Quantitative Radioimmunoassay Research Articles

B-011 Analytical Validation of a New Vasoactive Intestinal Peptide Radioimmunoassay

Abstract Background Vasoactive intestinal peptide (VIP), a potent vasodilator, is produced by neuronal cells located in the central nervous system, the digestive, respiratory, and urogenital tracts, and the exocrine, thyroid, and adrenal glands. Increased circulating VIP is clinically useful to detect VIP-producing tumors in patients with chronic diarrheal diseases. Objective Develop a sensitive replacement radioimmunoassay (RIA) for VIP quantitation in human plasma due to the diminished supply of the antibody used in the previous in-house method. Method Patient VIP in EDTA plasma competes with labeled (125I) VIP for a limited number of primary rabbit polyclonal antibody (BMA Biomedicals, Augst, Switzerland) binding sites. Antibody-bound VIP is then complexed with a goat anti-rabbit secondary antibody and precipitated out of the solution with the use of polyethylene glycol and centrifugation. After the supernatant is discarded, the 125I signal from the pellet is counted on the Wizard2 gamma counter (Perkin Elmer, Waltham, MA). The measured signal is inversely proportional to the amount of VIP present in the patient’s sample. Method validation of the new assay included determination of imprecision (pooled human EDTA plasma), limits of detection and quantification, analytical measurement range (AMR), accuracy by spike recovery, interferences, cross-reactivity, stability, reference interval determination, and agreement with the current Mayo laboratory-developed assay. Results Stability studies on freshly collected EDTA plasma showed VIP is stable for 3 days ambient, 7 days refrigerated, and 14 days frozen. Intra-assay imprecision studies showed 8.3, 2.3, and 1.3%CV for mean VIP concentrations of 41, 144, and 304 pg/mL. Inter-assay imprecision was 12.9, 7.6, 5.2, and 7.5%CV for mean VIP concentrations of 30, 81, 180, and 308 pg/mL. The assay limit of detection was 29 pg/mL. The limit of quantitation was 30 pg/mL (%CV = 13%) and was established via precision profile. The AMR was 30–500 pg/mL (slope of 0.98, intercept of 3.0 pg/mL, and R2 of 0.97). The mean % spike recovery using a different preparation lot of calibrator material was 89% (range 83%–93%). The assay was not affected by up to 1000 mg/dL hemoglobin, 1000 mg/dL triglycerides, or 10 mg/dL bilirubin. Peptide histidine methionine 27, neurotensin, gastric inhibitory peptide, motilin, secretin, and glucagon do not cross-react in the assay. The 97.5th reference limit was <86 pg/mL (95% CI 81–90, n = 127). Positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) with the current Mayo VIP assay were 97, 82, and 90% respectively. Conclusion We have developed a replacement VIP RIA. This assay has a lower limit of quantitation and a broader AMR than the current assay. Concordance with the current assay was acceptable.

The association between anti-acetylcholine receptor antibody level and clinical improvement in myasthenia gravis.

Background and purposeAnti‐acetylcholine receptor (AChR) antibodies (ab) in the serum are detected in most patients with generalized myasthenia gravis (MG) and used as a diagnostic tool. The aim of this study was to analyse a possible association between anti‐AChR‐ab serum levels and clinical improvement of MG.MethodsThe Maastricht University Medical Center is a centre of expertise for the treatment of MG. Between 1997 and 2020, more than 4000 anti‐AChR‐ab blood samples were measured for clinical care using a quantitative radioimmunoassay technique. These results, in combination with clinical status obtained from the patients’ electronic patient files, were retrospectively analysed by a single blinded clinician. Symptoms of MG were classified using the Myasthenia Gravis Foundation of America (MGFA) scale.ResultsIn total, 90 anti‐AChR‐ab‐positive MG patients with 837 blood samples were included. The median follow‐up time was 72 months. The majority of the included patients were women (61.1%), were on immunosuppressive drug therapy (88.9%), and underwent a thymectomy (54.4%). Multilevel logistic regression analysis showed a significantly inverse association between change in anti‐AChR‐ab level and the odds of MGFA improvement (per 10% decrease of anti‐AChR‐ab level: odds ratio 1.21, 95% confidence interval 1.12–1.31; p < 0.001).ConclusionsA change in anti‐AChR‐ab serum level is associated with clinical status in patients with MG. Analyses of anti‐AChR‐ab are not only useful for diagnostics but also in follow‐up of adult symptomatic patients with MG. The use of repetitive anti‐AChR‐ab serum levels might be valuable in long‐term monitoring for clinical improvement in patients with MG, however, further research is required for specific recommendations.

