Specific lysis of melanoma cells by receptor grafted T cells is enhanced by anti-idiotypic monoclonal antibodies directed to the scFv domain of the receptor

Malignant transformation of melanocytes is frequently associated with abnormalities in antigen processing and in human leukocyte antigen class I antigen expression. Here, we evaluated a human leukocyte antigen class I antigen-independent approach to target cytotoxic T lymphocytes to melanoma cells by grafting cytotoxic T lymphocytes with a chimeric receptor that consists of both a domain binding to high molecular weight-melanoma associated antigen and a cellular activation domain. The binding domain is a single-chain antibody fragment (scFv) derived from the monoclonal anti-high molecular weight-melanoma associated antigen antibody 763.74 by phage display techniques. The cellular activation domain is the signaling unit of the FcepsilonRI receptor gamma chain. Both domains constitute the chimeric receptor scFv763.74-gammaR. Cytotoxic MD45 T cells grafted with the scFv763.74-gammaR receptor bind specifically to high molecular weight-melanoma associated antigen-positive melanoma cells and lyse melanoma cells in a human leukocyte antigen class I independent fashion. Pre-incubation of receptor grafted T cells with immobilized anti-idiotypic (id) monoclonal antibody MK2-23 binding to the scFv domain of the receptor enhanced the lysis of melanoma cells indicating that the specific cytolytic activity of receptor grafted T cells can be increased by costimulation with cross-linked anti-idiotypic monoclonal antibodies that recognize the antigen binding domain of the chimeric receptor.     

皮肤肿瘤中的雌激素受体及皮肤恶性黑素瘤中雌激素和孕激素受体及C－erbB2癌基因的表达

通过对８个病种的２１例皮肤恶性肿瘤进行的雌激素受体的免疫组化标记（ＡＢＣ法），发现有２例皮肤恶性黑素瘤呈阳性表达。在１５例皮肤恶性黑素瘤中进行的免疫组化染色中（ＬＳＡＢ），雌激素受体阳性７例、孕激素受体阳性３例、Ｃ－ｅｒｂＢ２癌基因阳性７例。作者认为通过进一步研究有望获得恶性黑素瘤激素治疗和预后观察的生物学依据     

A mannose-binding receptor is expressed on human keratinocytes and mediates killing of Candida albicans

Human keratinocytes are known to kill Candida albicans in vitro, but the mechanism of killing is not yet understood. Here, we demonstrate that spontaneous, ultraviolet-B-light-induced, alpha-melanocyte-stimulating-hormone-induced, and interleukin-8-induced Candida killing by keratinocytes can be inhibited with mannan and mannosylated bovine serum albumin (Man-BSA). A polyclonal goat serum raised against the human macrophage mannose receptor stained suprabasal keratinocytes, but no staining was observed on keratinocytes with a monoclonal antibody (mAb15) specific for the human macrophage mannose receptor. Mannose-affinity chromatography of keratinocyte extract isolated a 200 kDa protein, and on the Western blot the goat antiserum reacted with a 200 kDa protein. In radioligand binding studies, the binding of 125I-Man-BSA to human keratinocytes was inhibited by mannan in a concentration-dependent manner. Analysis of the binding revealed a single class keratinocyte mannose receptor with a KD of 1.4 x 10(-8) M and a Bmax of 1 x 10(4) binding sites per cell. The binding of 125I-Man- BSA to keratinocytes proved to be time-dependent, acid-precipitable, and Ca2+- and trypsin-sensitive. After trypsinization the receptors underwent a rapid recovery at 37 degrees C. These results demonstrate the presence of mannose receptor on human keratinocytes, and its active involvement in the killing of Candida albicans.     

