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PubMed2006

Chick pancreatic B islets as an alternative in vitro model for screening insulin secretagogues.

Previously, we reported a simple technique to isolate functional B islets from chick pancreata with retention of their insulin secretory ability in response to glucose challenge. To test the hypothesis that chick B islets are equally good candidates as mammalian islets for screening hypoglycemics and insulin secretagogues, we compared the structural and functional status of chick B islets with those of normal and diabetic mouse islets. Pancreata from chick, normal (nondiabetic) mice, and diabetic mice were collected, fixed, and processed for histological analysis using Gomori stain to distinguish A and B cells from islets. Similarly isolated islets from these animals were treated with different concentrations of tolbutamide, a known insulin secretagogue, and glucose to study insulin release. Histological analysis of pancreata from chicks and normal mice revealed intact B cells, whereas those from diabetic mice were destroyed. The insulin secretory response of chick B islets against the tolbutamide and glucose challenge was comparable to that of normal mouse islets. However, diabetic mouse islets did not respond to glucose challenge, indicating impaired functionality. We have identified a critical window that lies within 5 to 6 d posthatching for isolating chick B islets showing maximum glucose responsiveness and insulin secretion. The previous reports on chicken pancreatic islets involve the use of 4- to 6-wk-old chicks in which islets were found to be nonresponsive to glucose and, hence, could not be used for testing insulin secretory activity. However, our data on B islets from 5- to 6-d-old chick pancreata is highly promising, as islets are responsive to insulin secretagogues. The present data thus indicates that chick B islets can be used as an alternative in vitro model for screening insulin secretagogue and hypoglycemics.

Animals↗

PubMed1990

Expression of 25-hydroxyvitamin D3-24-hydroxylase activity in Caco-2 cells. An in vitro model of intestinal vitamin D catabolism.

The C-24 oxidation pathway plays a major role in the degradation of vitamin D metabolites in kidney and other target tissues. The aim of the present study was to establish an intestinal cell culture system to study the mechanisms regulating the vitamin D catabolic pathway. 25-Hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the first enzyme in the catabolic sequence, was examined in Caco-2 cells, a human colon adenocarcinoma cell line which exhibits differentiated functions of absorbing intestinal epithelial cells. While untreated Caco-2 cells did not exhibit 24-hydroxylase activity, significant catabolic activity was induced by prior treatment of cell monolayers with 1,25-dihydroxyvitamin D3(1,25-(OH)2D3). Induced 24-hydroxylase D3 (25OHD3) and 1,25-(OH)2D3 was detected 6 h after treatment of cells with 10(-8)M 1,25-(OH)2D3, peaked at 16 h, and decreased thereafter. Treatment of cells with 10(-7) M 1,25-(OH)2D3 elicited a maximal 24-hydroxylase response. Comparable time courses of induction by 1,25-(OH)2D3 and 1,25-(OH)2D3-dose response curves were observed in cultured human skin fibroblasts and Caco-2 cells. 25OHD3 was not as good an inducer of the vitamin D catabolic pathway in Caco-2 cells as 1,25-(OH)2D3. Induction of 24-hydroxylase activity by 1,25-(OH)2D3 was inhibited by pretreatment of Caco-2 cells with either actinomycin D, alpha-amanitin, or cycloheximide suggesting that mRNA and protein synthesis are required for induction. The present study demonstrates that 1,25-(OH)2D3-treated Caco-2 cells express the vitamin D catabolic pathway and, therefore, constitute a useful in vitro model to study the mechanism of induction by 1,25-(OH)2D3.

Adenocarcinoma↗

PubMed1986

An in vitro model for clonal anergy in continuously growing antigen-specific B-cell lines.

