Use of a sensitive bioimmunoabsorbent assay to isolate and characterize monoclonal antibodies to biologically active human erythropoietin

At present, one of the most sensitive assays for human erythropoietin (Ep) is a bioassay that measures the Ep-dependent proliferation of spleen cells from phenylhydrazine-treated mice after 24 hours in culture. We describe how this assay can be used as the basis of a very sensitive method for detecting mouse antibodies to biologically active human Ep. In this procedure, microtiter wells are first coated with goat anti-mouse Ig antibody, then treated with mouse antibodies (serum or hybridoma culture supernatants), and finally incubated with a fixed amount of pure human Ep. Specific binding of anti-Ep antibodies is detected by adding spleen cells from phenylhydrazine-treated mice to the wells and measuring the ability of the cells to incorporate 3H- thymidine 24 hours later. This bioimmunosorbent assay (BISA) revealed the presence of anti-EP antibodies in sera from mice immunized with either pure human urinary Ep or a synthetic dodecapeptide corresponding to the aminoterminal region of Ep and in the culture supernatants from three of eight stable anti-Ep antibody-producing hybridoma cell lines that we have isolated. The three monoclonal antibodies showed similar reactivities in the BISA, but showed different affinities for Ep, with Kd values of approximately 0.7, 8, and 240 nmol/L, respectively. Further studies showed that all antibodies were capable of neutralizing Ep bioactivity and of binding 125I-labeled Ep in a radioimmunosorbent assay (RIA) but were virtually unreactive to Ep adsorbed to the bottom of enzyme-linked immunosorbent assay (ELISA) wells. Our results suggest that the BISA strategy may be an important complement to conventional RIA and ELISA techniques for identification of monoclonal antibodies specific for biologically active growth factors.     

The phytoconstituents and the comparative effects of aqueous extract of Irvingia gabonensis seeds and proviron on the biochemical parameters of male guinea pigs

ObjectiveTo investigate the phytochemical screening and the effects of the aqueous extracts of the seeds of Irvingia gabonensis on the biochemical parameters of male guinea pigs.MethodsThe biochemical parameters were assayed using Randox Diagnostic kits, Phenolphthalein method and colorimetric method. The phytochemical screening was carried out using standard procedures.ResultsPhytochemical investigations revealed the presence of flavonoids, tannins, carbohydrate, alkaloids, terpenoids, steroids, volatile oils, saponins and cardiac glycosides. The aqueous extract of Irvingia gabonensis seeds (50–400 mg/kg) caused a statistically significant (P<0.05 ANOVA) decrease in the levels of total cholesterol, urea, uric acid, total protein, prostatic, alkaline, and acid phosphatases. The highest reduction effect was obtained with uric acid at 400 mg/kg of Irvingia gabonensis extract while the least effect was observed in total cholesterol. These effects were dose-and time-dependent.ConclusionsThis shows that the seeds of Irvingia gabonensis have hepatoprotective, nephroprotective and cardio protective properties. The study therefore, supports the claims on the use of the seeds of this plant by traditional medicine practitioners as a hepatoprotective and nephroprotective agent. Although further studies need to be done to isolate, identify and characterize the active principles in the seeds of this plant.     

Cyclic AMP-mediated chloride secretion is induced by prostaglandin F2α in human isolated colon

Background and purpose:

Prostaglandin F_2α (PGF_2α) is implicated in the pathogenesis of inflammatory bowel disease and colorectal cancer. This study investigates the effects of PGF_2α on electrophysiological parameters in isolated human colonic mucosa.

Experimental approach:

Ion transport was measured as changes in short-circuit current across human colonic epithelia mounted in Ussing chambers. Colonic crypts were isolated by calcium chelation and cyclic adenosine monophosphate (cAMP) was measured by ELISA.

Key Results:

PGF_2α stimulated chloride secretion in a concentration-dependent manner with an EC₅₀ of 130 nM. The PGF_2α induced increase in chloride secretion was inhibited by AL8810 (10 µM), a specific PGF_2α receptor antagonist. In addition, PGF_2α (1 µM) significantly increased levels of cAMP in isolated colonic crypts.

Conclusions and implications:

PGF_2α stimulated chloride secretion in samples of human colon in vitro through a previously unrecognizd cAMP-mediated mechanism. These findings have implications for inflammatory states.     

BGC20-1531, a novel,potent and selective prostanoid EP4 receptor antagonist: a putative new treatment for migraine headache

Background and purpose:

Prostanoid EP₄ receptor antagonists may have therapeutic utility in the treatment of migraine since EP₄ receptors have been shown to be involved in prostaglandin (PG)E₂-induced cerebral vascular dilatation, which may be an important contributor to migraine pain. This study reports the pharmacological characterization of BGC20-1531, a novel EP₄ receptor antagonist.

Experimental approach:

BGC20-1531 was characterized in radioligand binding and in vitro functional assays employing recombinant and native EP₄ receptors. Changes in canine carotid haemodynamics were used to assess the pharmacodynamic profile of BGC20-1531 in vivo.

Key results:

BGC20-1531 exhibited high affinity at recombinant human EP₄ receptors expressed in cell lines (pK_B 7.6) and native EP₄ receptors in human cerebral and meningeal artery (pK_B 7.6–7.8) but showed no appreciable affinity at a wide range of other receptors (including other prostanoid receptors), channels, transporters and enzymes (pKi < 5). BGC20-1531 competitively antagonized PGE₂-induced vasodilatation of human middle cerebral (pK_B 7.8) and meningeal (pK_B 7.6) arteries in vitro, but had no effect on responses induced by PGE₂ on coronary, pulmonary or renal arteries in vitro. BGC20-1531 (1–10 mg·kg⁻¹ i.v.) caused a dose-dependent antagonism of the PGE₂-induced increase in canine carotid blood flow in vivo.

