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Zearalenone is a mycotoxin that is widespread in cereal food. We questioned whether this mycotoxin, administered during known critical exposure periods such as the fetal period and the first days of life, at doses compatible with mean daily intake in humans, could have an effect on mammary gland development in rodents. Wistar female rats were exposed to zearalenone (0.2 μg/kg to 5mg/kg) during the last 14 days of fetal life and the first 5 post-natal days (PND). The mammary tissue was examined for development and maturation by morphologic analyses and immunochemistry. At PND 30, the mean length of terminal buds was significantly enhanced in all of the zearalenone-exposed females (p<0.05). The mammary tissue, as evaluated by scoring of tissue slides, was significantly more differentiated in the 1mg/kg treated group than in controls (p<0.05). At PND 180, mammary tissue was more differentiated in all of the zearalenone treated groups (p<0.05). At six months, 4 of 18 females exposed to 5mg/kg of zearalenone presented mammary hyperplasia lesions. The induction of phenotypic alterations by zearalenone administered in utero and in the neonatal period at doses as low as 0.2 μg/kg suggests that zearalenone could contribute to the induction of breast endocrine disorders., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Transmission of Caprine Arthritis Encephalitis virus (CAEV) from the mother to offspring is principally mediated by infected cells from colostrum and milk. The infection of the dam is often sub-clinical, and results in increased cellularity of milk, sometimes exacerbated by bacterial co-infections. Although monocytes are the major viral host cells, several other cell types, including epithelial mammary cells, fibroblasts and endothelial cells show low levels of in vivo infection. In vitro, however, all phenotypes of mammary gland cells are individually highly sensitive to CAEV infection. This suggests that local mechanisms act to control viral expression. Our goal is to analyse the mechanisms regulating local virus infection, including the physiological status of the mammary gland and bacterial co-infections. In this work, we present the development of a model for the in vitro reconstitution of mammary gland tissue using 3D cultures in Matrigel. Mononuclear cells from the blood are added to the 3D cultures in vitro. In these experimental conditions, the mammary cells spontaneously organize into mammospheres. Blood leucocytes migrate into the culture gel, and localize particularly at the periphery of the mammospheres. Mammospheres were susceptible to infection in vitro by CAEV, as shown by a cytopathic effect and expression of late CAEV antigen p30. This model will allow the in vitro study of virus expression, transfer of infection to mammary gland cells and interactions between the mammary gland cells, infected monocytes and immunocompetent cells. It will allow the study of mechanisms participating in the control of passage of pathogens into milk, according to the physiological and CAEV-infection status of the animal, microenvironment and the presence of bacterial co-infections.

The caprine arthritis-encephalitis lentivirus (CAEV) causes a lifelong persistent infection in goats, and induces infiltrations of leucocytes and tissue reorganization in target organs, with a cyclical pattern of viral expression. The mammary gland is an important site of infection, associated with mother-to-kid transmission by infected cells in colostrum and milk. The monocyte/macrophage is the principal target cell, but other cell types, including epithelial and endothelial cells and fibroblasts, are susceptible to in vitro infection with varying levels of viral replication. Such cells, perhaps at specific differentiation states, might play a role in the regulation and transfer of in vivo infection in target organs. In this paper we describe the in vitro infection of endothelial cell monolayers by the transmigration of monocytes carrying the CAEV provirus. The infected endothelial cells progress to expression of the viral p30 capsid antigen, suggesting viral proliferation. Such a process occurring in vivo during angiogenesis and leucocyte homing to the mammary gland in the final third of mammogenesis, might contribute to viral spread in this crucial target organ.

