Elastic fibres: histological correlation with orcein and a new monoclonal antibody, HB8

HB8 is a new monoclonal antibody, prepared against human lymphocytes, which has been found to react with elastic fibres, possibly the microfibrillar component. Samples of normal human and animal skin and human skin from various pathological conditions were subjected to immunolabelling with HB8, and alternate sections from each sample were stained by a standard orcein procedure. The individual fibres were clearly marked by the HB8 and this was confirmed by comparison with the standard elastic tissue stain. HB8 provides a new direct, practical and accurate method for the study of elastic tissue.     

A monoclonal antibody recognizing desmosomes: use in human pathology

A mouse monoclonal IgG1 antibody that recognizes desmosomes in human tissues has been isolated and characterized. DP1 recognizes desmoplakin I (250 kD) in immunoblots of partially purified desmosome preparations. In immunofluorescence microscopy DP1 reacts with desmosomes in epithelial cells from a variety of human tissues including skin, esophagus, uterus, intestine, and in liver hepatocytes. In axilla skin, the epithelial cells in epidermis, sweat glands, and in the outer hair follicles are strongly stained whereas relatively little staining is seen in the epithelial cells of the apocrine glands. Nonepithelial cells are not stained, although decoration of myocardial desmosomes is seen. In cells in culture DP1 is also specific for desmosomes in epithelial cells. Thus the mouse epidermal line HEL, bovine MBCK cells, as well as the human lines A431, HT29, and HeLa are positively stained by DP1. Desmosomes are retained in tumors of epithelial origin such as adenocarcinoma and squamous cell carcinoma. Thus DP1 should be a useful diagnostic marker in human pathology.     

A monoclonal antibody recognizing plasminogen and plasmin—altered reactivity in psoriatic lesions

To study the organization of the plasminogen activator/plasminogen-plasmin (PA/PG-P) system in human epidermis we raised monoclonal antibodies with specificity for human plasminogen and plasmin (PG-P). Monoclonal antibody P2, which appeared most suitable for immunohistology, is a mouse monoclonal antibody of IgG1 subtype, specific for the precursor enzyme plasminogen and for the high molecular weight chain of the active enzyme, plasmin. Immunofluorescence analysis of normal human epidermis revealed that reactivity with P2 was confined to the basal cell layer. In psoriatic lesions, however, this regional organization was not found. Immunoreactivity in this case was scattered throughout all the hyperproliferating cell layers, which is taken as evidence for an altered distribution of PG-P in psoriatic lesions. In psoriasis other components of the PA/PG-P system have previously been found to be altered. In this context our findings add to the hypothesis that this system may be involved in the pathology or the pathogenesis of psoriatic lesions.     

Omalizumab: a recombinant humanized monoclonal IgE-blocking antibody

Omalizumab (Xolair) is a recombinant humanized monoclonal antibody that blocks the high-affinity Fc receptor of immunoglobulin E (IgE). It is used to treat patients with moderate-persistent to severe-persistent asthma; patients must be older than 12 years, have a positive skin test to a perennial aeroallergen (e.g., dust mites, cats, dogs, and mold), and be symptomatic with inhaled corticosteroids. Omalizumab has a low incidence of side effects, and it is very expensive. The exact dosage of omalizumab dosage is determined by body weight and pretreatment serum total IgE levels. Omalizumab may have a role in the treatment of atopic dermatitis when IgE plays a causal role.     

Michelin tyre syndrome: a congenital disorder of elastic fibre formation?

We describe a 15-month-old girl with Michelin tyre syndrome. She had hirsuties and marked skin folds. Histological examination showed fragmented elastic fibres in addition to smooth muscle hamartoma. On electron microscopy, decreased deposition of elastin was observed. We speculate that elastic fibre abnormafities may account for the characteristic skin changes in the Michelin tyre syndrome.     

Congenital agminated Spitz nevi: immunoreactivity with a melanoma-associated monoclonal antibody

Agminated, or grouped Spitz nevi are a curious expression of nevomelanocytic growth; and represent an uncommon manifestation of these nevi. We encountered an example of agminated Spitz nevi present at birth, which showed progression and rapid growth over months. All showed histologic Spitz nevus features. Avidin-biotin immunohistochemical techniques using anti S-100 protein, neuron-specific enolase (NSE), and melanoma-specific monoclonal antibody, HMB-45 labelled the nevi as follows: diffuse S-100 and NSE staining in most and HMB-45 positivity in sporadic cells deep in the dermis. HMB-45 reactivity occurred in both spindle cells and epithelioid forms.     

