Tissue distribution of a melanoma-associated antigen D-1 immunogenic in patients with melanoma.

A human melanoma-associated antigen D-1 was recently identified by screening an expression cDNA library derived from mRNA of cultured melanoma cells with sera of melanoma patients. The aim of this study is to present in vivo expression and precise distribution of D-1 in normal tissues and benign or malignant neoplasms. By in situ hybridization, we found that the D-1 mRNA was exclusively expressed in the cytoplasms of melanoma cells, but not in keratinocytes, fibroblasts and lymphocytes adjacent to melanoma nests. Further immunohistochemical studies revealed that the expression of D-1 antigen was distributed to both the surface and cytoplasm of melanoma cells, indicating that D-1 antigen can be recognized by killer T lymphocytes or antibodies in vivo. No significant mRNA nor peptide of D-1 was detected in basal cell carcinoma, squamous cell carcinoma and other benign tumors such as melanocytic nevi and seborrheic keratosis. We also confirmed that D-1 mRNA and peptide were not expressed in normal organs by dot blot hybridization and western blot analysis, respectively. These results will assess the suitability of recombinant D-1 protein to implement active specific immunotherapy against melanoma.

Molecular genetic analysis of HLA class II alleles in Japanese patients with melanoma.

Distribution of HLA-DQA, -DQB and -DPB alleles in ninety-six Japanese patients with melanoma was analyzed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, and the association between clinical parameters and the presence of certain HLA class II alleles investigated. The frequency of HLA-DQB1*0302 was increased, while those of DQA1*0101(04), -DQA1*0401 and DRB1*0802 were decreased in melanoma patients compared with controls. Moreover, the frequency of HLA-DQA1*0103 in patients with acral lentiginous melanoma was increased compared with controls. However, none of these HLA class II alleles showed significant positive or negative associations after correction of the P value. In addition, there was no correlation between these antigens and clinical parameters. These results indicate that HLA class II alleles may not contribute to a strong susceptibility to melanoma in the Japanese.

Induction of temporary beating in paralyzed flagella of Chlamydomonas mutants by application of external force.

To help understand the mechanism by which the sliding movement of outer-doublet microtubules in cilia and flagella is converted into bending waves, we examined the effect of mechanical force imposed on the flagella of Chlamydomonas mutants lacking the central pair or multiple dyneins. These mutants were almost completely nonmotile under normal conditions. A bend was produced in a flagellum either by holding a cell with a micropipette and quickly moving it with a piezoelectric actuator; or by pushing a flagellum with a microneedle. After removal of the external force, mutants lacking the central pair (pf18 and pf19) displayed beating at irregular intervals of > 1 second for one to several cycles. Similarly, a double mutant (ida2ida4) lacking four species of inner-arm dynein displayed beating at intervals of > 0.1 second for up to 80 cycles. However, paralyzed flagella of double mutants that lack the outer dynein arm in addition to the central pair or the inner dynein arm did not show cyclical movements upon application of external force. These results indicate that the central pair and the inner dynein arm are important for both stable bend formation at the base and efficient bend propagation along the flagellar length. They also suggest that the outer dynein arm, and not the inner dynein arm, enables the flagellar axoneme to propagate bends independently of the central pair. We propose that the axoneme is equipped with two independent motor systems for oscillatory movements: an outer-arm system controlled by the axonemal mechanical state independently of the central pair/radial spoke system, and an inner-arm system controlled by both the axonemal mechanical state and the central pair/radial spokes.

HLA class I antigens in Japanese patients with melanoma.

In this study, we analyzed the frequencies of human leukocyte antigen (HLA) class I alleles in 110 Japanese patients with melanoma using serological methods, and compared such frequencies with clinical parameters. As expected, frequencies of HLA allele distribution in patients with melanoma reflected the frequencies observed in the normal Japanese population. Because these are different from populations belonging to other races (e.g., white), it followed that the HLA allele distribution in melanoma patients varies among different races. This differences may have significant implications for T-cell-mediated, HLA-restricted therapeutic modalities. No significant associations between HLA and clinical parameters were noted in this study. This report may help design future clinical trials involving therapeutic approaches based on HLA-restricted mechanisms.

