A serum-free [3H]thymidine incorporation assay for the detection of transforming growth factors

Summary A rapid mitogenic assay suitable for the detection of transforming growth factors in the extracts of tissues or cells or in the medium conditioned by tumor cells in vitro is described. The method utilizes a nontumorigenic mouse embryo cell line (AKR-2B cells) maintained in serum-free conditions. Three classes of growth factors can be distinguished using this assay.     

Measurement of antigen specific lymphocyte proliferation using 5-bromo-deoxyuridine incorporation An easy and low cost alternative to radioactive thymidine incorporation

The classical in vitro assay for the determination of cell mediated immune responses is the lymphocyte transformation test (LTT) in which cell proliferation is measured by incorporation of radioactive labeled thymidine (³H-TdR). The LTT assay using ³H-TdR is less suited for modestly equipped laboratories as it is costly, laborious and involves the need to handle radioactive isotopes and specialized equipment.

Here we describe an improved alternative LTT method which is capable of detecting specific cellular immune reactions (CMI) against (mycobacterial) antigens in vitro. This assay, the bromodeoxyuridine-ELISA LTT test, is simple, less expensive, reproducible and is as sensitive as the ³H-TdR test. The specific advantages of the test are a simple denaturation step and the fact that no radioactive isotopes are needed. The test is specifically suited for research laboratories in tropical countries which study CMI in those human infectious diseases where this arm of the immune response plays a pivotal role in the generation of immunity, e.g., in tubercolosis, leprosy and leishmaniasis.     

Nonradioactive techniques for measurement of in vitro T-cell proliferation: alternatives to the [(3)H]thymidine incorporation assay

T-cell proliferation is an important in vitro parameter of in vivo immune function and has been used as a prognostic marker of human immunodeficiency virus type 1 (HIV-1) disease progression. The proliferative capacity of T cells in response to various stimuli is commonly determined by a radioactive assay based on incorporation of [(3)H]thymidine ([(3)H]TdR) into newly generated DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, two alternative methods were tested and compared to the [(3)H]TdR assay as a "gold standard." As an alternative, T-cell proliferation was measured by flow cytometric assessment of CD38 expression on T cells and by an enzyme-linked immunosorbent assay (ELISA) based on bromo-2'-deoxyuridine (BrdU) incorporation. Peripheral blood mononuclear cells (PBMCs), either in whole blood or Ficoll-Isopaque separated, from a total of 26 HIV-1-positive and 18 HIV-1-negative Dutch individuals were stimulated with CD3 monoclonal antibody (MAb) alone, a combination of CD3 and CD28 MAbs, or phytohemagglutinin. BrdU incorporation after 3 days of stimulation with a combination of CD3 and CD28 MAbs correlated excellently with the [(3)H]TdR incorporation in both study groups (HIV-1 positives, r = 0.96; HIV-1 negatives, r = 0.83). A significant correlation of absolute numbers of T cells expressing CD38 with [(3)H]TdR incorporation, both in HIV-1-positive (r = 0.96) and HIV-1-negative (r = 0.84) individuals, was also observed under these conditions. The results of this study indicate that determination of both the number of CD38-positive T cells and BrdU incorporation can be used as alternative techniques to measure the in vitro T-cell proliferative capacity. The measurement of CD38 expression on T cells provides the additional possibility to further characterize the proliferating T-cell subsets for expression of other surface markers.     

An evaluation of an assay for DNA synthesis in lymphocytes with [H]thymidine and harvesting on to glass fibre filter discs

Different grades of glass fibre filter paper have been compared for harvesting cells labelled with tritium using a semi-automatic harvester. Grade GF/C (Whatman Ltd.) was selected for routine use although GF/B was found to be useful for whole blood cultures.

The factors affecting the choice of thymidine concentration and activity are discussed and 1 μCi of [³H]thymidine (5 Ci/mMol)/0.2 ml culture is shown to give a linear relationship with total DNA synthesis for up to 22 h.     