Steroid receptors in malignant melanomas

Epidemiological and experimental investigations have led to the hypothesis that the growth of malignant melanoma is induced by hormones. The demonstration of free-hormone binding sites in melanoma tissue may help to determine the validity of this hypothesis, as receptors are necessary for the transformation of hormonal action. The DCC assay with subsequent saturation analysis was used for the demonstration of specific cytoplasmatic binding sites of steroid hormones. The presence of free-cytosolic estrogen-, progesterone- and glucocorticosteroid receptors was investigated in 50 melanoma samples from 46 patients. In 20 of the 50 specimens, free estrogen and progesterone receptors were found. Free-glucocorticosteroid receptors we found in 10 cases. The use of four fold-labeled estradiol (2,4,6,7-[3]-17 beta-estradiol) in the DCC assay produced a false-positive demonstration of free-estrogen receptors. Tyrosinase hydroxylates estradiol at the C2 level of the steroid. Using fourfold labeled estradiol, tritium is separated at the C2 position, thus forming radioactive water in the cytosol which mimics high, free-estrogen binding sites. The use of twofold-labeled estradiol or L-dopa in the experiments with fourfold labeled estradiol produced no false-positive determinations of estrogen receptors. The demonstration of receptors in malignant melanomas was unaffected by the sex of the patient. Also, the type of malignant melanoma, the invasion level, and the prognostic index did not show a correlation with the presence of the various hormone receptors. In metastases, free steroid hormone receptors were detected less often than in the primary tumor.     

Cutaneous leiomyomas lack estrogen and progesterone receptor immunoreactivity

Gonadotropin-releasing hormone analog therapy is useful in treating uterine and some extrauterine smooth muscle tumors. These smooth muscle tumors have been demonstrated to have estrogen receptor and progesterone receptor immunoreactivity. The estrogen receptor and progesterone receptor immunoreactivity of smooth muscle tumors of the skin has not been reported. We evaluated 15 examples of cutaneous leiomyomas for estrogen receptor and progesterone receptor with ER-1D5 antibody and PGR-1A6 antibody. None of the 15 cutaneous leiomyomas demonstrated positive staining by this method. The tumorigenesis of cutaneous leiomyomas does not appear to be related to estrogen or progesterone receptor-mediated effects.     

Regulation of human melanoma growth and metastasis by AGE-AGE receptor interactions

Advanced glycation end products (AGE), nonenzymatically glycated protein derivatives, have been implicated in the development and progression of diabetic angiopathies, including skin dermopathy. Nevertheless, the involvement of AGE in the development and progression of melanoma has not been fully elucidated. In this study we investigated the expression levels of their receptor for AGE (RAGE) in human melanoma and subsequently studied the effects of AGE on melanoma growth and migration. First, RAGE was detected in the cytoplasm of human melanoma cells (G361 and A375). Among the different types of AGE, glyceraldehyde- and glycolaldehyde-derived AGE significantly stimulated the growth and migration of human melanoma cells. Furthermore, tumor formation of melanoma cell xenografts in athymic mice was prevented by treatment with anti-RAGE neutralizing antibodies. In tumor-bearing mice, survival rates were prolonged, and spontaneous pulmonary metastases were inhibited by treatment using anti-RAGE neutralizing antibodies. In addition, all AGE were present in beds of human melanoma tumor, whereas they were barely detected in normal skin. These results suggest that AGE might be involved in the growth and invasion of melanoma through interactions with RAGE and represent promising candidates for assessing the future therapeutic potential of this therapy in treating patients with melanoma.     

The distribution of estrogen receptor beta is distinct to that of estrogen receptor alpha and the androgen receptor in human skin and the pilosebaceous unit

Both estrogens and androgens play important parts in skin and hair physiology, although studies of estrogen action in human skin have been rather limited. Recently, a second estrogen receptor (beta) has been identified in many nonclassical target tissues, including androgen-dependent tissues. Therefore, we have revisited the role of estrogens in human skin and hair by comparing the pattern of expression by immunohistochemistry for both estrogen receptors (alpha and beta) and the androgen receptor. Immunolocalization of androgen receptors was only seen in hair follicle dermal papilla cells and the basal cells of the sebaceous gland. Little specific staining of estrogen receptor alpha was seen anywhere except the sebaceous gland. In contrast estrogen receptor beta was highly expressed in epidermis, blood vessels, and dermal fibroblasts, whereas in the hair follicle it was localized to nuclei of the outer root sheath, epithelial matrix, and dermal papilla cells. Serial sections also showed strong nuclear expression of estrogen receptor beta in the cells of the bulge, whereas neither estrogen receptor alpha or androgen receptor was expressed. In the sebaceous gland, estrogen receptor beta was expressed in both basal and partially differentiated sebocytes in a similar pattern to estrogen receptor alpha. There was no obvious difference in the expression of either estrogen receptor in male or female nonbalding scalp skin. The results of this immunohistochemical study propose that estrogen receptor beta and not estrogen receptor alpha is the main mediator of estrogen action in human skin and the hair follicle. Further studies with androgen-dependent skin are required to determine whether estrogen receptor beta has a regulatory role on androgen receptor expression in the hair follicle in parallel with its role in other androgen-dependent tissues.     

