Tritiated Thymidine Incorporation Research Articles

Transcript Profiling of T Lymphocytes and Dendritic Cells in a Co–culture System Using Anti‐CD3 and Allergen Activation

The interaction of antigen‐specific T lymphocytes with hapten‐bearing dendritic cells (DC) and the subsequent activation and clonal expansion of specific T lymphocyte populations are critical steps in the induction of skin sensitization. Therefore, we have sought to characterize changes in gene expression in T lymphocytes stimulated by incubation with allergen‐treated DC compared with anti‐CD3‐treated T cell‐DC cocultures as a method to identify potential markers of skin sensitization. Human T cells and autologous, mature peripheral blood‐derived DC were co–cultured in the presence or absence of anti‐CD3 monoclonal antibody (mAb) for 6 hours at a 10:1 responder:stimulator ratio. In a separate experiment, autologous DC and T cells from a donor sensitized to the potent contact allergen dinitrochlorobenzene (DNCB) were isolated. T cells were cultured for 6 hours at a responder to stimulator ratio of 20:1 with mature DC that had been treated with either 1 mM 2,4‐dinitrobenzenesulfonic acid (DNBS; the water soluble analog of DNCB), or media alone for 15 minutes. Total RNA was prepared and changes in gene expression were analyzed using Affymetrix U95Av2 GeneChips®. Comparative analysis of Affymetrix mean signal values from triplicate control cultures with those from anti‐CD3‐treated samples revealed highly significant (p ≤ 0.001) changes in the expression of 344 transcripts of the total of approximately 12,000 represented on the chip. However, mean signal values for T cells cocultured with allergen‐treated DC compared to vehicle‐treated DC‐T cell co–cultures identified only 17 significant gene changes (p ≤ 0.001), 11 of which were also identified as having changed significantly in response to stimulation with anti‐CD3. In parallel assays, antigen‐specific T cell proliferative responses were assessed as a function of tritiated thymidine incorporation. Increased T cell proliferative responses were observed in the cultures that contained both DNBS‐treated DC and T cells as well as the anti‐CD3 treated cultures compared with their respective controls. These data suggest that this approach can be used to identify genes that might serve as indicators of contact allergy and may be used in an in vitro predictive assay for skin sensitization.

Cytokine Production, Lymphocyte Proliferation and T-Cell Receptor Vβ Expression in Primary Peripheral Blood Mononuclear Cell Cultures from Nickel-Allergic Individuals

Background: Clinical history and patch test constitute the two cornerstones in the diagnosis of nickel (Ni) allergy. Due to technical and interpretative limits of the patch test, the in vitro lymphocyte transformation test (LTT) has been developed for confirming contact allergy; however, most studies show an overlap in lymphocyte proliferation between Ni-allergic and nonallergic subjects using the LTT. The aim of this study was to see if the secretion of cytokines, especially interleukin (IL)-10 and IL-17, or the use of T-cell receptor (TCR) Vβ families in Ni-stimulated primary peripheral blood mononuclear cell (PBMC) cultures might be more useful for discriminating between allergic and nonallergic subjects. Methods: Ni²⁺-stimulated primary PBMC cultures derived from female subjects diagnosed as Ni-allergic (n = 5) or nonallergic (n = 5) on the basis of a positive or negative patch test were assessed for cell proliferation by tritiated thymidine incorporation and for production of interferon-γ, IL-4, IL-10 and IL-17 in the culture supernatant by ELISA. The immunophenotype and TCR-Vβ family affiliation of the Ni²⁺-induced lymphoblasts were determined by flow cytometry. Results: Lymphocytes from Ni-allergic individuals challenged with a high and a low concentration of Ni showed significantly higher cell proliferation than lymphocytes from nonallergic individuals, but all subjects showed a positive LTT result (stimulation index >2). We found a significantly higher release of IL-10 in Ni²⁺-treated cultures from Ni-allergic compared with nonallergic subjects that provided better separation between individuals in the two groups than did lymphocyte proliferation. The proliferating lymphoblasts were predominantly CD4+, and in 2 of the 5 Ni-allergic subjects, but in none of the 5 nonallergic subjects, the CD4+ lymphoblasts showed a dominance of TCR-Vβ17. Conclusions: Determination of IL-10 production in primary PBMC cultures is a potentially promising in vitro method for discrimination of Ni allergy in females, as compared with cell proliferation.

