercial/Medicare (N = 2,218); Medicaid (N = 824)) were compared to patients initiating bisphosphonates (commercial/Medicare (N = 97,570); Medicaid (N = 77,526)) in terms of age, provider specialty, comorbidities, prior use of osteoporosis medications, fractures, BMD screening, health status, and resource utilization. RESULTS: Teriparatide patients were older and in poorer health than bisphosphonate patients. Approximately 38% of teriparatide patients in both groups had fractured in the pre-period compared to 16% of commercial/Medicare and 15% of Medicaid bisphosphonate patients. Teriparatide patients were more likely to have used osteoporosis medications in the pre-period (79.9% versus 32.1% (commercial/Medicare); 82.2% versus 19.6% (Medicaid)). CONCLUSIONS: In these samples of patients, those initiating teriparatide differed from those initiating bisphosphonates. Teriparatide patients were older, in poorer health, and appeared to have more severe osteoporosis than bisphosphonate patients. Comparisons of treatment outcomes should take these differences in patient characteristics into consideration.

Expression of resistance markers to methotrexate predicts clinical improvement in patients with rheumatoid arthritis.

BACKGROUND: Methotrexate is transported into the cell by the reduced folate carrier (RFC) and out of the cell by members of the multidrug resistance protein family (MRP). Transport proteins may affect the therapeutic efficacy of this drug in patients with rheumatoid arthritis. OBJECTIVE: To investigate the potential benefit of the presence of RFC and the absence of functional MRP for the efficacy of methotrexate treatment. METHODS: The study involved 163 patients (116 female, 47 male; mean age 59.5 years) on methotrexate (mean weekly dose 12.2 mg). RFC was determined using reverse transcriptase polymerase chain reaction, and MRP function by flow cytometry, using a calcein acetoxymethylesther/probenecid assay. Clinical response to methotrexate was evaluated by the EULAR response criteria and the ACR 20% improvement criteria. The clinical data were obtained at the beginning of methotrexate treatment and at the time of blood sampling during ongoing therapy. Patients were divided into four groups according to the presence (+) or absence (-) of RFC and functional (f) MRP. RESULTS: fMRP+/RFC+ and fMRP-/RFC- patients more often had good EULAR response rates (60%, p = 0.014, and 53%, p = 0.035, respectively) in comparison with the fMRP-/RFC+ group (29%); fMRP+/RFC- patients had a low frequency of good disease activity responses. CONCLUSIONS: Absence of fMRP plus presence of RFC did not prove to be related to beneficial effects of methotrexate, but the lack or the presence of both fMRP and RFC led to a significantly better therapeutic outcome. Determination of these markers may predict responsiveness to methotrexate.

3D-VIEWER: an atlas-based system for individual and statistical investigations of the human brain.

3D-VIEWER is a new software tool for neurosurgical planning and population studies. It is based on digitized three-dimensional brain atlases derived from standard stereotactic atlases that can be adapted to an individual's brain and shown as a series of displayed images. If the patient's brain has been imaged in different modalities, the standardized anatomical information can be adapted to the individual images, which will bring the images into registration. The 3D-VIEWER can be used as a tool for combining multimodal information from the same patient. In addition, several tools are available that allow oblique views of anatomical structures or the view along the intended trajectory during a neurosurgical intervention. Furthermore, using the atlas transformation matrices, anatomical information can be determined when comparing an individual's brain to the anatomy of the atlas brain. Thus, standardized anatomical information from the atlas can be introduced into individual images. This standardization is used to perform individual-group and group-by-group comparisons between patients and normal controls in anatomical studies.

Expression of the lung resistance protein in primary colorectal carcinomas.

To assess whether the lung resistance protein (LRP) is of clinical significance in colorectal carcinomas, we immunohistochemically determined LRP expression of colorectal carcinoma specimens (n = 68) by means of the monoclonal antibody LRP-56 and compared this expression with clinical parameters. LRP expression was negative in 7 (10%), low in 36 (52%) and high in 25 (38%) carcinomas. LRP expression was independent of histological grade, tumor size, lymph node involvement and distant metastasis. Survival of the patients with LRP-positive tumors was similar to the survival of patients with LRP-negative tumors. However, patients with high LRP expression in their carcinomas had a prolonged survival. Thus LRP is frequently expressed in colorectal carcinomas and high expression might indicate improved survival.

