Fortune Kohen

25-Hydroxyvitamin D3-1α-Hydroxylase Is Expressed in Human Vascular Smooth Muscle Cells and Is Upregulated by Parathyroid Hormone and Estrogenic Compounds

Background—1,25(OH)2 vitamin D3 exerts multiple effects in human vascular smooth muscle cells (VSMCs). We therefore tested the possibility that VSMCs possess an endogenous 25-hydroxyvitamin D3-1α-hydroxylase system, the final enzyme in the biosynthetic pathway of 1,25(OH)2D3. Methods and Results—We assessed the expression and activity of 25-hydroxyvitamin D3-1α-hydroxylase by real-time polymerase chain reaction and the conversion of 25(OH)D3 into 1,25(OH)2D3. First, 25-hydroxyvitamin D3-1α-hydroxylase mRNA was identified in cultured VSMCs by real-time polymerase chain reaction. Second, in cells treated daily (3 days) with parathyroid hormone (66 nmol/L), estradiol-17β (30 nmol/L), raloxifene (3 μmol/L), and the phytoestrogens genistein (3 μmol/L), biochainin A (3 μmol/L), and 6-carboxy biochainin A (30 nmol/L), 25-hydroxyvitamin D3-1α-hydroxylase mRNA increased by 43±13%, (P<0.05) 7±24% (P=NS), 176±28% (P<0.01), 65±11% (P<0.05), 152±24% (P<0.01), and 71±9% (P<0.05), respectively. Third, production of 1,25(OH)2D3 from 25(OH)D3 was seen with a Km of 25 ng/mL and increased dose dependently after treatment with parathyroid hormone, genistein, and the phytosetrogen derivative 6-carboxy biochainin A. Estradiol-17β and biochainin A also increased the generation of 1,25(OH)2D3 by 40±23% (P<0.05) and 55±13% (P<0.05), respectively. Conclusions—We provide here the first evidence for the expression of an enzymatically active 25(OH)D3-1α-hydroxylase system in human VSMCs, which can be upregulated by parathyroid hormone and estrogenic compounds. Because exogenous vitamin D inhibits VSMC proliferation, the role of this system as an autocrine mechanism to curb changes in VSMC proliferation and phenotype is a subject for future investigation.

Interaction of the Estrogen Receptors with the Fas Ligand Promoter in Human Monocytes

The predominance of autoimmune diseases among women suggests that estrogen may modulate immune function. Monocytes and macrophages are important in initiating, maintaining, and resolving inflammatory responses through cell-signaling molecules, which control immune cell survival. One important mechanism of cell survival is mediated by the Fas/Fas ligand (FasL) system. In this study, the link between estrogen, monocytes/macrophages, and the Fas/FasL system was investigated. Estrogen treatment increased FasL expression in monocytes through the binding of the estrogen receptors (ER) to the estrogen recognizing elements and AP-1 motifs present at the FasL promoter. Furthermore, estrogen induced apoptosis in monocytes expressing ERβ, but not in monocyte-differentiated macrophages expressing ERα. The expression of either ERα or ERβ and their response to estrogen in monocytes was found to be dependent on the their stage of cell differentiation. Previously, we have shown that estrogen replacement therapy in postmenopausal women decreased the number of circulating monocytes. In this study, we have characterized the molecular mechanism by which estrogen regulates monocytes homeostasis. These findings indicate that estrogen may regulate immune cell survival through the Fas/FasL system. There is biological relevance to these findings in view of studies showing that accumulation of activated monocytes is involved in the pathogenesis of conditions such as vasculititis, arteriosclerosis, and rheumatoid arthritis.

Antivascular Treatment of Solid Melanoma Tumors with Bacteriochlorophyll–serine-based Photodynamic Therapy¶