Downregulation of apelin in the human placental chorionic villi from preeclamptic pregnancies.

The role of the endogenous apelin system in pregnancy is not well understood. Apelin's actions in pregnancy are further complicated by the expression of multiple forms of the peptide. Using radioimmunoassay (RIA) alone, we established the expression of apelin content in the chorionic villi of preeclamptic (PRE) and normal pregnant women (NORM) at 36-38 wk of gestation. Total apelin content was lower in PRE compared with NORM chorionic villi (49.7±3.4 vs. 72.3±9.8 fmol/mg protein; n=20-22) and was associated with a trend for lower preproapelin mRNA in the PRE. Further characterization of apelin isoforms by HPLC-RIA was conducted in pooled samples from each group. The expression patterns of apelin peptides in NORM and PRE villi revealed little or no apelin-36 or apelin-17. Pyroglutamate apelin-13 [(Pyr1)-apelin-13] was the predominant form of the peptide in NORM and PRE villi. Angiotensin-converting enzyme 2 (ACE2) activity was higher in PRE villi (572.0±23.0 vs. 485.3±24.8 pmol·mg(-1)·min(-1); n=18-22). A low dose of ANG II (1 nM; 2 h) decreased apelin release in NORM villous explants that was blocked by the ANG II receptor 1 (AT1) antagonist losartan. Moreover, losartan enhanced apelin release above the 2-h baseline levels in both NORM and PRE villi (P<0.05). In summary, these studies are the first to demonstrate the lower apelin content in human placental chorionic villi of PRE subjects using quantitative RIA. (Pyr1)-apelin-13 is the predominant form of endogenous apelin in the chorionic villi of NORM and PRE. The potential mechanism of lower apelin expression in the PRE villi may involve a negative regulation of apelin by ANG II.

Study of the Hormones Controlling Food Intake (Leptin and Agouti Related Protein “AgRP”) in Cannabis Smokers

Background: Leptin is an adipocyte derived hormone which is thought to be a key regulator of food intake and body weight. Agouti-related protein also called Agouti-related peptide (AgRP) is a neuropeptide produced in the brain by the AgRP/NPY neuron. It is one of the most potent and long-lasting of appetite stimulators. The appetite stimulating effects of AgRP are inhibited by the hormone leptin. There is a public perception that exposure to cannabis produces an increase of appetite (a phenomenon referred to as the 'munchies'). This phenomenon needs an exploration of the role of the endocannabinoid system in the regulation of obesity and associated metabolic syndrome Aim: This study aimed to find out the food intake style, of subjects smoking cannabis by studing the relationship between the most two popular hormones controlling food intake (leptin hormone, and agouti related protein hormone) compared with healthy subjects. Methods: The study compared two groups of personells; the test group (Group II) included ten males aged 37.40±2.67 years (mean age ±SD) with positive 9-carboxyTetrahydrocanabinol (THC) detected in their urine by qualitative analysis using Gas Chromatography-Mass spectrometry. And control group (GroupI) included ten healthy males aged 38.67±2.35 years (mean age ± SD) as volunteers with negative 9-carboxyOriginal Research Article British Journal of Medicine & Medical Research, 4(29): 4844-4854, 2014 4845 Tetrahydrocanabinol (THC). In both groups, age and body mass index (BMI) were matched, diabetic subjects were excluded. Plasma leptin concentration was measured by quantitative radio immunoassay (RIA). Human plasma AgRP hormone was quantitatively measured by using a kit supplied from Quantikine R&D systems INC. Results: In group I (GI), there was an increase in mean plasma leptin concentrations ±SD (6.39±1.93) ng/ml, than that found, in group II (GII) (4.49±1.40) ng/ml, but it's statistically insignificant (P>0.05). There was an elevation, in mean plasma AgRP concentrations (27.77±2.17) pg/ml in GII, comparing with mean plasma AgRP concentrations (25.84±1.93) pg/ml, in GI but it's statistically insignificant (P>0.05). There was a statistically significant inversely correlation between plasma leptin concentrations and plasma AgRP concentrations in GI (P 0.05). Conclusion: The relationship between plasma leptin concentrations and plasma AgRP concentrations affected by smoking cannabis, and that may change the mechanism of food intake in cannabis smokers.

