Frontal fibrosing alopecia.

Frontal fibrosing alopecia (FFA) is a patterned primary cicatricial alopecia that was first described in 1994. Once rare, the incidence of FFA has increased dramatically, representing the current most common cause of cicatricial alopecia worldwide. FFA typically begins in postmenopausal women with symmetrical, progressive recession of the frontotemporal hairline together with bilateral loss of the eyebrows. FFA has a distinctive clinical phenotype, which remains a challenge. The histology is identical to lichen planopilaris (LPP), but only a small number of patients have coincidental LPP, usually of the scalp. The vast majority of patients have no evidence of lichen planus elsewhere, and the symmetry and patterned nature of the hair loss are unusual for LPP. Familial cases of FFA are reported, and gene associations have been identified in population studies; however, the pathophysiology remains controversial. Without treatment, FFA is slowly progressive, and although many treatments have been prescribed, the response is often disappointing. We review the pathogenesis, epidemiology, clinical features, histology, and treatment of FFA.

Phospho-ß-catenin expression in primary and metastatic melanomas and in tumor-free visceral tissues, and associations with expression of PD-L1 and PD-L2.

ß-catenin (ßcat) is an important downstream effector in the Wnt signaling pathway and plays important roles in the development and progression of many cancers including melanoma. ßcat expression is regulated by GSK-3ß-mediated phosphorylation at positions 33, 37 and 41. In normal cells, phosphorylation at these sites triggers proteasomal degradation, which prevents accumulation of free cytoplasmic ßcat. In cancer cells, stabilized ß-catenin translocates into the nucleus, where it associates with TCF/Lef proteins to activate transcription of genes that promote tumorigenesis and metastasis, including PD-L1. It has been suggested that nuclear phospho-ßcat (pßcat) staining may be diagnostically useful in differentiating primary from metastatic melanoma. Also, a pßcat peptide (residues 30-39, with only S33 phosphorylated) is naturally presented by melanoma cells as a T-cell target. We evaluated expression of pS33-ßcat in primary and metastatic melanomas by immunohistochemistry and found its expression varied widely but was most commonly cytoplasmic. Nuclear staining was identified in only 18% of metastatic melanomas. Staining with antibodies to pS33-ßcat and pS33/37/T41-ßcat was most intense in mitotic melanoma cells; however, pS33-ßcat intensity was not significantly associated with AJCC stage, tumor location, BRAF mutation status, or immune infiltrates. Yet, PD-L1 and PD-L2 expression by tumor cells were significantly higher in tumors with high pS33-ßcat expression. The low rate of nuclear pS33-ßcat expression suggests that pS33-ßcat may have limited utility for identifying metastatic melanomas. However, high expression in dividing cells and strong associations with PD-L1 and PD-L2 expression may inform future personalized therapies for tumors with high pS33-ßcat expression.

Characterization and comparison of innate and adaptive immune responses at vaccine sites in melanoma vaccine clinical trials.

The strength and durability of systemic anti-tumor immune responses induced by cancer vaccines depends on adjuvants to support an immunogenic vaccine site microenvironment (VSME). Adjuvants include water-in-oil emulsions with incomplete Freund's adjuvant (IFA) and combinations of toll-like receptor (TLR) agonists, including a preparation containing TLR4 and TLR9 agonists with QS-21 (AS15). IFA-containing vaccines can promote immune cell accumulation at the VSME, whereas effects of AS15 are largely unexplored. Therefore, we assessed innate and adaptive immune cell accumulation and gene expression at the VSME after vaccination with AS15 and compared to effects with IFA. We hypothesized that AS15 would promote less accumulation of innate and adaptive immune cells at the VSME than IFA vaccines. In two clinical trials, patients with resected high-risk melanoma received either a multipeptide vaccine with IFA or a recombinant MAGE-A3 protein vaccine with AS15. Vaccine site biopsies were obtained after one or multiple vaccines. T cells accumulated early after vaccines with AS15, but this was not durable or of the same magnitude as vaccination in IFA. Vaccines with AS15 increased durable expression of DC- and T cell-related genes, as well as PD-L1 and IDO1, suggesting complex activation and regulation of innate and adaptive immune function with AS15. These changes were generally greater with vaccines containing IFA, but IFA induced reduction in myeloid suppressor cells markers. Evidence of tertiary lymphoid structure (TLS) formation was observed with both adjuvants. Our findings highlight adjuvant-dependent changes in immune features at the VSME that may impact systemic immune responses.