Drug resistance factors in acute myeloid leukemia: a comparative analysis.

To compare the clinical relevance of drug resistance factors in de novo acute myeloid leukemia (AML), we determined their relationship to both response to induction chemotherapy and survival of the patients in univariate as well as multivariate analyses. The drug resistance factors immunocytochemically studied in 111 patients at the time of diagnosis included the lung resistance protein (LRP), P-glycoprotein (P-gp), multidrug resistance protein (MRP1) and bcl-2. In the univariate analyses, age (P = 0.005), karyotype (P = 0.03), LRP (P = 0.003), P-gp (P = 0.02) and bcl-2 (P = 0.03) predicted for response to induction chemotherapy, whereas MRP1 had no predictive value. Age (P = 0.05), karyotype (P = 0.05) and LRP (P = 0.03) retained their predictive value in the multivariate logistic regression analyses. With regard to overall survival, age (P = 0. 008), karyotype (P = 0.006), LRP (P = 0.001) and P-gp (P = 0.01) were of prognostic value in the univariate Cox regression analyses but only age (P = 0.01), karyotype (P = 0.02) and LRP (P = 0.01) retained their prognostic significance in the multivariate analyses. A risk score based on the number of independent prognostic factors allowed division of patients into four groups with different outcome. In these groups, the complete remission rates were 93%, 75%, 47% and 33%, respectively, and median overall survival was 2.4, 1.2, 0.6 and 0.2 years, respectively. Thus, several drug resistance factors did predict outcome in the univariate analyses but LRP was the only drug resistance factor with independent predictive and prognostic significance. The proposed risk score might be useful for risk-adapted treatment in the future. Leukemia (2000) 14, 68-76.

Regression of colonic low grade B cell lymphoma of the mucosa associated lymphoid tissue type after eradication of Helicobacter pylori.

BACKGROUND: Lymphoma of the mucosa associated lymphoid tissue (MALT) arising in the stomach has been shown to be related to Helicobacter pylori infection, and total regression of gastric lymphoma after successful eradication of H pylori has consistently been reported. MALT-type lymphoma at other localisations, however, has to our knowledge not been linked to H pylori, and eradication of the bacteria has not been studied for management of such lymphomas. PATIENT/METHOD: A 67 year old man was diagnosed with MALT-type lymphoma simultaneously involving the stomach and the colon descendens. In addition to the presence of MALT-type lymphoma, H pylori associated chronic gastritis was diagnosed, and treatment with clarithromycin, metronidazole, and omeprazole was initiated, resulting in its successful eradication. RESULTS: Follow up performed four months later showed regression of the colonic manifestation, whereas the gastric lymphoma did not respond to antibiotic treatment, as assessed by regular follow up for 14 months, in spite of its restriction to mucosa and submucosa. The patient was therefore treated with oral cyclophosphamide (100 mg a day) resulting in partial remission after seven months of continuous treatment. Because of the presence of residual lymphoma, additional irradiation was performed, which led to complete remission of the gastric lymphoma. The patient remains in complete remission 40 months after diagnosis and 26 months after initiation of treatment. CONCLUSION: In the case of concurrent gastric and intestinal low grade MALT-type lymphoma, H pylori eradication may cause regression of the intestinal lesion.

Expression of the lung resistance protein predicts poor outcome in patients with multiple myeloma.

Expression of the lung resistance protein (LRP) is associated with resistance to various anticancer drugs including melphalan and, therefore, may affect the clinical outcome in multiple myeloma (MM). To determine the clinical significance of LRP, we have compared LRP expression in bone marrow plasma cells with clinical parameters including response to chemotherapy and survival of previously untreated patients with MM (n = 72). LRP expression immunocytochemically assessed by means of the LRP-56 monoclonal antibody was positive (> or =10% staining plasma cells) in 44 (61%) samples. There was no correlation between LRP expression and age, sex, type of the paraprotein, serum creatinine, stage, beta2-microglobulin, serum lactate dehydrogenase, or C-reactive protein. However, LRP expression was more frequently observed in patients with a p53 deletion than in those without such a deletion (P = 0.01). The overall response rate for all of the patients evaluable for response to induction chemotherapy (n = 58) was 67%. The response rate was 87% for patients without LRP expression but only 54% for patients with LRP expression (P = 0.01). Kaplan-Meier analysis revealed that patients with LRP expression had a shorter overall survival (median, 33 months) than those without LRP expression (median not reached; P = 0.04). These data show that LRP expression is an important marker for clinical drug resistance and predicts a poor outcome in MM.