Inhibition of human mast cell proliferation and survival by tamoxifen in association with ion channel modulation

Background Human lung mast cells (HLMCs) and the human mast cell line HMC-1 express a strongly outwardly rectifying Cl − current characteristic of that carried by the voltage-dependent Cl − channel ClC-5. A similar but distinct current has been implicated in the control of cell proliferation in astrocytes. Objective In this study, we have examined the effects of the Cl − channel blocker tamoxifen on ion channel activity and cell proliferation in both HMC-1 and HLMCs. Methods We used the whole-cell patch-clamp technique to characterize macroscopic ion currents in mast cells before and after addition of tamoxifen. HMC-1 proliferation was assessed by incorporation of tritiated thymidine, HLMC proliferation was determined by counting cells in long-term culture, and cell viability was assessed by annexin V binding and propidium iodide uptake. Results In HMC-1, tamoxifen reduced the outward Cl − current at +130 mV by 73% ± 9% at a concentration of 3 μmol/L and simultaneously opened a novel inwardly rectifying nonselective cation current with a mean inward current of 153 ± 18 pA at −130 mV. Tamoxifen produced a dose-dependent inhibition of HMC-1 proliferation (90.3% ± 4.0% inhibition at 30 μmol/L) without altering cell viability. Tamoxifen inhibited the outward ClC-5-like current in HLMCs, did not open an inward current, and produced a dose-dependent inhibition of HLMC proliferation in long-term culture. Conclusion Tamoxifen inhibits HMC proliferation, possibly through ion channel modulation. This suggests that tamoxifen might be useful in the treatment of mast-cell-mediated diseases, including mastocytosis, asthma, and pulmonary fibrosis.

Characterization of angiotensin II–receptor subtypes in podocytes

Glomerular podocytes play a key role in maintaining the integrity of the glomerular filtration barrier. This function may be regulated by angiotensin II (Ang II) through activation of cell-surface receptors. Although studies suggest that podocytes express receptors for Ang II, the Ang II binding site has not been characterized with radioligand binding techniques. We therefore used iodine 125–labeled Ang II to monitor Ang II–receptor density during differentiation of a mouse podocyte cell line. Scatchard analyses of equilibrium binding data revealed a single class of high-affinity binding sites (dissociation constant ∼ 3 nmol/L) in both differentiated and nondifferentiated cells. During differentiation, the density of Ang II–receptor sites increased roughly 15-fold in differentiated podocytes (maximal density of specific binding sites 881 fmol/mg protein) compared with that in nondifferentiated cells (52 fmol/mg protein; P < .005). Glomerular podocytes expressed messenger RNA for AT1A, AT1B, and AT2 receptor subtypes, and competitive binding studies found that differentiated podocytes expressed mostly AT1 receptors (∼75%) with lesser amounts of AT2 (∼25%). Up-regulation of Ang II–receptor number was associated with increased Ang II–receptor responsiveness, as evidenced by enhanced Ang II–stimulated inositol phosphate (IP) generation and incorporation of tritiated thymidine. Both [ 3H]thymidine incorporation and IP generation were mediated by AT1-receptor activation. These data suggest that glomerular podocytes express a high-affinity binding site for Ang II with pharmacologic characteristics of both AT1 and AT2 receptors. This receptor site is up-regulated during podocyte differentiation, and receptor activation induces both IP generation and DNA synthesis by AT1-dependent mechanisms. We speculate that activation of podocyte Ang II receptors contributes to glomerular damage in disease states.

Differential behavior of mesangial cells derived from 12/15-lipoxygenase knockout mice relative to control mice