Differential expression of Neuropeptide Y (NPY) and NPY receptors 1/2 between rat lung and kidney allografts

Recently, our group described a strong leukocytic expression of NPY in the rat. During acute renal graft rejection, we observed a drastical down-regulation of NPY by mononuclear leukocytes accumulating in blood vessels of rat kidneys. Concurrently, the expression of NPY receptors Y1 and Y2 was increased. NPY, a sympathetic co-mediator modulating immune functions, may regulate leukocyte accumulation in graft blood vessels. Lung transplantation is the therapeutic measure of last resort for patients with end-stage lung disease. Compared to kidney transplants, the long-term outcomes of lung grafts are worse. This study addresses the expression of NPY and its receptors in acute rejection of pulmonary grafts. Orthotopic left lung transplantations were performed in the Dark Agouti to Lewis rat strain combination. Animals were sacrificed 4 days after surgery, one day before irreversible graft destruction. Time-matched isografts and untreated organs served as controls. The expression of NPY and its receptors was analyzed by quantitative RT-PCR, Western Blotting, radioimmunoassay and/or immunohistochemistry. Surprisingly, in lung allografts, mononuclear intravascular leukocytes, obtained by vascular perfusion, and intraalveolar leukocytes from bronchoalveolar lavages, abundantly expressed NPY mRNA during acute rejection. In intraalveolar leukocytes, but not in leukocytes isolated from the blood vessels of allografts, we observed an increase in Y1 expression. NPY peptide concentrations in the lung lavage fluids were higher in transplanted allografts compared to control lungs. Our study verifies distinctive organ-specific features of the mononuclear leukocytes in lung allografts. The observed differences between lungs and kidneys may be responsible for the moderate number of leukocytes accumulating in pulmonary graft vessels compared to the massive number in renal grafts. The mechanisms involved in the regulations of the NPY/NPY receptor system are currently under investigation.

Adaptive peripheral immune response increases proliferation of neural precursor cells in the adult hippocampus

To understand the link between peripheral immune activation and neuronal precursor biology, we investigated the effect of T-cell activation on adult hippocampal neurogenesis in female C57Bl/6 mice. A peripheral adaptive immune response triggered by adjuvant-induced rheumatoid arthritis (2 microg/microl methylated BSA) or staphylococcus enterotoxin B (EC(50) of 0.25 microg/ml per 20 g body weight) was associated with a transient increase in hippocampal precursor cell proliferation and neurogenesis as assessed by immunohistochemistry and confocal microscopy. Both treatments were paralleled by an increase in corticosterone levels in the hippocampus 1- to 2-fold over the physiological amount measured by quantitative radioimmunoassay. In contrast, intraperitoneal administration of the innate immune response activator lipopolysaccaride (EC(50) of 0.5 microg/ml per 20 g body weight) led to a chronic 5-fold increase of hippocampal glucocorticoid levels and a decrease of adult neurogenesis. In vitro exposure of murine neuronal progenitor cells to corticosterone triggered either cell death at high (1.5 nM) or proliferation at low (0.25 nM) concentrations. This effect could be blocked using a viral vector system expressing a transdomain of the glucocorticoid receptor. We suggest an evolutionary relevant communication route for the brain to respond to environmental stressors like inflammation mediated by glucocorticoid levels in the hippocampus.