Characterization of the glucocorticoid receptor in human skin

Because of the profound importance glucocorticoids have in dermatologic therapy, we studied the glucocorticoid receptor in human skin. A cytosol fraction was prepared from frozen skin by homogenization and centrifugation. When reacted with [3H]dexamethasone, this cytosol contained saturable, low-capacity binding. The glucocorticoid binding was stabilized by a protease inhibitor, phenylmethylsulfonylfluoride, and by sodium molybdate and was destroyed by trypsin. Sedimentation analysis of the glucocorticoid binding protein showed an 8S to 4S transition in high salt, a property of many known steroid hormone receptors. The binding was steroid specific, supporting the conclusion that this binding protein was a glucocorticoid receptor. The receptor molecule had a frictional ratio of 1.60 and a Mr of about 226,000 under low-salt conditions (0.05 M KCl) and a frictional ratio of 1.86 and a Mr of about 100,000 under high-salt conditions (0.3 M KCl) consistent with a nonglobular, elongated molecule. Isoelectric focusing showed that the receptor had 2 molecular species with isoelectric points of approximately 5.8 and 7.5. Quantitation of receptor in human skin showed 4-7 times more receptors in the epidermis and papillary dermis than in the lower dermis and nearly equal numbers in epidermis and papillary dermis. The concentration of receptors varied in different anatomic areas, with male foreskin showing the highest concentration, followed by female face, breast, and abdominal skin. Interestingly, the concentration of glucocorticoid receptors also varied with age; the highest levels were present at the extremes of life and a significantly lower level at midlife.     

Human scalp hair: Modulation by various factors and hormones do estrogens inhibit or stimulate—A perplexing perspective

Several journal reports, reviews, and commentaries over the last 20‐25 years have pointed out the controversy attached to 17β‐estradiol's inhibitory or stimulatory influence on hair follicle growth/cycling citing rodent (murine) and human results. While 17β‐estradiol is the most potent sex steroid hormone in the body and has almost equal affinity for estrogen receptor (ER) alpha (α) and beta (β), there appears to be specific ER‐mediated effects on scalp hair follicles/growth, etc. Additionally, the newly discovered G protein‐coupled estrogen receptor (GPR30 or GPER) and the orphan receptor, estrogen‐related receptor (ERR) gamma (γ), in skin and other tissue sites have potential impacts of how estrogens via these receptors may alter scalp hair characteristics, but this remains to be elucidated. Conversely, the negative impact of the 5α‐reductase enzyme and its steroid product, 5α‐dihydrotestosterone, on scalp hair growth is clear. Less clear is how 17β‐estradiol is stimulatory in some scalp hair studies, but inhibitory in others. This brief summary examines the potential influences of steroidogenesis via aromatase (estrogen biosynthesis) and 5α‐reductase expression, their enzyme activities, and steroid products along with the concepts of how steroid acute regulatory protein (StAR) and estrone sulfate may be involved in the complex hormonal, cellular/molecular signaling cascade of the hair follicle in growth and cycling.     