The 12/15-lipoxygenase (12/15-LO) enzyme has been implicated in the pathogenesis of diabetic nephropathy since lipoxygenase products induce cellular hypertrophy and extracellular matrix deposition in mesangial cells. In this study, in order to determine the potential in vivo functional role of 12/15-LO in kidney disease, we compared mouse mesangial cells (MMCs) derived from 12/15-LO knockout mice with those from genetic control wild-type mice. MMCs were isolated from wild-type and 12/15-LO knockout mice. Cellular growth, activation of mitogen-activated protein kinases (MAPKs), transcription factors, superoxide levels, and fibronectin expression were compared in the two cell types. Levels of the 12/15-LO product and protein were lower in MMC from 12/15-LO knockout relative to wild-type. MMCs from 12/15-LO knockout mice grew slower than wild-type cells, and also showed lower rates of tritiated thymidine and leucine incorporation (21% and 15% of wild-type, respectively, P < 0.001). Levels of superoxide and the matrix protein fibronectin were also lower in 12/15-LO knockout mice cells. Serum and angiotensin II (Ang II)-stimulated activities of p38 or ERK1/2 MAPKs, and cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB) transcription factor were lower in 12/15-LO knockout relative to wild-type cells. In addition, DNA binding and transcriptional activities of activated protein-1 (AP-1) and CREB were lower in 12/15-LO knockout cells. Furthermore, stable 12/15-LO overexpression in MMC led to reciprocal increase in p38 MAPK activation and fibronectin expression. The differential activation of oxidant stress, specific signaling pathways, transcription factors, and growth and matrix genes may lead to reduced growth and growth factor responses in 12/15-LO knockout versus wild-type MMCs. These results provide ex vivo functional evidence for the first time that 12/15-LO activation plays a key role in mesangial cell responses associated with renal diseases such as diabetic nephropathy.

The functional CD40 antigen of fibroblasts may contribute to the proliferation of rheumatoid synovium.

This paper demonstrates that CD40 is expressed on rheumatoid synovial pannus and primary fibroblast cell lines established from rheumatoid and osteoarthritic synovium as well as normal skin. Among various tested cytokines, interferon-gamma (IFN-gamma) and to a lower extent, tumour necrosis factor-alpha (TNF-alpha) were found to upregulate CD40 expression on fibroblasts. Synovial and skin fibroblasts cultured over CD40 Ligand transfected L cells (L-CD40 L) demonstrate a CD40 specific increase of DNA synthesis as measured by tritiated thymidine incorporation. Cell-cycle analysis and enumeration of viable cells further show that CD40 induced fibroblast proliferation. Costimulation with L-CD40 L and IFN-gamma resulted in maximal proliferation. Engagement of fibroblasts CD40 increased the IL-1-induced production of granulocyte macrophage-colony stimulating factor and macrophage inflammatory protein-1 alpha MIP-1 alpha. CD40 L activated fibroblasts showed decreased levels of CD40, but only marginal alterations of other cell-surface antigens. Taken together, the present results indicate that fibroblasts express functional CD40 and suggest a possible role of CD40 L expressing cells, such as activated T cells and mast cells, in the development of synovium hyperplasia.

Effect of bladder outlet obstruction on detrusor smooth muscle cell: an in vitro study1, 2

BackgroundAlthough relieving obstruction is generally curative on bladder outlet obstruction (BOO), bladder dysfunction persists in some patients. Repetitive stretch and relaxation applied to cultured bladder smooth muscle (SM) cells in vitro have been used to mimic increases in urodynamic load experienced by the detrusor muscle under conditions of BOO. We first clarified the relationship between phenotype transformation and biomechanical properties of detrusor smooth muscle cell (DSMC) subjected to the cyclic mechanical stretch. Materials and methodsCultured rat DSMC were grown on collagen-coated silicone membranes and subjected to continuous cycles of stretch-relaxation. All experiments were performed on cells between passages 2 and 4. Each cycle consists of 5 seconds of stretch and 5 seconds of relaxation. The computer controlled vacuum induced 10% (1), 20% (2), and 30% (3) maximum elongation of the plate membrane at different designed pressures. The deoxyribonucleic acid synthesis rate was assessed by performing tritiated thymidine incorporation assay. The expression of SM-α-actin and proliferation of DSMC were analyzed by immunofluorescent assay and flow cytometry. The image analysis and micropipet aspiration systems were used to investigate the single cell contraction and viscoelasticity. Using the 3-element standard linear solid model, the elastic modulus K1, K2, and viscoelastic coefficient μ were determined, which show the passive deformation ability of detrusor cells. ResultsAs the basic structural changes to mechanical stretch, DSMC undergo phenotypic modulation from their normal contractile phenotype to a “synthetic” phenotype: the DSMC become more proliferative and the actin less organized along the cell’s long axis. The cell proliferation index of control and stretched group (10%, 20%, 30% elongation) are 0.24, 0.43, 0.58, and 0.65, respectively. The actin filaments in unstimulated cells were evident and orientated along the major axis of the cell. After mechanical stretch, the well-spread filaments changed their orientation. The function, such as contraction, and viscoelasticity of a single DSMC subjected to stretch both decreased significantly compared with control. The maximum contractile velocity and maximum cell length shortening rate of group 3 (30% elongation) showed significant decreases compared with unstretched control (P < 0.01). K1 and K2 were decreased with the increase of mechanical overload. However, there was no statistic difference between groups 2 and 3. ConclusionsFunctional abnormalities of BOO have the structural basis: phenotype transformation (i.e., remodeling) of the detrusor cells. Cyclic stretch and relaxation applied to DSMC in vitro can be used to model increases in urodynamic load experienced by the bladder detrusor muscle under conditions of BOO. Phenotype transformation is the structural basis of functional changes of DSMC subjected to periodic overload mechanical stretch.