Two continuously growing nonmalignant B-cell lines specific for the hapten DNP have been used to study tolerance in developing B cells. These cell lines have previously been shown to consist of small cells without sIgM but with cytoplasmic mu chains, and mature sIgM- and sIgD-bearing cells. When the sIgM-negative cells are placed in culture, mature DNP-specific B cells begin to appear. The studies reported here have shown that when these cell lines were propagated in the presence of either 200 micrograms/ml or 1 mg/ml of the tolerogen DNP-MGG there was no inhibition of cell line growth as measured by thymidine incorporation, and no inhibition of receptor expression by maturing B cells. The cell line lymphocytes propagated in the presence of 200 micrograms/ml DNP-MGG for 7, 30, 45, or 60 days became tolerant and the tolerance persisted for at least 6 days after removal of DNP-MGG. However, tolerance was lost between 6 and 10 days after removal of DNP-MGG. Propagation of the cell lines for 30 days in either DNP-KLH or DNP-Ficoll produced the same results. Limiting dilution cultures of cell line lymphocytes made tolerant by growing them for 30 days in the presence of DNP-MGG demonstrated that there was a marked decrease in precursor frequency compared to controls. However, cell line lymphocytes made tolerant by a 48-hr incubation with DNP-MGG did not have a significant decrease in precursor frequency. These data suggest that tolerance induced by growing these cell lines in the presence of DNP-MGG is a valid in vitro model of tolerance in developing antigen-specific B cells. Tolerance induced in this model is consistent with the clonal anergy hypothesis, but requires the continued presence of DNP-MGG to maintain unresponsiveness. This suggests that clonal anergy can occur in B cells but may not be the sole mechanism of self tolerance for those antigens which are sequestered from the immune system.

Animals↗

PubMed2005

The novel use of the human nasal epithelial cell line RPMI 2650 as an in vitro model to study the influence of allergens and cytokines on transforming growth factor-beta gene expression and protein release.

BACKGROUND: The epithelial accumulation of mast cells is a feature of allergic rhinitis and this has been linked to the expression of the known mast cell chemoattractant transforming growth factor-beta (TGF-beta) at this site. Little is known concerning the regulation of TGF-beta gene expression or protein release by nasal epithelial cells. To address this we have utilized the RPMI 2650 human nasal epithelial cell line, which has some features that closely resemble normal nasal epithelium and has been reported to secrete a TGF-beta-like molecule. OBJECTIVES: To investigate the regulation of TGF-beta gene expression and protein secretion in RPMI 2650 nasal epithelial cells following exposure to allergens (house dust mite (HDM) and grass pollen) and mast cell associated T-helper type 2 (Th2) cytokines (IL-4, IL-13, and TNF-alpha). Methods Light and scanning electron microscopy was used to evaluate the morphology of RPMI 2650 cells in culture, enzyme-linked immunosorbent assay was used to investigate their TGF-beta secretory capacity and the identification of the TGF-beta isotype(s) involved, flow cytometry was used to demonstrate the presence of TGF-beta receptors on the RPMI 2650 cells, and the quantitative real-time TaqMan PCR was used to measure TGF-beta gene expression. RESULTS: TGF-beta(2) was identified as the main isotype secreted by the RPMI 2650 cells. HDM allergens and TNF-alpha increased both TGF-beta gene expression and protein release from these cells, whereas grass pollen, IL-4, and IL-13 were without effect. CONCLUSIONS: The RPMI 2650 nasal epithelial cell line represents a valid in vitro model to evaluate the regulation of TGF-beta biology. In this system HDM allergens have stimulatory activity that is fundamentally different from that of grass pollen allergens, and the Th2 cytokines IL-4 and IL-13 are without effect. The ability of TNF-alpha to up-regulate both TGF-beta gene expression and protein release indicates that mast cell-epithelial interactions concerning TGF-beta are bi-directional and this may be fundamental to epithelial immunoregulation. The availability of a model system, such as the RPMI 2650 cells, will enable the early evaluation of future novel and targeted interventions directed toward the aberrant responses of upper airway structural cells.

Allergens↗

PubMed1990

Autocrine regulation of human tumor cell proliferation by insulin-like growth factor II: an in-vitro model.