Serum factors affecting the incorporation of [3H]thymidine by lymphocytes stimulated by antigen: I. Serum concentration

Lymph node cells from immunized rabbits were cultured with varying concentrations of antigen in preheated (56°, 30 minutes) autologous serum which had been collected before immunization. [³H]Thymidine was present for the last 6 hours of the 24-hour culture period and the radioactive labelling of acid-precipitable material was then determined.

Changes in labelling due to variations of culture conditions were interpreted according to whether they were specific for control or antigen-treated cultures or non-specific. Cell concentration and serum concentration were predominantly non-specific variables influencing the labelling in control and antigen-treated cultures to a proportionate extent. However, at serum concentrations below 5 per cent labelling was disproportionately inhibited in antigen-treated cultures; there were further minor disproportionate inhibitions at higher serum concentrations.

Labelling was inhibited by increasing the concentration of serum from 25 to 50 per cent, mainly due to a non-diffusible competitive inhibitory activity. Isotope-dilution analysis of the effects of serum on labelling over a wide range of serum concentrations indicated that the relationship was a complex one with at least three step-wise stimulations of the maximum labelling rate being produced by increasing the serum concentrations from 0 to 25 per cent. Labelling in antigen-treated cultures containing post-immunization serum was less than labelling in cultures containing an equal volume of preimmunization serum, but labelling in control cultures was enhanced by post-immunization serum.

These results are shown to be compatible with the proposals (i) that labelling in control cultures reflects the response of cells to low concentrations of endogenous antigens, and (ii) that preimmunization serum and post-immunization serum contain `natural' and `acquired' antibodies respectively, which normally buffer cell-borne receptor sites against reaction with endogenous and exogenous antigens.

     

Serum factors affecting the incorporation of [3H]thymidine by lymphocytes stimulated by antigen: II. Evidence for a role of complement from studies with heated serum

Lymph node cells from preimmunized rabbits were cultured with varying concentrations of antigen in autologous serum which had been collected before immunization. [³H]Thymidine was added after 18 hours of culture and the cells were harvested at 24 or 66 hours for the determination of the radioactive labelling of acid-precipitable material.

Preheating serum (56°, 20 minutes) enhanced labelling in both control and antigen-treated cultures. This `heat effect' had an early (24 hour) non-specific component, independent of antigen concentration, and a late (66 hour) specific component which was most evident at high antigen concentrations. The conditions of preheating serum (temperature and time) required to produce the heat effect were similar to those required to remove haemolytic activity against rat erythrocytes. However, at certain temperatures and times there were discrepancies. These discrepancies, and data from experiments in which preheated and unheated sera were mixed in varying proportions, or interchanged in different sequences, were explicable on the basis of (i) a requirement for complement in stoichiometric quantities dependent on the number of cells being inhibited, (ii) the involvement of the majority of the cultured cells in the early non-specific component of the heat effect, but only cells capable of proliferating in response to added antigen in the late specific component, (iii) the secretion of complement by cultured cells.

Preheating serum (66°, 20 minutes) depressed labelling in control and antigen-treated cultures and reduced agglutinating activity against both autologous and heterologous erythrocytes.

The results are discussed in relationship to models which require that the size of a specific lymphocyte clone be positively or negatively regulated by the concentration of antigen specific for that clone. With increasing antigen concentration three effects on cells bearing specific receptor sites are distinguished. (i) Cell stimulation under conditions of antigen concentration and cell receptor specificity such that only a few antigen molecules can bind to cells. (ii) Complement-dependent inhibition under conditions such that more antigen molecules can bind to cells. (iii) Complement-independent inhibition under conditions, possibly unphysiological, such that very large quantities of antigen molecules can bind to cells.

     

Serum factors affecting the incorporation of [3H]thymidine by lymphocytes stimulated by antigen: III. Evidence for a role of complement from studies with specific complement inhibitors

To obtain further evidence that complement is involved in both the early, non-specific and the late, specific, components of the heat effect (Forsdyke, 1973b), lymph node cells from preimmunized rabbits were cultured with varying concentrations of antigen in autologous preimmunization serum treated with one of three specific complement inhibitors, inulin, Zymosan or cobra venom factor. The Zymosan particles were removed before use of the serum in cultures, but the other inhibitors were not removed.

