Effects of all-trans retinoic acid and Ca++ on human skin in organ culture.

In this study, we have established an organ culture model of human skin and examined the effects of both all-trans retinoic acid (RA) and extracellular Ca++ on the epidermal and dermal components of the organ-cultured skin. Our data show that while organ cultures maintained in serum-free, growth factor-free culture medium containing 0.15 mM Ca++ degenerated rapidly, those treated with concentrations of RA that have been shown previously to stimulate fibroblast and keratinocyte proliferation in monolayer culture (J Invest Dermatol 1989, 93:449; 1990, 94:717; Am J Pathol 1990, 136:1275) demonstrated a healthy appearance for up to 12 days. Degeneration of the control cultures was characterized by separation of the epidermis from the underlying dermis, progressive cell necrosis leading to a complete absence of viable cells from both the dermal and epidermal compartments, disintegration and fibrillation of the dermal connective tissue, and a cessation of protein synthesis. RA-treated organ cultures contained large numbers of healthy-appearing cells in both the epidermal and dermal compartments. One or several layers of viable basal cells in the epidermis could be seen at least through day 12. However, the upper layers of the epidermis frequently separated from the cells in the basal layer. The dermal connective tissue was histologically well-preserved. Furthermore, the level of protein synthesis was higher in the RA-treated cultures than in the control cultures. In addition to treating organ cultures with RA, other cultures were exposed to serum-free, growth factor-free culture medium containing 1.4 mM Ca++. The presence of the elevated Ca++ concentration also preserved cellular and connective tissue structures in the dermal and epidermal compartments. In comparison to RA there was better preservation of the overall epidermal structure. The upper layers of epidermal cells did not separate from the basal cells, and the various stages of epithelial differentiation could be seen. Histologically, the dermis was well-preserved in the presence of elevated extracellular Ca++. Specimens treated with a combination of Ca++ and RA demonstrated features consistent with the features induced by each treatment separately. This included an expanded basal layer of epithelial cells and a prominent keratotic layer with a fairly orderly pattern of differentiation. The tendency of the upper epidermis to separate from the basal cells was partially mitigated. Taken together, these data indicate that both RA and extracellular Ca++ act to prevent the degeneration of human skin in organ culture but probably do so through different mechanisms.     

Human skin in organ culture and human skin cells (keratinocytes and fibroblasts) in monolayer culture for assessment of chemically induced skin damage

Human skin cells (epidermal keratinocytes and dermal fibroblasts) in monolayer culture and human skin in organ culture were exposed to agents that are known to produce irritation (redness, dryness, edema and scaly crusts) when applied topically to skin. Among the agents used were three well accepted contact irritants (i.e., all-trans retinoic acid [RA], sodium lauryl sulfate [SLS] and benzalkonium chloride) as well as the corrosive organic mercury compound, aminophenyl mercuric acetate (APMA), and 5 contact sensitizers (oxazolone, nickel sulfate, eugenol, isoeugenol and ethylene glycol dimethacrylate [EGDM]). As a group, the contact irritants (including the corrosive mercuric compound) were cytotoxic for keratinocytes and fibroblasts and suppressed growth at lower concentrations than the contact sensitizers. The contact irritants also produced histological changes (hyperplasia, incomplete keratinization, loss of the granular layer, acantholysis and necrosis) in organ-cultured skin at dose levels at which the contact sensitizers appeared to be inert. Finally, the profile of secreted molecules from organ-cultured skin was different in the presence of contact irritants versus contact sensitizers. Taken together, these data suggest that the use of organ-cultured skin in conjunction with cells derived from the skin in monolayer culture may provide an initial approach to screening agents for deleterious changes in skin.     

