Cutaneous adverse effects of BRAF inhibitors in metastatic malignant melanoma, a prospective study in 20 patients.

BACKGROUND: BRAF inhibitors frequently cause significant cutaneous adverse reactions. OBJECTIVE: To study the timing, prevalence and response to treatment of skin lesions in patients receiving V-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors. METHODS: We prospectively studied the cutaneous side-effects of patients with a BRAF mutant (V600E, V600K, V600R) metastatic malignant melanoma treated with a BRAF inhibitor. We systematically registered prevalence, timing of onset and response to treatment. RESULTS: Twenty patients were treated for 2-52 weeks with a BRAF inhibitor. Eleven patients on vemurafenib (58%) developed cutaneous side-effects and 10 patients (42%) had more than one cutaneous adverse event. Verrucous papillomas were observed in eight patients (42%), after 1-12 weeks. We diagnosed four keratoacanthomas in two patients (11%) after 6-10 weeks and two squamous cell carcinomas in two patients (11%) after 10-16 weeks. Seven patients (37%) developed a hyperkeratotic, folliculocentric eruption after 2-8 weeks, resolving quickly under topical steroids. Four patients (21%) presented a facial erythema, two patients (11%) a seborrhoeic dermatitis-like eczema on the scalp. Three patients (16%) developed cystic lesions after 2-11 weeks. Three patients (16%) presented a hand-foot skin reaction after 4-6 weeks, which was successfully treated with topical steroids and keratolytics. Hyperkeratosis of the nipples was seen in one patient (5%). We observed phototoxic reactions after UV exposure in five patients (26%) and alopecia in two patients (11%) after 8-10 weeks. One patient on dabrafenib developed curly hairs (24 weeks), keratotic papules (1 and 36 weeks), a keratoacanthoma (4 weeks) and a hand-foot skin reaction (31 weeks). CONCLUSION: Multiple cutaneous toxicities were observed in patients under BRAF inhibitors, mostly well controlled with adequate treatment. We recommend a multidisciplinary approach with regular assessments of the skin by a dermatologist. This allows early identification and adequate treatment to avoid premature discontinuation of a life-prolonging therapy.

Activity and mechanism of action of HDVD, a novel pyrimidine nucleoside derivative with high levels of selectivity and potency against gammaherpesviruses.

A novel nucleoside analogue, 1-[(2S,4S-2-(hydroxymethyl)-1,3-dioxolan-4-yl]5-vinylpyrimidine-2,4(1H,3H)-dione, or HDVD, was evaluated against a wide variety of herpesviruses and was found to be a highly selective inhibitor of replication of the gammaherpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). HDVD had also a pronounced inhibitory activity against murine herpesvirus 68 (MHV-68) and herpes simplex virus 1 (HSV-1). In contrast, replication of herpesvirus saimiri (HVS), HSV-2, and varicella-zoster virus (VZV) was weakly inhibited by the compound, and no antiviral activity was determined against human cytomegalovirus (HCMV) and rhesus rhadinovirus (RRV). The HDVD-resistant virus phenotype contained point mutations in the viral thymidine kinase (TK) of HSV-1, MHV-68, and HVS isolates. These mutations conferred cross-resistance to other TK-dependent drugs, with the exception of an MHV-68 mutant (E358D) that exhibited resistance only to HDVD. HSV-1 and HVS TK-mutants isolated under selective pressure with bromovinyldeoxyuridine (BVDU) also showed reduced sensitivity to HDVD. Oral treatment with HDVD and BVDU was assessed in an intranasal model of MHV-68 infection in BALB/c mice. In contrast to BVDU treatment, HDVD-treated animals showed a reduction in viral DNA loads and diminished viral gene expression during acute viral replication in the lungs in comparison to levels in untreated controls. The valyl ester prodrug of HDVD (USS-02-71-44) suppressed the latent infection in the spleen to a greater extent than HDVD. In the present study, HDVD emerged as a highly potent antiviral with a unique spectrum of activity against herpesviruses, in particular, gammaherpesviruses, and may be of interest in the treatment of virus-associated diseases.