We have shown that a pleomorphic cell line of abnormal human karyotype derived from a stomach carcinoma (LIM-1839) proliferates in serum-free medium, expresses insulin-like growth factor II (IGF-II) mRNA, and secretes IGF-II (up to 56 ng/ml in serum-free conditioned medium, as measured in a rat liver RRA. No detectable levels of IGF-I can be measured in serum-free conditioned medium by RIA. These cells also secrete IGF-binding proteins, detected by a charcoal adsorption assay. The release of IGF-II and IGF binding proteins into serum-free conditioned medium (1.7 pmol/10(6) cells.24 h and 0.8 pmol binding sites/10(6) cells.24 h for 3 days, respectively) is inhibited 80% by cycloheximide (10 micrograms/ml). The LIM-1839 cells have type I and type II IGF receptors, determined by affinity cross-linking and competition binding studies. These cells proliferated 1.6-fold over 4 days in serum-free medium, with fresh medium changes on days 0 and 2: their growth was inhibited 56% by 40 micrograms/ml Sm 1.2, a monoclonal antibody which recognizes IGF-I and IGF-II. The addition of 20 and 50 ng/ml multiplication stimulating activity (rat IGF-II) caused 1.8- and 1.7-fold increases in cell growth between days 0 and 4 compared to controls, while [Thr59]IGF-I, at 20 and 50 ng/ml, caused 1.6- and 2.0-fold increases. Insulin, at 2 and 10 micrograms/ml, had no significant effect. The stimulatory effects of endogenous and exogenous IGFs on LIM-1839 cell proliferation were inhibited by a monoclonal antibody to the type I IGF receptor, alpha IR-3. These results suggest that the LIM-1839 cells are biologically responsive to endogenously produced IGF-II, and may thereby provide an in vitro model for autocrine regulation of human tumor growth by IGF-II.

Antibodies, Monoclonal↗

PubMed1993

Sensitivity of Sertoli and Leydig cells to xenobiotics in in vitro models.

Different chemicals are known to cause testicular damage in the human male and experimental animals. However, the ability to assess the potential and mechanism of action leading to chemically-induced damage in men has been hampered by a lack of good predictive models. Although many of these chemicals were found to impair reproductive capacity in various laboratory animals, only some have caused reproductive damage in men. Mammalian spermatogenesis takes places within the avascular seminiferous tubules of the testis. Specialized tight junctions, which form between adjacent Sertoli cells at the time of puberty, divide the tubular space into the basal and adluminal compartments, and create a "blood-testis" barrier that restricts passage of substances and ions from the circulation. Thus, the completion of meiosis and post-meiotic germ cell differentiation, which take place in the adluminal compartment, are isolated from circulating substances unable to cross the blood-testis barrier. It seems feasible, therefore, that damage to the germ cells induced by testicular toxicants may be mediated through other cells in the testis such as the Sertoli, peritubular, or Leydig cells. A recently developed two-compartment system for culture of testicular cells can simulate, to some degree, the normal physiologic conditions. In principle, Sertoli cells isolated from mammalian testes are cultured on a permeable support (that is millipore filter) between two fluid compartments. They form a highly polarized epithelial layer with characteristic tight junctions that restrict the passage of substances between the two compartments, in analogy to the blood-testis barrier. We believe this system provides an excellent in vitro model for determining the ability of chemicals to: a) alter the permeability of the blood-testis barrier, b) impair the secretory function of Sertoli cells, or c) affect their viability, all of which could indirectly affect the germ cells. We have utilized this system for examining the effects of cadmium chloride (CdCl2) and other toxic substances known to affect the testis. The Leydig cell toxicity was investigated in testicular perfusion system or cultures of isolated Leydig cells.

Animals↗

PubMed1997

Calcium-dependent DNA damage and adenosine 3',5'-cyclic monophosphate-independent glycogen phosphorylase activation in an in vitro model of acetaminophen-induced liver injury.