Inulin only slowly removed haemolytic activity from serum and enhanced the late response to high concentrations of specific antigen. The inulin concentrations required to remove haemolytic activity were similar to those required to enhance the response to antigen. Zymosan-pretreated serum enhanced labelling with [³H]thymidine in both control and antigen-treated cultures to a proportionate extent. No late specific enhancement of labelling was detected, probably because of the secretion of complement by cultured cells. The interpretation of data obtained when Zymosan was left in cultures was complicated by Zymosan acting as an antigen and inducing a primary response to itself. Cobra venom factor reduced the labelling of both control and antigen-treated cultures so that the non-specific component of the heat effect was not detectable. However, the response to high concentrations of specific antigen was enhanced.

It is concluded that although each complement inhibitor shows unique individual characteristics in the system, on balance the results support the view that the activity responsible for both components of the heat effect is complement.

     

A comparative study of [3H]thymidine and [125I]iodo-2'-deoxyuridine uptake as indicators of in vivo cell proliferation induced by graft-versus-host reaction in the mouse

Since [3H]thymidine ([3H]TdR) and [125I]iododeoxyuridine ([125I]UdR) appear to label very different cell populations in different tissues, we carried out the present investigation to determine the value and limitations of the use of [125I]UdR as a DNA precursor in a study of grafted cell proliferation during the course of a graft-versus-host reaction in mice. Results showed that the iodine radioisotope was as efficient as [3H]thymidine when endogenous thymidylate synthetase was inhibited by fluorodeoxyuridine. The use of 125I as an in vivo labelling compound constitutes a simple technique for evaluating DNA synthesis, provided the unfixed labelled iodine is eliminated by in vitro ethanol washing of tissues. The optimal conditions for elution are defined but may vary from one tissue to another. [125I]UdR and [3H]TdR give similar pictures of the very intricate phenomena accompanying a chronic graft-versus-host reaction (GVHR) induced in mice by minor histoincompatible antigens.     

Serum factors affecting the incorporation of [3H]thymidine by lymphocytes stimulated by antigen. IV. Comparison of enhancement by heated (56 degrees C) serum and by 2-mercaptoethanol.

Serum preheated to 56 degrees C and 2-mercaptoethanol (ME) both enhance the stimulation (%) by antigen of [3H]thymidine incorporation by lymph node cells from immunized rabbits. The enhancements show several similar features suggesting a common mechanism. Both enhancements show (i) non-specific components, affecting [3H]thymidine incorporation in control and antigen-treated cultures proportionately, and (ii) antigen-specific components, having a greater proportionate effect on antigen-treated cultures than on control cultures. The non-specific components, in combination, are approximately additive. The specific components, in combination, are non-additive (mutually exclusive). With both preheated serum and ME the specific component of the enhancement is most evident late in the culture period and is greater at high antigen concentrations. The ME concentration needed for optimum enhancement is reduced if the serum concentration is reduced or if serum is preheated.     

A simple quantitative protein A micro-immunoprecipitation method; assay of antibodies to the N and H antigens of poliovirus

Staphylococcus aureus (Cowan strain I) was used to absorb immune complexes from antiserum to poliovirus to which labeled N or H poliovirus antigens had been added, and the radioactivity in the pelleted organisms and in the supernatant was measured. Excellent agreement was obtained between values calculated separately from the pellet and supernatant readings, validating the use of supernatant measurements from a microtitration plate method.     

Interaction of subpopulations of murine lymph node lymphocytes in antigen-induced [14C]-thymidine incorporation: T and B cell synergy in the response to antigen.

The antigen-induced [14C]-thymidine incorporation of murine lymph node cells (LNC) that were non-adherent (NAD) or adherent (AD) to nylon wool was studied. In contrast to NAD-LNC, AD-LNC responded like unfractionated LNC, and these responses were T lymphocyte dependent. By co-culturing NAD-LNC with subpopulations of AD-LNC the cellular requirements and interactions necessary for maximal incorporation of [14C]-thymidine were determined. A synergistic effect was observed when NAD-LNC and AD-LNC were co-cultured. Synergism was not dependent on T lymphocytes or macrophages in the AD-LNC population but was associated with the B lymphocyte subpopulation. These results indicate that the number of B lymphocytes present in a population of LNC can significantly influence the magnitude of the response to antigen.     