A characteristic lesion in goats infected by the lentivirus CAEV is mastitis with lymphoid hyperplasia. In order to investigate the mechanism of lesion formation, cultures highly enriched in microvascular endothelial cells, mature and immature luminal epithelial cells, fibroblasts and myoepithelial cells were established from goat mammary gland biopsies. Their susceptibility to in vitro infection with two distinct types of CAEV was investigated by PCR, antigen expression and cytopathy. The capacity of infected mammary gland cells to bind uninfected caprine leukocytes was determined by flow cytometry. All cell types tested were susceptible to CAEV infection in vitro, with different levels of sensitivity according to cell phenotype. Our results suggest that the limited extent of natural infection of mammary gland cells reflects a protective local immune response, and that the myoepithelial cell could act as a reservoir cell. After infection, the mature luminal cell acquires the capacity to bind leukocytes in vitro, which could indicate a facilitation of cellular interactions. The distinct reactions of the different cell types to CAEV infection may be correlated with events leading to progressive lesion development during the natural infection.

Infection of goats by caprine arthritis-encephalitis virus (CAEV) provides a convenient example of the infiltration of various tissues by leukocytes following a natural lentiviral infection. This event is important in determining organ susceptibility and local immunity. Caprine vascular endothelial cells are susceptible to infection by CAEV in vitro, so we have investigated the consequences of this infection on the transmigration of uninfected leukocytes in an in vitro model. After in vitro infection by CAEV or stimulation by TNFalpha, the endothelial cells allowed the passage of tenfold more leukocytes from uninfected donors than did the uninfected endothelial cells. The transmigrating leukocytes were enriched in CD8+ lymphocytes, and the leukocytes appeared to have been activated during transmigration, as demonstrated by their expression of IL2R, MHC class II antigens and gamma-delta T-lymphocyte markers. CD4+, CD8+ and B-lymphocytes all proliferated in culture after transmigration. These results suggest that any possible infection or specific stimulation of endothelia in an infected animal could profoundly influence the choice of target organs and could activate the cells involved in local mucosal immune responses.

Little is known about the functional interactions between digestive neuroendocrine tumor cells and their stromal microenvironment. The focus of our study is whether mesenchymal cells modulate peptide expression, cell proliferation, and invasiveness in digestive neuroendocrine tumor cells. We designed an experimental in vivo and in vitro study using the mouse enteroendocrine cell line STC-1. In vivo, STC-1 cells were injected subcutaneously in 18 immunosuppressed newborn rats. At day 21, all animals presented poorly differentiated neuroendocrine tumors with lung metastases. Subcutaneous tumors were usually limited by a capsule containing basement membrane components and myofibroblasts that presented a low mitotic index. Lung tumors were devoid of capsule and poor in myofibroblasts, and their mitotic index was high. The profile of peptide expression in STC-1 tumors was different from that of cultured STC-1 cells. In vitro, STC-1 cells were cultured with fibroblasts of different origins, including dermis, lung, digestive tract, and liver. Based on their origin, myofibroblasts differentially modulated hormone synthesis, proliferation, spreading, and adhesion of STC-1 cells. In conclusion, our results show that site-specific functional interactions between mesenchymal and neuroendocrine cells may contribute to modulating the behavior of digestive neuroendocrine tumors, depending on their growth site.

During the invasive process, tumor cells must move through the extracellular matrix. They have to adhere to the extracellular matrix components, then proteolyse them and migrate on their fragments. This implicates integrins and proteinases, namely metalloproteinases. Numerous experiments which had been performed on various models, namely malignant melanomas proved that integrins have a major role in the transduction of signals from the outside to the inside of the cells, such signals enhancing the expression of the metalloproteinases or, in the contrary, inhibiting it. The modifications of this expression are dependent of extracellular matrix components and may be induced by the linking of specific antibodies to integrins. In some instances, the integrins localized on the tumor cell surface may act as receptors for extracellular matrix proteins and metalloproteinases at once, that may give to tumor cells an higher efficiency in the invasive process. Such mechanisms may result in interesting clinical perspectives for the control of metalloproteinases regulation in pathological processes.

