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PubMed1991

Human immunodeficiency virus infection in the US Air Force: seroconversions, clinical staging, and assessment of a T helper cell functional assay to predict change in CD4+ T cell counts.

As of January 1990, 933 persons with human immunodeficiency virus type 1 (HIV-1) infection were clinically evaluated at Wilford Hall US Air Force (USAF) Medical Center. The Walter Reed HIV staging system was used in these evaluations to describe disease status and progression. Most persons were diagnosed through mandatory HIV testing in the USAF and were asymptomatic at the time of diagnosis. As of May 1990, 161 HIV-positive seroconverters (estimated overall seroconversion rate of 0.156/1000 person-years between 30 June 1988 and 1 July 1990) had been identified among active-duty USAF personnel, as they had previously tested negative for antibody to HIV. Men constitute 95% of the USAF HIV-positive population. An in vitro T helper cell functional assay was assessed to predict rate of CD4+ T cell decline over the subsequent year (mean, 15 months) in patients with greater than 200 CD4+ T cells/mm3. This assay may prove useful for prognostication and comparisons of patients in clinical trials of anti-HIV interventions.

Adult↗

PubMed2000

Recombinant human receptors and functional assays in the discovery of altinicline (SIB-1508Y), a novel acetylcholine-gated ion channel (nAChR) agonist.

Neuronal nicotinic acetylcholine receptors (nAChRs) are a class of ion channels with significant potential as molecular targets for the design of drugs to treat a variety of CNS disorders. The discovery that neuronal nAChRs are further subdivided into multiple subtypes suggests that drugs which act selectively at specific nAChR subtypes might effectively treat Parkinson's disease (PD), Alzheimer's disease (AD), schizophrenia, ADHD, depression, anxiety or pain without the accompanying adverse side effects associated with non-selective agents such as nicotine (1) and epibatidine. Altinicline (SIB-1508Y) is a novel, small molecule designed to selectively activate neuronal nAChRs and is undergoing clinical evaluation for the treatment of PD. It was selected from a series of compounds primarily on the basis of results from functional assays, including (a) measurement of Ca2+ flux in stable cell lines expressing specific recombinant human neuronal nAChR subtypes; (b) determination of in vitro and in vivo neurotransmitter release; (c) in vivo models of PD. Biological data on both altinicline and the series of compounds from which it was selected are reported.

Calcium↗

PubMed1996

Functional assay for HER-2/neu demonstrates active signalling in a minority of HER-2/neu-overexpressing invasive human breast tumours.

Overexpression of HER-2/neu in human breast carcinomas correlates with poor prognosis, although its strength as a prognostic indicator varies widely in different reports. Variability may be due to active signalling by HER-2/neu in a subset of the tumours in which it is overexpressed. To study this hypothesis, we have developed an activation state-specific anti-HER-2/neu monoclonal antibody. In this report, we use this antibody to analyse the signalling status of HER-2/neu in a large series of invasive breast carcinomas. Overexpression of HER-2/neu was detected in 9% of 223 cases. Of the cases demonstrating overexpression, active signalling by HER-2/neu was detected in only 35%. The clinicopathological characteristics of these cases are described. This functional assay is predicted to improve the utility of HER-2/ neu as a prognostic indicator.

Antibodies, Monoclonal↗

PubMed2006

Functional assays for BRCA1 and BRCA2.

Genetic testing for the two major breast cancer susceptibility genes has now been available for several years with more than 70,000 people tested in the USA alone. While the current genetic testing identifies many sequence alterations there are problems with both sensitivity and specificity of the assay. In particular, the genetic testing is limited in its ability to determine which of the many missense mutations identified in BRCA1 and BRCA2 actually predispose to cancer and which are simply neutral alterations. Here we will focus on the limitations in test result interpretation and we will explore how biochemistry and cell biology can help to clarify these issues. Although we limit our discussion to genetic testing of BRCA1 and BRCA2, the problem is common to an expanding group of genes, including ATM and MSH2, in which germ-line missense mutations may also confer increased risk of cancer. Here we advocate the use of functional assays to complement genetic data in the analysis of unclassified missense mutations and propose a set of standards to conduct and interpret these assays.