Acetaminophen (N-acetyl-p-aminophenol [APAP]) hepatotoxicity is a process characterized by Ca2+ deregulation. Cellular functions utilizing Ca2+ as a second messenger molecule affect both cytosolic and nuclear signal transduction. Many studies have independently shown Ca2+-related effects on target molecules in response to toxic doses of APAP; however, the primary Ca2+ target resulting in liver necrosis has not been determined. We hypothesize that Ca2+-dependent DNA damage is a critical event in liver necrosis caused by alkylating hepatotoxins. In this study, Ca2+-dependent endonuclease activity was determined from DNA single-strand lesions measured by fluorometric analysis of DNA unwinding. The status of cytosolic Ca2+ was determined by measuring Ca2+-dependent activation of glycogen phosphorylase a. Primary cultures of mouse hepatocytes exposed to a toxic concentration of APAP showed twofold and greater increases in glycogen phosphorylase a stimulation at 6 hours, which was reversible with Ca2+-chelating agents. Cell death was preceded by a large decline in intact, double-stranded DNA. Following toxic administration of APAP, the percentage of total double-stranded DNA was significantly reduced by 2 hours. At 6 and 24 hours, genomic integrity was compromised by 26% and 37%, respectively, compared with untreated controls. Hepatotoxic effects of APAP-mediated Ca2+ deregulation were confirmed in both primary mouse hepatocytes and the human hepatoblastoma HepG2 cell line by lactate dehydrogenase (LDH) release and tetrazolium reduction using the 3-4,5-dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide thiazol blue(MTT) assay. The Ca2+ chelator, ethylene glycol-bis (beta-aminoethyl ether) N',N',N', N'-tetraacetic acid (EGTA), blocked APAP-induced phosphorylase a activation and necrotic cell death, but failed to inhibit phosphorylase a activation by the adenosine 3',5'-cyclic monophosphate (cAMP) analogue, dibutyryl cAMP, indicating little or no contribution of the cAMP pathway to phosphorylase a stimulation during APAP-induced necrotic death. Results with these in vitro models of liver injury are interpreted as supporting the hypothesis that increased Ca2+ availability plays a major role in the progression of APAP-dependent cellular necrosis, and that the nucleus is a critical target for APAP hepatotoxicity.

Acetaminophen↗

PubMed1999

Predictivity of an in vitro model for acute and chronic skin irritation (SkinEthic) applied to the testing of topical vehicles.

An in vitro human reconstructed epidermis model (SkinEthic) used for screening acute and chronic skin irritation potential was validated against in vivo data from skin tolerability studies. The irritation potential of sodium lauryl sulfate (SLS), calcipotriol and trans-retinoic acid was investigated. The in vitro epidermis-like model consists of cultures of keratinocytes from human foreskin on a polycarbonate filter. The modulation of cell viability, the release and gene expression of proinflammatory cytokines, interleukins 1alpha and 8, and morphological changes were evaluated during 3 days as endpoints representative for an inflammatory reaction. The cumulative irritation potential of the topical products was evaluated in a human clinical study by visual scoring and biophysical measurement of inflammatory skin reaction after repeated 24 h applications over 3 weeks under Finn chamber patches. All topical products that were nonirritating in the human study were noncytotoxic and did not induce cytokine expression in the in vitro acute model (day 1 exposure). All irritating controls exhibited specific cell viability and cytokine patterns, which were predictive of the in vivo human data. The ranking of mild to moderate skin irritation potential was based on the lack of cytotoxicity and the presence of cytokine patterns including gene expression specific for each irritant, using the chronic in vitro model (up to 3 days exposure). The human reconstructed epidermis model SkinEthic was shown to be a reliable preclinical tool predicting the irritation potential of topical products. Moreover, it is a useful model in a two-step tiered strategy for screening acute and chronic irritation potential for the selection of vehicles for new topical drugs.