Effect of soy isoflavones on circulating C-reactive protein in postmenopausal women: meta-analysis of randomized controlled trials

Strong evidence suggests that C-reactive protein (CRP) is a novel risk factor for cardiovascular disease. We aimed to examine the effect of soy isoflavones on circulating CRP concentrations in postmenopausal women by conducting a meta-analysis of randomized controlled trials. We performed a literature search using PubMed, the Cochrane Central Register of Controlled Trials, and ClinicalTrials.gov databases in December 2010 for randomized controlled trials conducted in postmenopausal women, using soy foods with isoflavones or isoflavone extracts as treatment, and with a report of CRP change. A meta-analysis was performed using a fixed-effects model or a random-effects model to calculate the combined effect size. In addition, subgroup and metaregression analyses were carried out to examine the influences of study designs and participant characteristics on the effect estimates. A pooled analysis of 14 trials showed a slight, but not significant, reduction of 0.17 mg/L (95% CI, -0.38 to 0.04; P = 0.12) in CRP concentrations among postmenopausal women with soy isoflavone intervention compared with controls. No substantial heterogeneity was observed. Subgroup analyses showed that soy isoflavones significantly lowered CRP by 0.70 mg/L (95% CI, -1.17 to -0.23; P = 0.003) among women with baseline CRP concentrations greater than 2.2 mg/L. No significant changes in CRP were observed in the other subgroups. Metaregression analysis further revealed that baseline CRP was a potential effect modifier of isoflavone treatment in lowering CRP. The present meta-analysis found insufficient evidence that soy isoflavones significantly reduce CRP concentrations in postmenopausal women. However, soy isoflavones may produce a significant reduction in CRP among postmenopausal women with elevated CRP.     

Expression of serine proteinases and metalloproteinases in organ-cultured human skin. Altered levels in the presence of retinoic acid and possible relationship to retinoid-induced loss of epidermal cohesion.

Neonatal human foreskin obtained at circumcision was cut into 2 x 2-mm pieces and placed in organ culture. Culture medium consisted of a serum-free, growth factor-free basal medium containing either 0.15 mmol/L Ca2+ or 1.4 mmol/L Ca2+. Some cultures were left as control, whereas others were treated with 3 mumol/L all-trans retinoic acid (RA). In the presence of RA, epidermal cohesion was disrupted and the upper layers separated from the viable epidermis beneath. This effect was observed under both low Ca2+ and high Ca2+ conditions. At 2-day intervals, culture fluids were collected and analyzed for serine and metalloproteinase activities. Serine proteinase activity was detected in the culture fluids and virtually all of the detected activity was dependent on the presence of plasminogen. Activity was elevated in the RA-treated tissues and this was due to increased amounts of both urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA). Elastase and cathepsin G were not detected in either control or RA-treated cultures. Increased plasminogen activator levels were also detected in RA-treated keratinocytes and fibroblasts in monolayer culture. Significant amounts of t-PA (though not u-PA) were found in fibroblast culture fluids, whereas both t-PA and u-PA were detected in culture fluids from keratinocytes. Metalloproteinase activity was also detected in the culture fluids of control and RA-treated tissues but in contrast to plasminogen activator, metalloproteinase activity decreased in the presence of RA. Casein and gelatin zymographic studies indicated the presence of both 92- and 72-kd gelatinases and stromelysin-1 and suggested that the decreased activity was primarily due to reduction in the 92- and 72-kd gelatinases. When serine proteinase inhibitors (aprotinin and soybean trypsin inhibitor) were included in the culture medium throughout the incubation period, epidermal discohesion was reduced. A metalloproteinase inhibitor, tissue inhibitor of metalloproteinase-2, did not have this effect. Taken together, these data show that a number of proteolytic enzymes are produced during organ culture of human skin. They suggest that these proteases may influence the structural integrity of the tissue.     