We describe here a strategy for photodynamic eradication of solid melanoma tumors that is based on photoinduced vascular destruction. The suggested protocol relies on synchronizing illumination with maximal circulating drug concentration in the tumor vasculature attained within the first minute after administrating the sensitizer. This differs from conventional photodynamic therapy (PDT) of tumors where illumination coincides with a maximal concentration differential of sensitizer in favor of the tumor, relative to the normal surrounding tissue. This time window is often achieved after a delay (3–48 h) following sensitizer administration. We used a novel photosensitizer, bacteriochlorophyll–serine (Bchl–Ser), which is water soluble, highly toxic upon illumination in the near‐infrared (λmax 765–780 nm) and clears from the circulation in less than 24 h. Nude CD1 mice bearing malignant M2R melanotic melanoma xenografts (76–212 mm3) received a single complete treatment session. Massive vascular damage was already apparent 1 h after treatment. Changes in vascular permeability were observed in vivo using contrast‐enhanced magnetic resonance imaging (MRI), with the contrast reagent Gd‐DTPA, by shortening spin–spin relaxation time because of hemorrhage formation and by determination of vascular macromolecular leakage. Twenty‐four hours after treatment a complete arrest of vascular perfusion was observed by Gd‐DTPA–enhanced MRI. Histopathology performed at the same time confirmed primary vascular damage with occlusive thrombi, hemorrhage and tumor necrosis. The success rate of cure of over 80% with Bchl–Ser indicates the benefits of the short and effective treatment protocol. Combining the sensitizer administration and illumination steps into one treatment session (30 min) suggests a clear advantage for future PDT of solid tumors.

Regulation of fas ligand expression in breast cancer cells by estrogen: functional differences between estradiol and tamoxifen.

During neoplastic growth and metastasis, the immune system responds to the tumor by developing both cellular and humoral immune responses. In spite of this active response, tumor cells escape immune surveillance. We previously showed that FasL expression by breast tumor plays a central role in the induction of apoptosis of infiltrating Fas-immune cells providing the mechanism for tumor immune privilege. In the present study, we showed that FasL in breast tissue is functionally active, and estrogen and tamoxifen regulate its expression. We identified an estrogen recognizing element like-motif in the promoter region of the FasL gene, suggesting direct estrogen effects on FasL expression. This was confirmed by an increase in FasL expression in both RNA and protein levels in hormone sensitive breast cancer cells treated with estradiol. This effect is receptor mediated since tamoxifen blocked the estrogenic effect. Interestingly, tamoxifen also inhibited FasL expression in estrogen-depleted conditions. Moreover, an increase in FasL in breast cancer cells induces apoptosis in Fas bearing T cells and, tamoxifen blocks the induction of apoptosis. These studies provide evidence that tamoxifen inhibits FasL expression, allowing the killing of cancer cells by activated lymphocytes. This partially explains the protective effect of tamoxifen against breast cancer.

An assay procedure for plasma progesterone based on antibody‐enhanced chemiluminescence

Existing radioimmunoassay (RIA) procedures have the advantage of extreme sensitivity, but depend on the availability of a suitable radiolabeled ligand and of expensive equipment. Furthermore, such RIAs require a time-consuming phase-separation step [ 11. Several approaches have been suggested to overcome the drawbacks of RIA while retaining the specificity of an immunoassay [2-41, but the procedures proposed thus far suffer from inadequate sensitivity. We have explored the possibility of using chemiluminescent markers, since chemlluminescent compounds are commonly detectable at pM levels [5-71. In the method described here, an isoluminol derivative is attached covalently to a carboxy derivative of a steroid. The resulting steroid-chemiluminescent marker conjugate emits light upon oxidation with hematin compounds and HzOz. When the steroidchemiluminescent marker conjugate is bound to a specific binding protein, the total light production of the conjugate is enhanced. This binding and the consequent enhancement of light emission is prevented by the addition of unaltered steroid in a competitive manner. A ‘homogeneous’ immunoassay, viz. one requiring no separation of bound and free hormone, can thus be designed. To illustrate this approach, the results of an immunoassay for plasma progesterone based on the monitoring of chemiluminescence are reported in the following sections.

An assay for urinary estriol-16α-glucuronide based on antibody-enhanced chemiluminescence

An immunoassay for estriol-16 alpha-glucuronide in pregnancy urine is described that utilizes antibody-enhanced chemiluminescence. The steroid glucuronide was covalently conjugated with the chemiluminescent marker aminobutyl-ethyl-isoluminol. The light yield of this conjugate upon oxidation was augmented by specific antibody, and this effect was inhibited by addition of the homologous steroid glucuronide (10-100 pg) in a dose-dependent manner. The assay does not require separation of bound and free ligand and proved satisfactory with respect to sensitivity, precision and accuracy. Assay results obtained by radioimmunoassay and chemiluminescence immunoassay were in good agreement (r = 0.98; n = 25).

Monoclonal immunoglobulin G augments hydrolysis of an ester of the homologous hapten: An esterase-like activity of the antibody-containing site?