Expression of estrogen-related receptor gamma (ERRgamma) in human skin

Skin is a non-classical target for estrogens. Despite evidence showing that estrogen receptors (ER) are expressed in skin, there are still extensive gaps in our understanding of how estrogens exert their action in non-reproductive tissues. Estrogen-related receptor gamma (ERRgamma), an orphan member of the nuclear receptor superfamily, shows a strong sequence homology with estrogen receptor alpha but it does not bind estradiol. Here, for the first time, we demonstrate the expression of ERRgamma in adult human skin. ERRgamma mRNA was detected in the keratinocytes and fibroblasts of 8 female donor skins using RT-PCR. The presence of the protein was confirmed using immunohistochemistry on 11 adult human skins and Western Blotting on monolayer-cultures of fibroblasts and keratinocytes from respectively 4 and 2 donors. This study shows that ERRgamma is expressed in human skin and could intervene in a potentially new estrogen signaling pathway in the skin.     

Estrogen receptor status in malignant melanoma

Estrogen receptor (ER) positivity demonstrated in malignant melanomas by histochemical and biochemical assays suggested the possibility of hormonal management and improved prognosis as for breast carcinoma patients. We studied the ER status of 5 primary and 28 metastatic malignant melanomas with a commercial immunohistochemical kit (ER-ICA monoclonal), that utilizes monoclonal anti-ER and a peroxidase-antiperoxidase technique, and by a histochemical method using fluorescein-conjugated estradiol (Fluoro-Cep Estrogen assay), on frozen sections. In addition, we conducted a biochemical assay [dextran-coated charcoal cytosolic assay (DCC)] in 16 cases. All 33 cases were ER negative by ER-ICA and Fluoro-Cep: 11 biochemical assays were negative (less than 3 fmol ER/mg protein), four were in the borderline range (3 to 10 fmol ER/mg protein), and one was positive (greater than 10 fmol ER/mg protein) at 11 fmol. The melanomas in 97% of the cases we studied were ER negative by two or three different assays. Low-level estrogen binding of MM tissues may be the result of interactions other than with Type I true ER. The low frequency of ER positivity of malignant melanomas appears to preclude the clinical use of ER status as an indicator for response to hormonal manipulation in patients with malignant melanoma.     

Steroid hormone receptors in human melanoma

Summary Human melanomas were investigated for the presence of highaffinity estrogen-, gestagen-, and glucocorticoid-binding proteins. A statistically significant difference was found for mean estrogen receptor (ER) concentrations in melanomas of male versus female origin: female origin 37.6 (0–107) fmol/mg protein, male origin 3.9 (0–8.3) fmol/mg protein. No significant difference between sexes was found for gestragen receptors: 41.5 (0–194) fmol/mg protein for melanomas of female origin versus 99 (0–362) fmol/mg protein for male.Sucrose density gradient analyses revealed specific binding for both receptor types in the 4–5 S region as well as in the 8 S region. The binding affinities were in the same order of magnitude as reported for receptors found in typical steroid target organs.No significant difference in receptor values depending on sex was found for the glucocorticoid receptor: 19.2 (0–43) fmol/mg protein.     

Fibroblast matrix and surface components that mediate cell-to-cell interaction with lymphocytes

The interaction between lymphocytes and fibroblasts in vitro has been examined using a quantitative ELISA assay to measure the binding of T and B cells to monolayer cultures of human dermal fibroblasts. This was carried out on microtiter culture plates, using an anti-Thy-1 monoclonal antibody, to determine the attachment of murine T lymphocytes and an affinity-purified polyclonal anti-IgM antibody to measure B cell binding. Both types of lymphocyte were found to adhere strongly to intact human fibroblasts, and also had high levels of attachment to purified fibroblast plasma membranes and extracts of the fibroblast extracellular matrix. Attachment, particularly of B lymphocytes, also took place onto plastic surfaces coated with fibronectin, but not to collagens or to intact fibroblasts that had been fixed with a low concentration of paraformaldehyde. Lymphocyte binding to fibroblasts was partially prevented by a monoclonal antibody against fibroblast MHC class II antigens, but not against the class I membrane complex, or by polyclonal antiserum to the cell surface mannose 6-phosphate receptor. In addition, although both lymphocyte types were able to adhere to fibro-nectin, the presence of antibody against fibronectin or the synthetic peptide Arg-Gly-Asp-Ser, had no effect on their attachment to fibroblasts. Thus, lymphocyte adhesion may occur by fibronectin, but other types of interactions with fibroblasts also appear to take place.     