Constitutively activating mutation in WASP causes X-linked severe congenital neutropenia.

The Wiskott-Aldrich syndrome protein (WASP; encoded by the gene WAS) and its homologs are important regulators of the actin cytoskeleton, mediating communication between Rho-family GTPases and the actin nucleation/crosslinking factor, the Arp2/3 complex. Many WAS mutations impair cytoskeletal control in hematopoietic tissues, resulting in functional and developmental defects that define the X-linked Wiskott-Aldrich syndrome (WAS) and the related X-linked thrombocytopenia (XLT). These diseases seem to result from reduced WASP signaling, often through decreased transcription or translation of the gene. Here we describe a new disease, X-linked severe congenital neutropenia (XLN), caused by a novel L270P mutation in the region of WAS encoding the conserved GTPase binding domain (GBD). In vitro, the mutant protein is constitutively activated through disruption of an autoinhibitory domain in the wild-type protein, indicating that loss of WASP autoinhibition is a key event in XLN. Our findings highlight the importance of precise regulation of WASP in hematopoietic development and function, as impairment versus enhancement of its activity give rise to distinct spectra of cellular defects and clinical phenotypes.

Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis, and thrombolysis.

The effects of plasminogen activator inhibitor-1 (PAI-1) gene inactivation on hemostasis, thrombosis and thrombolysis were studied in homozygous PAI-1-deficient (PAI-1-/-) mice, generated by homologous recombination in D3 embryonic stem cells. Diluted (10-fold) whole blood clots from PAI-1-/- and from PAI-1 wild type (PAI-1+/+) mice underwent limited but significantly different (P < 0.001) spontaneous lysis within 3 h (6 +/- 1 vs 3 +/- 1%, respectively). A 25-microliters 125I-fibrin-labeled normal murine plasma clot, injected into a jugular vein, was lysed for 47 +/- 5, 66 +/- 3, and 87 +/- 7% within 8 h in PAI-1+/+, heterozygous PAI-1-deficient (PAI-1+/-), and PAI-1-/- mice, respectively (P = 0.002 for PAI-1+/+ vs PAI-1-/- mice). Corresponding values after pretreatment with 0.5 mg/kg endotoxin in PAI-1+/+ and PAI-1-/- mice, were 35 +/- 5 and 91 +/- 3% within 4 h, respectively (P < 0.001). 11 out of 26 PAI-1+/+ but only 1 out of 25 PAI-1-/- mice developed venous thrombosis (P = 0.004) within 6 d after injection of 10 or 50 micrograms endotoxin in the footpad. Spontaneous bleeding or delayed rebleeding could not be documented in PAI-1-/- mice after partial amputation of the tail or of the caecum. Thus, disruption of the PAI-1 gene in mice appears to induce a mild hyperfibrinolytic state and a greater resistance to venous thrombosis but not to impair hemostasis.

Gene expression profiling of primary cutaneous melanoma.