The effects of soy extract on the uterus of castrated adult rats

OBJECTIVE: The aim of this study was to evaluate the effects of different doses of a standardized soy extract on the uterus of castrated rats. METHODS: Fifty-six adult castrated female Wistar rats were randomly divided into seven groups (eight animals in each) that received: GI--drug vehicle (propylene glycol); GII--soy extract 10mg/kg per day; GIII--soy extract 50mg/kg per day; GIV--soy extract 100mg/kg per day; GV--soy extract 300mg/kg per day; GVI--soy extract 600mg/kg per day; GVII-conjugated equine estrogens (CEE) 200microg/kg per day. After 21 days of treatment, all animals were sacrificed and fragments of the uterine horns were immediately removed, fixed in 10% formaldehyde and submitted to routine histological techniques for morphometric study. The endometrial cell proliferation index was determined with the PCNA antibody PC-10 and expressed as the percentuals of the PCNA-positive nuclei relative to the total countings. Other fragments were immediately frozen in liquid nitrogen for RNA extraction and VEGF analysis using RT-PCR technique. RESULTS: The minimal dose of soy extract that produced a significant increase of the morphometric parameters was 100mg/kg (GIV). The maximum effects on endometrial and myometrial morphometry were detected in the groups treated with 300 and 600mg/kg of soy extract (groups V and VI) and CEE (GVII). The expression of PCNA in the endometrial epithelium and stroma was increased by treatment with 100-600mg/kg per day of soy extract (groups IV-VI) or with CCE (group VII). Doses equal to or higher than 50mg/kg of soy extract (groups III-VI) and CEE stimulated the expression of VEGF. CONCLUSION: The treatment of adult castrated rats during 21 days with doses of 100mg/kg per day or higher of soy extract may determine significant proliferation in the endometrium and myometrium.     

Modulation of Ca2+ levels in keratinocytes by all-trans retinoic acid.

Human epidermal keratinocytes, that have been growth-arrested by removal of epidermal growth factor from the culture medium, are stimulated to proliferate by all-trans retinoic acid (RA). The same treatment inhibits the onset of differentiated features and reduces cell-substrate adhesion. In the present study we show that the same treatment results in a decrease in total cell-associated Ca2+ as measured by changes in the amount of 45Ca2+ bound to cells at equilibrium following RA treatment and by a decrease in intracellular free Ca2+ levels as measured with the Ca(2+)-sensitive dye, Indo-1. The alterations in Ca2+ levels were evident within an hour after RA treatment, were in the range of 30-35% and occurred over the same RA concentration range that stimulated proliferation (i.e., 0.25-1.0 micrograms/ml). When the extracellular Ca2+ concentration was elevated from the normal level of 0.15-1.4 mM, intracellular free Ca2+ increased by a factor of 2 while total cell-associated Ca2+ increased approximately 6-fold. Even under conditions of high extracellular Ca2+, RA was able to reduce cell-associated and intracellular free Ca2+. These data indicate that RA has the capacity to lower Ca2+ levels in keratinocytes concomitantly with its effects on biological behavior.     

An Altered Response by Psoriatic Keratinocytes to Gamma Interferon

To determine whether psoriatic keratinocytes differ from normal keratinocytes in their response to gamma interferon, epidermal cell suspensions from normal and from lesional and uninvolved psoriatic skin were cultured in the presence of gamma interferon and the induction of HLA-DR expression and inhibition of cell growth were measured. The addition of 10(2) units of gamma interferon/ml during a 7-day culture period significantly increased mean HLA-DR+ cell numbers in 21 epidermal suspensions of normal from 3.9 to 24.1% (P less than 0.0001), uninvolved psoriatic from 8.4 to 33.1% (P less than 0.0001), and to a lesser extent lesional psoriatic biopsies from 12.6 to 18.3% (P less than 0.01). However, the increase in HLA-DR+ cell numbers in these latter cultures was significantly less than that observed in either normal or uninvolved psoriatic epidermal cell cultures (P less than 0.0001). Furthermore, [3H]thymidine incorporation was substantially decreased by gamma interferon in 16 out of 22 (73%) cultures of normal epidermal cells; this decrease was statistically significant (P less than 0.01). In contrast, only 4 out of 11 (36%) lesional and 9 out of 21 (43%) uninvolved psoriatic epidermal cultures showed comparable inhibition of proliferation. These findings suggest that psoriatic keratinocytes have an altered response to gamma interferon; this could explain the infrequency of keratinocyte HLA-DR expression in psoriatic plaques in vivo and may also contribute to the increased epidermal proliferation that characterizes this disease.     