Influence of pro-inflammatory (IL-1α, IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-4) cytokines on chondrocyte function

Objective: Cytokines produced by inflammatory cells play a pivotal role in synovial inflammation and joint destruction in rheumatoid arthritis. To investigate the influence of pro-inflammatory cytokines (IL-1α, IL-6, TNF-α, IFN-γ) and subsequently the possible beneficial role of an anti-inflammatory cytokine (IL-4) on chondrocyte viability (necrosis/apoptosis), proliferation and nitric oxide (NO) production. Methods: Primary bovine chondrocytes were cultured until monolayers were obtained. Cells were incubated with cytokines (IL-1α, IFN-γ, TNF-α, IL-4, IL-6) at 0.1, 1, 10 and 100 ng/mL. After 48 h, the viability of the chondrocytes was measured flow cytometrically with propidium iodide. Proliferation was determined by the incorporation of tritiated thymidine. The morphology of the chondrocytes, including presence of apoptotic nuclei, was evaluated by a May-Grünwald–Giemsa staining. In addition, the number of apoptotic chondrocytes was detected flow cytometrically with a TUNEL technique and annexin-V/propidium iodide staining. NO production was evaluated using a spectrophotometric assay, based upon the Griess reaction. Results: The viability and proliferation of bovine chondrocytes decreased after incubation with 100 ng/mL IL-1α, TNF-α or IFN-γ. In contrast, incubation of chondrocytes with IL-4 or IL-6 had no influence on the viability or the proliferation of cells. IL-1α was able to enhance NO production in a dose dependent manner. IFN-γ and TNF-α induced NO production only at the highest concentration (100 ng/mL), whereas IL-4 and IL-6 did not. There was a dose dependent increase in apoptosis of bovine chondrocytes cultured in the presence of IL-1α and TNF-α. This effect could not be prevented by preincubation with IL-4. Preincubation with IL-4 diminished IL-1α and TNF-α induced NO production and increased proliferation of chondrocytes. In an additional experiment, incubation of human chondrocytes with anti-Fas did not induce apoptosis as measured by annexin-V/propidium iodide staining. Conclusions: Pro-inflammatory cytokines are able to induce apoptosis, whereas IL-4 as an anti-inflammatory cytokine can inhibit the effect of IL-1α and TNF-α on NO production and proliferation of bovine chondrocytes.

Milk Yield and Mammary Growth Effects Due to Increased Milking Frequency During Early Lactation