A new, simple and rapid method for enumerating human T lymphocytes in full blood: the E. coli (ATCC 11303) rosette test

A new bacterial rosette technique for enumerating T lymphocytes is described. E. coli (strain B; ATCC 11303), fixed in formaldehyde after overnight growth in thioglycolate medium, are mixed with washed whole blood cells (100 μl) and after incubation at 4°C, slides are made, stained and counted. The nature of the lymphocytes forming E. coli rosettes was demonstrated by comparing their cytochemical staining characteristics with those of E rosetted lymphocytes, and by mixed E. coli and E, mouse E rosette and Fc receptor tests, and by mixed E. coli rosette tests and anti-Ig staining. E. coli and E rosette tests in controls and pediatric patients were also compared. The results show that T_μ and T_γ cells rosette with E. coli.     

Neurogenesis of the nucleus accumbens septi and neighboring septal nuclei in the rhesus monkey: A combined [3H]thymidine and electron microscopic study

The time of origin and spatio-temporal pattern of neuron distribution in the nucleus accumbens septi and the lateral and medial septal nuclei were determined in the rhesus monkey. Autoradiographic analysis of 2 to 5 month old monkeys that had been exposed to a pulse of [³H]thymidine during embryonic or early postnatal days showed that neurons destined for the nucleus accumbens septi are generated over a period of approximately 50 days (between embryonic day 36 and 85 of the 165 day gestational period) whereas the neurons destined for the medial and lateral septal nuclei are produced for only 25 days (from embryonic day 36 to 62). The peak of proliferation of neurons destined for all three nuclei occurs synchronously around embryonic day 45. A number of small cells with round, densely stained nuclei were labeled in specimens injected after embryonic day 85; however, following combined light- and electron-microscopic analysis, these late generated cells were classified as glia.Neurons destined for the nucleus accumbens septi generated on different embryonic days did not exhibit spatio-temporal gradients in their distribution. In contrast, neurons destined to populate the lateral and medial septal nuclei displayed a prominent ‘outside-to-inside’ spatio-temporal gradient in which earlier generated neurons occupied positions closer to the pial surface and later forming neurons were positioned closer to the ventricular surface. Based on this evidence regarding the time span of neuronal production and the absence of spatio-temporal gradients, the mode of development of the nucleus accumbens septi more closely resembles the pattern of neurogenesis reported for the primate neostriatum than that in the adjacent septal nuclei.These findings indicate that the nucleus accumbens septi and neostriatum in primates may have a common embryological origin.     

A pure likelihood approach to the analysis of genetic association data: an alternative to Bayesian and frequentist analysis

Investigators performing genetic association studies grapple with how to measure strength of association evidence, choose sample size, and adjust for multiple testing. We apply the evidential paradigm (EP) to genetic association studies, highlighting its strengths. The EP uses likelihood ratios (LRs), as opposed to P-values or Bayes'' factors, to measure strength of association evidence. We derive EP methodology to estimate sample size, adjust for multiple testing, and provide informative graphics for drawing inferences, as illustrated with a Rolandic Epilepsy (RE) fine-mapping study. We focus on controlling the probability of observing weak evidence for or against association (W) rather than type I errors (M). For example, for LR⩾32 representing strong evidence, at one locus with n=200 cases, n=200 controls, W=0.134, whereas M=0.005. For n=300 cases and controls, W=0.039 and M=0.004. These calculations are based on detecting an OR=1.5. Despite the common misconception, one is not tied to this planning value for analysis; rather one calculates the likelihood at all possible values to assess evidence for association. We provide methodology to adjust for multiple tests across m loci, which adjusts M and W for m. We do so for (a) single-stage designs, (b) two-stage designs, and (c) simultaneously controlling family-wise error rate (FWER) and W. Method (c) chooses larger sample sizes than (a) or (b), whereas (b) has smaller bounds on the FWER than (a). The EP, using our innovative graphical display, identifies important SNPs in elongator protein complex 4 (ELP4) associated with RE that may not have been identified using standard approaches.     

