Carole Yee

The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus E1A

Clinical and epidemiological data have implicated the human papillomaviruses (HPVs) as having an etiologic role in some anogenital malignancies, with HPV-16 being most frequently (greater than 60%) detected in cervical carcinoma. HPV-16 is actively transcribed in the cancers; the most abundant transcripts map to the E6 and E7 early open reading frames. Evidence is presented that the HPV-16 E7 open reading frame encodes transcriptional transactivation and cellular transformation functions analogous to those of adenovirus E1A proteins. Specifically, the HPV-16 E7 gene product could transactivate the adenovirus E2 promoter and cooperate with an activated ras oncogene to transform primary baby rat kidney cells. The E7 transforming function differed somewhat from that of adenovirus E1A in that E7 was also able to transform established mouse cells. Examination of the amino acid sequence of HPV-16 E7 revealed striking similarities with conserved domains 1 and 2 of adenovirus E1A proteins.

Mutations in serines 15 and 20 of human p53 impair its apoptotic activity.

Phosphorylation of the p53 tumor suppressor protein is likely to play an important role in regulating its activity. To study the regulatory role of potential phosphorylation sites within the N-terminal transactivation domain of human p53 (hp53), a series of p53 serine mutants were evaluated for transcriptional transactivation and sequence specific DNA binding. The role of these mutations in regulating p53-mediated growth suppression and programmed cell death was examined. This mutational analysis comprised serine residues located at positions 6, 9, 15, 20, 33 and 37 of human p53. Substitution of serine for alanine, either at individual residues or at all six residues together, did not affect the suppression of cell growth and cell transformation, or the ability to bind DNA specifically and to transactivate different promoters, nor did it alter p53 expression. However, the ability of p53 to induce apoptosis was impaired by specific serine substitutions. Mutations in all six N-terminal serines together reduced the apoptotic activity of p53 in H1299 cells by 50%. Analysis of individual mutants revealed that mutations in serine 15 and 20 are primarily responsible for this impairment. Our results suggest that these serines play a role in the regulation of p53-mediated apoptosis.

Identification and characterization of autoreactive T cell responses to bullous pemphigoid antigen 2 in patients and healthy controls.

Antibodies against the extracellular domain of bullous pemphigoid antigen 2 (BPAG2) are thought to play a key role in the pathogenesis of bullous pemphigoid (BP), the most frequent autoimmune bullous disease of the skin. Autoreactive T cell responses to BPAG2 were investigated in 16 BP patients and 24 healthy controls by coculture of PBMC with two recombinant BPAG2 proteins (extracellular domain of BPAG2). Primary in vitro T cell responses to BPAG2 were observed in 10/12 BP patients expressing the BP-associated HLA-DQB1*0301 allele and 8/10 DQB1*0301 positive healthy individuals. DQB1*0301 also restricted three autoreactive T cell lines from two BP patients and a healthy donor. In contrast, PBMC from 14 normal patients carrying HLA class II alleles other than DQB1*0301 were not stimulated by BPAG2. Autoreactive BPAG2-specific CD4(+) T cell lines and clones from five BP patients produced both Th1 and Th2 cytokines, whereas three autoreactive T cell lines from three DQB1*0301 positive normal patients produced exclusively IFN-gamma. The absence of BPAG2-specific Th2 cells in healthy individuals strongly suggests that autoreactive Th2 responses to BPAG2 are restricted to BP patients and may thus be critical in the pathogenesis of BP.

Passive Transfer of Anti-Laminin 5 Antibodies Induces Subepidermal Blisters in Neonatal Mice

Patients with a recently identified subepithelial blistering disease have IgG anti-laminin 5 autoantibodies. To determine if such antibodies can be pathogenic in vivo, we developed and characterized rabbit anti-laminin 5 IgG, and passively transferred these antibodies to neonatal mice. Immune rabbit IgG specifically bound human and murine epidermal basement membranes, immunoblotted and immunoprecipitated all laminin 5 subunits from extracts of human and murine keratinocytes, and showed no reactivity to other keratinocyte proteins or epithelial basement membranes that do not contain laminin 5. Mice (n = 29) receiving purified anti-laminin 5 IgG developed, in a dose-related fashion, circulating anti-laminin 5 antibodies, deposits of rabbit IgG and murine C3 in epidermal basement membranes, and subepidermal blisters of skin and mucous membranes. No alterations developed in controls (n = 14) receiving identical amounts of normal rabbit IgG. Passive transfer of anti-laminin 5 (but not control) IgG to neonatal C5- (n = 3) or mast cell-deficient (n = 3) mice produced subepidermal blisters with the same clinical, histologic, and immunopathologic features as those documented in BALB/c mice. These studies establish an animal model of a human blistering disease that can be used to define disease mechanisms and treatment modalities.