Cutaneous Malignant Melanoma (CMM) is the most malignant skin tumour in humans, the incidence of which is rising rapidly in most fair-skinned populations, without apparent decline in mortality. Both hereditary, constitutional and environmental factors play a role in its etiology. CMM arises from melanocytes in the epidermis, and proceeds through discrete steps of tumor-progression that consist histologically of the radial growth phase (RGP), vertical growth phase (VGP) and metastatic phase. The underlying molecular mechanisms that govern the transition between these growth phases are hardly known. The prognosis of patients with VGP melanoma depends on several clinical and histological parameters; the latter include thickness, mitotic activity, presence or absence of ulceration and regression, and pattern of lymphocytic host response. However, there is still need for new prognostic parameters. To obtain insight in the molecular mechanisms of tumor progression in CMM, and in search of new prognostic markers, we performed global gene-expression profiling using 44K oligonucleotide micro-arrays on a unique retrospective series of 83 frozen primary MM with VGP, 9 metastases and 23 benign nevi. Unsupervised analysis allowed us o identify clusters of melanoma patients with different outcome based on their gene expression profile only. Supervised analysis resulted in the identification of a genomic signature of 254 genes with prognostic significance. The large majority of the 254 enes was correlated with thickness, thereby stressing the importance of thickness in he prognosis of CMM. This signature was validated on a separate series of melanoma patients, and proved to have a predictive accuracy comparable to what can be obtained by tumour thickness and ulceration. On an immunohistochemical level, we identified 8 new markers that may help in the prognostication of melanoma patients; three of these markers, i.e. the mini-chromosome maintenance (mcm) proteins mcm3, 4 and 6, hat are involved in DNA-replication, had independent prognostic value. Additionally, upervised analysis showed similarities in gene expression profile between primary CMM and their metastases. In conclusion, our data provide new information regarding the molecules that are operative in the progression of CMM. CMM is notorious for its resistance to chemotherapy, and disseminated CMM is a uniformly fatal disease. As several of the progression-related genes, encode molecules that have been the target of established or xperimental cancer therapies, our results may hopefully contribute to the treatment of end-stage CMM-patients.

A rare conjunctival Spitz nevus: a case report and literature review.

A conjunctival Spitz nevus is a very rare, benign melanocytic lesion, which can be mistaken for a malignant melanoma. We present a case of a 28-year old man, who suffered from a rapidly growing, non-pigmented mass in the left caruncular area, extending to the nasal conjunctiva. The lesion was excised and pathologic examination showed nests of large, polygonal, non-pigmented epithelioid cells, located in the stroma. The overlying epithelium showed focal erosions. At the base, there was a lymphocytic infiltrate. Immunohistochemical techniques, with stainings for S-100 protein, HMB-45 and MIB-1, were used for further investigation and showed the melanocytic origin of the lesion (S-100 staining) as well as many cells in cell cycle (MIB-1 staining). However, no mitoses were seen. The clinical image, combined with pathologic and immunohistochemical findings, provided the diagnosis of a Spitz nevus localised in the conjunctiva. Although the cutaneous location of Spitz nevi is well known, conjunctival Spitz nevi are very rare and because of their mucosal origin, some of the histological features are different.

A novel N-ras mutation in malignant melanoma is associated with excellent prognosis.

Mutations in the ras gene are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in malignant melanoma (MM). We searched for point mutations in the N-ras gene in a large series of primary and metastatic MM from 81 different retrospectively selected patients using the very sensitive denaturing gradient gel electrophoresis technique, followed by sequencing. The classical codon 12 and codon 61 mutations were found in 21 and 17% of the cases, respectively. No codon 13 mutation was found. A novel mutation at codon 18 of exon 1, consisting of a substitution of alanine (GCA) by threonine (ACA), was found in 15% of the primary MMs but in none of the metastatic MMs. All of the other cases were free of mutations. Using microdissected cells from distinctive MM growth phases as source of DNA for mutation analysis, this particular N-ras exon 1 mutation at codon 18 was already present in the radial growth phase and preserved throughout the successive growth phases; it was also found in a dysplastic nevi in continuity with a MM, indicating a clonal relationship between both lesions. Our findings also illustrate the clonal relationship between the distinctive growth phases in MM and suggest the codon 18 mutation to occur early in MM development. The MM in patients with this mutation were significantly thinner than those without a codon 18 mutation (P = 0.0257). Statistical analysis, comparing the group of codon 18 patients with the group of patients with the classical mutations and without mutations, revealed a highly significant difference in overall outcome. The cumulative probability of developing metastasis was significantly lower for the group patients with a codon 18 mutation (P = 0.0130). We can thus conclude that this codon 18 mutation identifies a group of patients with better prognosis than patients with melanoma that harbor wild-type sequence or classical activating point mutations in codon 12 or 61. Preliminary nucleotide binding measurements could not detect a difference between wild-type Ras protein and the mutant Ras(A18T) protein. However, for a precise elucidation of the role of the N-Ras(A18T) mutant in melanoma, additional studies aimed to measure the affinity to guanine nucleotide exchange factors and GTPase-activating proteins are needed.