Antigenicity of the proteins in soy lecithin and soy oil in soybean allergy

BACKGROUND: Soy lecithin and soy oil are usually produced from the hexane extract of soybean. Some of the soybean proteins are included in the extract and are therefore present in small amounts in both soy lecithin and soy oil. The antigenicity of the proteins present in defatted soybean has been studied with respect to soybean allergy, but the antigenicity of those found in the extract is yet to be investigated. OBJECTIVE: The antigenicity of soy lecithin and soy oil proteins with regard to soybean allergy were investigated. METHODS: The proteins present in soy lecithin and soy oil were determined according to already established method and analysed by SDS-PAGE. The IgE- and IgG4-binding abilities of the soy lecithin proteins were investigated by immunoblotting with sera from 30 soybean-sensitive patients, including seven with a positive challenge test. Immunoblotting of soy oil proteins was performed with the sera from some of these patients. RESULTS: In 100 g of sample, the soy lecithin and soy oil contained 2.8 mg and 1.4-4.0 microg of proteins, respectively. The results of SDS-PAGE demonstrated the presence of only three proteins, with molecular weights of about 58-67 kDa in soy oil, and suggested that soy lecithin also contains these proteins. The soy lecithin also contained many proteins besides these. In the soy lecithin, the detection rate of only one protein, with a molecular weight of 31 kDa, by the serum IgE of patients was significantly different compared with controls (detection rate: 40%). The proteins with molecular weights of 58-67 kDa rarely bound to serum IgE. Only one of the patients who presented a positive challenge test had IgE antibodies to soy lecithin proteins. IgG4-binding proteins were found only rarely in soy lecithin. Neither the IgE nor the IgG4 present in the patients' sera reacted to any soy oil protein. CONCLUSION: Proteins present in soy lecithin and soy oil have little antigenicity with regard to soybean allergy.     

The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro

The effects of chitin [(1 --> 4)-2-acetamido-2-deoxy-beta-D-glucan] and its partially deacetylated derivatives, chitosans, on the proliferation of human dermal fibroblasts and keratinocytes were examined in vitro. Chitosans with relatively high degrees of deacetylation strongly stimulated fibroblast proliferation while samples with lower levels of deacetylation showed less activity. Fraction, CL313A, a shorter chain length, 89% deacetylated chitosan chloride was further evaluated using cultures of fibroblasts derived from a range of human donors. Some fibroblast cultures produced a positive mitogenic response to CL313A treatment with proliferation rates being increased by approximately 50% over the control level at an initial concentration of 50 microg/ml, whilst others showed no stimulation of proliferation or even a slight inhibition (< 10%). The stimulatory effect on fibroblast proliferation required the presence of serum in the culture medium suggesting that the chitosan may be interacting with growth factors present in the serum and potentiating their effect. In contrast to the stimulatory effects on fibroblasts, fraction CL313A inhibited human keratinocyte mitogenesis with up to 40% inhibition of proliferation being observed at 50 microg/ml. In general highly deacetylated chitosans were more active than those with a lower degree of deacetylation. These data demonstrate that highly deacetylated chitosans can modulate human skin cell mitogenesis in vitro. Analysis of their effects on cells in culture may be useful as a screen for their potential activity in vivo as wound healing agents, although in the case of fibroblasts it is important to select appropriate strains of cells for use in the screen.     

Transforming growth factor beta (TGF-beta) potentiates the inhibitory effect of retinoic acid on human breast carcinoma (MCF-7) cell proliferation

Anchorage-dependent and -independent MCF-7 cell growth was dose-dependently inhibited by retinoic acid (RA) but was insensitive to TGF-beta (from 1 to 100 pM). Growth of MCF-7 monolayer cultures was inhibited (50%) when exposed to 10(-6) M RA. RA was unable to completely inhibit MCF-7 cell proliferation, as concentrations above 10(-6) M were rapidly cytotoxic. However, the combination of TGF-beta and RA resulted an increase in RA inhibitory effect on MCF-7 monolayer growth and a 80% reduction in colony formation in soft agar. These results demonstrate that although TGF-beta does not inhibit the growth of MCF-7 cells, it potentiates the antiproliferative effect of RA, suggesting that it may play a part, albeit indirect, in the regulation of MCF-7 cell growth.     