The lung resistance protein (LRP) predicts poor outcome in acute myeloid leukemia.

To determine the clinical significance of the lung resistance protein (LRP) in acute myeloid leukemia (AML), we have studied LRP expression of leukemic blasts and its association with clinical outcome in patients with de novo AML. LRP expression of leukemic blasts was determined by immunocytochemistry by means of monoclonal antibody LRP-56. LRP expression at diagnosis was detected in 31 out of 86 (36%) patients and correlated with white blood cell count (p = 0.01). The complete remission rate of induction chemotherapy was 72% for all treated patients (n = 82). The complete remission rate was 81% for patients without LRP expression but only 55% for patients with LRP expression (p = 0.01). Overall survival and disease-free survival were estimated according to Kaplan-Meier in 82 and 59 patients, respectively. At a median follow-up of 16 months, median overall survival was 17 months for LRP-negative patients but only 8 months for LRP-positive patients (p = 0.006). Disease-free survival was 9 months for LRP-negative patients and 6 months for LRP-positive patients (p = 0.078). Thus LRP predicts for poor outcome indicating that the LRP gene is a clinically relevant drug resistance gene in AML.

MRP expression in acute myeloid leukemia. An update.

To determine the clinical significance of the multidrug resistance protein (MRP) in patients with de novo AML, we have studied MRP expression of leukemic cells at diagnosis and its association with clinical outcome in 127 patients. MRP expression was determined by immunocytochemistry by means of monoclonal antibodies QCRL-1/QCRL-3. MRP expression was low, intermediate and high in 30%, 46% and 24% of the patients, respectively. MRP expression was independent of age and sex of the patients, white blood cell count, FAB subtype, serum lactate dehydrogenase levels and karyotype aberrations. MRP expression had no impact no response to induction chemotherapy. The complete remission rates were 75%, 70% and 64% for patients with low, intermediate and high expression, respectively. Patients with intermediate or high MRP expression showed a trend toward shorter overall survival (p = 0.09) as compared to patients with low MRP expression. MRP does not predict for response to induction chemotherapy but intermediate or high MRP expression might be associated with shorter overall survival of the patients.

Effect of ipratropium bromide treatment on oxygen saturation and sleep quality in COPD.

STUDY OBJECTIVES: Patients with COPD are at risk of experiencing a deterioration in arterial oxygen saturation (SaO2) during sleep, which is generally most pronounced during rapid eye movement (REM) sleep. Increased cholinergic tone has been suggested as a contributing factor to this decrease in SaO2. Therefore, we investigated whether 4-week treatment with ipratropium bromide inhalation solution 0.02% (qid) could improve sleep characteristics in COPD. DESIGN: Randomized, placebo-controlled, double-blind, two-arm parallel study of 4 weeks of treatment with ipratropium bromide solution or placebo. SETTING: Multicenter investigation. PATIENTS: Thirty-six patients with moderate-to-severe COPD (FEV1 < 65% of predicted). MEASUREMENTS AND RESULTS: Evaluation included polysomnographic, pulmonary function, and subjective quality of sleep (visual analog scale [VAS]) assessments. It was found that 4 week of treatment with ipratropium bromide solution in patients with COPD led to the following: (1) a significant (p = 0.05) improvement in mean nocturnal SaO2 with the more severe the nocturnal desaturation, the greater the improvement in SaO2; (2) significant (p = 0.03) improvement in perceived sleep quality (VAS: 5.5 +/- 0.5 after placebo; 7.2 +/- 0.5 after ipratropium); (3) a significant (p = 0.05) increase in REM sleep time (48.6 +/- 6.3 min after placebo; 66.5 +/- 6.4 min after ipratropium) with no effect on other sleep stages or total sleep time; and (4) a significant (p = 0.01) increase in pre-sleep FVC and flow rate at 50% of the vital capacity. CONCLUSIONS: These findings demonstrate that ipratropium bromide therapy can improve sleep SaO2 as well as sleep quality in patients with moderate-to-severe COPD.

Efficacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection: a randomized clinical trial.