Analysis of N- and K-ras mutations in the distinctive tumor progression phases of melanoma.

Mutations in the ras genes are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in cutaneous melanoma. To investigate whether these mutations occur in early or late tumor progression phases, we searched for point mutations in the N- and K-ras genes in 69 primary cutaneous melanoma, 35 metastases, and seven nevocellular nevi in association with cutaneous melanoma. Lesions were microdissected in order to procure pure tumor samples from the distinctive growth phases of the cutaneous melanoma; the very sensitive denaturing gradient gel electrophoresis technique was used to visualize the mutations, and was followed by sequencing. Point mutations in the N-ras gene but not in the K-ras gene were detected on denaturing gradient gel electrophoresis. Twenty-three primary (33%) and nine metastatic (26%) melanomas showed bandshifts for N-ras. In the majority of cases, mutations occurring in early growth phases (i.e., the "intraepidermal" radial growth phase), were preserved in later growth phases (i.e., the invasive radial growth phase, vertical growth phase, and metastatic phase), which proves the clonal relationship between the successive growth phases. In three cases, however, the mutations differed between the distinctive growth phases within the same cutaneous melanoma, due to the occurrence of an additional mutation (especially in codon 61) in a later tumor progression phase. Our approach also permitted us to analyze the mutational status of nevi, associated with cutaneous melanoma. Six out of seven associated nevi carried the same sequence (mutated or wild-type) as the primary cutaneous melanoma, whereas in one case the sequence for N-ras differed between the primary melanoma and the associated nevus. In conclusion, this approach allowed us to demonstrate the clonal relationship between subsequent growth phases of melanoma and associated nevi; our results suggest that N-ras exon 1 mutations preferentially occur during early stages of tumor progression and hence may be involved in melanoma initiation, whereas those in N-ras exon 2 are found preferentially during later stages and hence are more probably involved in metastatic spread of cutaneous melanoma.

Multiple immunoenzyme staining techniques. Use of fluoresceinated, biotinylated and unlabelled monoclonal antibodies.

Simultaneous detection of multiple tissue epitopes with an overlapping distribution pattern by monoclonal antibodies is sometimes needed for routine immunohistological evaluations. Therefore, multistep double and triple immunoenzymatic methods using antibodies from the same species or Ig (sub)class have been developed. Since only commercially available monoclonal antibodies (either unlabelled, biotinylated or as fluorescein conjugate) have been used, the techniques may be regarded as generally applicable. The staining protocol for double staining consists of six incubation steps: (1) unlabelled monoclonal antibody 1; (2) enzyme I-conjugated anti-mouse Ig; (3) normal mouse serum--for blocking; (4) fluoresceinated monoclonal antibody 2; (5) rabbit anti-fluorescein isothiocyanate--employing the fluorochrome as hapten; (6) enzyme II-conjugated anti-rabbit Ig. For enzymes I and II, peroxidase, alkaline phosphatase and beta-galactosidase can be applied; excellent results were obtained with the following colour combinations: peroxidase activity in red/alkaline phosphatase in blue and beta-galactosidase in green/alkaline phosphatase in violet. Moreover, this double staining method can be extended to provide an immunoenzyme triple staining technique by mixing biotinylated monoclonal antibody 3 and avidin-biotin enzyme III complex with the steps 4 and 5 reagents, respectively. In this way three tissue epitopes can simultaneously be detected clearly and selectively in green (beta-galactosidase), blue (alkaline phosphatase) and red (peroxidase).

Suppurative granulomatous lymphadenitis. Immunohistochemical evidence for a B-cell-associated granuloma.