Deletion of specific protein kinase C subspecies in human melanoma cells.

It has been shown that tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulates the proliferation of normal human melanocytes, whereas it inhibits the growth of human melanoma cell lines. The expression of protein kinase C (PKC) subspecies, the major intracellular receptors for TPA, was examined in normal melanocytes and the four melanoma cell lines HM3KO, MeWo, HMV-1, and G361. PKC was partially purified and then separated into subspecies by column chromatography on Mono Q and hydroxyapatite successively, and finally subjected to immunoblot analysis using antibodies specific for the PKC subspecies. Of the PKC subspecies examined, delta-, epsilon-, and zeta-PKC were detected in both normal melanocytes and the four melanoma cell lines. In contrast, both alpha-PKC and beta-PKC were expressed in normal melanocytes, whereas either alpha-PKC or beta-PKC was detected in melanoma cells. Specifically, HM3KO, MeWo, and HMV-1 cells were shown to contain alpha-PKC but not beta-PKC, while G361 cells expressed beta-PKC but not alpha-PKC. The growth of these melanoma cells was suppressed by TPA treatment, and the growth of the G361 cells lacking alpha-PKC was inhibited more efficiently than the other melanoma cell lines which lacked beta-PKC. It was further shown that beta-PKC was not detected in freshly isolated human primary or metastatic melanoma tissues. These results suggest that the expression of alpha-PKC or beta-PKC may be altered during the malignant transformation of normal melanocytes and that loss of alpha-PKC or beta-PKC may be related to the inhibitory effect of TPA on the growth of melanoma cells.

Tissue distribution of a melanoma-associated antigen immunogenic in patients with melanoma as analyzed by polyclonal antibodies to recombinant peptide antigen.

This study aimed to detect in vivo expression of human melanoma-associated antigen D-1, which was identified by screening an expression cDNA library constructed from mRNA extracted from cultured melanoma cells with sera from patients with melanoma. The tissue distribution of D-1 antigen was then analyzed. Murine anti-D-1 recombinant peptide polyclonal antibodies were raised by immunization of in vitro synthesized D-1 peptide against Balb/c mice and applied immunohistochemically on paraffin-embedded tissue specimens. D-1 antigen was found to be restrictedly expressed on melanoma cells, but not on normal melanocytes, adjacent keratinocytes, fibroblasts, lymphocytes and adnexal structures of skin. The reactivities of anti-D-1 antibodies did not correlate with histogenesis of the lesions, their ability to produce melanin, and/or their primary or metastatic nature. There was no positive reactivity of anti-D-1 antibodies with other skin tumors, including squamous cell carcinoma, basal cell epithelioma, seborrheic keratosis, and nevus cell nevus. Further, cytoplasmic expression of D-1 antigen in melanoma cells was observed only in a certain subgroup of patients with melanoma. This indicates that the cell surface expression of D-1 peptide requires specific transporting proteins, such as HLA molecules.

Tissue distribution of an immunogenic melanoma associated antigen, D-1.

A human melanoma-associated antigen immunogenic in patients was recently identified by screening an expression cDNA library constructed from cultured human melanoma cell line with sera from patients with melanoma. The nucleic acid sequence of the cloned D-1 cDNA has no significant homology with previously reported mammalian genes. The cDNA D-1 encodes a peptide of about 37 kDa, which showed fivefold higher reactivity with sera from patients with melanoma than with sera from normal donors. In order to detect D-1 gene expression in vivo, in-situ hybridization and immunostaining with cRNA probe and murine anti-D-1 sera were carried out on surgically removed tissues. Digoxigenin-labeled cRNA D-1 was exclusively hybridized with mRNA in the cytoplasm of melanoma cells but not with keratinocytes and fibroblasts adjacent to melanoma nests. Polyclonal anti-D-1 antibodies were obtained by immunization of Balb/c mice with recombinant D-1 peptide and clearly reacted with melanoma cells but not with keratinocytes and fibroblasts, similar to the results of in-situ hybridization. The above information will help to assess the suitability of recombinant D-1 peptide to implement active specific immunotherapy in patients with melanoma.