Increasing epidermal growth factor receptor expression in human melanocytic tumor progression.

Different results have been reported on the expression of epidermal growth factor receptor (EGFR) in human melanocytic lesions, which may be due to different methodologic approaches. Therefore, we compared EGFR expression in six human melanoma cell lines by utilizing the monoclonal antibodies 2E9, 425, and 225, applying four immunocytochemical staining procedures. The results were compared with those obtained by a multiple point ligand binding assay. In addition, Northern blot analysis was performed. A three-step immunoperoxidase method using the monoclonal antibody 2E9 proved most sensitive. Staining intensities, estimated semiquantitatively, correlated well with the quantitative data obtained by the ligand-binding assay. Expression on the mRNA level was also in agreement with these results. Immunohistochemical staining of a large series of human cutaneous melanocytic lesions using the method selected showed differential EGFR expression in various stages of melanocytic tumor progression: 19% of common nevocellular nevi; 61% of dysplastic nevi, 89% of primary cutaneous melanomas, and 91% of melanoma metastases showed staining of the melanocytic cells. Intralesional heterogeneity of EGFR expression was present. Although the mean percentage of positive melanocytic cells in positive lesions did not increase with progression, mean staining intensity was stronger in malignant lesions compared to benign lesions. Ligand binding assays showed that EGFR expression in the highly metastasizing cell lines MV3 and BLM was at least 40 times higher than in the cell lines IF6, 530, M14, and Mel57, which do not or only sporadically metastasize after subcutaneous inoculation in nude mice. Although the differences between the various stages of progression are not absolute, we provide further evidence that EGFR expression increases in human melanocytic tumor progression.     

Agonists of peroxisome proliferator-activated receptor gamma inhibit cell growth in malignant melanoma

Peroxisome proliferator-activated receptor gamma is a member of the nuclear receptor superfamily involved in adipocyte differentiation and glucose homeostasis. There is evidence that peroxisome proliferator-activated receptor gamma may also act as a tumor suppressor. Here, we demonstrate expression of peroxisome proliferator-activated receptor gamma in benign melanocytic naevi, different variants of primary cutaneous melanomas, and melanoma metastases. Peroxisome proliferator-activated receptor gamma protein and peroxisome proliferator-activated receptor gamma1 mRNA were also detected in human melanoma cell lines. The peroxisome proliferator-activated receptor gamma specific agonists 15-deoxy-Delta12,14-prostaglandin J2, troglitazone, and rosiglitazone dose-dependently inhibited cell proliferation in four melanoma cell lines, whereas a specific agonist of peroxisome proliferator-activated receptor alpha had no such effect. At a concentration of 50 microM rosiglitazone, the most potent peroxisome proliferator-activated receptor gamma agonist tested suppressed cell growth by approximately 90%. Apoptosis could be induced in melanoma cell lines by incubation with tumor-necrosis-factor-related apoptosis-inducing ligand. In contrast, the growth inhibitory effect of peroxisome proliferator-activated receptor gamma activation was independent of apoptosis and seemed to occur primarily through induction of cell cycle arrest. Our data indicate that melanoma cell growth may be modulated through peroxisome proliferator-activated receptor gamma.     

大麻素2型受体在皮肤恶性黑素瘤中的表达

目的 探讨大麻素2型受体(CB2受体)在色素痣及皮肤恶性黑素瘤中的表达规律及其意义.方法 免疫组化、RT-PCR检测色素痣和恶性黑素瘤组织中CB2受体在蛋白和mRNA不同水平的表达情况.结果 CB2受体在色素痣和恶性黑素瘤均有表达;在色素痣主要表达分布于痣细胞和表皮层的基底细胞层,在皮下组织中表达不明显;皮肤恶性黑素瘤与色素痣表达CB2的强度间差异有统计学意义(P<0.05).结论 恶性黑素瘤中CB2受体在蛋白和基因水平表达均升高,恶性黑素瘤CB2受体表达强度明显大于色素痣.     