The cellular composition of suppurative granulomas was investigated by the application of monoclonal and polyclonal antibodies to paraffin sections and compared with nonsuppurative, hypersensitivity-type granulomas. Macrophages, T cells, and dendritic cells showed a similar distribution in both types of granulomas. In addition to the presence of granulocytes, a major difference between suppurative granulomas and hypersensitive-type granulomas concerned their relationship with B lymphocytes. Hypersensitive-type granulomas were surrounded by small mantle B cells, but they did not contain any B lymphocytes. In contrast, variable numbers of B cells were found either at the periphery or in the center of suppurative granulomas. In view of their morphology (medium size, pale cytoplasm, irregular nuclear shape) and phenotype (L26 +/LN1 -/MB2 +/MT2 +) these B lymphocytes closely resembled monocytoid B cells. The monocytoid B cells might have a role in the recruitment of polymorphonuclear granulocytes and in the development of the necrosis, which occur within suppurative granulomas.

Mantle zone lymphoma. Immuno- and enzymehistochemical studies on the cell of origin.

Using in-situ immuno- and enzymehistochemical techniques, the phenotype of the neoplastic cells in seven cases of mantle zone lymphoma (MZL) was compared to that in seven cases of nodular poorly-differentiated lymphocytic lymphoma (NPDLL). The neoplastic nodules in MZL consisted of medium-sized lymphoid cells with slightly irregular nuclei and finely dispersed chromatin, expressing monoclonal surface IgM or IgM plus IgD, and displaying membranous alkaline phosphatase (ALP) activity. These cells proliferated around follicular centers that demonstrated a polyclonal pattern of reactivity for both types of light chains and a distorted meshwork of dendritic reticulum cells. The neoplastic nodules in NPDLL consisted of small lymphoid cells with markedly irregular nuclei and coarsely granulated chromatin, expressing monoclonal surface IgM and lacking ALP-activity. These tumor cells also frequently expressed transferrin receptor and common acute lymphoblastic leukemia-antigen (CALLA). The neoplastic nodules showed an undistorted meshwork of dendritic reticulum cells, and were occasionally bordered by remnants of polyclonal lymphocytic coronas. These results confirm the previous suggestion that NPDLL arises from a cell type that is a normal constituent of follicular centers, whereas MZL arises from the lymphocytic corona. The morphological, enzyme- and immunohistochemical features of MZL cells strongly suggest that MZL arises from marginal zone lymphocytes, a subset of corona lymphocytes that expresses ALP-activity, high IgM and low IgD-levels.

Leukemia-associated lymph node infiltrates of plasmacytoid monocytes (so-called plasmacytoid T-cells). Evidence for two distinct histological and immunophenotypical patterns.

The medium-sized mononuclear cell with plasmacytoid features, formerly known as "T-associated plasma cell" or "plasmacytoid T cell," has recently been shown to express several myelomonocyte and monocyte-macrophage associated antigens, suggesting a monocytic origin, and it has been renamed "plasmacytoid monocyte." The present study describes the clinical and pathological features of two patients with generalized lymphadenopathy and leukemia (chronic myelomonocytic leukemia in Case 1, and acute non-B, non-T lymphoblastic leukemia in Case 2), and whose lymph node biopsies showed large numbers of plasmacytoid monocytes associated with the leukemic infiltrates. Case 1 was strikingly similar to three previously reported cases of so-called "plasmacytoid T cell" lymphoma, all associated with a myeloproliferative disorder. In our case, the destructive growth pattern of the plasmacytoid monocytes and the de novo expression of CD5 on these cells favored their neoplastic nature; the sharing of some markers of plasmacytoid monocytes with the myelomonocytic infiltrate suggested they were part of the tumoral proliferation. In Case 2, plasmacytoid monocytes displayed an immunophenotype guide similar to that reported in reactive conditions and were antigenically unrelated to the leukemic cells; plasmacytoid monocyte clusters occurred also in the lymphoid parenchyma spared by the leukemic infiltrate. These findings led us to interpret the large numbers of plasmacytoid monocytes in this second case as a tumor-associated host reaction.