Development of fungal mycelia as a skin substitute: characterization of keratinocyte proliferation and matrix metalloproteinase expression during improvement in the wound-healing process

SACCHACHITIN membranes, prepared from the waste residue of the fruiting body of Ganoderma taugae, were used in our previous study to enhance skin wound healing in animal models. In the present study, the effects of the membrane on the growth of keratinocytes and the activity of matrix metalloproteinases (MMPs), as well as on the healing of skin wounds in humans, were estimated. Fresh human foreskin was employed as the source of the keratinocyte culture, and a modified keratinocyte-SFM medium supplemented with 0.2 ng/mL of recombinant epidermal growth factor and 30 microg/mL bovine pituitary extract was used to enhance the successful growth of keratinocytes under an atmosphere of 5% CO2, at 37 degrees C. The results indicated that 0.01% SACCHACHITIN enhanced the proliferation of keratinocytes in the culture on the fourth and fifth days, and cells showed neither morphological alteration nor disordered proliferation. This evidence clearly indicated that SACCHACHITIN was not cytotoxic to and was safe for the growth of keratinocytes. Thus, SACCHACHITIN might play a positive role in the proliferation and differentiation of keratinocytes around wounds and in accelerated wound healing of epidermal tissue. In addition, microscopic observations during the growth of keratinocytes showed that normal proliferation and differentiation took place along the margin of the SACCHACHITIN membrane. This indicates that SACCHACHITIN is possibly cytocompatible with keratinocytes. Electrophoretic analysis and inhibition tests for the binding effect of SACCHACHITIN on MMPs showed that SACCHACHITIN reduced MMPs in extracellular matrix degradation and facilitated establishment of an extracellular matrix around wounds; these effects resulted in rapid wound healing. SACCHACHITIN was used as a skin dressing for patients who had skin chronicle ulcer, which had not healed for over 7 months. Preliminary clinical observations showed that the wound improved and began to heal. An analysis of MMPs by ELISA in tissue of the wound indicated a significant decrease in MMP levels.     

Evaluation of modified trypticase soy broth versus supplemented peptone broth in the detection of bacteremia and fungemia

In vitro, 1.2% gelatin counteracts the inhibition of growth of bacterial species by sodium polyanetholsulfonate in blood culture media. Additionally, 1% yeast extract has been used to promote bacterial growth. We compared the performance of supplemented peptone broth and Trypticase soy broth, both of which contained sodium polyanetholsulfonate, gelatin and yeast extract. Trypticase soy broth with gelatin and yeast extract inhibited (p less than 0.001) and delayed growth, especially of gram-positive (p less than 0.01) and gram-negative (p less than 0.005) anaerobic bacteria. Although the recovery of organisms usually inhibited by sodium polyanetholsulfonate was similar in supplemented peptone and Trypticase soy broths, supplemented peptone broth clearly was superior in the recovery of other organisms commonly found in blood cultures.     

Human skin in organ culture. Elaboration of proteolytic enzymes in the presence and absence of exogenous growth factors.