Augmented myocardial methionine-enkephalin in a murine model of cardiac angiotensin II-overexpression

The endogenous opioid system has been reported to interact with both the cardiac sympathetic and renin-angiotensin systems in exerting a local regulatory action on the heart. The goal of this investigation was to examine how cardiac levels of enkephalin production are altered in the development of normotensive primary hypertrophy due to elevated intra-cardiac angiotensin II (Ang II) production. Atrial and ventricular methionine-enkephalin (ME) levels were measured by quantitative radioimmunoassay in 14 and 28-week-old male transgenic mice (TG1306/1R) and control mice. The TG1306/1R exhibit cardiac specific Ang II overexpression and cardiac hypertrophy, but not hypertension. TG1306/1R mice had significantly higher heart/body weight ratios (15-20%) than control littermates at both 14 (p=0.02) and 28 weeks (p=0.04). Relative to controls, ME content was significantly elevated (approximately two-fold) in atria and ventricles in the older 28-week TG1306/1R mice only. A significant inverse correlation between heart size and ME level was observed for 28-week TG1306/1R only. We have provided evidence that a marked elevation of myocardial enkephalin level is observed in the established (but not early) phase of cardiac hypertrophy associated with cardiac-specific Ang II-overexpression. This study identifies a potentially important relationship between two endogenous peptidergic signalling systems involved in the regulation of growth and function of the hypertrophic heart.

Increased expression of rapsyn in muscles prevents acetylcholine receptor loss in experimental autoimmune myasthenia gravis

Myasthenia gravis is usually caused by autoantibodies to the acetylcholine receptor (AChR). The AChR is clustered and anchored in the postsynaptic membrane of the neuromuscular junction (NMJ) by a cytoplasmic protein called rapsyn. We previously showed that resistance to experimental autoimmune myasthenia gravis (EAMG) in aged rats correlates with increased rapsyn concentration at the NMJ. It is possible, therefore, that endogenous rapsyn expression may be an important determinant of AChR loss and neuromuscular transmission failure in the human disease, and that upregulation of rapsyn expression could be used therapeutically. To examine first a potential therapeutic application of rapsyn upregulation, we induced acute EAMG in young rats by passive transfer of AChR antibody, mAb 35, and used in vivo electroporation to over-express rapsyn unilaterally in one tibialis anterior. We looked at the compound muscle action potentials (CMAPs) in the tibialis anterior, at rapsyn and AChR expression by quantitative radioimmunoassay and immunofluorescence, and at the morphology of the NMJs, comparing the electroporated and untreated muscles, as well as the control and EAMG rats. In control rats, transfected muscle fibres had extrasynaptic rapsyn aggregates, as well as slightly increased rapsyn and AChR concentrations at the NMJ. In EAMG rats, despite deposits of the membrane attack complex, the rapsyn-overexpressing muscles showed no decrement in the CMAPs, no loss of AChR, and the majority had normal postsynaptic folds, whereas endplates of untreated muscles showed typical AChR loss and morphological damage. These data suggest not only that increasing rapsyn expression could be a potential treatment for selected muscles of myasthenia gravis patients, but also lend support to the hypothesis that individual differences in innate rapsyn expression could be a factor in determining disease severity.

IgG antibodies against bovine serum albumin in humans—their prevalence and response to exposure to bovine serum albumin

Human exposure to bovine serum albumin (BSA) is very common and occurs through dietary and medicinal routes. Although great effort has been made to reduce exposure to BSA in pharmaceuticals to eliminate the threat of bovine spongiform encephalopathy, less attention has been given to assessing the human immune response after exposure to BSA. A sensitive quantitative radioimmunoassay was therefore developed to measure anti-BSA IgG antibodies in healthy subjects and in cancer patients participating in a randomized, placebo controlled clinical trial where they were exposed to BSA as an intrathoracic surgical sealant during pneumonectomy. Anti-BSA antibodies were detected in 55% of 60 healthy blood donors and 51% of 83 patients before lung cancer resection. The median antibody levels were the same in both cohorts; 0.086 μg/mL (range 0.016–19.5 μg/mL) for health blood donors and 0.062 μg/mL (range 0.009–44 μg/mL) for cancer patients. Six months after exposure of the cancer patients to BSA, the percentage of patients with anti-BSA antibody rose to 96% and the median antibody level rose to 19 μg/mL (range 0.009–258 μg/mL). Placebo-treated cancer patients showed no significant increase in the percentage of patients with anti-BSA antibody (41%) or the median antibody level (0.047 μg/mL; range 0.008–1.58) over 6 months. Western blot analysis confirmed the presence of anti-BSA antibody. Elevated levels of anti-BSA antibody were not associated with any detectable clinical events in either the healthy blood donors or the cancer patients.