Anti-epiligrin cicatricial pemphigoid and relative risk for cancer

It is not known whether patients with anti-epiligrin cicatricial pemphigoid (AECP) have an increased risk of malignancy. We calculated the expected numbers of cancers in a cohort of 35 such patients based on respective incidence rates for all cancers in the National Cancer Institutes Surveillance, Epidemiology, and End Results (NCI SEER) Registry. Ten patients in this cohort had solitary solid cancers; eight patients developed cancer after onset of AECP (seven within 14 months). The relative risk (RR) for cancer in this cohort was 6.8 (95% confidence intervals [CI]: 3.3-12.5). AECP seems to be associated with an increased relative risk for cancer.

Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression

The wild-type (wt) p53 protein has transcriptional activation functions which may be linked to its tumor suppressor activity. Many mutant p53 proteins expressed in cancers have lost the ability to function as transcriptional activators and furthermore may inhibit wt p53 function. To study the mechanisms by which mutant forms of p53 have lost their transactivation function and can act in a dominant negative manner, a structure-function analysis of both mutant and engineered truncated forms of p53 was carried out. We show that different mutant p53 proteins found in cancers vary in the ability to inhibit the transcriptional transactivation and specific DNA binding activities of wt human p53. This transdominant effect was mediated through the carboxy-terminal oligomerization region. The role of the transactivation activity in transformation suppression by wt p53 was also examined by constructing an N-terminal deletion mutant lacking the transactivation domain. This mutant was unable to transactivate but could bind specifically to DNA. Although it was impaired in its ability to suppress transformation of primary rat embryo fibroblasts by adenovirus E1A plus activated ras, the N-terminal deletion mutant still had some suppression activity, suggesting that additional functions of p53 may contribute to transformation suppression.

Structural and functional properties of the "hairy" cells of leukemic reticuloendotheliosis.

Structural and functional studies were performed on „hairy”︁ cells from 7 patients with leukemic reticuloendotheliosis („hairy cell leukemia”︁) (HCL). In all cases tartrate‐resistant acid phosphatase‐containing cells were demonstrated. The abnormal cells displayed complement receptors in 6 cases although there was variation in the number of abnormal cells expressing the receptor. Receptors for IgG were present in all 7 cases on a high number of abnormal cells. In 6 cases the „hairy”︁ cells showed surface immunoglobulins (SIg) when examined immediately after isolation. Procedures to eliminate in vivo bound protein substantially decreased the number of SIg‐bearing cells, indicating that most SIg represented cytophilic protein. In 2 cases, however, SIg restricted to a single light chain type remained on the abnormal cells, suggesting that in these 2 cases the SIg may have been an intrinsic cellular product. Attempts to demonstrate immunoglobulin synthesis were unsuccessful and there was no evidence that the „hairy”︁ cells contained cytoplasmic immunoglobulin. In vitro phagocytosis of latex particles by the abnormal cells was observed in all cases by transmission electron microscopy although the number of phagocytic „hairy”︁ cells varied widely from case to case. In 4 of 5 spleens with HCL, normal macrophages detected by the presence of nonspecific esterase were abundant and markedly enlarged. The electronic size distribution of HCL suspensions demonstrated a characteristic double‐peaked curve and modal volumes seldom seen in other chronic leukemias or lymphomas. Quantitative scanning electron microscopic analysis of HCL populations corroborated that the peculiar „hairy”︁ appearance of the abnormal cells was due to extensive surface ruffles which are not observed in normal or neoplastic lymphocytes. Our findings suggest that the „hairy”︁ cells are structurally and functionally unique elements, different than any other normal or abnormal cell of the lymphoreticular system known at present. Studies of cellular DNA quantitation and thymidine incorporation indicated that the growth rate of the „hairy”︁ cells is exceedingly low.

Anti-epiligrin cicatricial pemphigoid: clinical findings, immunopathogenesis, and significant associations.