CONTEXT: Attachment of most rhinovirus subtypes to cells depends on a cellular receptor, the intercellular adhesion molecule 1 (ICAM-1). A recombinant soluble ICAM-1 (tremacamra, formerly BIRR 4) has shown possible efficacy in early studies. OBJECTIVE: To determine the efficacy and safety of intranasal administration of tremacamra in experimental rhinovirus colds in humans. DESIGN: Four randomized, double-blind, placebo-controlled trials conducted in January to March 1996. SETTING AND SUBJECTS: Volunteers between the ages of 18 and 60 years who had an antibody titer of 1:4 or less to the challenge virus. Subjects were isolated in a hotel room during study days 0 to 8; symptoms were recorded through day 14. A total of 198 subjects were randomized, of whom 196 received drug or placebo and were included in the safety analysis. A total of 177 subjects were included in the efficacy analysis. INTERVENTIONS: Tremacamra or placebo was given beginning 7 hours before inoculation with rhinovirus type 39 (preinoculation studies) or 12 hours after (postinoculation studies). Tremacamra as an inhaled solution or as a powder (each given preinoculation and postinoculation for a total of 4 studies) and placebo were given in 6 doses at 3-hour intervals daily during days 1 through 7. Recipients of active treatment received 367 microg of tremacamra per nostril per dose for a total of 4.4 mg/d. MAIN OUTCOME MEASURES: Effect of tremacamra on infection, as determined by virus isolation and seroconversion, and on illness, as determined by symptom scores, clinical colds, and nasal mucus weights. Treatment-by-study interaction was not significant, so results were pooled for the main analysis. RESULTS: A total of 88 (92%) of the 96 subjects in the placebo groups and 69 (85%) of the 81 subjects in the active treatment groups were infected (P=.19). For placebo vs tremacamra, respectively, the total symptom score (+/- 95% confidence interval [CI]) was 17.6 (+/- 2.7) vs 9.6 (+/- 2.9), the proportion of clinical colds was 64/96 (67% +/- 9%) vs 36/81 (44% +/- 11%), and the nasal mucus weight was 32.9 (+/- 8.8) g vs 14.5 (+/- 9.4) g (P<.001 for all comparisons). Tremacamra was not associated with adverse effects or evidence of absorption through the nasal mucosa and did not interfere with development of neutralizing antibody. CONCLUSION: Tremacamra reduced the severity of experimental rhinovirus colds. Whether tremacamra will be useful clinically will require further study.

Expression of the multidrug resistance protein (MRP1) in breast cancer.

BACKGROUND: The multidrug resistance protein (MRP1) is expressed in human breast carcinomas but its clinical significance remains unclear. The aim of the present study was to determine the clinical significance of MRP1 in breast cancer patients. MATERIALS AND METHODS: MRP1 expression of primary carcinomas from 100 breast cancer patients was immunohistochemically determined by means of the monoclonal antibodies QCRL-1/QCRL-3. RESULTS: MRP1 was negative in 20 (20%) and positive in 80 (80%) breast carcinomas. MRP1 expression was more frequent in both estrogen receptor-negative carcinomas and progesterone receptor-negative carcinomas (p = 0.1 in both cases), but was independent of tumor size and lymph node involvement. Patients with MRP1-negative carcinomas had prolongations of overall survival (p = 0.01 for death due to any cause, p = 0.04 for breast cancer-related death) and disease-free survival (p = 0.07) as compared to those with MRP1-positive carcinomas. Also in subsets of patients (negative lymph nodes; positive lymph nodes; positive estrogen receptor; T1/T2 tumors), overall survival was longer for patients with MRP1-negative carcinomas. In univariate Cox regression analyses, MRP1 positivity was associated with relative risks of 4.9 (95% CI 1.2-20.6; p = 0.03) for death due to any cause, 6.4 (95% CI 0.9-48.0; p = 0.07) for breast cancer-related death and 3.5 (95% CI 0.8-14.9; p = 0.09) for relapse. In multivariate Cox regression analyses, MRP1 positivity had relative risks of 5.1 (95% CI 1.2-21.7; p = 0.03) for death due to any cause, 6.5 (95% CI 0.8-50.1; p = 0.07) for breast cancer-related death and 3.4 (95% CI 0.8-15.1; p = 0.1) for relapse. CONCLUSIONS: Our results suggest that MRP1 might be an important factor in breast cancer indicating excellent prognosis for patients with MRP1-negative carcinomas.

Expression of the lung resistance protein (LRP) in primary breast cancer.