Conclusions and implications:

BGC20-1531 is a potent and selective antagonist at EP₄ receptors in vitro and in vivo, with the potential to alleviate the symptoms of migraine that result from cerebral vasodilatation. BGC20-1531 is currently in clinical development for the treatment of migraine headache.     

On the limits of effort testing: symptom validity tests and severity of neurocognitive symptoms in nonlitigant patients

Modern symptom validity tests (SVTs) use empirical cutoffs for decision making. However, limits to the applicability of these cutoffs may arise when severe cognitive symptoms are present. The purpose of the studies presented here was to explore these limits of applicability. In Experiment 1, a group of 24 bona fide neurological patients without clinically obvious cognitive symptoms was compared to a group of 24 patients with rather severe symptoms. A comprehensive test battery was employed, which included four SVTs (the Test of Memory Malingering, TOMM, the Word Memory Test, WMT, the Bremer Symptomvalidierung, BSV, and the Amsterdam Short-Term Memory Test, ASTM). In Experiment 2, a group of 20 patients with mild Alzheimer's disease was compared to 14 healthy controls. Results of both studies showed that cognitive impairment may significantly interfere with SVT performance. Correlation analyses revealed dissimilar relationships between SVTs and neuropsychological test measures. Whereas TOMM and WMT correlated mainly with tests of declarative memory, the BSV correlated with tests of attention, and ASTM correlated with tests of working memory. Intercorrelations between symptom validity measures were relatively low. The published cutoffs of the TOMM would be suitable for estimating effort in patients with Mini-Mental State Examination scores of 24 or above. More research will be necessary to investigate how performance in SVTs is related to cognitive functioning in populations of severely impaired patients.     

Added Value of Use of a Purified Protein Derivative-Based Enzyme-Linked Immunosorbent Spot Assay for Patients with Mycobacterium bovis BCG Infection after Intravesical BCG Instillations

In this case series, we describe four cases in which the use of gamma interferon release assays with purified protein derivative (PPD) as a stimulating antigen was able to demonstrate PPD-specific immune activation. This may help to improve the adequate diagnosis of (systemic) Mycobacterium bovis BCG infections after intravesical BCG instillations for bladder carcinoma.     

COMBINATION ANTI-PARATUBERCULOSIS THERAPY WITH CLARITHROMYCIN, RIFABUTIN and CLOFAZIMINE IN THE TREATMENT OF CROHN'S DISEASE. A PRELIMINARY REPORT ON THE AUSTRALIAN MULTICENTRE TRIAL

Mycobacterium paratuberculosis has been proposed as the cause of Crohn's disease, although this remains controversial. Observational studies of antibiotics with activity against this organism have led to the establishment of an Australian multicentre placebo-controlled trial of clarithromycin, rifabutin and clofazimine in patients with active Crohn's disease. All subjects receive treatment with a tapering regimen of prednisolone for the initial 16 weeks, combined with either antibiotics or matching placebos. If remission is achieved at the end of this period and prednisolone has been ceased they continue the trial medications for 2 years. The primary outcomes – remission rates at 1, 2 and 3 years – aim to determine whether these antibiotics are of long-term benefit in Crohn's disease. Secondary outcomes include rate of remission at 4 months, safety and quality of life.
As of May 2001, 171 of the required 212 subjects have been enrolled. Eighty-seven of 146 potential subjects have reached 16 weeks in remission (60%). Eighty of these are ongoing (92%). Only five withdrawals from the trial have occurred for adverse events thought probably related to trial medications – four subjects with raised LFTs and one who developed a rash. Other withdrawals have been mainly due to lack of response or worsening of Crohn's disease (37), hypomania from steroids (one patient) and pregnancy (one patient on placebo). There have been 11 withdrawals for protocol violations. An Independent Data Monitoring Committee has analysed progress of the trial and unanimously recommended that it continue.
This study is the largest and longest suitably powered randomised controlled trial of antibiotics undertaken in Crohn's disease. It will determine whether this antibiotic combination alters the natural history of this disorder.
This study is being supported by Pharmacia Australia Pty Limited.     

Diagnostic potential of an enzyme-linked immunospot assay in tuberculous pericarditis

Tuberculous pericarditis is a rare disease in developed countries. The diagnosis is difficult to set since there are no robust rapid tests, and culture of pericardial fluid for Mycobacterium tuberculosis is often negative. T-SPOT.TB, an enzyme-linked immunospot (ELISPOT) test, measures the gamma interferon response of lymphocytes against tuberculosis antigens and can be performed on blood and body fluids. We describe a patient with tuberculous pericarditis for which the diagnosis was rapidly set by positive T-SPOT.TB results, which were confirmed by isolation of Mycobacterium tuberculosis in pericardial fluid culture. We performed a literature search to assess the diagnostic potential of ELISPOT testing in tuberculous pericarditis. The limited data on this subject indicate that T-SPOT.TB aids in diagnosing active tuberculosis (TB) infection and results in a more rapid decision to start antituberculosis treatment. Enumerating TB-specific lymphocytes and testing blood/compartmental fluid simultaneously can provide useful information on active tuberculous pericarditis.