Prodynorphin knockout mice demonstrate diminished age-associated impairment in spatial water maze performance

Dynorphins, endogenous κ-opioid agonists widely expressed in the central nervous system, have been reported to increase following diverse pathophysiological processes, including excitotoxicity, chronic inflammation, and traumatic injury. These peptides have been implicated in cognitive impairment, especially that associated with aging. To determine whether absence of dynorphin confers any beneficial effect on spatial learning and memory, knockout mice lacking the coding exons of the gene encoding its precursor prodynorphin ( Pdyn) were tested in a water maze task. Learning and memory assessment using a 3-day water maze protocol demonstrated that aged Pdyn knockout mice (13–17 months) perform comparatively better than similarly aged wild-type (WT) mice, based on acquisition and retention probe trial indices. There was no genotype effect on performance in the cued version of the swim task nor on average swim speed, suggesting the observed genotype effects are likely attributable to differences in cognitive rather than motor function. Young (3–6 months) mice performed significantly better than aged mice, but in young mice, no genotype difference was observed. To investigate the relationship between aging and brain dynorphin expression in mice, we examined dynorphin peptide levels at varying ages in hippocampus and frontal cortex of WT 129SvEv mice. Quantitative radioimmunoassay demonstrated that dynorphin A levels in frontal cortex, but not hippocampus, of 12- and 24-month mice were significantly elevated compared to 3-month mice. Although the underlying mechanisms have yet to be elucidated, the results suggest that chronic increases in endogenous dynorphin expression with age, especially in frontal cortex, may adversely affect learning and memory.

Inter-laboratory reproducibility and inter-assay correlation of the Turbo-MP™ radioimmunoassay for quantitative measurement of allergen-specific immunoglobulin E

Rationale Turbo-MP is a quantitative radioimmunoassay that was developed for the measurement of allergen-specific immunoglobulin E. We examined the intra- and inter-laboratory reproducibility of this assay, and its correlation to another commercially available test (Pharmacia UniCAP ® 100e). Methods Thirty serum samples were selected from a serum bank and were tested on two different days at two laboratories using the same lots of reagents. Quantitation of specific IgE was done for 13 allergens ( D. pteronyssinus, D. farinae, dog epithelium, cat dander, egg white, cow's milk, cod fish, peanut, box elder, Bermuda grass, P. notatum, A. fumigatus, and A. tenuis). Correlation of quantitative results was determined by linear regression. Testing of samples using UniCAP (Pharmacia FEIA) was conducted at one laboratory. Results The data from one laboratory exhibited a correlation coefficient of 0.94 (slope 1.1) and data from the other laboratory had a coefficient of 0.99 (slope 1.1). Inter-laboratory comparisons of the data sets resulted in correlation coefficients of 0.94 to 0.96 (slope 0.9 to 1.2). The comparison of Turbo-MP and UniCAP quantitative results resulted in a correlation coefficient of >0.90 (slope 0.8 to 1.2). Conclusions We demonstrate that the Turbo-MP radioimmunoassay exhibited excellent quantitative reproducibility in runs conducted within and between laboratories and that the Turbo-MP produces results that are essentially interchangeable with the Pharmacia UniCAP system.

Smokeless tobacco or nicotine replacement therapy has no effect on serum immunoglobulin levels

Background: Tobacco smokers have lower serum levels of immunoglobulin (Ig)G, mainly due to lower levels of IgG2, than non-smokers. The component(s) in tobacco smoke responsible for this effect is unknown, but animal studies have implicated nicotine as a major contributor to the immunologic effects of smoking. Does nicotine exposure due to use of smokeless tobacco (oral moist snuff) or nicotine replacement therapy influence serum Ig levels in humans? Methods: Serum content of Ig classes and IgG subclasses was analysed in 77 non-smoking nicotine consumers, including 48 users of oral moist snuff (smokeless tobacco users) and 29 ex-smokers on nicotine replacement therapy, and compared with 44 healthy controls. Former smokers in any group had quit smoking at least 6 months prior to study entry. Ig class and IgG subclass levels were determined by radial immunodiffusion. Systemic nicotine exposure was excluded and confirmed by measuring urine content of cotinine using a quantitative radioimmunoassay. Results: Ig class and IgG subclass levels did not differ significantly between the groups, with the sole exception of IgG4, which was significantly lower in nicotine consumers than in healthy subjects (0.4±0.3 vs. 0.6±0.4 g/l, mean± sd, 95% confidence interval {−0.3;−0.05}). There was no correlation between any Ig variable and cotinine concentration. Conclusions: The decreased levels of IgG and IgG2 seen in tobacco smokers do not seem to be an effect of systemic exposure to nicotine.