Proteinase levels were assessed in organ culture fluids from human neonatal foreskin maintained under growth factor-free conditions and in the presence of a combination of growth factors (ie, epidermal growth factor, insulin, hydrocortisone, pituitary extract, and all-trans-retinoic acid). Analysis of culture fluids by gelatin zymography revealed the presence of 92-kd and 72-kd gelatinases. There was a greater amount of 92-kd gelatinase activity in the presence of growth factors whereas the levels of 72-kd gelatinase were similar in growth factor-free and growth factor-containing media. Experiments with keratinocytes and fibroblasts in monolayer culture and with isolated dermal tissue in organ culture indicated that the epithelial component was responsible for most of the 92-kd gelatinase activity whereas fibroblasts were primarily responsible for the 72-kd gelatinase activity. Activation with aminophenyl mercuric acetate, requirement for divalent cations, inhibition with EDTA, and insensitivity to inhibition with phenylmethyl sulfonyl fluoride indicated that both gelatinases were metalloproteinases. In additional studies, culture fluids were examined for the presence of plasminogen activator activity. This was detected in culture fluids from tissues maintained under both conditions but was increased in the growth factor-containing medium. The increased amount seen in the growth factor-containing medium appeared to be due almost entirely to a single factor, ie, all-trans-retinoic acid. In monolayer culture, both keratinocytes and fibroblasts produced plasminogen activator; the level was higher in keratinocyte culture fluids than in culture fluids from fibroblasts.     

Phenotypes and interactions of human melanocytes and keratinocytes in an epidermal reconstruction model

The morphologic and antigenic phenotype of normal human melanocytes and keratinocytes was investigated in monolayer and 3-dimensional cultures in an effort to develop an epidermal model that resembles the normal human epidermis. When cultured for several passages in optimal growth medium, pure cultures of either cell type could be established as demonstrated by light and electron microscopy and with monoclonal antibodies defining melanocyte- and keratinocyte-associated antigens. Three-dimensional growth of keratinocytes on polycarbonate filters was induced by increasing calcium concentrations in the culture medium and exposing cultures to air. After 30 to 35 days incubation, the 3-dimensional keratinocyte cultures reached a total of 12 to 25 layers and keratinocytes of various stages of differentiation formed three morphologically and antigenically different strata. The basal layer of these constructs consisted of ovoid cells with desmosomes and hemidesmosome-like structures. These cells expressed low molecular weight cytokeratins similar to basal cells in situ. The intermediate layer, representing the stratum spinosum in situ, contained flat cells with keratohyaline granules and many desmosomes. These cells expressed gp 80 kilodaltons, gp 40 to 50 kilodaltons, involucrin, and filaggrin. The upper layer, the stratum corneum equivalent, contained large, flattened cells with keratohyaline granules. The majority of these cells were anucleate. When melanocytes were cocultured with keratinocytes in monolayer or in epidermal reconstructs, they assumed a multidendritic morphology and donated pigment to surrounding keratinocytes. The majority of pigmented cells localized singly within the basal layer of the reconstructs and their dendrites were intimately associated with keratinocyte plasma membranes. Pigment donation to keratinocytes appeared to occur through the uptake of melanosome-containing dendrite fragments and phagocytosis of individual melanosomes by keratinocytes. It is hypothesized that keratinocytes produce unique microenvironmental factors that regulate the melanocytic phenotype.     

Apoptosis and necrosis induced by cyclic mechanical stretching in alveolar type II cells

Alveolar type II (ATII) cells are exposed to mechanical stretch during breathing and mechanical ventilation. Increased stretch may contribute to lung injury. The influence of three stretching patterns (characterized by frequency [min(-1)] - increase in surface area [%]: S40-13, S60-13, S40-30) on parameters of apoptosis, necrosis, and membrane integrity of rat ATII cells was compared with that in static cultures. The S40-13 stretching pattern simulated normal breathing. The other patterns were chosen to study increased amplitude and frequency. There were no significant differences between the S40-13 group and static cultures. Lactic acid dehydrogenase (LDH) release and early apoptotic cells were significantly increased in S60-13 and S40-30 in comparison with static cultures (LDH: 0.089 +/- 0.014 microg/ml and 0.177 +/- 0.050 microg/ml versus 0.050 +/- 0.011 microg/ml; early apoptosis: 17 +/- 3.5% and 23 +/- 3.1% versus 9.7 +/- 1.4%) at 24 h. Necrosis was significantly increased only in the S40-30 group (13 +/- 2.4% versus 6.1 +/- 0.9% in static culture at 24 h). Captopril as well as L-Arginine prevented apoptosis and reduced apoptotic cells to static culture levels in the S40-30 group, but did not influence necrosis and LDH release. Increased mechanical stretch may contribute to lung injury by induction of apoptosis and necrosis in ATII cells. Apoptosis induced by high-amplitude mechanical stretch is prevented by captopril and L-Arginine.     