BACKGROUND: To determine the clinical significance of the lung resistance protein (LRP) in breast cancer, we have studied its expression in primary breast carcinomas (n = 99) and assessed the association of this expression with clinical parameters of the patients. MATERIALS AND METHODS: LRP expression was immunohistochemically determined by means of the monoclonal antibody LRP-56 on frozen tumor sections. RESULTS: LRP expression was negative in 12%, low in 20%, intermediate in 47% and high in 21% of the carcinomas. LRP expression was independent of age of the patients, histology, tumor grade, estrogen receptor as well as progesterone receptor status, tumor size and lymph node involvement. Kaplan-Meier analyses revealed that both overall survival and disease-free survival were independent of the degree of LRP expression. CONCLUSIONS: LRP is frequently expressed in breast carcinomas, but is neither associated with known prognostic factors, nor a prognostic factor by itself.

Expression of the lung resistance protein predicts poor outcome in de novo acute myeloid leukemia.

The 110-kD lung resistance protein (LRP) is overexpressed in P-glycoprotein-negative multidrug-resistant cell lines and most likely involved in the multidrug resistance (MDR) of these cell lines. To determine the clinical significance of LRP, we have studied LRP expression of leukemic blasts and its association with clinical outcome in patients with de novo acute myeloid leukemia (AML). LRP expression of leukemic blasts obtained from peripheral blood or bone marrow of previously untreated patients (n = 86) was determined by immunocytochemistry by means of monoclonal antibody LRP-56. LRP expression at diagnosis was detected in 31 (36%) patients. LRP expression was independent of age and sex of the patients, French-American-British subtype, cytogenetic abnormalities, and lactate dehydrogenase levels, but correlated with white blood cell count (P = .01). Eighty-two patients received standard induction chemotherapy that included cytarabine and MDR drugs (daunorubicin in most patients, additional etoposide in the majority of patients). The complete remission rate of induction chemotherapy was 72% (95% confidence interval [CI] = 61% to 82%) for the total study population. The complete remission rate was 81% (95% CI = 67% to 91%) for patients without LRP expression but only 55% (95% CI = 36% to 74%) for patients with LRP expression (P = .01). Overall survival and disease-free survival were estimated according to Kaplan-Meier in 82 and 59 patients, respectively. Overall survival was significantly longer in patients without LRP expression than in patients with LRP expression. At a median follow-up of 16 months, median overall survival was 17 months (95% CI = 12 to 38 months) for LRP-negative patients but only 8 months (95% CI = 4 to 12 months) for -positive patients (P = .006). Disease-free survival was 9 months (95% CI = 7 to 11 months) for LRP-negative patients and 6 months (95% CI = 5 to 8 months) for -positive patients (P = .078). Outcome was best in patients lacking both LRP and P-glycoprotein expression. In conclusion, LRP predicts for poor outcome and thus the LRP gene appears to be another clinically relevant drug resistance gene in AML.

Combination of chemotherapy and antiviral therapy for Epstein-Barr virus-associated non-Hodgkin's lymphoma of high grade malignancy in cases of HIV infection.

Two HIV patients with Epstein-Barr virus (EBV)-associated B cell lymphoma of high grade malignancy enjoyed prolonged remission after therapy with COPBLAM and the antiviral agent Acyclovir. After 3, respectively 5 cycles of treatment, the patients (stage C3 according to CDC) responded to the administered drugs by achieving complete remission. Under maintenance therapy with Acyclovir for 32, respectively 31 months, both patients still remain free of lymphoma as of today.

Sendai virus-induced IFN-alpha production analysed by immunocytochemistry and computerized image analysis.

IFN-alpha production in Sendai virus-stimulated human buffy coat cultures could readily be demonstrated in individual cells at a protein level by the use of a novel immuno-enzymatic staining procedure. A distinctive rounded, juxtanuclear staining pattern was generated in producer cells by the accumulation of the intracellularly synthesized IFN-alpha in the Golgi stacks. The technology is based upon acquiring a video image of stained monolayers of cells, viewed in a microscope by a colour camera, which then transfers binary images directly into a computer-controlled operating system. The characteristic appearance of the immunocytochemical staining enabled a computerized image-analysis system to measure IFN-alpha producing cells based on defined criteria set for morphology, intensity, colour and size. The automated system could accurately and reproducibly register a range of 0.1-7.0% of the total cell population as IFN-alpha producing cells during the kinetic studies of the response. Congruent results were obtained with manual microscopy and image analysis concerning the assessment of the incidence of IFN-alpha producing cells in the total cell populations. All IFN-alpha producing cells expressed surface HLA-DR molecules and 95% of these cells belonged to the myelomonocytic lineage. The image analysis system provided, in contrast to conventional microscopy, an opportunity to assess and document differences of signal intensity and cell size of individual IFN-alpha producing as well as non-producing cells.