3F11单克隆抗体检测人类疱疹病毒8型潜伏相关核抗原的表达

人类疱疹病毒8型(HHV-8)又称卡波西肉瘤(KS)相关疱疹病毒(KSHV),由Chang于1994年从AIDS患者的卡波西肉瘤组织中首次鉴定[1].该病毒与KS、原发性渗出性淋巴瘤(PEL)等肿瘤的形成密切相关,其中与KS关系最为密切.由HHV-8潜伏期主要基因开放阅读框73(ORF73)编码的潜伏相关核抗原(LANA)是一个多功能蛋白,在HHV-8感染的各种类型KS细胞中均有表达[3],LANA一方面长期维持病毒DNA在宿主细胞中的传代,另一方面通过控制宿主细胞转录调节蛋白、抑癌基因产物如p53、pRb、NF-kB等表达,促进KS形成[3].     

A monoclonal antibody labelling the keratinocyte membrane: a marker of epidermal differentiation

A murine hybridoma secreting an IgM monoclonal antibody (KL3) was produced by cell fusion of mouse myeloma cells with spleen cells from mice immunized with human epidermal keratins. On normal human epidermis KL3 stained the intercellular spaces from the stratum germinatum to the stratum granulosum with a fluorescence intensity increasing from the basal layer to the upper layers. Basal cells were not stained on the side facing the basement membrane. About 90% of free keratinocytes isolated after trypsinization were labelled by KL3 in a punctate staining. Immunoelectron microscopy allowed us to show that the antigen recognized by KL3 was exclusively localized on the keratinocyte membrane especially in the desmosomal plaques. KL3 reactivity was not modified by preincubation of skin sections with lectins showing a selective intercellular labelling of upper layers of epidermis or pemphigus antisera, nor by adsorption of the antibody on NP40 soluble proteins of the epidermis. Though KL3 reactivity was completely abolished after adsorption of purified keratins, no immunological reactivity of KL3 was detected with epidermal keratin polypeptides blotted on nitrocellulose paper. In psoriatic epidemis and epidermal tumors KL3 reactivity was drastically modified. These results suggest that KL3 recognized a keratinocyte membrane antigen implied in the epidermal differentiation process.     

Anti-elastofibril monoclonal antibody NKH-1: production and application

A new monoclonal antibody NKH-1 was developed using human subepidermal basement membrane zone substances as immunogen. NKH-1, IgG1 kappa light chain, labeled proteins in the subbasement membrane zone in a linear fashion. It also labeled oxytalan fibers and elaunin fibers in the papillary dermis. Mature elastic fibers were labeled only in their peripheral microfibrils (elastofibrils) and the center core of elastin was nonreactive. Basal lamina itself was not decorated with NKH-1 even at the immunoelectron microscopic level. Skin appendages such as eccrine and apocrine glands, arrector pili muscle, hair follicle, and sebaceous gland were surrounded with NKH-1-positive substances. This substance was in linear fashion closely associated with the basal lamina but deposited linearly outside of it. Species specificity tests were performed only in nonprimates: mouse and guinea pig skins were nonreactive with NKH-1. NKH-1 seems to recognize a new substance in the subbasal lamina region (subbasal lamina proteins) which crossreact with elastic fiber microfibrils.     

MON-150, a versatile monoclonal antibody against involucrin: characterization and applications

Summary A monoclonal antibody, designated MON-150, was found serendipitously to react strongly with the granular layer of normal human epidermis and with the upper spinous layers of psoriatic epidermis. From analysis by flow cytometry of cultured human keratinocytes, it appeared that the percentage of MON-150-positive cells strongly increased when the cells reached confluence and the growth fraction declined. To identify the antigen recognized by MON-150, a lysate of human keratinocytes was subjected to affinity chromatrography using a MON-150 Sepharose column. This yielded a single protein of approximately 350 kDa as measured on Superose 6 FPLC gel permeation chromatography using nondenaturing conditions. In Western blot analysis under denaturing and reducing conditions, a 140-kDa protein was detected. The subcellular localization and the molecular weight of the antigen recognized by MON-150 suggested that the antigen involved might be involucrin. This was confirmed using a commercial polyclonal antiserum against involucrin. We conclude that MON-150 is a new, versatile antibody against human involucrin.     