Adult↗

PubMed1995

Leukemia cell lines: in vitro models for the study of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) serves as a paradigm in clinical and biological leukemia research. Firstly, APL represents a model for the new therapeutic approach of differentiation therapy, taking advantage of the ability of APL cells to respond to retinoic acid treatment by terminal differentiation. Secondly, the 15;17 chromosomal translocation specific for APL leads at the molecular genetic level to a chimeric gene fusing the PML and RAR alpha genes and appears to be an instrumental, if not actually the causative event, in the neoplastic process. These unique characteristics of an otherwise rather rare disease have recently attracted intense research interest. As in other types of leukemia where continuous cell lines are powerful research tools, studies using APL-derived cell lines have contributed a large body of relevant data in efforts to unravel the pathobiology and leukemogenesis of APL. Three cell lines have been reported to be derived from patients with APL: HL-60, NB-4 and PL-21. Both HL-60 and PL-21 lack t(15;17) while NB-4 carries this cytogenetic hallmark pathognomonic for APL. Morphological and immunophenotypical examinations of the cell lines do not permit a clear assignment to any stage of myelomonocytic differentiation. Some additional data, such as expression of myeloperoxidase, monocyte-specific esterase and annexin VIII, together with the cytogenetic and molecular biological information, suggest that NB-4 is the only genuine promyelocytic leukemia cell line, whereas HL-60 may represent a discrete stage of differentiation between the late myeloblasts and the promyelocyte; PL-21 has distinct features associated with monocytic cells. These cell lines provide unique in vitro model systems for studying the cellular and molecular events involved in the proliferation and differentiation of normal and leukemic myelomonocytic cells.

Cell Differentiation↗

PubMed1990

Percutaneous absorption of benzyl acetate through rat skin in vitro. 1. Validation of an in vitro model against in vivo data.

The percutaneous absorption in vitro of the fragrance agent benzyl acetate has been evaluated in flow-through diffusion cells using shaved full-thickness skin from male Fischer 344 rats. After the application of neat [methylene-14C]benzyl acetate to the epidermal surface of the skin and occlusion with parafilm 1.3 cm above the skin surface, the absorption of the chemical across the skin and into the receptor fluid was rapid and extensive, commencing within 1 hr of application, and reaching 49.8 +/- 3.2% (mean +/- SD, n = 4) of the applied dose after 48 hr. The coefficient of variation for absorption at 48 hr between four identical experiments was 6.4%. The extent of absorption at 48 hr of benzyl acetate applied in 50% (v/v) ethanol was not significantly different from that after application neat, although absorption at earlier times was enhanced, with a maximum increase of 8.5% of the applied dose at 12 hr. Over the dose range studied there was a linear relationship (r = 0.996) between the amount of benzyl acetate applied to the skin (1.66-33.13 mg benzyl acetate/cm2) and the amount absorbed into the receptor fluid at 24 hr (0.66 +/- 0.04-10.27 +/- 0.51 mg/cm2). The extent of absorption of benzyl acetate through rat skin in vitro was compared with the extent of absorption in vivo at 24 hr and a correlation coefficient of 0.993 was obtained. These data support the use of this in vitro system as a model to predict in vivo absorption and indicate the suitability of the system to study factors influencing the disposition of topically applied benzyl acetate.

Animals↗

PubMed2004

Primary culture of rat growth plate chondrocytes: an in vitro model of growth plate histotype, matrix vesicle biogenesis and mineralization.