Serum concentrations of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol in clinically normal dogs and dogs with acute and chronic renal failure.

To compare serum concentrations of 1,25-dihydroxycholecalciferol (1,25-[OH]2D3) and 25-hydroxycholecalciferol (25-[OH]D3) in healthy control dogs and dogs with naturally occurring acute renal failure (ARF) and chronic renal failure (CRF). 24 control dogs, 10 dogs with ARF, and 40 dogs with CRF. Serum concentrations of 1,25-(OH)2D3 were measured by use of a quantitative radioimmunoassay, and serum concentrations of 25-(OH)D3 were measured by use of a protein-binding assay. Mean +/- SD serum concentration of 1,25-(OH)2D3 was 153 +/- 50 pmol/L in control dogs, 75 +/- 25 pmol/L in dogs with ARF, and 93 +/- 67 pmol/L in dogs with CRF. The concentration of 1,25-(OH)2D3 did not differ significantly between dogs with ARF and those with CRF and was in the reference range in most dogs; however, the concentration was significantly lower in dogs with ARF or CRF, compared with the concentration in control dogs. Mean +/- SD concentration of 25-(OH)D3 was 267 +/- 97 nmol/L in control dogs, 130 +/- 82 nmol/L in dogs with ARF, and 84 +/- 60 nmol/L in dogs with CRF. The concentration of 25-(OH)D3 was significantly lower in dogs with ARF or CRF, compared with the concentration in control dogs. The concentration of 1,25-(OH)2D3 was within the reference range in most dogs with renal failure. Increased serum concentrations of parathyroid hormone indicated a relative deficiency of 1,25-(OH)2D3. A decrease in the serum concentration of 25-(OH)D3 in dogs with CRF appeared to be attributable to reduced intake and increased urinary loss.

Evidence of nidogen-2 compensation for nidogen-1 deficiency in transgenic mice.

Previous studies have shown that inhibition of nidogen-laminin binding interferes with basement membrane stabilization in various mouse organ cultures while no overt phenotype has been observed following inactivation of the nidogen-1 gene in mice. We have now used recombinant mouse nidogen-1 and nidogen-2 in order to evaluate a possible compensation between the two isoforms in the knock-out mice. Essentially, a comparable in vitro binding of nidogens-1 and -2 to the same laminin gamma1 chain structure and to several other basement membrane proteins has been revealed. Quantitative radioimmuno-assays have demonstrated high concentrations of nidogen-1 exceeding those of laminin gamma1 and nidogen-2 by factors of 5 and 20-50, respectively, in tissue extracts of wild-type mice. A three- to sevenfold increase in nidogen-2 was observed in heart and muscle of mice with nidogen-1 deficiency and confirmed by a similar increase in the intensity of immunogold staining of these tissues. However, a few of the tissues from mice with the gene knock-out still contained some nidogen-1-like immunoreactivity (1% of wild-type). Furthermore, both nidogen isoforms showed a similar distribution in various organs during embryonic development which, however, as shown previously, changed in some adult tissues. The data support the nidogen-2 compensation hypothesis to explain the limited phenotype observed following elimination of the nidogen-1 gene.

Development of Quantitative Enzyme Immunoassay and Radioimmunoassay of λ-Free Light Chains of Immunoglobulins

New quantitative techniques of radioimmunoassay (RIA) and enzyme immunoassay (EIA) were developed for determination of free light λ-chains of immunoglobulins (immunometric sandwich-like variants) in biological fluids using two types of monoclonal specific antibodies (MAB-1 and MAB-2) to different epitopes of the antigen molecule: MAB-1 were immobilized on a solid phase (polystyrene beads), whereas MAB-2 were labeled with iodine or with the enzyme. The test systems prepared can be used for determination of concentrations from 25 to 1000 ng/ml, are very sensitive (25 ng/ml), and the analysis time is 5 h. The two methods were compared, and their clinical and diagnostic validity was evaluated in patients with various diseases associated with disorders in the antibody synthesis by the immune system.