Increased milking frequency (IMF) at the beginning of lactation has been shown to increase milk yield not only during IMF but also after its cessation. The objectives of this experiment evaluated the effects of increased milking frequency initiated during early lactation on mammary growth and effects on milk yield (MY). Thirty-one cows were divided into treatment groups: 1) 2X: cows milked twice daily (2X) beginning at parturition (d 1), 2) IMF1: cows milked four times daily (4X) from d 1 to 21 postpartum (PP) and 3) IMF4: cows milked 2X d 1 to 3 and 4X d 4 to 21 PP. The 4X cows were milked immediately before 2X cows and again approximately 3h later, at the end of the normal milking routine. All cows were milked 2X from d 21 to 305 postpartum. Milk yields were 34.5, 37.8 and 37.6 kg/d during wk 1 to 44 for 2X, IMF1 and IMF4, respectively. Mammary biopsies from four cows per treatment were obtained on d 7 and 14 PP to evaluate mammary cell proliferation. Tritiated-thymidine incorporation tended to increase on d 7 in IMF1 cows, and arithmetic means of the percentage of cells expressing Ki-67 proliferation antigen were consistent with a proliferative response to IMF though not significant. Blood was sampled three times per wk during the first 2 wk and then once per wk during wk 3, 4, 5, 6, 8, and 10. Plasma insulin-like growth factor-1 (IGF-1) averaged 20.1ng/ml in IMF cows vs. 24.2 in 2X but was not accompanied by a change in bST. Prolactin was also not affected by treatment. Neither milk yield nor potential effects on mammary cell proliferation were correlated with systemic IGF-1. Implementing an IMF routine increases MY during treatment and elicits a carryover effect on the remainder of lactation. Milk yield responses after an IMF routine may be the result of increased mammary cell proliferation.

Production of genetically engineered biotinylated interleukin-2 and its application in a rapid nonradioactive assay for T-cell activation.

The development of reliable assay systems that can measure lymphocyte activation in vitro has been a major goal of immunodiagnostics. Traditionally, tritiated thymidine incorporation has been used to monitor T-cell activation. Other methods include enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay, and colorimetric assays. We have established a lymphocyte activation assay that utilizes fluorescein isothiocyanate (FITC)-streptavidin bound to recombinant biotinylated human interleukin-2 (IL-2). Utilizing recombinant DNA technology, a unique monobiotinylated human IL-2 has been created and isolated using the Promega PinPoint vector system. ELISA has been used to demonstrate streptavidin binding and recognition by a human IL-2-specific antibody. IL-2 function has been demonstrated using a murine IL-2-dependent T-cell line, CTLL-2, responsive to human IL-2. Recombinant biotinylated human IL-2 conjugated to streptavidin-FITC or streptavidin-horseradish peroxidase has been used to monitor T-cell activation in the presence of antigen as well as mitogen. The sensitivity and convenience of this method make this lymphocyte activation assay an attractive alternative to tritiated thymidine incorporation as a method for monitoring T-cell activation. In addition, the availability of a recombinant biotinylated human IL-2 will permit the production of a uniform product suitable for diagnostic and clinical application.

Initial in vitro interaction of osteoblasts with nano-porous alumina

In the present study we have used a characterised primary human cell culture model to investigate cellular interactions with nano-porous alumina. This material, prepared by anodisation, is being developed as a coating on titanium alloy implants. The structure of the alumina, as determined by X-ray diffraction and transmission electron microscopy, was amorphous. When studying cell/material interactions we used both biochemical and morphological parameters. Cell viability, proliferation and phenotype were assessed by measurement of redox reactions in the cells, cellular DNA, tritiated thymidine ([ 3H]-TdR) incorporation and alkaline phosphatase (ALP) production. Results showed a normal osteoblastic growth pattern with increasing cell numbers during the first 2 weeks. A peak in cell proliferation was seen on day 3, after which cell growth decreased, followed by an increase in ALP production, thus indicating that the osteoblastic phenotype was retained on the alumina. Cell adhesion was observed, the osteoblast-like cells having a flattened morphology with filipodia attached to the pores of the material. SDS-PAGE and western blot measurements showed that the nano-porous alumina was able to adsorb fibronectin. Trace amounts of aluminium ions were measured in the surrounding medium, but no adverse effect on cell activity was observed.

Tritiated thymidine incorporation does not enhance sensitivity of the popliteal lymph node assay

The popliteal lymph node (PLN) assay has been proposed as a tool to predict drugs and chemicals with the potential to induce systemic autoimmune reactions in man. In this assay, weight and cellularity indices typically are the measured endpoints. The present study was conducted to test whether incorporation of tritiated thymidine could improve sensitivity of the PLN assay. Male and female Balb/c mice were injected with 20 μCi of [ 3H]-methyl-thymidine intravenously 7 days after receiving 0.5, 1 or 2 mg of diphenylhydantoin, streptozotocin, sulfamethoxazole, ofloxacin, phenobarbital, or metformin intradermally. Results obtained with incorporation of tritiated thymidine were compared to weight indices. No consistent or marked differences in these endpoints were noted whatever the compound used. This study shows that incorporation of tritiated thymidine does not improve sensitivity of the PLN assay.