BRCA1 Protein↗

PubMed1987

Platelet-type von Willebrand disease platelet aggregating factor: a novel functional assay of von Willebrand factor.

Blood platelets from patients with platelet-type von Willebrand disease (vWD) aggregate upon the addition of human von Willebrand factor (vWf) in the absence of ristocetin or other stimulating factors. We measured quantitatively the ability of vWf to induce directly aggregation of platelet-type vWD platelets (platelet-type vWD platelet aggregating factor [PT-PAF]). Cryoprecipitate and factor VIII concentrates were used as a source of vWf of various multimeric composition. The PT-PAF activity was dependent on the multimer size of vWf, like in the case of ristocetin cofactor (RCof) activity. However, PT-PAF activity was not equivalent to RCof activity and the relative PT-PAF/RCof ratio ranged from 1.00 to 0.18 in the materials studied. The preparations containing the higher-molecular-weight multimers had higher PT-PAF/RCof ratio. These findings suggest that PT-PAF activity is a functional expression of more highly polymerized multimers of vWf as compared with RCof activity. Measurement of PT-PAF would serve as a novel functional assay of vWf.

Factor VIII↗

PubMed2000

Transcriptional slippage of p53 gene enhanced by cellular damage in rat liver: monitoring the slippage by a yeast functional assay.

The Long-Evans Cinnamon (LEC) rat is a mutant strain characterized by abnormal copper metabolism and a high incidence of hepatitis and hepatoma. Using a yeast-based assay which scores mutants in p53 gene transcripts as red colonies, we detected frequent mutations in the liver of LEC rats. The majority (50-60%) of these were frameshift mutations caused by the insertion of an extra adenine (A) in the regions containing six consecutive adenines. The rate of A insertion was calculated to be 6.9-9.0% of the total p53 cDNA. Insertions of an extra adenine were found almost exclusively in the mRNA (cDNA), especially in the (A)(6) tract located at the most 5'-side (exon 4) among the three (A)(6) tracts (exons 4, 7, and 8), but rarely in the corresponding sites of genomic DNA. Wild-type p53 cDNA was transcribed in vitro into mRNA with the use of SP6 RNA polymerase and tested by the yeast functional assay. Subsequent sequencing detected A insertions at an overall rate of 1.6% in exons 7 and 8 but none in exon 4. This indicates that the A insertion in the exon 4 (A)(6) tract was an in vivo phenomenon rather than an artifact in reverse transcription or polymerase chain reaction. The percentage of red colonies increased sharply to about 20% of the liver samples in the acute hepatitis stage, and returned to control level of those in the chronic hepatitis stage, and increased again slightly to those in the neoplastic stage. The percentage of red colonies correlated with the serum GOT level (r=0.96, p<0.001) but not with the contents of copper and 8-hydroxydeoxyguanosine in the liver of LEC rats. Ethanol treatment of hepatic cell lines also increased the rate of transcriptional slippage at the (A)(6) tract. These findings indicate that cellular damage is responsible for the increase in the rate of mutation at the transcriptional level, and suggest that cellular damage degrades transcriptional fidelity, thereby further impairing cellular functions.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2005

Unscheduled DNA synthesis: a functional assay for global genomic nucleotide excision repair.

The unscheduled DNA synthesis (UDS) assay measures a cell's ability to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique in living cells by creating 6-4 photoproducts and pyrimidine dimers using UVC irradiation, then allowing for their repair. Repair is quantified by the amount of radioactive thymidine incorporated after this insult, and the length of time allowed for this incorporation is specific for repair of particular lesions. Radioactivity is evaluated by grain counting after autoradiography. The results are used to diagnosis repair-deficient disorders clinically and provide a basis for investigation of repair deficiency in human tissues or tumors. At the present time, no other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation, as discussed in Chapter 28, is not an equivalent technique, as it specifically measures transcription-coupled repair at active genes, a subset of total NER.

Autoradiography↗

PubMed1982

Suppressor cell activity after major injury: indirect and direct functional assays.

