Alice A. Roberts

Differential regulation of beta-defensin expression in human skin by microbial stimuli

In response to infection, epithelia mount an innate immune response that includes the production of antimicrobial peptides. However, the pathways that connect infection and inflammation with the induction of antimicrobial peptides in epithelia are not understood. We analyzed the molecular links between infection and the expression of three antimicrobial peptides of the β-defensin family, human β-defensin (hBD)-1, hBD-2, and hBD-3 in the human epidermis. After exposure to microbe-derived molecules, both monocytes and lymphocytes stimulated the epidermal expression of hBD-1, hBD-2, and hBD-3. The induced expression of hBD-3 was mediated by transactivation of the epidermal growth factor receptor. The mechanisms of induction of hBD-1 and hBD-3 were distinct from each other and from the IL-1-dependent induction of hBD-2 expression. Thus during inflammation, epidermal expression of β-defensins is mediated by at least three different mechanisms.

By IL-1 Signaling, Monocyte-Derived Cells Dramatically Enhance the Epidermal Antimicrobial Response to Lipopolysaccharide

Epithelia react to microbial pathogens by mounting a defensive response that includes the production of antimicrobial peptides. In this study, we show that, in human epidermal cultures, Escherichia coli LPS was a very weak direct inducer of human β-defensin (HBD)-2 mRNA and peptide, but the induction was greatly amplified when monocyte-derived cells (MoDeC) acted as intermediaries between LPS and the epidermis. IL-1R antagonist largely reversed the effect of MoDeC on epidermal HBD-2, indicating that, from among the many products of MoDeC, IL-1 was the dominant inducer of HBD-2 synthesis. In normal fresh human skin, which contains Langerhans cells and other myeloid cell types, in addition to keratinocytes, LPS also induced HBD-2 in an IL-1-dependent manner. In DNA microarray expression studies, HBD-2 was one of the most abundant mRNAs induced in epidermis by LPS-treated MoDeC, and its induction was reversed by IL-1Ra. Thus, epidermal response to LPS is potently amplified by MoDeC through IL-1-mediated signaling, leading to a selective increase in the synthesis of the antimicrobial peptide HBD-2. This pattern of responses establishes a key role for both IL-1 and HBD-2 in the host defense reaction of the epidermis.

Injury-induced innate immune response in human skin mediated by transactivation of the epidermal growth factor receptor

We found that sterile wounding of human skin induced epidermal expression of the antimicrobial (poly)peptides human beta-defensin-3, neutrophil gelatinase-associated lipocalin, and secretory leukocyte protease inhibitor through activation of the epidermal growth factor receptor. After skin wounding, the receptor was activated by heparin-binding epidermal growth factor that was released by a metalloprotease-dependent mechanism. Activation of the epidermal growth factor receptor generated antimicrobial concentrations of human beta-defensin-3 and increased the activity of organotypic epidermal cultures against Staphylococcus aureus. These data demonstrate that sterile wounding initiates an innate immune response that increases resistance to overt infection and microbial colonization.