A new tool to assess and monitor the burden of chronic cough on quality of life: Chronic Cough Impact Questionnaire

INTRODUCTION: Chronic cough, one of the most frequent causes for a patient to consult a medical practitioner, limits the course of normal activities in everyday life of the patient affected (work, physical activities, social relations, night sleep). By now, there are few validated questionnaires for the evaluation of the impact of this symptom in the patient's quality of life (QoL). For this reason, we created a new questionnaire for the assessment of QoL in patients affected by chronic cough (Chronic Cough Impact Questionnaire, CCIQ). MATERIALS AND METHODS: In the development procedure of CCIQ an initial questionnaire of 40 items was compiled and given to a first pool of 170 patients, each coming to our attention because of chronic cough; then the 25 most significant items were detected and converted into questions evaluating the answers on a Likert scale of five steps. Consequently, this final questionnaire underwent a validation procedure to assess its construct validity, internal consistency, reliability, and responsiveness. 95 patients (44.2% F, 55.8% M) were evaluated (age 53.69 +/- 11.7 years). RESULTS: Following a statistical analysis, CCIQ showed a four-dimensional structure and good levels of internal consistency for the extracted factors: sleep/concentration (79.98), relationship (86.98), daily life impact (69.04), and mood (65.41). In stable conditions CCIQ showed a good reliability, ranged between 0.67 and 0.88. Responsiveness to clinical changes was accomplished. DISCUSSION: These results provide evidence that CCIQ has specificity enough for being a valid tool for detecting the relative burden of cough on subjective well-being, and for obtaining a global evaluation both of chronic cough impact and of treatments for it, taking into account the patient's point of view. The CCIQ was easily and quickly filled in by the patients while waiting, and it was accepted by the patients.     

The nature of the immune response (Ir) gene defect for pigeon cytochrome c in [B10.A(4R) x B10.PL]F1 mice: A Comparison Between Thymic Selection and Antigen Presentation

[B10.A(4R) x B10.PL]F₁ mice are low responders to pigeon cytochromec, while [B10.A(2R) x B10.PL]F₁ and B10.A mice are high responders.The in vivo site at which the different aliomorphs of the E_aIa molecule exert their Ir gene effect on the immune responseto pigeon cytochrome c was examined by creating two differentsets of radiation-induced bone marrow chimeras. [B10.A(4R) xB10.PL]F₁(b.m.) B10.A(lrr.) chimeras, which possess antigen-presentingcells (APC) of the low responder, but whose T cells are educatedin a high responder environment, were found to be low respondersto pigeon cytochromec. In contrast, B10.A(b.m.) [B10.A(4R)x B10.PL]F₁(lrr.) chimeras, which possess APC of the high respondertype, but whose T cells are educated in a low responder environment,responded to pigeon cytochrome c Addition of B10.A APC to thefirst type of chimera, both prior to antigen priming and atthe time of the secondary challenge in vitro, converted 50%of the animals to responders Furthermore, [B10.A(4R) x B10.PL]F₁mice responded to pigeon cytochrome c If they were primed witha 10-fold greater antigen dose and restimulated in vitro Inthe presence of B10.A APC. These results suggest that the primarysite of the Ir gene defect in this system is at the level ofantigen presentation and not in the T cell repertoire.     