In vitro cellular response to titanium electrochemically coated with hydroxyapatite compared to titanium with three different levels of surface roughness.

The in vitro response of primary human osteoblast-like (HOB) cells to a novel hydroxyapatite (HA) coated titanium substrate, produced by a low temperature electrochemical method, was compared to three different titanium surfaces: as-machined, Al(2)O(3)-blasted, plasma-sprayed with titanium particles. HOB cells were cultured on different surfaces for 3, 7 and 14 days at 37 degrees C. The cell morphology was assessed using scanning electron microscopy (SEM). Cell growth and proliferation were assessed by the measurement of total cellular DNA and tritiated thymidine incorporation. Measurement of alkaline phosphatase (ALP) production was used as an indicator of the phenotype of the cultured HOB cells. After three days incubation, the electrochemically coated HA surface produced the highest level of cell proliferation, and the Al(2)O(3)-blasted surface the lowest. Interestingly, as the incubation time was increased to 7 days all surfaces produced a large drop in tritiated thymidine incorporation apart from the Al(2)O(3)-blasted surface, which showed a small increase. Cells cultured on all four surfaces showed an increased expression of ALP with increased incubation time, although there was not a statistically significant difference between surfaces at each time point. Typical osteoblast morphology was observed for cells cultured on all samples. The HA coated sample showed evidence of a deposited phase after three days of incubation, which was not observed on any other surface. Cells incubated on the HA coated substrate appeared to exhibit the highest number of cell processes attaching to the surface, which was indicative of optimal cell attachment. The crystalline HA coating, produced by a low temperature route, appeared to result in a more bioactive surface on the c.p. Ti substrate than was observed for the other three different Ti surfaces.

Control of fibroblast-like synoviocyte proliferation by macrophage migration inhibitory factor.

The hyperplasia of fibroblast-like synoviocytes (FLS) is considered essential to the evolution of joint destruction in rheumatoid arthritis (RA), but the mechanisms underlying FLS proliferation remain poorly understood. Macrophage migration inhibitory factor (MIF) is a cytokine that has recently been shown to exert proinflammatory effects on RA FLS. This study sought to identify the mechanisms of activation of FLS by MIF, and to assess the effects of MIF on synovial cell proliferation. Human RA FLS were treated with recombinant MIF, interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and/or anti-MIF monoclonal antibodies (mAb). Proliferation was measured with tritiated thymidine incorporation. Nuclear factor kappa B (NF-kappa B) and mitogen-activated protein (MAP) kinase activation were measured with immunohistochemistry and Western blotting, respectively. FLS proliferation was significantly increased by MIF. IL-1 beta and TNFalpha also induced proliferation, but these effects were prevented by neutralization with anti-MIF mAb. Activation of NF-kappa B was induced by IL-1 beta, but not by MIF. Anti-MIF mAb had no effect on IL-1 beta-induced NF-kappa B nuclear translocation. By contrast, MIF induced phosphorylation of extracellular signal-regulated kinase (ERK) MAP kinase. ERK antagonism, but not NF-kappa B antagonism, prevented the effect of MIF on FLS proliferation. These data suggest that MIF may regulate RA synovial hyperplasia by acting directly and via involvement in the effects of IL-1 beta and TNFalpha. In addition, the effects of MIF on FLS activation are independent of NF-kappa B, and dependent on ERK MAP kinase. These data suggest an important therapeutic potential for MIF antagonism in RA.

Expression of 11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue is not increased in human obesity.