Pseudomyxoma Peritonei Is a Disease of MUC2-Expressing Goblet Cells

Pseudomyxoma peritonei, a syndrome first described by Karl F. Rokitansky in 1842, is an enigmatic, often fatal intra-abdominal disease characterized by dissecting gelatinous ascites and multifocal peritoneal epithelial implants secreting copious globules of extracellular mucin. Although past interest in the syndrome has focused on the questions of the site of origin (appendix versus ovary), mechanisms of peritoneal spread (multicentricity, redistribution phenomenon, or metastasis), and the degree of malignant transformation present (adenoma, borderline tumor, or carcinoma), another important question is the mechanism behind the accumulation of extracellular mucin, the real cause of the diseases morbidity and mortality irrespective of the site of origin, mechanism of peritoneal spread, or transformed status of its epithelium. Taking advantage of the recently cloned human mucin genes, we decided to investigate this question. Our studies revealed that pseudomyxoma peritonei is a disease of MUC2-expressing goblet cells. These cells also express MUC5AC but the latter mucin is not specific for pseudomyxoma peritonei. MUC2 expression accounts for the voluminous deposits of extracellular mucin (mucin:cell ratios exceeding 10:1) and distinguishes pseudomyxoma peritonei secondarily involving the ovary from primary ovarian mucinous tumors with peritoneal implants. Because mucinous tumors of the appendix similarly express MUC2, the MUC2 expression profile also supports an appendiceal rather than ovarian origin for pseudomyxoma peritonei. Increased steady-state mRNA is observed in pooled cases of pseudomyxoma peritonei but does not occur on the basis of gene rearrangement or gene amplification. Primary epithelial cell cultures obtained from pseudomyxoma peritonei express MUC2 whose levels can be epigenetically regulated. These lines up-regulate MUC2 expression in response to both methylation inhibition by 5-azacytidine and exposure to Pseudomonas aeruginosa lipopolysaccharide, both of whose effects can be suppressed by genistein pretreatment. Both immunocytochemical as well as in situ hybridization studies with ancillary digital image analysis reveal that MUC2 expression in cases of pseudomyxoma peritonei is independent of the degrees of malignant transformation that are present and, in fact, reflects the constitutive levels of expression observed in normal goblet cells of the appendix. Extracellular mucin accumulates dramatically in pseudomyxoma peritonei because the number of MUC2-secreting cells dramatically increase and because this MUC2 has no place to drain. These studies suggest that pseudomyxoma peritonei should be regarded as a disease of MUC2-expressing goblet cells whose MUC2 expression might be susceptible to pharmacological targeting.

The place of lymphatic mapping and sentinel node biopsy in oncology.

The techniques of sentinel lymphatic mapping (LM) and sentinel lymph node biopsy (SLNB) have become almost routine for the staging of clinically node-negative patients with high-risk cutaneous melanoma. The techniques are also widely applied to staging of the axilla in breast cancer. Investigations of the use of LM and SLNB for other solid malignancies have also shown promise. LM/SLNB requires a multidisciplinary effort involving experienced surgeons, nuclear medicine physicians, and surgical pathologists. The techniques require a learning curve for all involved personnel, requiring experience with at least 30 cases followed by complete nodal dissection after SLNB to achieve full competency. Surgical pathologists play a pivotal role in determining optimum sentinel node analysis. The techniques have lower morbidity and greater accuracy than traditional complete regional node dissection. Pathologists are receiving increasing numbers of SLN specimens and are expected to evaluate the results of the application of the LM/SLNB techniques to a range of solid tumors. We have reviewed LM/SLNB in regard to melanoma and breast cancer and other types of malignancies. The techniques have much to offer, but despite their seeming simplicity, need considerable technical skill and clinical judgment if they are to be effectively applied. They also provide unique opportunities for basic and translational research.

Update on lymphatic mapping and sentinel node biopsy in the management of patients with melanocytic tumours

Aims: To communicate best practices for sentinel lymph node evaluation and assessment of prognosis for patients with melanoma. Methods: Description and justification of approaches derive from experience with management of more than 2000 melanoma patients evaluated by lymphatic mapping and sentinel node biopsy (LMSNB). Results: Pathologists, by detecting blue dye or carbon particles or alterations in nodal cell populations should attempt to confirm that a node submitted as sentinel is truly sentinel. Pathologists must adequately sample the node by examining multiple tissue sections and determine the presence or absence of metastatic melanoma using sections stained by H&E and immunocytochemistry. Approximately 20% of patients have melanoma in the sentinel node (SN) and accurate evaluation of SN tumour status is the most precise technique for staging clinically localised cutaneous melanoma. The remaining non‐sentinel nodes (NSN) in the basin are tumour‐free in 67% of patients with melanoma in the SN. Breslow thickness of the primary, the area of tumour in the SN (relative to total nodal area) and density of dendritic leukocytes in the SN paracortex (factors that are combinable in prognostic algorithms) predict metastases in the NSN and the likelihood of recurrence and melanoma‐specific death. Conclusions: Careful pathological analysis is essential to determine the presence or absence of metastatic melanoma in sentinel nodes, findings that indicate whether completion lymphadenectomy is required. Quantitative analysis of the primary melanoma and the amount of tumour in the sentinel node, with evaluation of the dendritic cells in that node, provide invaluable information that predicts non‐sentinel node tumour status with increased accuracy and the likelihood of future recurrence and death from melanoma. While these activities require considerable effort from pathologists, their clinical impact justifies the increased workload.