Characterization of nontypeable rotavirus strains from the United States: identification of a new rotavirus reassortant (P2A[6],G12) and rare P3[9] strains related to bovine rotaviruses

Among 1316 rotavirus specimens collected during strain surveillance in the United States from 1996 to 1999, most strains (95%) belonged to the common types (G1 to G4 and G9), while 5% were mixed infections of common serotypes, rare strains, or not completely typeable. In this report, 2 rare (P[9],G3) and 2 partially typeable (P[6],G?; P[9],G?) strains from that study were further characterized. The P[6] strain was virtually indistinguishable by hybridization analysis in 10 of its 11 gene segments with recently isolated P2A[6],G9 strains (e.g., U.S.1205) from the United States, but had a distinct VP7 gene homologous (94.7% a.a. and 90.2% nt) to the cognate gene from P1B[4],G12 reference strain L26. Thus, this serotype P2A[6],G12 strain represents a previously unrecognized reassortant. Three P3[9] strains were homologous (97.8-98.2% aa) in the VP8 region of VP4 to the P3[9],G3 feline-like reference strain AU-1, but had a high level of genome homology to Italian bovine-like, P3[9],G3 and P3[9],G6 rotavirus strains. Two of the U.S. P3[9] strains were confirmed to be type G3 (97.2-98.2% VP7 aa homology with reference G3 strain AU-1), while the other was most similar to Italian bovine-like strain PA151 (P3[9],G6), sharing 99.0% a.a. homology in VP7. Cross-neutralization studies confirmed all serotype assignments and represented the first detection of these rotavirus serotypes in the United States. The NSP4 genes of all U.S. P3[9] strains and rotavirus PA151 were most closely related to the bovine and equine branch within the DS-1 lineage, consistent with an animal origin. These results demonstrate that rare strains with P and G serotypes distinct from those of experimental rotavirus vaccines circulate in the United States, making it important to understand whether current vaccine candidates protect against these strains.     

Chronic uridine treatment reduces the level of [3H]spiperone-labelled dopamine receptors and enhances their turnover rate in striatum of young rats: relationship to dopamine-dependent behaviours

The effect was studied of chronic uridine treatment on the recovery of striatal D-2 dopamine (DA) receptors after their irreversible blockade by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in young (40 days old) and adult (14 months old) male rats using [³H]spiperone as radioligand. Chronic uridine treatment (15 mg kg^-1 day^-1, i.p., 14 days) causes a reduction of [³H]spiperone binding sites in striatum of young rats. This treatment also produces an increase in the rate of recovery of striatal [³H]spiperone-labelled DA receptors in young, but not in adult rats. Catalepsy and exploratory locomotor activity, two behaviours associated with blockade versus activation of DA receptors, were evaluated in the same rats. The behavioural recovery from the EEDC^induced syndrome is more rapid in the young rats treated with uridine than in the saline-treated group. The behavioural recovery in old rats was not affected by chronic uridine treatment. Thus, in young rats the pyrimidine nucleoside uridine may modulate the steady state and the turnover rate of striatal D-2 DA receptors.     

The Trypanosoma lewisi immunofluorescence test: A new simple technique for simultaneous determination of total antinuclear antibodies and the detection of antibodies to double-stranded DNA

An indirect immunofluorescence technique was developed for the detection of antibodies to dsDNA and the simultaneous assessment of antinuclear antibodies ‘in toto’ (ANA). This assay was based upon the use as substrate of smears of peripheral blood derived from rats infected with Trypanosoma lewisi. T. lewisi possesses a giant kinetoplast posteriorly to the nucleus. Enzyme digestion and absorption experiments provided strong evidence that T. lewisi kinetoplast contains dsDNA uncontaminated by other nuclear antigens. The T. lewisi immunofluorescent test was evaluated on a total of 130 sera (30 from patients with SLE) and compared with radioimmunoassays for antibodies to dsDNA ([¹²⁵I]dsDNA-RIA) and antibodies to ssDNA ([¹²⁵I]ssDNA-RIA). Excellent correlation was found between kinetoplast immunofluorescence and [¹²⁵I]dsDNA-RIA, whereas no non-SLE sera showing significant ssDNA binding activity gave kinetoplast staining. With a single exception, only SLE sera reacted with T. lewisi kinetoplast. Sera containing auto-antibodies other than ANA did not induce fluorescene of any part of the parasite, including the flagellum and its base. These results indicated that the T. lewisi immunofluorescence test is specific and reliablem and combines the advantages of Crithidia luciliae with those of Trypanosoma gambiense. It may be used routinely for evaluating of total ANA and simultaneous detection of antibodies against dsDNA.