Clone C5 of the human colon adenocarcinoma LoVo cell line was subcutaneously injected with or without exogenous laminin-1 (EHS laminin) into immunosuppressed newborn rats. Cultures were initiated from lung metastases obtained with or without laminin-1 and gave rise to the C5 sublines LM and M4, respectively. The LM subline was mainly composed of spreading cells whereas most C5 and M4 cells remained round and aggregated. The mesenchymal marker vimentin was expressed by very rare C5 and M4 cells (< 1%), and by many LM cells (about 35%). On the opposite, the epithelial markers villin and dipeptidylpeptidase IV were well expressed by C5 cells but not by LM cells. In in vitro migration and invasion assays, LM cells migrated and invaded basement membrane extract twice as much as the parental C5 clone and the M4 subline, probably in association with vimentin-expressing cells, because invasion of basement membrane extract Matrigel by LM cells gave rise to 100% vimentin-positive cells (sublines LM 22, LM 23 and LM 24). When subcutaneously injected, C5 cells induced tumors limited by an interrupted but well organized basement membrane, whereas LM cells induced tumor masses, occasionally limited by a very irregular basement membrane, as observed when C5 cells were injected with laminin-1. Gelatin zymographic analysis clearly showed an increased expression of matrix metalloproteinase-2 by LM cells. Our results suggest a specific role of laminin-1 on the in vivo proliferation of highly invasive vimentin-expressing colon carcinoma cells. This proliferation may result from the initial interaction of C5 cells with large amounts of laminin-1, leading to a selection of vimentin-expressing cells during the metastatic cascade.

To investigate the nonsaturable, paracellular pathway of intestinal Ca absorption, the luminal contents of 12-cm segments of the intestine of 8-wk-old male Sprague-Dawley rats were analyzed for pH, sojourn time and soluble and insoluble Ca over a 24-h period. The rats had been fed one of two high Ca diets for 2 wk: 1.5% Ca (diet group 3a) and 3.1% (diet group 5a). The pH of the small intestine increased from < 6.6 to > 8.0 from duodenum to ileum; transit time increased from 2.5 min in the duodenum to 58 min in the distal ileum, with the entire ileum accounting on the average for 74% of the transit time of 3 h. The amount of Ca solubilized throughout the intestine was 32 +/- 3.3 mumol in diet group 3a and 53 +/- 5.3 mumol in diet group 5a, i.e., 2.7% and 2.0% of the total luminal Ca. Because absorption by diet group 3a was 1.45 +/- 0.23 mmol/d and that by diet group 5a was 2.50 +/- 0.18 mmol/d, the amounts absorbed were 45.3 and 47.1 times greater than present in the lumen in soluble form at any one time. Thus, over a 24-h period, an average of 3.2% (46.2/1440) of the soluble Ca present in the lumen at any time was absorbed per min. Calculations involving the gradient between luminal and plasma Ca show that the rate of Ca diffusion from lumen to blood is < 2% of what it would be if the paracellular path were unrestricted. Thus, intestinal sojourn time, Ca solubility and mucosal permeability to Ca are factors that determine the rate of passive Ca absorption.

Six-week-old male rats were placed on two high calcium regimens: one with calcium carbonate and monobasic calcium phosphate, with calcium content increased via calcium carbonate; and another with calcium phosphate and calcium gluconate, with calcium gluconate the source of increased calcium. Animals fed the gluconate-containing diets absorbed 29% of the ingested calcium over the entire calcium intake range, whereas those fed the calcium carbonate diets absorbed 25% over an intake range of 225 to 450 mg Ca/d, but at calcium intakes above 450 mg Ca/d their absorption reached a plateau at approximately 109 mg/d. Active calcium transport decreased with increased calcium intake in both the calcium carbonate- and calcium gluconate-fed groups. Nonsaturable transport was unchanged as a result of increasing calcium intake and did not differ among the diet groups. Because the absorptive processes were unaffected by the calcium source, events in the lumen must have been responsible for the observed differences. Because phosphate is nearly 18 times more soluble than carbonate, very little calcium of calcium carbonate origin can have been solubilized in the presence of phosphate and this, we conclude, accounts for the limit on calcium absorption observed in diets high in calcium carbonate. Moreover, when intake is expressed as soluble calcium, absorption approaches 50%, the value expected when intestinal transit time (approximately 3 h) is multiplied by 16%/h, the experimental value of nonsaturable absorption.