Regulation of embryonic chick insulin cells: effect of retinoic acid and insulin-like growth factor 1

We are interested in the regulation of early pancreatic differentiation, particularly with regard to factors that enhance insulin cell proliferation. Both retinoic acid and insulin-like growth factor 1 (IGF-1) are known to be important in the proliferation and differentiation of insulin cells. Individually, they have the ability to increase the proportion of insulin cells when added to cultures of chick dorsal pancreatic buds. The aim of this study was to define the action of retinoic acid (RA) in combination with IGF-1 on the proportion of insulin cells. The dorsal pancreatic bud of 5-day-old chick embryos was excised and the endodermal component, with minimal adherent mesenchyme, was explanted onto Matrigel. RA (10(-6) M) and IGF-1 (50 ng/ml) were added to Ham's F12 culture medium containing transferrin (5 microg/ml) and selenium (10(-10) M) (F12.TS). Control explants were cultured in F12.TS alone or in F12.TS containing dimethyl sulphoxide (DMSO) [F12.TS (DMSO)]. After 7 days in culture, insulin and glucagon cells were localized immunocytochemically; numbers of insulin cells were expressed as a percentage of insulin plus glucagon cell counts. Addition of RA plus IGF-1 to the medium increased the proportion of insulin cells markedly (23.43%) compared with the proportions in control explants (11.3% with F12.TS (DMSO), 13.2% with F12.TS). This increase represents a more than twofold increase in the proportion of insulin cells over that of control explants.     

Potentiation of IL-2-induced t-cell proliferation by retinoids.

We evaluated the capacity of retinoids to potentiate proliferative responses of murine T-cells to recombinant human interleukin 2 (rIL-2). Concanavalin A (Con A) prestimulated spleen cells responded in a dose-dependent manner to added rIL-2. All-trans-retinoic acid (RA) at 10(-8) M potentiated the proliferative response by fivefold at saturating levels of IL-2. In similar experiments, two closely related retinamides, all-trans-(phenyl)retinamide (PR) and N-(4-hydroxyphenyl)retinamide (4-HPR), also potentiated murine splenocyte rIL-2 responses. Potentiation of IL-2-induced proliferation was dose-responsive to the concentration of added retinoid with peak potentiation occurring at 10(-10) - 10(-8) M in the presence of 10 U/ml rIL-2. Significant potentiation was observed at retinoid concentrations as low as 10(-14) M. Fluorescence flow cytometry of the responding cells revealed that among L3T4+, Lyt-2+ or total T-cells, at 72 h following Con A stimulation, essentially all of the cells expressed IL-2 receptors (IL-2R). This apparently represents near maximum IL-2R expression and treatment of the cells with retinoids did not increase IL-2R expression at that time point. The potentiation of IL-2 responses by retinoids was also observed with IL-2-dependent HT-2 cells, 98% of which were IL-2R positive. HT-2 proliferative responses to rIL-2 were potentiated as much as fourfold by 10(-10) M RA. HT-2 proliferative responses to rIL-2 were potentiated by all three retinoids dose dependently. Significant potentiation was observed with as little as 10(-14) M retinoid. Retinoids in the absence of IL-2 induced no proliferative responses. These data suggest that retinoids can augment the capacity of IL-2 to induce T-cell proliferation using Con A-activated murine splenic T-cell blasts and a long-term-cultured T-cell line.     