During endochondral ossification (EO), cartilage is replaced by bone. Chondrocytes of growth plate undergo proliferation, maturation, hypertrophy, matrix vesicle (MV) biogenesis and programmed cell death (PCD, apoptosis). The in vitro system presented here provides a potential experimental model for studying in vitro differentiation and MV biogenesis in chondrocyte cultures. Chondrocytes were obtained from collagenase-digested tibial and femoral growth plate cartilage of 7-week-old rachitic rats. The isolated chondrocytes were plated as monolayers at a density of 0.5 x 10(6) cells per 35-mm plate and grown for 17 days in BGJ(b) medium supplemented with 10% fetal bovine serum, 50 microg/ml ascorbic acid. Light microscopy revealed Sirius red-positive, apparent bone matrix in layers at the surfaces of cartilaginous nodules that developed in the cultures. The central matrix was largely alcian blue staining thus resembling cartilage matrix. Electron microscopy revealed superficial areas of bone like matrix with large banded collagen fibrils, consistent with type I collagen. Most of the central matrix was cartilaginous, with small fibrils, randomly arranged consistent with type II collagen. The presence of peripheral type I and central type II and type X collagen was confirmed by immunohistochemical staining. Immunohistochemistry with anti-Bone morphogenetic proteins 2, 4 and 6 showed that BMP expression is associated with maturing hypertrophic central chondrocytes, many of which were TUNEL positive and undergoing cell death with plasma membrane breaks, hydropic swelling and cell fragmentation. During early mineralization, small radial clusters of hydroxyapatite-like mineral were associated with matrix vesicles. Collagenase digestion-released MVs from the cultures showed a high specific activity for alkaline phosphatase and demonstrated a pattern of AMP-stimulated nonradioactive (40)Calcium deposition comparable to that observed with native MVs. These studies confirm that primary cultures of rat growth plate chondrocytes are a reasonable in vitro model of growth plate histotype, MV biogenesis and programmed cell death.

Alkaline Phosphatase↗

PubMed1996

Antigen stimulation of human pulmonary smooth muscle: an in vitro model of inflammation.

Human airways in vitro contract when stimulated by anti-IgE, whereas human pulmonary vessels relax. Leukotriene D4 (LTD4) induced a contractile response in the airways, while in pulmonary vessels both contractions and relaxations were observed. The LTD4 contractions in airways were blocked by cysLT1 receptor antagonists (MK 571, ICI 198615, and BAY x7195). In contrast none of the compounds affected the LTD4 contractions of pulmonary veins. These results suggest that the leukotrienes which are released during antigen challenge of airways and pulmonary vessels may be acting at distinct receptors in the human lung.

Antigens↗

PubMed1998

The human gallbladder increases cholesterol solubility in bile by differential lipid absorption: a study using a new in vitro model of isolated intra-arterially perfused gallbladder.

In this study, we first developed and validated a new in vitro isolated, intra-arterially perfused, gallbladder model and then applied the method to investigate the absorption of biliary lipids by the gallbladder wall and the effect of this process on the composition of human bile. Oxygenated and glucose-added buffer was perfused through the cystic artery to maintain organ viability. A standard pooled natural bile, radiolabeled with H3-cholesterol and C14-palmitoyl-linoleoyl-phosphatidylcholine, was instilled in the lumen via a cystic duct catheter. Changes in bile volume and lipid concentrations were monitored at time intervals to evaluate the disappearance of lipids from bile caused by gallbladder absorptive function. Organ viability was demonstrated by stable lactate dehydrogenase (LDH) organ release and oxygen consumption throughout the experiments. In the pig, disappearance rates of lipids from bile were similar in vitro and in vivo, demonstrating the validity of the isolated in vitro model for functional studies. By applying our in vitro isolated preparation to the human gallbladder, we found that 23% of cholesterol and 32% of phosphatidylcholine, but only 9% of bile salts, disappeared from bile in 5 hours. As a consequence, at the end of the experiments, cholesterol (P < .05) and phospholipid (P < .05) molar percentages were significantly reduced, while the bile salt (P < .05) molar percentage was significantly increased with respect to values at the beginning of the studies. Our findings are of pathophysiological relevance and support the concept that the human gallbladder modifies the relative composition of biliary lipids in such a way as to increase cholesterol solubility in bile.

Absorption↗

PubMed1997

The dissected aorta: part I. Early anatomic changes in an in vitro model.