An in situ ligated loop procedure was applied to dissect transmural calcium transport in the intestine into two components, a saturable and a nonsaturable process. The existence of two such processes was confirmed in the duodenum, but ileal calcium transport was devoid of the saturable component. There was a small saturable component in the upper jejunum. The level of CaBP, the vitamin D-dependent cytosolic calcium-binding protein (Mr, approximately or equal to 9,000), corresponded to the magnitude of the saturable component. No CaBP was detected in the ileum. Vitamin D dependence of the saturable component was established by inducing it in the duodenum of vitamin D-deficient animals following intraperitoneal injection of 1,25-dihydroxyvitamin D3. In these same animals, conversely, the ileum did not respond to exogenous 1,25-dihydroxyvitamin D3. This confirms the absence in the ileum of the saturable component of transmural calcium movement and the fact that the nonsaturable component is not vitamin D dependent. Everted sac experiments also showed that duodenal sacs from vitamin D-replete or -repleted animals transported calcium against a chemical gradient, whereas ileal sacs did not. Vitamin D regulation of intestinal calcium absorption thus occurs only in the proximal intestine, even though calcium is absorbed down its chemical gradient all along the small intestine.

Vitamin D-replete (+D) and vitamin D-deficient (-D) rats received by intraperitoneal injection varying amounts of 1,25-dihydroxyvitamin D3, and 4 h (+D) or 9 h (-D) later everted duodenal sacs were prepared to evaluate active calcium transport, i.e., the amount of calcium found in the serosal fluid. At the same time, duodenal calcium-binding protein (CaBP) content was measured. Calcium transport was a close positive function of CaBP content. It was not detectable when CaBP content was zero and increased linearly without plateauing as CaBP content increased to 100 nmol calcium bound/g mucosa. Trifluoperazine (TFP) inhibited active calcium transport in a concentration-dependent manner. Experiments using vesicles prepared from brush-border or basolateral membranes indicated that TFP inhibited the calcium-extrusion process, with virtually no effect on calcium entry. It is concluded that vitamin D exerts its major regulation of active calcium transport in the rat duodenum via CaBP on transport steps beyond brush-border entry.

Duodenal calcium transport was resolved into a saturable and a nonsaturable process by means of an in situ ligated loop procedure applied to Wistar rats at 3, 12, 19, 24, 30, 40, 60, 110, and 150 days of age. All postweaning animals were males that had been placed on a 1.5% calcium, 1.5% phosphorus semisynthetic diet. Duodenal calcium-binding protein (CaBP) levels were determined at all ages. The newborn rat had no saturable transport component and no CaBP. Its nonsaturable component was very high. With increasing age the saturable component and CaBP varied biphasically, increasing steeply until the animals were about 35 days old; thereafter, each decreased to low but detectable values. The nonsaturable component, on the other hand, decreased in near-linear fashion in the first 35 days; in animals beyond that age it remained invariant. The difference in age dependence between the saturable and nonsaturable components may be considered to constitute additional evidence for the existence of the two transport processes. CaBP and the saturable transport process were highly correlated, further proof that both are vitamin D dependent. Histological studies have revealed the presence of many vacuoles in the intestinal cells of the very young rats; these vacuoles were absent in rats older than 35 days. It is suggested that these vacuoles may be implicated in a pinocytosislike nonsaturable transport that is superimposed on the nonsaturable, non-vitamin D-dependent calcium transport found in all enterocytes.

Rats were fed a purified diet containing 30% lactose and calcium absorption was measured in duodenal loops in situ following instillation of 1.25 or 10 mM CaCl2 solutions. Lactose feeding caused absorption to be depressed from 88 to 69% (1.25 mM Ca solution) and from 71 to 43% (10 mM Ca solution). The effect of lactose feeding was more pronounced in 5-month old rats than in 2-month old rats. In the lactose-fed rats, calcium-binding protein (CaBP), measured by a competitive binding assay following partial purification, was depressed on the average from 24 to 10 nmoles Ca bound per mg protein. The effect of the lactose ingestion can be likened to the effect expected from continued high calcium intake, i.e., a decrease in the efficiency of calcium absorption and a decrease in CaBP.