Autoantibodies to human melanocyte-specific protein pmel17 in the sera of vitiligo patients: a sensitive and quantitative radioimmunoassay (RIA).

In the present study we describe the in vitro transcription-translation of human melanocyte-specific protein Pmel17 cDNA and subsequent use of the resulting 35S-labelled Pmel17 in an RIA to analyse vitiligo sera for the presence of Pmel17 antibodies. Of 53 vitiligo sera examined in the assay, three (5.9%) were found to be positive for Pmel17 antibodies. In contrast, sera from 20 healthy controls, 10 patients with Hashimoto's thyroiditis and 10 patients with Graves' disease (GD) were all negative for Pmel17 antibodies. All three patients positive for Pmel17 antibodies (aged 50-63 years) had had vitiligo of the symmetrical type for > 1 year and all of them also had an associated autoimmune disorder: GD in one and autoimmune hypothyroidism in two. In addition, all three patients had antibodies to the melanogenic enzymes tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) in their serum. Absorption studies indicated that preincubation with COS-7 cell extract containing expressed Pmel17 absorbed out the immunoreactivity of the three sera positive in the RIA, confirming the anti-Pmel17 reactivity of the sera from these patients. In contrast, COS-7 cell extracts containing either expressed tyrosinase, TRP-1 or TRP-2 did not remove the anti-Pmel17 reactivity of the three sera in the RIA. This lack of cross-reactivity suggests that the humoral response to Pmel17 in these patients is specific and independent of the antibody reactivity to tyrosinase, TRP-1 and TRP-2.

Laparoscopic Surgical Treatment of Ovarian Ectopic Pregnancy Using the Harmonic Scalpel

The object of this study was to review five ovarian pregnancies that occurred during an 8-year period (1991–1998). The prevalence of the disease is higher than expected. Early diagnosis is possible owing to the development of ultrasonography and quantitative radioimmunoassay for β-HCG. Etiologic factors such as pelvic inflammatory disease, endometriosis, and intrauterine device usage are considered. The authors present their experience in the management of that rare gynecologic condition treated by operative laparoscopy with the harmonic scalpel.

Serum p97 levels as an aid to identifying Alzheimer's disease.

Background: The application of formal clinical diagnostic criteria for the identification of Alzheimer's Disease (AD) has improved diagnostic sensitivity. However, there remains a need for non-invasive biological markers and laboratory tests, which can facilitate case identification, and the assessment of treatment response. The p97 protein is a secreted protein specifically expressed by amyloid plaque associated reactive microglia that may have AD diagnostic ability. Methods: A quantitative radioimmunoassay was developed to measure serum p97. This study, under a double blind protocol, evaluated the utility of serum p97 as diagnostic test for AD. All subjects were referred to the UBC Clinic for Alzheimer's Disease and Related Disorders (CADRD) for clinical assessment of dementia. A serum p97 sample was obtained at the time of assessment but diagnosis of disease was determined independently of p97 examination. Results: "Possible" and "probable" AD cases (n = 41) and cognitively normal controls (n = 64) showed a highly significant difference in mean p97 concentration (41 vs. 20 ng/ml, p<0.001). There was some overlap in p97 distributions between AD cases and control subjects. The area under the curve (AUC) for the receiver operator curve (ROC) was 0.812. Conclusions: These results further support the specificity of high serum p97 levels in AD and its potential utility as a biological marker in AD. The reproducible elevation of serum p97 in AD underlines the need to further determine its role as a biological marker and diagnostic adjunct for AD.

Expression of apoptosis proteins in chronic myelogenous leukemia: associations and significance.