PURPOSE: To define early anatomic changes in the aorta in an in vitro hydrostatic model of acute aortic dissection. MATERIALS AND METHODS: Aortic dissections were created in explanted aortas. A small portion of the dissection flap was resected to allow free communication between the true and false lumina. The aortas were suspended in a saline bath and distended by means of a saline fluid column. By using intravascular ultrasonography, the arc lengths of the outer walls of the true and false lumina and of the dissection flap were measured at increasing hydrostatic pressures and at increasing fractions of dissected wall circumference. RESULTS: The dissected aorta immediately became ectatic. At a given fraction of wall circumference involved in dissection, total aortic cross-sectional area increased with increased hydrostatic pressure. At a given hydrostatic pressure, this area increased with increased fraction of wall circumference dissected. Most of the increase was due to dilatation of the false lumen. There were minimal changes in true-lumen size. Dissection flap length, after initial contraction at the time of dissection, changed minimally with increasing pressure. CONCLUSION: Anatomic changes in the dissected aorta depend on hydrostatic pressure and on the percentage of aortic wall involved in the dissection.

Acute Disease↗

PubMed2001

An in vivo/in vitro model to assess endocrine disrupting activity of xenoestrogens in uterine leiomyoma.

Xenoestrogens with endocrine disrupting activity have been associated with the dysregulation of reproductive function and promotion of malignancies in experimental animals and human populations. The high incidence of uterine leiomyomas, a benign estrogen-responsive neoplasm of the uterine myometrium, calls into question the potential influence of xenoestrogens in the pathogenesis of these tumors. An in vivo/in vitro animal model, the Eker rat, that can be used to assess the estrogen-like agonist activity of potential endocrine disruptors in the uterine myometrium is discussed. Using this model, several in vitro assays are developed that demonstrate that compounds from three major classes of xenoestrogens can mimic the effect of estrogen on leiomyoma cells and act as estrogen receptor (ER) agonists: phytoestrogens, organochlorine pesticides and pharmacologic agents. These compounds can stimulate transcription via the ER and upregulate the expression of an estrogen-responsive gene in uterine leiomyoma cells. The use of these in vitro assays has also advanced our ability to predict the agonist activity of potential therapeutic agents in the uterine myometrium. Selective estrogen receptor modulators (SERMs), while able to act as agonists in some tissues such as the bone and uterine endometrium, act as antagonists in vivo in the uterine myometrium and in our in vitro assays. This antagonist activity in the uterine myometrium suggests that SERMs may be useful in the treatment of uterine leiomyoma.

Animals↗

PubMed1983

Effect of Bordetella pertussis leukocytosis (lymphocytosis)-promoting factor (LPF) on the physical lymphoepithelial-cell association studied with the use of an in vitro model of mouse thymus.

The effect of highly purified leukocytosis (lymphocytosis)-promoting factor (LPF) of Bordetella pertussis on physical lymphocyte and reticuloepithelial (RE) cell association was studied in an in vitro thymus model. First, a simplified in vitro system to assess the lympho-RE-cell association was developed. A completely confluent layer of thymic RE cells was formed by cultivating trypsinized thymus cell suspensions from 2- to 7-day-old mice. When thymic lymphoid cells were seeded on this cell layer and cultivated overnight, a significant proportion of them were found underneath the RE cell layer. This physical lympho-RE-cell association was quantitated by counting the lymphoid cells underneath the RE cell layers. Second, the effect of LPF on this physical lympho-RE-cell association phenomenon was investigated. Addition of LPF to the culture markedly inhibited the formation of the lympho-RE-cell complex; that is, it inhibited the infiltration of lymphoid cells under the RE cell layer. LPF rendered a nearly maximal level of inhibitory effect at a dose of 0.1 ng/ml. Furthermore, LPF enhanced the liberation of lymphoid cells from preformed lympho-RE-cell complexes. On the other hand, LPF had no direct cytotoxic effect on lymphoid cells at doses below 1 microgram/ml. In order to investigate whether LPF produced the effect by acting on lymphoid cells, RE cells, or both, the following experiments were performed. When lymphoid cells were pretreated with LPF and added to normal RE cell layers, the lympho-RE-cell association was maximally inhibited above the dose of 1 ng/ml. Treatment of these LPF-treated lymphoid cells with anti-LPF antibodies failed to abrogate the effect of LPF. When RE cell layers were similarly pretreated with LPF and were cultivated with normal lymphoid cells, however, much higher doses of LPF, above 100 ng/ml, were required for maximal inhibition. Furthermore, treatment of these LPF-treated RE cells with anti-LPF antibodies abrogated the effect of LPF. Therefore, the apparent effect of LPF on RE cells was considered to be due to the carry-over by RE cells of LPF, which should directly act on lymphoid cells at extremely low doses. On the basis of these results, it was concluded that LPF acted directly on lymphoid cells without mediation of RE cells. These in vitro results appear to parallel the effects of LPF in vivo, where it induces a depletion of cells in the thymus. The model may be useful to study this phenomenon and the concomitant accumulation of blood lymphocytes.