Molecular recognition through the interaction of specific binding sites is a characteristic property of antibodies and enzymes [l]. Both types of proteins bind ligands and discriminate between closely related compounds. However, while enzymes are capable of causing cleavage of covalent bonds, it is generally held that antibodies lack this property [2]. Slobin [3] examined the effect of rabbit IgG and of the homologous antibody, on the hydrolysis of p-nitrophenylacetate, and found no specific enhancement in the rate of hydrolysis of the labile ester by the specific IgG. However, the studies in [4-71 indicate that specific antibody directed against the hapten may enhance the rate of hydrolysis of labile esters containing the homologous hapten. Further, we have investigated the quasi-esterase properties of monoclonal IgG directed against the well-explored haptenic group, 2,4-dinitro-phenyl(DNP), towards a fluorescentdye conjugate of the homologous ligand with a view to elucidate the specificity, the generality and the mechanism of the hydrolytic reaction. Here, DNP-e-aminocaproic acid was conjugated covalently through an ester bond with the fluorescent compound 7-hydroxycoumarin to give the nonfluorescent conjugate 7-[N-(2,4-dinitrophenyl)6-aminohexanoxyl] -coumarin (DNP-e-aminocaproyl-umbelliferone conjugate; DNP-CU) (fig.1). On incubation with monoclonal anti-DNP IgG from SPE-2 1 in a solution buffered at pH 8, this conjugate is hydrolyzed to yield umbelliferone which yCCH,-CH,-CH2-CH,-Cl-l,

The role of the Fas/Fas ligand system in estrogen-induced thymic alteration

PROBLEM: Estrogen induces atrophy in the thymus by an unknown mechanism. Since the Fas/FasL system is one of the main pathways in T cell apoptosis, we tested the hypothesis that estrogen‐induced thymic atrophy is mediated by the Fas/FasL system.
 METHODS OF STUDY: In vivo experiments were done using ovariectomized female rats treated with estrogen or saline. In vitro experiments were performed using isolated thymocytes. Estrogen receptor (ER) α and β expression was characterized using flow cytometry, RT‐PCR and immunofluorescence. Fas and FasL mRNA and protein expression was evaluated using RT‐PCR and Western blot analysis respectively.
 RESULTS: ERα and ERβ are present in thymocytes and stromal cells. ER expression is mainly localized in the Double Positive CD4+CD8+ thymocytes. Estrogen treatment decreases thymus size and increase FasL expression.
 CONCLUSION: CD4+CD8+ thymocytes and thymic stroma cells express ERα and ERβ. In vivo and in vitro we showed that estrogen treatment increases FasL expression while decreasing thymus cell number. These findings support the hypothesis that estrogen‐induced thymic atrophy occurs as a result of apoptosis and is mediated by estrogen‐induced FasL expression.

A thiophilic adsorbent for the one-step high-performance liquid chromatography purification of monoclonal antibodies

Thiophilic adsorption chromatography, developed originally by Porath and colleagues (1985, FEBS Lett. 185, 306-310) for conventional chromatographic techniques, was transformed to the HPLC mode upon preparation of new and improved sulfur-containing silica beads. The new thiophilic adsorbent is of high capacity and is suitable for the rapid and single-step purification of all subclasses of monoclonal and polyclonal antibodies. Due to its broad specificity, the thiophilic silica column is an efficient, stable, and inexpensive substitute for protein A and protein G columns used today to purify antibodies.

An immunoassay for plasma cortisol based on chemiluminescence

An immunoassay procedure for the determination of cortisol in human plasma is described, which utilizes chemiluminescence as the end point. A cortisol-isoluminol conjugate serves as the chemiluminescent marker. The light emission by this conjugate upon oxidation is delayed by prior incubation with anti-cortisol IgG, but not by unrelated gamma-globulin. This delayed light emission was inhibited by cortisol in a dose-dependent manner, with a linear range of 20-1000 pg steroid/assay tube. A competitive protein binding assay based on this procedure was applied to methylene chloride extracts of cortisol from normal and pathological human plasma (2-40 micrograms/100 ml). Cortisol values obtained by this procedure agreed well with those obtained by radioimmunoassay, using the same antiserum with tritiated cortisol as the label (r = 0.98). The chemiluminescence immunoassay is comparable to radioimmunoassay with regard to sensitivity, specificity, precision and accuracy. The advantage of the new assay procedure is that it obviates the need for counting radioactivity and for separation of bound and free ligand.