Modulative effect of endogenous granuloid inhibitors on cell proliferation

The particle-free crude extract of differentiated granulocytes (GCE) and the GI-1, GI-2, GI-3 proliferation inhibitory fractions (M.W. larger than or equal to 70,000 approximately 11,500 and less than or equal to 4000) were studied for the effects they exert on cultured cells. As shown by the curve, in bone marrow suspension cultures, in the dose range of 0.01-10.0 microng/ml, GCE maximally inhibits 3H-TdR incorporation into the DNA for 5.5 hours. In agar-gel cultures, 1.0-5.0 microng/ml of GCE reduces both the number (by 50.6-54.8%) and the size of the colonies formed. The manifestation of the inhibitory effect of GI-1, GI-2 and GI-3 fractions requires 2.5-3.5 hours. The inhibitors designated GI-2 and GI-2 are target-cell specific but they do not reduce 3H-TdR incorporation either in thymocyte suspension or HeLa monolayer cultures. On the other hand GI-1 inhibis the proliferation in HeLa cultures, as well. These endogenous inhibitors exert their optimum effect, even in a partially purified state at 2-4 orders or magnitude lower concentrations than does 1,2/5,6-dianhydrogalactitol (DAD), a cell-aspecific inhibitor used for comparison in the same system.     

Isoflavones made simple - genistein's agonist activity for the beta-type estrogen receptor mediates their health benefits

Soy isoflavones, the focus of much research and controversy, are often referred to as "weak estrogens". In fact, genistein is a relatively potent agonist for the recently characterized beta isoform of the estrogen receptor (ERbeta). The low nanomolar serum concentrations of unconjugated free genistein achieved with high-nutritional intakes of soy isoflavones are near the binding affinity of genistein for this receptor, but are about an order of magnitude lower than genistein's affinity for the "classical" alpha isoform of the estrogen receptor (ERalpha). Moreover, these concentrations are far too low to inhibit tyrosine kinases or topoisomerase II, in vitro activities of genistein often cited as potential mediators of its physiological effects. The thesis that these physiological effects are in fact mediated by ERbeta activation provides a satisfying rationale for genistein's clinical activities. Hepatocytes do not express ERbeta; this explains why soy isoflavones, unlike oral estrogen, neither modify serum lipids nor provoke the prothrombotic effects associated with increased risk for thromboembolic disorders. The lack of uterotrophic activity of soy isoflavones reflects the fact that ERalpha is the exclusive mediator of estrogen's impact in this regard. Vascular endothelium expresses both ERalpha and ERbeta, each of which has the potential to induce and activate nitric oxide synthase; this may account for the favorable influence of soy isoflavones on endothelial function in postmenopausal women and ovariectomized rats. The ERbeta expressed in osteoblasts may mediate the reported beneficial impact of soy isoflavones on bone metabolism. Suggestive evidence that soy-rich diets decrease prostate cancer risk, accords well with the observation that ERbeta appears to play an antiproliferative role in healthy prostate. In the breast, ERalpha promotes epithelial proliferation, whereas ERbeta has a restraining influence in this regard - consistent with the emerging view that soy isoflavones do not increase breast cancer risk, and possibly may diminish it. Premenopausal women enjoy a relative protection from kidney failure; since ERbeta is an antagonist of TGF-beta signaling in mesangial cells, soy isoflavones may have nephroprotective potential. Estrogen also appears to protect women from left ventricular hypertrophy, and recent evidence suggests that this effect is mediated by ERbeta. In conjunction with reports that isoflavones may have a modestly beneficial impact on menopausal symptoms - perhaps reflecting the presence of ERbeta in the hypothalamus - these considerations suggest that soy isoflavone regimens of sufficient potency may represent a safe and moderately effective alternative to HRT in postmenopausal women. Further clinical research is required to characterize the impact of optimal genistein intakes on endothelial and bone function in men. Studies with ERbeta-knockout mice could be helpful for clarifying whether ERbeta does indeed mediate the chief physiological effects of low nanomolar genistein. S-equol, a bacterial metabolite of daidzein, has an affinity for ERbeta nearly as high as that of genistein; whether this compound contributes meaningfully to the physiological efficacy of soy isoflavones in some individuals is still unclear.