A recommended procedure for ultrastructural immunohistochemistry on small human tissue samples.

Various fixation and staining procedures for the demonstration of surface and cytoplasmic antigens have been described. An immunostaining procedure was sought that would allow the demonstration of these antigens, especially in small human tissue samples at the ultrastructural level. A modification and adaptation of the technique of Eldred, Zucker, Karten, and Yazula (J Histochem Cytochem 31:285, 1983) was applied on several varieties of human tissue, including liver, skin, and lymphoid tissue, using monoclonal and polyclonal antibodies in an indirect peroxidase procedure. In this way a reliable and generally applicable procedure was developed that satisfied the following demands: Use of a universal fixative that allows preservation of the antigenicity of various antigens; Adequate penetration of the tissue by the immunological reagents; Optimal preservation of subcellular structures; and Possibility to store the tissue samples for considerable periods of time.

Binding of biotin to hepatitis B surface antigen: a possible pitfall in immunohistochemistry.

We report on the binding of biotin, and hence of biotinylated antibodies and lectins, to ground glass hepatocytes and liver cell membranes in chronic hepatitis B viral infection. This binding is of low affinity, and was proved to be directed at the hepatitis B surface antigen, presumably at its disulfide bonds. To avoid false-positive results, this affinity should be considered in the interpretation of immunohistochemical stainings of hepatitis B virus-infected liver tissue with biotinylated reagents.

The transitional zone between limbus and peripheral cornea. An immunohistochemical study.

PURPOSE: The authors investigated the phenotypic characteristics of basally located "transitional cells" in peripheral superior cornea, characterized previously by their coexpression of cytokeratin 19 and vimentin and their negativity for AE5. METHODS: Twenty adult human corneas were studied, using in situ immunohistochemical techniques and a panel of specific monoclonal antibodies against various surface and cytoplasmic molecules. RESULTS: The transitional cells shared staining characteristics with limbal basal cells in their expression of alpha 6 beta 4-integrin, metallothionein, AE1, and transferrin receptor. CONCLUSIONS: These "transitional" epithelial cells exhibit a unique phenotype differing from that of the surrounding basal epithelial cells in the peripheral cornea but analogous to that of limbal basal cells. These findings further corroborate the hypothesis that, at least from an immunohistochemical point of view, transitional cells in the superior peripheral cornea exhibit stem cell characteristics.

Heterogeneous induction of major histocompatibility complex class II antigens on corneal endothelium by interferon-gamma.

The expression and distribution of major histocompatibility complex (MHC) class II gene products, HLA-DR, HLA-DQ, and the HLA-DR invariant chain, were studied on flat mounts of human corneal endothelial cells (HCEC) after in vitro incubation of donor corneas with interferon-gamma (IFN-gamma), interleukin-1 (IL-1), and IL-6, using a sensitive immunoperoxidase technique with monoclonal antibodies. Control HCEC and endothelium treated with IL-1 or IL-6 completely lacked MHC class II antigens. After treatment with 50 U/ml, 100 U/ml, 500 U/ml, and 5000 U/ml of human IFN-gamma, a mosaic-like, patchy staining for all MHC class II products was observed: part of the HCEC showed membranous and/or cytoplasmic positivity; other endothelial cells were negative. In addition, a dose-dependent response to IFN-gamma was observed: the proportion of cells expressing class II products rose with increasing doses of IFN-gamma. The induction of MHC class II antigen expression on HCEC by IFN-gamma was completely inhibited by the addition of a neutralizing antibody directed to IFN-gamma but not by IL-1 beta. The significance of these findings with respect to corneal transplantation immunology is discussed.

Distribution of very late activation integrins in the human cornea. An immunohistochemical study using monoclonal antibodies.