The intestinal absorption of calcium involves an active transport against an electrochemical gradient, a saturable and a nonsaturable transfer following the gradient. The active and the saturable components are transcellular, the nonsaturable component is partly paracellular. Calcium transfer through the intestinal cell includes three steps: 1) the "down-hill" crossing of the brush-border implies binding to specific sites, carrier-mediated transport using channels or carrier proteins specific to Ca and dependent upon composition phosphorylations alkaline phosphatases; the phospholipid composition of the brush-border also plays a role; 2) the intracellular transfer is characterized by an uptake by such organelles as mitochondria, lysosomes and Golgi vesicles and by a transfer on a specific calcium-binding protein; 3) the "up-hill" transfer across the baso-lateral membrane requires energy and energy is mediated by an ATP-activated Ca2+ pump and a Na+/Ca2+ antiport. 1.25 dihydroxycholecalciferol, steroid hormone synthetized from vitamin D3 is the major direct regulator of Ca absorption: in the vitamin D-deprived animal, it increases the selective permeability for Ca of the brush-border, induces the synthesis of proteins after genomic transcription, activates the Ca-ATPases, and acts as a trophic hormone. The other hormones principally act by modulation of the renal biosynthesis of 1.25 dihydroxycholecalciferol. The efficiency of Ca absorption depends on site, with duodenum greater than jejunum greater than caecum greater than ileum. Dietary constituents such as carbohydrates and amino acids increase Ca absorption whereas phytic acid and excess of phosphorus decrease it. They act by modifying Ca bioavailability and perhaps brush-border permeability. Adaptation to increased demand occurs during growth, pregnancy and lactation in normal states but disappears during vitamin D deficiency. In man, lowered efficiency with increasing age is often aggravated by a low calcium diet.

Theophylline, when added to the incubation medium of everted duodenal sacs prepared from rats on a low-calcium diet, was found to inhibit transcellular Ca transport in a concentration-dependent manner, with an inhibitor constant (Ki) of 10.8 mM theophylline. Neither the rate of cellular Ca entry, as evaluated with the aid of brush-border membrane vesicles, nor the rate of cellular Ca extrusion, assessed by measuring ATP-dependent Ca uptake of basolateral membrane vesicles, was significantly altered by the addition of theophylline to the uptake media. However, Ca-binding by calcium-binding protein (CaBP; calbindin D9k), Mr approximately 8,800) was depressed by theophylline in a concentration-dependent manner, with Ki = 3.2 mM theophylline. Theophylline had no effect on Ca binding by calmodulin and the theophylline-induced inhibition of transcellular calcium transport was independent of adenosine 3',5'-cyclic monophosphate levels. Theophylline also had no effect on paracellular Ca movement. Since the theophylline-induced inhibition of Ca-binding by CaBP paralleled the inhibition of transcellular Ca transport, it is concluded that CaBP functions in transcellular Ca transport via its ability to bind Ca.

Calcium absorption was studied by an in situ ligated-loop procedure in 9-wk-old male Wistar rats that had been placed from weaning on one of three semisynthetic regimens, 0.17% Ca, 0.44% Ca, or 0.44% Ca plus lactose. Lactose was added because it is known to increase intestinal calcium retention. When the amount of calcium absorbed was expressed as a function of calcium instilled in the loop, it became possible to describe absorption as the sum of a hyperbolic and a linear function, equivalent to a saturable and a nonsaturable process, respectively. The slope of the nonsaturable component was independent of prior calcium intake, while the maximum saturable flux (Jmax) decreased as calcium intake increased. Analysis of the duodenal content of the vitamin D-dependent calcium-binding protein (CaBP, Mr congruent to 10(4)) revealed a positive relation between Jmax and CaBP. Thus, vitamin D appears to be implicated in the saturable, but not in the nonsaturable, component of calcium absorption.