The altered expression of alpha-smooth muscle actin in basal cell epithelioma and its surrounding stroma: with special reference to proliferating cell nuclear antigen expression and adenoid differentiation.

Altered expression of alpha-smooth muscle actin (alpha-SMA) is known to indicate the morphological, tumorigenic and immunological changes occurring in tumour and stromal cells. The purpose of this study was to analyse the dynamics of alpha-SMA expression in human basal cell epithelioma (BCE) cells and their surrounding stromal cells, in the process of differentiation towards cutaneous appendages such as hair, sebaceous, apocrine and eccrine glands. Using anti-alpha-SMA specific monoclonal antibody (MAb), 17 of 36 BCEs (47%) were shown to express alpha-SMA, despite the usual absence of alpha-SMA in all eukaryotic cells except muscle cells. Solid, adenoid and sclerosing types of BCE expressed alpha-SMA more frequently, and in greater amount, than cystic, keratotic and superficial types. Furthermore, the expression of alpha-SMA in BCE cells significantly paralleled the expression of proliferating cell nuclear antigen (PCNA) in these cells. Thus, the altered expression of alpha-SMA may reflect the growing properties of BCE cells under the specific cellular regulations for differentiation. In addition, we have found anti-alpha-SMA MAb-positive fibroblasts with smooth muscle differentiation (myofibroblasts) in desmoplastic stroma surrounding BCE nests in 13 of 36 cases (36%). Coincidental expression of alpha-SMA in both BCE cells and stromal cells was found in nine of the 13 cases (69%), indicating the possibility of the induction of myofibroblastic stromal changes in surrounding tissues by cytokines secreted from BCE cells [e.g. basic fibroblast growth factor (bFGF)-like factor].

Head movement changes apparent depth order in a motion-parallax display.

The hypothesis that the apparent visual depth is determined by the proximal velocity relative to the position of the head was examined in three experiments. Apparent protrusion/recession changed when subjects observed a moving random-dot pattern with their heads tilted sideways or rotated in the horizontal plane. This is ascribed to lateral head movement, which increases the proximal velocity when the dots and the subjects' heads are moving in opposite directions, and decreases the proximal velocity when both are moving in the same direction. Changes in the direction of movement of the stimulus caused a reversal of the apparent protrusion/recession. The resultant proximal velocity of the stimulus determined the order of depth of surfaces when the movement of the stimulus was linked to the subject's head movement.

Dysplastic nevus syndrome: melanoma-prone disease.

Regardless of subsequent clinical courses of patients with dysplastic nevi (DN), substantial evidence supporting DN as one of the melanoma-prone diseases is not yet available, especially in sporadic DN, due to the lack of genetic information other than retrospective studies in clinical observation. This study aimed at the immunohistological characterization of sporadic DN distinct from common nevi (CN) and at the evaluation of the potentially of sporadic DN for malignant transformation. We considered our results together with previous immunological and epidemiological reports. We noted the following three immunohistological characteristics. 1) Proliferating cell nuclear antigen (PCNA), one of the markers for active cell division, could be detected on DN cells in junctional nests of only one among ten DN examined but not on CN cells at all. 2) The altered expression of alpha-smooth muscle actin (alpha-Sma), often observed in melanoma cells, could not be detected in DN cells. However, anti-alpha-Sma monoclonal antibody (MoAb) clearly demonstrated distinctive hypervascularity in the stroma surrounding DN when compared with CN. 3) ME491 antigen, which is known to be expressed mainly in the radial growth phase of melanoma, was detected with similar intensity on both DN and CN. These data indicate that DN has a somewhat higher potentiality than CN for cell division and secretion of some cytokines which can induce hypervascularity in the surrounding stroma, but that DN has not yet undergone the significant phenotypic changes observed in melanoma cells. Further advancements in understanding molecular events in DN cells will be of great benefit in setting DN in the multiple oncogenic spectrum from pigment cells to melanoma.