There is growing evidence that cellular adhesion mechanisms characterized by cell-cell and cell-matrix interactions are a fundamental process in the immunobiology of the cornea. Interactions with various extracellular matrix components are mediated by the very late activation (VLA) subgroup of the integrin superfamily of adhesion molecules. The six different VLA dimers known thus far consist of a common beta 1 subunit and a variable alpha (1 to 6) subunit. They serve as receptors for laminin (alpha 3 and alpha 6), collagen (alpha 2 and alpha 3), and fibronectin (alpha 4 and alpha 5). Using in situ immunohistochemistry and monoclonal antibodies, the distribution of the common beta 1 and the variable alpha-chains of VLA molecules was studied in normal human cornea and in cases with scarring or subepithelial/retrocorneal fibrous tissue. Epithelial cells were VLA-beta 1 and VLA-alpha 2, -alpha 3, -alpha 4, -alpha 5, and -alpha 6 positive. This is consistent with their intercellular adhesion and may aid in their attachment to the basement membrane which is composed of collagen, laminin, and fibronectin. Keratocytes in normal stroma expressed only the common beta 1-chain and no detectable alpha-chains. In regions of scar or fibrous tissue, however, an upregulated expression of the alpha-chains was detected. The VLA- alpha 1, -alpha 3, -alpha 4, and -alpha 5 were expressed in young fibrous tissue; in older lesions, VLA- alpha 1, -alpha 2, -alpha 3, -alpha 4, and -alpha 5 could be detected. The corneal endothelium showed a strikingly strong positivity for all VLA integrins.(ABSTRACT TRUNCATED AT 250 WORDS)

A new epithelial cell type in the human cornea.

PURPOSE: To study the expression of intermediate filaments in the human cornea. METHODS: Light and electron microscopic and immunohistochemical studies were performed on 20 corneas from subjects of various ages. RESULTS: A hitherto unrecognized epithelial cell population emerged from the immunohistochemical studies. Epithelial cells were invariably present in the superior cornea, whereas the nasal, temporal, and inferior segments almost lacked these cells. They were situated at the transition between peripheral cornea and limbus, and occurred as small groups in the basal epithelium. On electron microscopy, they were recognized by their marginated nuclear chromatin, large nucleoli, prominent bundles of intermediate filaments, and numerous hemidesmosomes and desmosomes. Immunohistochemistry on frozen sections revealed a unique intermediate filament make-up: ie, strong co-expression of vimentin and cytokeratin 19; other intermediate filaments, including cytokeratins 3, 4, 6, 7, 8, 10, 13, and 18 were negative. Finally, the cells lacked ultrastructural and immunohistochemical features of melanocytes, neuroendocrine cells, Langerhans' cells, and leukocytes. CONCLUSIONS: A new epithelial cell type in the human cornea is described with characteristic morphologic and immunohistochemical features. According to their particular segmental distribution, restricted localization at the junction between cornea and limbus, and expression of an "early" intermediate filament profile, it is tempting to speculate that they represent stem cells of the human cornea. Further studies are aimed to characterize their phenotype and function more extensively.

Anti-high endothelial venule monoclonal antibody HECA-452 recognizes plasmacytoid T cells and delineates an "extranodular" compartment in the reactive lymph node.

So-called plasmacytoid T cells represent a subset of monocyte related cells, which share with endothelium the CD36+ CD11b- (OKM5+ OKM1-) phenotype. The reactivity of plasmacytoid T cells with rat monoclonal antibody HECA-452, highly specific for high endothelial venules, was analyzed in reactive lymph nodes. In all cases, HECA-452 not only labelled the endothelium of high endothelial venules, but also strongly reacted with singular and clustered plasmacytoid T cells. The HECA-452 positivity for high endothelial venules and plasmacytoid T cells visualized a lymph node compartment extending from the subcapsular sinus to the corticomedullary junction. This compartment surrounded the composite nodule and was designated the "extranodular" compartment. The co-occurrence of plasmacytoid T cells and high endothelial venules in this extranodular compartment, together with their immunophenotypical similarities, may be indicative of functional co-operations.