Central obesity is associated with increased morbidity and mortality. Preadipocyte proliferation and differentiation contribute to increases in adipose tissue mass, yet the mechanisms that underlie these processes remain unclear. Patients with glucocorticoid excess develop a reversible form of central obesity, but circulating cortisol levels in idiopathic obesity are invariably normal. We have hypothesized that the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), by converting inactive cortisone to active cortisol in adipose tissue, might be an important autocrine regulator of fat mass. Paired omental and sc fat biopsies were obtained from 32 women (median age, 43 yr; range, 28-65; median body mass index, 27.5 kg/m(2); range, 19.7-39.2) undergoing elective abdominal surgery. 11beta-HSD1 activity and mRNA levels were assessed in whole tissue and in isolated preadipocytes and adipocytes using specific enzyme assays and real-time PCR. Preadipocyte proliferation was measured using tritiated thymidine incorporation. Whole adipose tissue 11beta-HSD1 mRNA levels did not differ between omental and sc samples (P = 0.73). In addition, mRNA levels did not correlate with body mass index (omental: r = 0.1; P = 0.6; sc: r = 0.15; P = 0.4). In keeping with earlier studies, 11beta-HSD1 mRNA levels were higher in omental compared with sc preadipocytes. However, in cultured omental preadipocytes, 11beta-HSD1 activity inversely correlated with body mass index (r = -0.47; P = 0.03). In omental preadipocytes, both cortisol and cortisone decreased proliferation (P < 0.05). Inhibition of 11beta-HSD1 with glycyrrhetinic acid partially reversed the cortisone-induced decrease in preadipocyte proliferation (P < 0.05). Enhanced preadipocyte proliferation within omental adipose tissue as a consequence of decreased 11beta-HSD1 mRNA levels and activity may contribute to increases in visceral adipose tissue mass in obese patients.

Selective estrogen receptor modulators inhibit the effects of insulin-like growth factors in hyperparathyroidism

Background. Primary hyperparathyroidism (HPT) predominately affects perimenopausal women, leading to speculations that an estrogen imbalance may be liable. We have previously demonstrated the importance of the insulin-like growth factor (IGF) axis in HPT. Because the antiestrogen tamoxifen has been shown to modulate the IGF axis, we examined the interactions of selective estrogen receptor modulators (SERMs) and IGF in HPT. Methods. Estrogen receptors were evaluated by Western immunoligand blotting. Sixteen parathyroid glands from 19 patients were included. After adhesion, the cells were treated with IGF (I or II) ± estrogen ± SERMs (tamoxifen, ICI 182,780) for 96 hours in serum-free media. Proliferation was assessed by measuring tritiated thymidine incorporation. Results. Both primary and secondary HPT express estrogen receptors α and β. Primary and secondary HPT had comparable responses to SERMs, they were analyzed together. Compared with control (100%), IGFs (I and II) induced a significant increase in DNA synthesis. Estradiol at 10−8 and 10−7 mol/L (physiologic range) had no significant effects on IGF (I and II, P >.05). Both tamoxifen and ICI 182,780 inhibited basal DNA synthesis (P <.05) and abolished the effects of both IGF I and II (P <.05). Conclusions. SERMs are capable of reducing basal and IGF-stimulated DNA synthesis. This reduction in proliferation has implications for cancer biology and therapeutic potential for SERMs in HPT. (Surgery 2002;132:998-1007.)

Identification of an abnormal beryllium lymphocyte proliferation test

The potential hazards from exposure to beryllium or beryllium compounds in the workplace were first reported in the 1930s. The tritiated thymidine beryllium lymphocyte proliferation test (BeLPT) is an in vitro blood test that is widely used to screen beryllium exposed workers in the nuclear industry for sensitivity to beryllium. The clinical significance of the BeLPT was described and a standard protocol was developed in the late 1980s. Cell proliferation is measured by the incorporation of tritiated thymidine into dividing cells on two culture dates and using three concentrations of beryllium sulfate. Results are expressed as a ‘stimulation index’ (SI) which is the ratio of the amount of tritiated thymidine (measured by beta counts) in the simulated cells divided by the counts for the unstimulated cells on the same culture day. Several statistical methods for use in the routine analysis of the BeLPT were proposed in the early 1990s. The least absolute values (LAV) method was recommended for routine analysis of the BeLPT. This report further evaluates the LAV method using new data, and proposes a new method for identification of an abnormal or borderline test. This new statistical–biological positive (SBP) method reflects the clinical judgment that: (i) at least two SIs show a ‘positive’ response to beryllium; and (ii) that the maximum of the six SIs must exceed a cut-point that is determined from a reference data set of normal individuals whose blood has been tested by the same method in the same serum. The new data is from the Y-12 National Security Complex in Oak Ridge (Y-12) and consists of 1080 workers and 33 non-exposed control BeLPTs (all tested in the same serum). Graphical results are presented to explain the statistical method, and the new SBP method is applied to the Y-12 group. The true positive rate and specificity of the new method were estimated to be 86% and 97%, respectively. An electronic notebook that is accessible via the Internet was used in this work and contains background information and details not included in the paper.