In vitro responses to streptokinase-streptodornase (SKSD), mumps, and mixed lymphocyte culture (MLC) in 19 burned and 13 multitrauma patients were studied sequentially by lymphocyte tritiated thymidine incorporation and compared to responses of 28 normals. Mean responses to SKSD remained significantly depressed from normal for up to 14 to 28 days following injury: MLC responses, significantly depressed at 48 hours, recovered promptly to normal levels. Because of evidence that the proliferative capacity of the T-cell population to soluble antigens is contained within the inducer subpopulation while both inducer and suppressor subpopulations respond in MLC, the observed increase in MLC responses, coupled with a sustained depression of SKSD and mumps responses, suggests activation of a population of suppressor cells. In a direct assay of suppressor cell function, lymphocytes from three or four multitrauma patients incubated in a two-way MLC with normal lymphocytes significantly suppressed PHA responsiveness, confirming the findings of the direct assay.

Adolescent↗

PubMed2002

Polyadenylate polymerase (PAP) and 3' end pre-mRNA processing: function, assays, and association with disease.

Polyadenylate polymerase (PAP) is one of the enzymes involved in the formation of the polyadenylate tail of the 3' end of mRNA. Poly (A) tail formation is a significant component of 3' processing, a link in the chain of events, including transcription, splicing, and cleavage/polyadenylation of pre-mRNA. Transcription, capping, splicing, polyadenylation, and transport take place as coupled processes that can regulate one another. The poly(A) tail is found in almost all eukaryotic mRNA and is important in enhancing translation initiation and determining mRNA stability. Control of poly(A) tail synthesis could possibly be a key regulatory step in gene expression. PAP-specific activity values are measured by a highly sensitive assays and immunocytochemical methods. High levels of PAP activity are associated with rapidly proliferating cells, it also prevents apoptosis. Changes of PAP activity may cause a decrease in the rate of polyadenylation in the brain during epileptic seizures. Testis-specific PAP may play an important role in spermiogenesis. PAP was found to be an unfavorable prognostic factor in leukemia and breast cancer. Furthermore, measurements of PAP activity may contribute to the definition of the biological profile of tumor cells. It is crucial to know the specific target causing the elevation of serum PAP, for it to be used as a marker for disease. This review summarizes the recently accumulated knowledge on PAP including its function, assays, and association with various human diseases, and proposes future avenues for research.

Breast Neoplasms↗

PubMed1985

Radioligand binding and functional assays demonstrate postsynaptic alpha 2-receptors on proximal tubules of rat and rabbit kidney.

We have explored the localization of renal alpha 2-adrenoceptors by radioligand binding techniques and studies of 22Na+ influx into isolated renal tubular cells. Initially, we validated the use of the alpha 2-adrenergic antagonist [3H]rauwolscine as a high-affinity (Kd = 2.5 +/- 0.65 nM) probe that binds to a single class (Hill slope = 0.94 +/- 0.06) of alpha 2-adrenoceptors in rat renal cortical membranes (Bmax = 340 +/- 50 fmol/mg). [3H]Rauwolscine and [3H]yohimbine identified an identical number of sites, but [3H]rauwolscine bound with a several-fold higher affinity. Treatment of rats with 6-hydroxydopamine depleted renal norepinephrine greater than 80% but failed to alter the number or affinity of renal cortical alpha 2-receptors, thus suggesting that these alpha 2-receptors are not located on presynaptic sites. Further evidence for the postsynaptic localization of these receptors was obtained by in vitro autoradiographic studies, which showed that [3H]rauwolscine sites were preferentially located on proximal tubular cells, and by functional assays with isolated rabbit proximal tubular cells. In the latter studies, Na+-H+ antiport activity was stimulated up to 2-fold by the selective alpha 2-adrenergic agonist guanabenz, and this stimulation was blocked by yohimbine but not by the alpha 1-blocker prazosin. Although amiloride was able to block this response to guanabenz, radioligand binding studies demonstrated that amiloride can compete for [3H]rauwolscine sites at the concentrations used to block Na+-H+ antiport activity. These data suggest that alpha 2-adrenoceptors are present on proximal tubular cells in rats and rabbits and that these receptors appear to stimulate Na+-H+ antiport activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

PubMed1998

Monitoring adenoviral p53 transduction efficiency by yeast functional assay.