Galectin-1–Mediated Apoptosis in Mycosis Fungoides: The Roles of CD7 and Cell Surface Glycosylation

Sezary cells, the malignant T cells in mycosis fungoides/Sezary syndrome, resist a variety of apoptosis-inducing agents, a feature that contributes to the poor response to therapy in mycosis fungoides. Galectin-1 is a mammalian lectin that triggers T cell apoptosis. For T cells to be susceptible to galectin-1–induced apoptosis, the T cells must express specific glycoprotein receptors, such as CD7, that bear the specific oligosaccharides recognized by galectin-1. Because Sezary cells are characteristically CD7−, lack of CD7 expression has been proposed to render Sezary cells resistant to galectin-1–induced death. However, the role played by aberrant cell surface glycosylation in resistance of Sezary cells to galectin-1 has not been examined. In this study, we demonstrated abundant galectin-1 in mycosis fungoides skin lesions, indicating that Sezary cells are exposed to galectin-1 in vivo. To determine specific characteristics of Sezary cells that contribute to galectin-1 resistance, we assessed CD7 expression and cell surface glycosylation of Sezary cells in mycosis fungoides lesions and of four Sezary T cell lines. Sezary cells in primary lesions and Sezary T cell lines demonstrated a characteristic “glycotype” with sialylated core 1 O-glycans that promote galectin-1 resistance. Expression of CD7 was necessary but not sufficient for galectin-1–induced death of Sezary cell lines. In addition, CD7− Sezary cell lines, and Sezary cells within mycosis fungoides lesions, expressed galectin-1, whereas CD7-positive Sezary cell lines did not express galectin-1. We propose that both loss of CD7 expression and altered cellular glycosylation contribute to apoptosis resistance of malignant T cells in mycosis fungoides.

Optimized assessment of sentinel lymph nodes for metastatic melanoma: implications for regional surgery and overall treatment planning.

Correct identification of the sentinel node (SN) and accurate evaluation of this nodes tumor status constitute the most precise technique for staging clinically localized cutaneous melanoma. However, even if tumor is present in the SN (as in approximately 20% of patients), the remaining nodes in the basin are often tumor-free. We have found that the Breslow thickness of the primary, the relative area of tumor in the SN (with respect to the area of the SN), and the density of tendritic leukocytes in the SN paracortex not only can predict the likelihood of nonsentinel node metastases but also are correlated with likelihood of tumor recurrence and melanoma-specific survival. The most robust of these predictors is relative tumor area, and this may be used as the basis of practical predictive algorithms.

Study of Sentinel Lymph Nodes in the Staging of Malignant Neoplasms

Surgeons have long appreciated the significance of lymph node metastases to cancer prognosis. As far back as the 6th century, Actus of Amida observed that “malignant phlegmons” develop in the armpits of advanced breast cancer patients (1). The importance of lymph node metastases was recognized early on, and this naturally led to investigations of the underlying physiology. In 1622, Aselius demonstrated lymphatic drainage into lacteals of a postprandial dog, thus laying the groundwork for centuries of attempts to map lymphatic pathways. The most common approach was to inject dye into peripheral sites and dissect adjacent tissues looking for colored lymphatics. Various dyes, including India ink and mercury, were utilized to map lymphatic drainage in human cadavers and research animals. In the mid-20th century, radioisotopes were employed as markers of lymph drainage; some early experiments in humans employed radioactive colloid gold (2), and later the less active technetium99 (TC-99) came into use (3). Peritumoral injections of radiocolloid are now used in sentinel node biopsy to visualize draining nodes preoperatively by lymphoscintigraphy and intraoperatively using a handheld Geiger counter. The presence of lymph node metastases overrides all other prognostic factors for solid tumors and the surgical approach is usually altered by the presence of metastatic tumor in a lymph node. Staging for most solid tumors depends on knowing the tumor status of the draining lymph nodes. As the surgeon Moynihan aptly stated, “surgery of malignant disease is not the surgery of organs; it is the anatomy of the lymphatic system” (4).