Melanoma-associated antigen synthesized in vitro for active specific immunotherapy.

The immunogenicity of the antigen molecule is a prerequisite for active specific immunotherapy for melanoma. Since most of the melanoma-associated antigens recognized by the murine immune system are known to be not immunogenic in man, a detection and analysis system for melanoma-associated antigens is required to reflect in vivo immune responses in patients with melanoma. One of the promising approaches, an attempt to develop human monoclonal antibodies from B lymphocytes of patients with melanoma, has met with limited success due to the difficulties of producing large amounts of antibodies and using them in immunochemical assays, because most of them belong to the IgM class and have low affinity. Our approach is to utilize the screening of a cDNA expression library constructed from mRNA extracted from cultured melanoma cells with antibodies from patients with melanoma. The cloned cDNA, designated as D-1, had 1029 bp and showed no significant homology with viral and mammalian sequences stored in GENETYX. cDNA D-1 hybridized to a 2.0 kb mRNA species from 3 different cell lines of human melanoma, neuroblastoma, erythroleukemia, B lymphoid, and T lymphoid cells, but not from a renal carcinoma cell line, normal peripheral lymphocytes, or normal fibroblasts. The in vivo expression and distribution of mRNA related to cDNA D-1 has been examined in tissue specimens by in situ hybridization and shown to be rather restricted on melanoma cells. The polypeptide antigen encoded by cDNA D-1 may be a valuable immunogen for implementing active specific immunotherapy in patients with melanoma.

Alpha-smooth muscle actin expression in tumor and stromal cells of benign and malignant human pigment cell tumors.

We examined the altered expression of alpha-smooth muscle actin (alpha-Sm) in human benign, pre-malignant, and malignant pigment cell tumors by immunohistochemical as well as biochemical (Western blot) analysis using anti-alpha-Sm monoclonal antibody (anti-alpha-Sm MoAb). The expression of alpha-Sm has been revealed immunohistochemically to be associated with mesodermal cells rather than with pigment cells. Western blot analysis using anti-alpha-Sm MoAb detected alpha-Sm expression as a 43-kD band in the extracts from normal papillary dermis, nevus cell nevus, and metastatic melanoma with stromal tissues, but not from primary melanoma with stromal tissues examined. The above findings of alpha-Sm expression by Western blot analysis were further characterized immunohistochemically in terms of the localization at the cellular level as follows. 1) In normal papillary dermis, pericytes encircling capillary vessels showed only positive staining with anti-alpha-Sm MoAb. 2) In nevus tissues, nevus cells were not shown to be positively stained, despite similar positivity of pericytes in normal papillary dermis. 3) In melanoma tissues, alpha-Sm expression of metastatic melanoma detected by Western blot analysis was found to be derived from fibroblasts with smooth-muscle differentiation (myofibroblasts), but not from melanoma cells. Such myofibroblastic stromal changes could not be found on primary melanoma tissue sections, which showed no reactivity in Western blot analysis. We conclude that the major sources of alpha-Sm in benign and pre-malignant pigment cell tumors are capillary pericytes, whereas alpha-Sm found in malignant melanoma tissue is primarily from melanoma-surrounding stromal fibroblasts that were changed to myofibroblasts by some cytokine factor(s), presumably secreted from melanoma cells.

Cloning and in vitro expression of a melanoma-associated antigen immunogenic in patients with melanoma.