Monitoring the transduction efficiency is of paramount importance in gene therapy. To monitor adenovirus-mediated wild-type p53 gene transfer, we have used a quantitative assay which tests the ability of human p53 to activate transcription in yeast. Selective amplification of cellular and viral p53 transcripts followed by quantitative assessment of mutant p53 content with the assay permits measurement of the wild-type p53 transduction efficiency into SF-188, U251MG and HUG31 glioblastoma cells. One reverse transcription primer tracks the wild-type/mutant ratio of endogenous p53 mRNA (P2), and the other the wild-type/mutant ratio of both endogenous and exogenous p53 mRNA (P1). Following infection of cell lines homozygous for mutant p53, the apparent transduction efficiency calculated (tau 0 = [P1-P2]/[1 + P2]) correlated with the level of p21 expression. Transduction efficiency in heterozygous wild-type/mutant HUG31 cells increased linearly with multiplicity of infection (MOI) for tau 0 values between 0.5 and 5.9, and admixture of normal cell-derived RNA produced only a modest reduction in tau 0 value, in keeping with theoretical predictions. These results suggest that the yeast p53 functional assay may be a useful tool for monitoring p53 gene therapy.

Adenoviridae↗

PubMed2001

A functional assay for mutations in tumor suppressor genes caused by mismatch repair deficiency.

The coding sequences of multiple human tumor suppressor genes include microsatellite sequences that are prone to mutations. Saccharomyces cerevisiae strains deficient in DNA mismatch repair (MMR) can be used to determine de novo mutation rates of these human tumor suppressor genes as well as any other gene sequence. Microsatellites in human TGFBR2, PTEN and APC genes were placed in yeast vectors and analyzed in isogenic yeast strains that were wild-type or deletion mutants for MSH2 or MLH1. In MMR-deficient strains, the vector containing the (A)(10) microsatellite sequence of TGFBR2 had a mutation rate (mutations/cell division) of 1.4 x 10(-4), compared to a mutation rate of 1.7 x 10(-6) in the wild-type strain. In MMR-deficient strains, mutation rates in PTEN and APC were also elevated above background levels. PTEN mutation rates were higher in both msh2 (4.4 x 10-5) and mlh1 strains (2.3 x 10-5). APC mutation rates in the msh2 strain (2.4 x 10-6) and the mlh1 strain (1.7 x 10-6) were also significantly, but less dramatically, elevated over background. Mutations selected for in the yeast screen were identical to those previously observed in human tumor samples with microsatellite instability (MSI). This functional assay has applicability in providing quantitative data about microsatellite mutation rates caused by MMR deficiency in any human tumor suppressor gene sequence. It can also be applied as a genetic screen to identify new genes that are vulnerable to such microsatellite mutations and thus may be involved in the neoplastic development of tumors with MSI.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Possible incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats on standard toxicology study.

The objective of this study was to examine the feasibility of conducting an immunotoxicological assay for assessing humoral immunity in rats on standard toxicology study. Male CD rats were untreated or dosed intraperitoneally daily for 30 or 90 days, excluding weekends, with vehicle or 2 mg/kg cyclophosphamide (CY). Six days prior to sacrifice, selected rats were injected intravenously with sheep red blood cells (SRBC). One day prior to necropsy, blood samples for hematological and clinical chemical measurements were collected from each rat. On the day of necropsy standard protocol tissues were collected, weighed, processed to slides, and examined microscopically. One-half of each spleen was used to prepare a single cell suspension in order to assess spleen cell numbers. Serum was analyzed for anti-SRBC IgM antibody using an enzyme-linked immunosorbent assay. A second set of studies was performed to examine further the effect of SRBC administration on lymphoid organ weights using 30- and 90-day study age-equivalent naive male CD rats. Exposure of animals to 2 mg/kg CY for 30 or 90 days resulted in a 28% and 61% decrease, respectively, in SRBC-specific serum IgM levels. CY treatment also caused mild alterations in some leukocytic parameters, with significant decreases of 35% and 33% in white blood cell and lymphocyte counts, respectively, observed in 30-day CY-treated animals receiving SRBC. Injection of SRBC alone did not alter hematological or clinical chemistry parameters. With the expected exception of the spleen (increased number and size of germinal centers), administration of SRBC did not significantly alter the weights or morphology of routine protocol tissues. Furthermore, administration of SRBC did not mask the immunosuppressive effects of CY treatment under the conditions of this study. Based on our preliminary findings, a functional assay for assessing humoral immunity may be conducted in animals on standard toxicology study.