Marked extension of proliferation of rat Sertoli cells in culture using recombinant human FSH.

Previous studies indicate that proliferation of rat Sertoli cells in culture can only be maintained until the equivalent of days 10-12 after birth, irrespective of the age of the donor animal. This report describes methods for the isolation and culture of Sertoli cells from day 6 rat testes, which can proliferate in culture for 20-24 days (that is, until the equivalent of days 26-30 after birth). Cells were isolated by enzymatic digestion of seminiferous cords followed by selective depletion of contaminating peritubular cells by adhesion to a polystyrene surface. The purity of the Sertoli cells was assessed using a combination of markers to be > 99.5%. Proliferation was assayed using tritiated thymidine incorporation and further verified by bromodeoxyuridine histochemistry and flow cytometry. Sertoli cells proliferated at basal levels in Dulbecco's modified Eagle's medium (DMEM)-F12 media alone, and proliferation was stimulated further by addition of recombinant human FSH to the culture media. After 20-24 days in culture, proliferation rapidly ceased, and cells assumed abnormal morphology and detached from the culture vessel; these events are consistent with the cells undergoing classic rodent cell senescence. The method described provides a useful tool for investigating the control of Sertoli cell division. Furthermore, these findings indicate that the timely differentiation of Sertoli cells is not dependent solely on an intrinsic timing mechanism, as has been suggested previously.

Circadian Variation of Topoisomerase II-α in Human Rectal Crypt Epithelium: Implications for Reduction of Toxicity of Chemotherapy

Topoisomerase II-alpha is a target of common chemotherapeutic agents such as doxorubicin and etoposide, which induce DNA damage by altering the activity of this enzyme. We took rectal biopsies at 4-hour intervals over a 24-hour period (seven total) from each of 10 healthy volunteers and examined immunoperoxidase-stained coded anti-topoisomerase II-alpha-stained sections. A significant circadian periodicity was seen in the number of rectal crypt epithelial cell nuclei that were stained (P = .01). Mean peak staining was at 7:23 a.m. ± 45 minutes, and the mean rate of change (difference between peak and trough expression) was 40%. Topoisomerase II-alpha expression in rectal epithelium has a significant circadian variation similar to that of tritiated thymidine incorporation. Although direct confirmation is needed, giving topoisomerase II-targeted chemotherapeutic agents at the proper time of day might reduce their mucositis side effects.

Loss of growth factor sensitivity and development of mitoxantrone resistance in transplanted recurrent SA7HD myeloid leukemia.

Recurrent SA7HD leukemia cells have also "lost" the response to stimulation by L-929-conditioned medium (L-929-CM) and indeed were significantly insensitive to proliferative stimulation by combinations of WEHI-conditioned medium (WEHI-CM) and L-929-CM (P = 0.04) in contrast to untreated SA7HD leukemia, which displayed dose-dependent stimulation by L-929-CM and exhibited synergistic proliferative response to combinations of the two growth factors. This altered growth-factor sensitivity, which was a function of both the route of leukemia inoculation and drug administration, was detected by both the colony and the tritiated thymidine (3HTdR) incorporation assays. In contrast to transplant in normal mice, recurrent leukemia transplanted in mitoxantrone pretreated mice remained growth-factor insensitive. In addition, no growth delay of the recurrent leukemia occurred during this passage in pretreated mice. However, recovery of growth-factor sensitivity still occurred after a single subsequent transplant in normal mice. The magnitude of resistance developed by the recurrent leukemia was not increased by passage in mitoxantrone pretreated mice. Rather, the extent of resistance was a function of the dose of mitoxantrone employed in the treatment of the leukemia before recurrence. These data interpreted along with previously published observations suggest that the development of altered growth-factor sensitivity by SA7HD recurrent leukemia was directly linked to the presence of mitoxantrone. In addition, this phenomenon possibly confers survival advantage to the leukemia cells and this probably accounts for the observation that most of the drug-treated mice ultimately developed recurrent disease despite adequate treatment.