Binding of tissue plasminogen activator to endothelial cells. The effect on functional properties. Localization of a ligand in the B-chain of tPA.

The binding of 125I-labelled tissue plasminogen activator (tPA), the tPA A- or B-chain to endothelial cells (EC) were studied in suspensions of cultured human umbilical vein EC (HUVEC) or immortalized microvascular EC (HMEC). By determinations of the concentration-dependent binding it was shown that both the A-chain and the B-chain, which were isolated after partial reduction of two-chain tPA, contain ligands for binding to EC. The affinity for the B-chain was much higher than for the A-chain according to Scatchard analysis (Kd 24 and 515 nM, respectively), whereas the number of binding sites was higher for the A-chain than for the B-chain (Bmax 8 x 10(5) and 1.2 x 10(5), respectively). There were no cross interactions between the A- and B-chains and their binding sites. The binding of tPA to EC induced an almost 100-fold increase of the activation rate when compared to the same amount of enzyme in free solution, which in contrast to the fibrin-induced stimulation was not inhibited by antibodies against fibrin. The enzymatic activity of the B-chain was much less affected by the association to the cells. Both tPA and the tPA B-chain were largely protected against inhibition by an excess plasminogen activator type-1 (PAI-1) when bound to EC, whereas the same amount of free tPA was totally inactivated. The competition studies strongly indicated that an N-terminal segment in the B-chain, AKHRRSPGER, may be the ligand part of the B-chain. It is interesting to note that this polypeptide segment also participates in a binding site for PAI-1, necessary for effective inhibition. This implies a possible competition between PAI-1 and a tPA-receptor for binding of tPA. High molecular weight urokinase had no quenching effect on the binding of the B-chain to EC.

Plasminogen activator inhibitor type-1 interacts exclusively with the proteinase domain of tissue plasminogen activator.

Two different techniques have been used to study the complex formation of recombinant human plasminogen activator inhibitor type-1, PAI-1, with either recombinant human two-chain tissue plasminogen activator, tc tPA (EC 3.4.21.68), or the tPA deletion variants tc K2P, containing the kringle 2 domain and the proteinase domain, and P, containing only the proteinase domain. The same value for Kon, 2.10(7) M-1s-1 for binding of PAI-1 was found for the three tPA forms by direct detection of the complex formation in real time by surface plasmon resonance, BIAcore, or indirectly by monitoring the time course of the inhibition of tPA using the chromogenic substrate N-methylsulfonyl-D-Phe-Gly-Arg-4-pNA-acetate. Apparently, no conformational change is involved in the rate-limiting step, since the kon value was found to be independent of the temperature from 20 to 35 degrees C. By the BIAcore technique, it was found that the complex between PAI-1 and tPA covalently coupled to the surface, was stable at 25 degrees C, since no dissociation was seen in buffer. However, extended treatment with 1 M NH4OH destroyed the complex with t 1/2 = 5 h. The same kon values and complex composition were found by measuring either the binding of tPA to PAI-1 captured on the monoclonal antibody MAI-11 or the binding of PAI-1 to tPA captured on the monoclonal antibody 2:2 B10. Quantification of the complex composition between PAI-1 captured on the monoclonal antibody MAI-11 with either tPA, K2P or P gave a one-to-one ratio with the fraction of active PAI-1, consistent with the results from SDS-PAGE and the specific activity of PAI-1. The complexes of the three tPA forms with PAI-1 captured on a large surface of MAI-11 dissociated more rapidly from MAI-11, with the same apparent koff, kdis, = 2.10(-3) s-1, compared with 0.7-10(-3) s-1 for the dissociation of PAI-1 alone. In consistance, the Kd, calculated from the direct determination of the kon and koff for the association of different form of PAI-1 to a small surface of MAI-11, was found to be higher for PAI-1 in complex with tPA than for free active PAI-1. Apparently, upon complex formation, a change is induced in PAI-1 at the binding epitope for MAI-11.(ABSTRACT TRUNCATED AT 400 WORDS)