Animals↗

PubMed1991

A functional assay to measure postsynaptic gamma-aminobutyric acidB responses in cultured spinal cord neurons: heterologous regulation of the same K+ channel.

The stimulation of postsynaptic gamma-aminobutyric acid (GABA)B receptors leads to slow inhibitory postsynaptic potentials due to the influx of K(+)-ions. This was studied biochemically, in vitro in mammalian cultured spinal cord neurons by using 86Rb as a substitute for K+. (-)-Baclofen, a GABAB receptor agonist, produced a concentration-dependent increase in the 86Rb-influx. This effect was stereospecific and blocked by GABAB receptor antagonists like CGP 35 348 (3-aminopropyl-diethoxymethyl-phosphonic acid) and phaclofen. Apart from the GABAB receptors, both adenosine via adenosine1 receptors and 5-hydroxytryptamine (5-HT) via 5-HT1 alpha agonists also increased the 86Rb-influx. These agonists failed to show any additivity between them when they were combined in their maximal concentration. In addition, their effect was antagonized specifically by their respective antagonists without influencing the others. These findings suggest the presence of GABAB, adenosine1 and 5-HT1 alpha receptors in the cultured spinal cord neurons, which exhibit a heterologous regulation of the same K(+)-channel. The effect of these agonists were antagonized by phorbol 12,13-didecanoate, an activator of protein kinase C, and pretreatment with pertussis toxin. This suggests that these agonists by acting on their own receptors converge on the same K(+)-channel through the Gi/Go proteins. In summary, we have developed a biochemical functional assay for studying and characterizing GABAB synaptic pharmacology in vitro, using spinal cord neurons.

Animals↗

PubMed2006

Functional assay of type I interferon in systemic lupus erythematosus plasma and association with anti-RNA binding protein autoantibodies.

OBJECTIVE: Peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) have increased expression of genes typically induced by type I interferon (IFN). However, it has been difficult to identify and quantify the factors responsible for activation of the IFN pathway in SLE. To characterize these mediators, we developed an assay that measures the functional effects of plasma or serum components on the gene expression of cultured target cells. METHODS: WISH epithelial cell line cells were cultured with medium, with recombinant IFNalpha, IFNbeta, or IFNgamma, or with 50% plasma from SLE patients (n = 73), rheumatoid arthritis (RA) patients (n = 19), or healthy donors (n = 30). Real-time quantitative polymerase chain reaction was used to determine WISH cell expression of IFN target genes, including PRKR, IFIT1, IFI44, MX1, and C1orf29 (preferentially induced by IFNalpha) and CXCL9 (Mig) (preferentially induced by IFNgamma). RESULTS: IFNalpha-regulated genes were induced by SLE plasma samples, but not by most of the RA or healthy control plasma samples. The activity in SLE plasma was inhibited >90% by anti-IFNalpha antibody, but not by anti-IFNbeta or anti-IFNgamma antibodies. The expression of each IFNalpha target gene induced by SLE plasma correlated with the expression of that gene studied ex vivo in PBMCs from the same patients and with the titer of anti-RNA binding protein (anti-RBP)-specific autoantibodies. Plasma activity paralleled PBMC expression of IFNalpha-inducible genes over time. CONCLUSION: IFNalpha in SLE plasma is a major stimulus of IFN target gene expression and is related to expression of those genes in PBMCs from SLE patients and to the titer of anti-RBP autoantibodies. These data provide additional support for the view that IFNalpha mediates immune system activation and dysregulation in SLE.

Antibodies↗

PubMed2025

CryoSCAPE: Scalable immune profiling using cryopreserved whole blood for multi-omic single cell and functional assays.