We report the clinical and immunopathologic findings in a cohort of 35 patients with anti-epiligrin cicatricial pemphigoid (AECP). These patients have a mucosal predominant subepithelial blistering disease that is clinically indistinguishable from other forms of cicatricial pemphigoid. The mucosal surfaces of the mouth and eye are most commonly involved. The skin is also involved in most patients, but usually this is less severe than mucosal involvement. AECP is characterized by the binding of circulating IgG autoantibodies to the dermal side of 1M NaCl split human skin on indirect immunofluorescence microscopy. These IgG antibasement membrane autoantibodies target laminin 5, a heterotrimeric protein consisting of α3, β3, and γ2 subunits. IgG autoantibodies predominantly target the G domain within the α subunit. The presence of circulating IgG autoantibodies are specific for the diagnosis of AECP and are not seen in patients with other autoimmune blistering diseases or normal volunteers. Furthermore, we expand on data previously reported on the finding of an increased relative risk for solid cancer in patients with AECP, especially in the first year after blister onset. The majority of cancers documented in a cohort of 35 patients assembled over 12 years of study were adenocarcinomas that were at an advanced stage at their time of detection. This circumstance is thought to account for a high incidence of mortality among AECP patients who develop an associated cancer. AECP patients also demonstrate a significant risk for mortality as a consequence of treatment with systemic immunosuppressives. The current longitudinal study suggests that only a minority of AECP patients go into remission.

Antiepiligrin cicatricial pemphigoid represents an autoimmune response to subunits present in laminin 5 (α3β3γ2)

Sera from 20 patients with antiepiligrin cicatricial pemphigoid were studied to define the specific reactivity of their IgG autoantibodies. IgG from all patients bound exclusively to the dermal side of 1 mol/L NaCl split skin and immunoprecipitated laminin 5 (α3β3γ2) from extracts of human keratinocytes (HKs). Immunoblot studies on purified laminin 5 subunits demonstrated that patient IgG bound α3 alone in 16 patients. In two patients, IgG autoantibodies were directed predominantly to the γ2 subunit, yet showed trace reactivity to α3 as well. Sera from two patients did not immunoblot any laminin 5 subunits, their IgG presumably immunoprecipitating laminin 5 via a conformational epitope. Sera from patients with α3 subunit‐specific IgG immunoprecipitated all subunits of laminin 5 as well as polypeptides of 190 and 200 kDa from the conditioned media of HKs. Preclearance studies and experiments utilizing affinity‐purified patient IgG demonstrated that the latter signified laminin 6 (α3β1γ1) that was bound by cross‐reactive α3 subunit‐specific patient IgG. Sera from patients with γ2 subunit‐specific IgG showed no reactivity to laminin 6, except for faint reactivity provided by low levels of their α3 subunit‐specific IgG. Taken together, these findings indicate that antiepiligrin cicatricial pemphigoid signifies an autoimmune response to subunits present in laminin 5.

HOX decoy peptide enhances the ex vivo expansion of human umbilical cord blood CD34+ hematopoietic stem cells/hematopoietic progenitor cells.

The isolation and characterization of living human epithelial stem cells is difficult because distinguishing cell surface markers have not been identified with certainty. Side population keratinocytes (SP‐KCs) that efflux Hoechst 33342 fluorescent dye, analogous to bone marrow‐derived side population (SP) hematopoietic stem cells, have been identified in human skin, but their potential to function as keratinocyte stem cells (KSCs) in vivo is not known. On the other hand, human keratinocyte populations that express elevated levels of β1 and α6 integrins and are distinct from SP‐KCs, which express low levels of integrins, may be enriched for KSCs based on reported results of in vitro cell culture assays. When in vitro assays were used to measure total cell output of human SP‐KCs and integrin‐bright keratinocytes, we could not document their superior long‐term proliferative activity versus unfractionated keratinocytes. To further assess the KSC characteristics in SP‐KCs and integrin‐bright keratinocytes, we used an in vivo competitive repopulation assay in which bioengineered human epidermis containing competing keratinocyte populations with different human major histocompatibility (MHC) class I antigens were grafted onto immunocompromised mice, and the intrinsic MHC class I antigens are used to quantify expansion of competing populations. In these in vivo studies, human SP‐KCs showed little competitive expansion in vivo and were not enriched for KSCs. In contrast, keratinocytes expressing elevated levels of α6 integrin and low levels of CD71 (α6‐bright/CD71‐dim) expanded over 200‐fold during the 33‐week in vivo study. These results definitively demonstrate that human α6‐bright/CD71‐dim keratinocytes are enriched with KSCs, whereas SP‐KCs are not.