Skin as a potential organ for ectopic monoclonal antibody production

The therapeutic potential of monoclonal antibodies for treating a variety of severe or life-threatening diseases is high. Although intravenous infusion appears the simplest and most obvious mode of administration, it is not applicable to many long-term treatments. It might be advantageously replaced by gene/cell therapies, however, rendering treatments cost-effective and eliminating the short- and long-term side-effects associated with injection of massive doses of antibodies. We have tested whether skin can potentially be used as an organ for production and systemic delivery of ectopic antibodies. Normal human primary keratinocytes were shown to be capable of synthesis and secretion of a model monoclonal antibody directed against human thyroglobulin upon retroviral gene transduction in vitro. Neo- epidermis reconstructed in vitro, either in cell culture inserts or on dermal substrates, from such modified keratinocytes also produced the monoclonal antibody. Interestingly, the latter could cross the epidermis basal layer and be released in culture fluids. Finally, grafting of epidermis reconstituted in vitro on dermal substrates to SCID mice permitted sustained monoclonal antibody delivery into the bloodstream to be achieved. Our data thus show that genetically engineered keratinocytes can potentially be used for genetic antibody-based immunotherapies. They also indicate that proteins as big as 150 kDa, after release by engineered keratinocytes into skin intercellular spaces, can migrate to the general circulation, which is potentially important for a number of other gene-based therapies.     

Detection of Treponema pallidum by a fluorescent monoclonal antibody test

Definitive diagnosis of early syphilis currently requires dark-field microscopy and/or a newly reactive serologic test for syphilis. The efficacy of dark-field microscopy depends on the availability of a microscope, the skill of the clinician in obtaining a specimen, and the expertise of the microscopist. Serologic diagnosis may be affected by the delay between the appearance of the primary chancre and the onset of serologic reactivity. We used a pathogen-specific fluorescein-conjugated monoclonal antibody to examine lesion exudates from 128 consecutive patients and compared these data with results of dark-field microscopy, the rapid plasma reagin (RPR) test, and the fluorescent treponemal antibody-absorbed (FTA-Abs) test. The monoclonal antibody test demonstrated Treponema pallidum in 48 (73%) of 66 patients with infectious syphilis, while dark-field microscopy was positive for 52 (79%) of 66 patients. None of 62 patients without syphilis was positive by either test. The FTA-Abs test was reactive for 61 patients (92%) of the 66 with infectious syphilis. Thus the fluorescent monoclonal antibody test for detection of T. pallidum in direct smears is as sensitive and specific as dark-field microscopy for the diagnosis of infectious syphilis. It has the potential to provide a convenient, accurate means for definitive diagnosis of genital ulcer disease by health care personnel without ready access to dark-field microscopy.     

Colloid and elastic fibre: ultrastructural study on the histogenesis of colloid milium

Tissues peripheral to well-developed papules of colloid milium were chosen for biopsy and electron microscopy examination. Evidence was presented that colloid is derived from elastic fibres through sequential degenerative changes. In the upper to middle dermis of the peripheral tissue, a number of abnormal masses in the vicinity of small colloid depositions were observed. Ultrastructurally, those abnormal masses were degenerating elastic fibres in which "electron-dense layers" of normal elastic fibre increased in amount, although their electron density diminished. Electron-light layers were gradually diminished in quantity. Degenerated elastic fibres finally became granulo-fibrillar and were indistinguishable from colloid. Many fibrils which closely resembled 10 nm tubular microfibrils of normal elastic fibre were observed in the granulo-fibrillar substance. Since one stage of the degeneration of elastic fibre which eventually leads to the formation of colloid milium was very similar to that of actinic elastosis, a direct role of sunlight in the formation of colloid milium is suggested.     

Pigmentation-associated glycoprotein of human melanomas and melanocytes: definition with a mouse monoclonal antibody

Pigmented melanoma cells and cultured melanocytes express a differentiation-related glycoprotein designated as pigmentation-associated antigen (PAA) of Mr 70,000-80,000. As described previously, PAA was initially defined by reactivity with antibodies in the serum of a patient with melanoma. Here we describe the production and characterization of a mouse monoclonal antibody to PAA. This antibody (TA99, an IgG2a) was shown by sequential immunoprecipitation experiments to react with the same component as the human antibody. Ab TA99 immunoprecipitated PAA from lysates of cells radiolabeled with [35S]methionine, [3H]glucosamine, [3H]fucose, and [3H]mannose as well as 125I. Using Ab TA99, the distribution of PAA was examined in frozen sections of 19 normal tissues and quantitatively in 68 tissue culture cell lines. In frozen sections, only melanin-containing cells were positive, including epithelial cells in the basal layer of the epidermis, in which pigment originates from melanocytes by transfer of melanosomes, and pigmented cells of the eye. In tissue culture cell lines, only pigmented melanoma cells were positive. PAA is an intracellular antigen, with a distribution very similar to that of melanosomes. This evidence confirms the close association of PAA with melanin production, and suggests that PAA may be a melanosome component. PAA was shown to be different from tyrosinase, the enzyme involved in melanin synthesis, but it was found to be identical to the previously recognized glycoprotein, gp75, characteristic of pigmented melanomas and melanocytes.