BACKGROUND: The field of single cell technologies has rapidly advanced our comprehension of the human immune system, offering unprecedented insights into cellular heterogeneity and immune function. While cryopreserved peripheral blood mononuclear cell (PBMC) samples enable deep characterization of immune cells, challenges in clinical isolation and preservation limit their application in underserved communities with limited access to research facilities. We present CryoSCAPE (Cryopreservation for Scalable Cellular And Proteomic Exploration), a scalable method for immune studies of human PBMC with multi-omic single cell assays using direct cryopreservation of whole blood. RESULTS: Comparative analyses of matched human PBMC from cryopreserved whole blood and density gradient isolation demonstrate the efficacy of this methodology in capturing cell proportions and molecular features. The method was then optimized and verified for high sample throughput using fixed single cell RNA sequencing and liquid handling automation with a single batch of 60 cryopreserved whole blood samples. Additionally, cryopreserved whole blood was demonstrated to be compatible with functional assays, enabling this sample preservation method for clinical research. CONCLUSIONS: The CryoSCAPE method, optimized for scalability and cost-effectiveness, allows for high-throughput single cell RNA sequencing and functional assays while minimizing sample handling challenges. Utilization of this method in the clinic has the potential to democratize access to single-cell assays and enhance our understanding of immune function across diverse populations.

Humans↗

PubMed2004

A functional assay for measuring activated thrombin-activatable fibrinolysis inhibitor in plasma.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase found in plasma that is activated by thrombin, the thrombin-thrombomodulin complex, or plasmin. The active carboxypeptidase, TAFIa, attenuates fibrinolysis by removing newly exposed carboxy-terminal lysine residues on fibrin. The half-maximal effect of TAFIa on clot lysis occurs at 1 nM and the maximal effect occurs at 20 nM. Since the circulating concentration of the procarboxypeptidase is approximately 75 nM, only a small portion needs to be activated to have a significant effect on clot lysis. Several assays to measure total plasma TAFI levels and plasma TAFIa levels after it is fully activated exist. However, no currently available assay is sufficiently sensitive and specific to measure endogenous TAFIa in plasma. We have devised a new sensitive and specific assay for TAFIa in plasma that is based on physiologic function. This assay is based on the fact that TAFIa decreases the cofactor activity of high-molecular-weight fibrin degradation products in the stimulation of plasminogen cleavage in a concentration-dependent fashion. With this assay, we can measure TAFIa concentrations as low as 10 pM in plasma and it is not affected by variability in other hemostatic factors. This assay is reliable and repeatable with intra- and interassay variabilities of 6.5 and 6.1%, respectively.

Carboxypeptidase B2↗

PubMed1994

Effect of time, temperature and additives on a functional assay of C1 inhibitor.

There are different recommendations for the handling of blood samples for analyses of the kallikrein-kinin or complement system, respectively. C1 inhibitor (C1-INH) takes a crucial part in both systems. In order to establish recommendations for blood specimen collection and transport for making the diagnosis of hereditary angioedema (HAE), the effect of time, temperature and different additives on C1-INH function and antigen was determined. We used blood samples from normals and patients suffering from HAE type I. Plasma containing EDTA, heparin, sodium citrate or polybrene-EDTA, and serum were assayed after incubations at 4 degrees C or 37 degrees C for 6 or 24 h. In addition, pooled serum was incubated for up to 5 days at room temperature. A modest decrease in C1-INH function was observed as an effect of storage-time in samples from normals (p = 0.039) and a substantial decrease was seen for the HAE patients (p = 0.0002). No significant effect of temperature (4 degrees C or 37 degrees C) was found. Clotting did not reduce C1-INH activity. Plasma containing heparin or polybrene interfered with the functional assay, yielding falsely high and low values, respectively. C1-INH functional assay performed within 24 h in serum, EDTA-treated or citrated plasma discriminated well between HAE patients and normals. This was also the case for serum kept at room temperature for up to 5 days, although a modest fall in C1-INH function was seen in the incubation period. For practical purposes we recommend serum as the sample of choice, preferably received within 48 h.

Angioedema↗