Melanosomal antigenic expression on the cell surface and intracellular subunits within melanogenic compartments of pigment cells: analysis by antimelanosome-associated monoclonal antibody

Antimelanosome-associated monoclonal antibody has recognized the common antigenic determinant of melanosomes and cell surface of pigment cells, and it is suggested that melanosomes play a significant role as an antigen in progressive depigmentary disorders, in which melanocytes are selectively altered and disappear presumably by auto-antibodies in vivo. Mouse myeloma cells were fused with spleen cells from BALB/c mice immunized with a melanosomal fraction separated from human melanotic melanoma cells (Mm-1-JCK). The monoclonal antibody (MoAb) A4F11 has been found to react with premelanosomes, melanosomes, and probably with Golgi-associated endoplasmic reticulum lysosomes, but not with mitochondria, nuclei, and cytosol from human melanoma cells, by immunoelectron microscopy using the saponin permeation method, which was carried out together with indirect radioimmunoassay and quantitative absorption assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using melanosome preparations have revealed the antigen(s) reactive with the MoAb A4F11 in 3 bands corresponding to Mr 50,000, 18,000, and 17,000. Cell binding assay has shown the reactivity of the MoAb A4F11 with the cell surface of human normal melanocytes and melanoma cells, but not with other mammalian melanoma cells or with human nonpigment cells examined. Indirect immunofluorescence on cultured cells and frozen sections has revealed distinct granular reactivity not only with human melanotic melanoma, but also with junctional and intradermal nevi, cultured malignant blue nevus cells, as well as normal melanocytes. The above evidence has indicated the presence of an antigenic determinant common to the intracellular melanogenic compartments and to the cell surface of human pigment cells, regardless of their oncogenic differentiation status.     

Expression of p16 protein in acral lentiginous melanoma

Background Acral lentiginous melanoma (ALM) is a clinicopathologic subtype of cutaneous malignant melanoma. ALM is the most common type of melanoma amongst Asians, Africans, and patients with mixed ancestry. In Brazil, ALM comprises 10% of cutaneous melanoma. ALM develops on palmar, plantar, and subungual skin, and its biology is different from that of other cutaneous melanomas, where sunlight is the major known environmental determinant. Alterations and inactivation of the p16INK4 gene that encodes a specific inhibitor of cyclin‐dependent kinase have been related to melanoma genesis and progression. Few studies, however, have addressed p16 expression in ALM. Methods In order to verify and compare p16 protein expression, 32 paraffin‐embedded ALM specimens were subjected to a immunohistochemical technique using a monoclonal anti‐p16 antibody. The tumors were classified according to thickness (up to 1.0 mm and thicker than 1.0 mm) and the presence of ulceration. Results Twenty‐five (78%) ALMs displayed positive p16 protein expression: 21 of the 25 (84%) with a thickness of more than 1.0 mm, and four of the seven (57%) with a thickness of 1.0 mm or less. Thirteen of the 17 (76%) nonulcerated lesions and 12 of the 15 (80%) ulcerated lesions displayed positive p16 protein expression. Conclusion The data obtained suggest that p16 protein expression per se may not represent a marker of retinoblastoma protein (pRb) pathway disturbance in ALM tumorigenesis.     

Distribution of an estrogen receptor-related protein (P29) in normal skin and in cultured human keratinocytes

A monoclonal antibody, ERD5, which recognizes a 29Kd phosphoprotein associated with human estrogen receptor of myometrium was used to study the expression of this protein in normal skin and in cultured human keratinocytes. By indirect immunofluorescence, both in vivo and in vitro keratinocytes showed a variable cytoplasmic staining which increased with cell differentiation. SDS gel electrophoresis of soluble extracts of cultured keratinocytes and normal epidermis showed that P29 was a minor protein. Immunoblot analysis demonstrated that ERD5 strongly reacted only with a 29Kd polypeptide band without any cross-reactivity. These data suggest that keratinocytes might be estrogen sensitive like other cells in which P29 has already been located. The exact role of this protein in the keratinocyte differentiation process and its relationship with estrogen receptors remain to be elucidated.