The purpose of this study was to identify human melanoma-associated Ag (MAA) that are immunogenic in patients, because these molecules may be useful immunogens to implement active specific immunotherapy. To this end, an expression cDNA library constructed from the human melanoma cell line A375 was screened with sera from patients with melanoma. A 1029-bp cDNA (designated D-1) was isolated. Its nucleotide sequence showed no significant homology with viral and mammalian sequences stored in GE-NETYX. cDNA D-1 hybridized to a 2.0-kb mRNA species from human melanoma, neuroblastoma, erythroleukemia, B lymphoid, and T lymphoid cell lines but not from a renal carcinoma cell line, PBL, and cultured skin fibroblasts. The D-1 clone produced a fusion protein that displayed a significantly higher reactivity with sera from patients with melanoma than from healthy controls. Furthermore, D-1 fusion protein induced in mice antibodies that immunoprecipitated a 50-kDa component from cultured human melanoma cells. The structural properties of D-1 MAA are different from those of previously described MAA. These results suggest that the approach we have applied may be useful to identify novel MAA expressed by melanoma cells. Furthermore, the immunogenicity of recombinant D-1 protein suggests that it may be a valuable immunogen to implement active specific immunotherapy in patients with melanoma, if additional experiments show that it has the appropriate tissue distribution.

Reversals of visual depth caused by motion parallax.

The role of the velocity and direction of retinal movement in the determination of apparent depth from motion parallax was examined. Motion parallax was produced either by linking the movement of random-dots to head movement or by making this motion independent of the head movement. The results show that apparent depth was largely estimated from the velocity difference between the stimuli. The direction of retinal movement in the absence of head movement did not determine whether the pattern appeared to protrude or recede. Information about direction linked to head movement was able to stabilize protrusion/recession by providing a cue for the location of the fixation point. Depth reversal occurred less frequently in the presence than in the absence of head movement. When the fixation point shifted from the apparently protruding pattern to the apparently receding pattern, in both the presence and absence of head movement, depth reversal was readily observed.

A heterogeneous double antibody enzyme-linked immunoassay to measure beta-galactosidase fusion protein.

A rapid and sensitive enzyme-linked immunoassay (ELISA) to quantitate recombinant fusion proteins encoded by cloned cDNA in the bacteriophage lambda gt11 is described. Since the fusion protein is expressed in an equimolar ratio to beta-galactosidase, the assay derives the concentration of the recombinant protein in total bacterial lysates or pure preparations from the measurement of beta-galactosidase with an enzyme-linked immunoassay. This assay is a useful technique to measure the recombinant proteins for subsequent immunological and biochemical characterization.

Chicks' maze learning reinforced by visual pitfall extending downward.

The present study examined whether visually evoked fear of depth could reinforce a particular response of animals, i.e., to special maze learning. The maze was composed of four units of Y-shaped alley. In this maze, the visual pitfalls were set behind corners of the alley in place of a physical barrier. The experiments showed that eight of 13 male chicks could achieve the initial learning and that three successful ones could also achieve reversal learning. The results suggest that the visually evoked fear of depth provided by motion parallax can act as a reinforcer.

Japanese monkeys' cardiac responses to visual depth.

The present study examined the heart-rate changes which occurred in the visual-depth situation of Japanese monkeys (Macaca fuscata). Four animals were tested over the first four weeks of age in the visual pitfall designed as a modification of the visual cliff. The infant monkeys showed heart-rate reduction in the depth condition. This reduction was observed from the first week and became remarkable at the third week. There were no differences in heart-rate change between monocular and binocular vision. These findings suggest that the Japanese monkey can discriminate visual depth shortly after birth and that the heart-rate reduction depends upon the fear of visual depth, not upon the novelty of the situation.

Tyrosinase-positive melanocyte distribution and induction of pigmentation in human piebald skin.

White forelock and hypomelanotic macules of piebaldism have been revealed to have almost regularly distributed, dopa-positive melanocytes, though with lower density than normal, on separated epidermis despite previous reports describing few or no melanocytes in piebald spots. The melanocytes observed in piebald hypomelanotic spots seem to be classified into the following two types: (1) strongly dopa-positive and markedly hyperdendritic large cell type and (2) moderately dopa-positive and slightly hyperdendritic, oversized cell type. The former are primarily seen in hypomelanotic lesions, while the latter are seen in transitional lesions. The above difference seems to be associated with compensatory melanogenic function of melanocytes in vivo. Moreover, we have induced new hyperpigmented spots in hypomelanotic lesions, with the exception of white forelock, following therapy with oral methoxsalen plus ultraviolet A light.