Animals↗

PubMed2006

Effect of trabecular bone loss on cortical strain rate during impact in an in vitro model of avian femur.

BACKGROUND: Osteoporotic hip fractures occur due to loss of cortical and trabecular bone mass and consequent degradation in whole bone strength. The direct cause of most fractures is a fall, and hence, characterizing the mechanical behavior of a whole osteopenic bone under impact is important. However, very little is known about the mechanical interactions between cortical and trabecular bone during impact, and it is specifically unclear to what extent epiphyseal trabecular bone contributes to impact resistance of whole bones. We hypothesized that trabecular bone serves as a structural support to the cortex during impact, and hence, loss of a critical mass of trabecular bone reduces internal constraining of the cortex, and, thereby, decreases the impact tolerance of the whole bone. METHODS: To test this hypothesis, we conducted cortical strain rate measurements in adult chicken's proximal femora subjected to a Charpy impact test, after removing different trabecular bone core masses to simulate different osteopenic severities. RESULTS: We found that removal of core trabecular bone decreased by ~10-fold the cortical strain rate at the side opposite to impact (p < 0.01), i.e. from 359,815 +/- 1799 microm/m per second (mean +/- standard error) for an intact (control) specimen down to 35,997 +/- 180 microm/m per second where 67% of the total trabecular bone mass (approximately 0.7 grams in adult chicken) were removed. After normalizing the strain rate by the initial weight of bone specimens, a sigmoid relation emerged between normalized strain rate and removed mass of trabecular bone, showing very little effect on the cortex strain rate if below 10% of the trabecular mass is removed, but most of the effect was already apparent for less than 30% trabecular bone loss. An analytical model of the experiments supported this behavior. CONCLUSION: We conclude that in our in vitro avian model, loss of over 10% of core trabecular bone substantially altered the deformation response of whole bone to impact, which supports the above hypothesis and indicates that integrity of trabecular bone is critical for resisting impact loads.

Animals↗

PubMed1991

Incorporation of liposomal phytosterols into human cells in culture: a potential in vitro model for investigating pathological effects of phytosterolemia.

A potential in vitro cell culture model was developed for studies concerning the pathological effect of phytosterolemia in which liposomal phytosterols were incorporated into human skin fibroblasts and hepatoblastoma (HepG2) cells. After incubation with phytosterols, fibroblasts and HepG2 cells contained a significant amount (20-27%) of phytosterols (campesterol and beta-sitosterol). Phytosterol accumulation caused a significant reduction in the cholesterol content of cells. Labeled sitosterol and cholesterol showed similar uptake with lower esterification of sitosterol when compared to cholesterol. Labeled sitosterol incorporated into LDL was esterified to a greater extent than sitosterol added as straight liposome. About 23% of the labeled sitosterol was converted into acidic products and 5.6% was present as 5 alpha-stanols in HepG2 cells.

Cells, Cultured↗