The mechanisms favoring the growth advantage of Philadelphia chromosome positive cells over normal cells in chronic myelogenous leukemia (CML) are not fully elucidated but could be due partly to altered apoptosis and longer survival of CML clones. Also, little is known about the biologic characteristics of disease progression in CML. Bcl-2 expression has been demonstrated to exert an antiapoptotic effect resulting in increased cell survival. Other proteins such as Bax and Bad are proapoptotic proteins. Fas, a cell surface protein, can be triggered by an appropriate death-promoting ligand (FasL) to activate downstream caspases pivotal in initiation of programmed cell death. Although the mechanisms underlying cellular proliferative and apoptotic pathways are complex, with involvement of multiple interlocking proteins, the relative expression of pro- and antiapoptotic proteins may have an influence on disease progression. This study aimed to determine whether the changes in the cellular expression of Bcl-2, Bax, and Fas correlate with caspase-3 activity and disease progression in CML, or with response to interferon (IFN)-alpha therapy and prognosis in early chronic phase CML. Bcl-2, Bax, and Fas expression were measured on whole cell lysates from bone marrow mononuclear cell fractions by Western blot analysis and quantitative radioimmunoassay. Caspase-3 activity was determined using the DEVD system. Specimens from 203 patients with CML were examined. These included 130 patients in early chronic phase disease (ECP; diagnosis to therapy, < or =12 months), 33 patients in late chronic phase (diagnosis to therapy, > 12 months), 27 patients in accelerated phase, and 13 patients in blastic phase. Correlations between apoptosis proteins and CML phases, risk groups in ECP, and response to IFN-alpha therapy and survival in ECP were investigated by standard statistical methods, and positive findings were assessed by multivariate analysis. Levels of Bcl-2, Fas, Bax, and caspase-3 activity did not correlate with disease progression. Among patients in ECP, higher Fas levels correlated with poorer risk groups (P = 0.05) and higher caspase-3 activity correlated with better risk groups (P = 0.048). With IFN-alpha therapy, major cytogenetic responses were noted in 30% of patients with high Fas and 53% with low Fas (P = 0.016) and failure to achieve a complete hematologic response (CHR) in 25% versus 2% (P = 0.0001). Survival was shorter with high Fas levels (5-year rates, 71% vs. 52%; P = 0.002), and the independent poor prognostic significance of high Fas levels was confirmed by multivariate analysis (P = 0.014). Response to IFN-alpha therapy and survival were not significantly different by different levels of Bcl-2, Bax, or caspase-3 activity. High Fas levels were associated with intrinsically worse disease at diagnosis, whereas high caspase-3 activity was associated with good risk disease. In ECP CML, high Fas levels were associated with significantly worse response to IFN-alpha therapy and with significantly worse survival. The influence of these cellular proteins and caspase-3 activity on apoptosis in CML is complex and merits further investigation.

Plasma C3a and C4a levels in liver transplant recipients: a longitudinal study.

Liver transplant patients were enrolled in a study designed to investigate correlations between plasma complement C3a or C4a levels and various postoperative complications. Longitudinal EDTA-plasma levels of C3a and C4a were measured by quantitative radioimmunoassay. Acute rejection gave a characteristic and marked increase in blood C3a, C4a and gamma-glutamyl transferase (gammaGT) levels, which rapidly resolved after high dose steroid treatment. Cytomegalovirus (CMV) infections in two of three patients gave an initial small increase only in C3a levels (i.e., alternative pathway activation) followed approximately 6 weeks later by a marked increase in C4a levels (i.e., classical or lectin pathway activation). In a third patient diagnosed for CMV infection, the complement activation profile was complicated by a coincident minor rejection episode. However, a late stage elevation in C4a was also noted. Two patients experiencing biopsy proven recurrent hepatitis C infections following transplantation exhibited increases in both gammaGT and C4a levels, without a significant increase in the level of C3a. Several hepatitis C and one hepatitis B patient had multiple late activation episodes involving marked elevation in both plasma C3a and C4a levels without detectable increases in the liver enzymes conventionally used to monitor organ function. We also showed that ex vivo activation of complement in EDTA plasma from all transplant patients was abnormally high. The classical or lectin pathway is believed to be responsible for this excessive ex vivo complement activation in the plasma of these patients. Therefore, subclinical rejection episodes and/or viral infections may be effectively detected or monitored by measuring C3a and C4a levels in plasma samples from liver transplant patients. Routine measurement of plasma complement products may provide an early non-invasive mode for detecting infections and also serve to monitor chronic or acute changes in the patient's immune system.