Patterns of immune-cell infiltration in murine models of melanoma: roles of antigen and tissue site in creating inflamed tumors.

Immune-cell infiltration is associated with improved survival in melanoma. Human melanoma metastases may be grouped into immunotypes representing patterns of immune-cell infiltration: A (sparse), B (perivascular cuffing), and C (diffuse). Immunotypes have not been defined for murine melanomas, but may provide opportunities to understand mechanism-driving immunotype differences. We performed immunohistochemistry with immune-cell enumeration, immunotyping, and vascular density scoring in genetically engineered (Braf/Pten and Braf/Pten/ß-catenin) and transplantable (B16-F1, B16-OVA, and B16-AAD) murine melanomas. The transplantable tumors were grown in subcutaneous (s.c.) or intraperitoneal (i.p.) locations. Braf/Pten and Braf/Pten/ß-catenin tumors had low immune-cell densities, defining them as Immunotype A, as did B16-F1 tumors. B16-OVA (s.c. and i.p.) and B16-AAD s.c. tumors were Immunotype B, while B16-AAD i.p. tumors were primarily Immunotype C. Interestingly, the i.p. location was characterized by higher immune-cell counts in B16-OVA tumors, with counts that trended higher for B16-F1 and B16-AAD. The i.p. location was also characterized by higher vascularity in B16-F1 and B16-AAD tumors. These findings demonstrate that spontaneously mutated neoantigens in B16 melanomas were insufficient to induce robust intratumoral immune-cell infiltrates, but instead were Immunotype A tumors. The addition of model neoantigens (OVA or AAD) to B16 enhanced infiltration, but this most often resulted in Immunotype B. We find that tumor location may be an important element in enabling Immunotype C tumors. In aggregate, these data suggest important roles both for the antigen type and for the tumor location in defining immunotypes.

Human melanomas and ovarian cancers overexpressing mechanical barrier molecule genes lack immune signatures and have increased patient mortality risk.

We have identified eight genes whose expression in human melanoma metastases and ovarian cancers is associated with a lack of Th1 immune signatures. They encode molecules with mechanical barrier function in the skin and other normal tissues and include filaggrin (FLG), tumor-associated calcium signal transducer 2 (TACSTD2), and six desmosomal proteins (DST, DSC3, DSP, PPL, PKP3, and JUP). This association has been validated in an independent series of 114 melanoma metastases. In these, DST expression alone is sufficient to identify melanomas without immune signatures, while FLG and the other six putative barrier molecules are overexpressed in a different subset of melanomas lacking immune signatures. Similar associations have been identified in a set of 186 ovarian cancers. RNA-seq data from 471 melanomas and 307 ovarian cancers in the TCGA database further support these findings and also reveal that overexpression of barrier molecules is strongly associated with early patient mortality for melanoma (p = 0.0002) and for ovarian cancer (p < 0.01). Interestingly, this association persists for FLG for melanoma (p = 0.012) and ovarian cancer (p = 0.006), whereas DST overexpression is negatively associated with CD8+ gene expression, but not with patient survival. Thus, overexpression of FLG or DST identifies two distinct patient populations with low immune cell infiltration in these cancers, but with different prognostic implications for each. These data raise the possibility that molecules with mechanical barrier function in skin and other tissues may be used by cancer cells to protect them from immune cell infiltration and immune-mediated destruction.

Porcine acellular dermal matrix (PADM) vascularises after exposure in open necrotic wounds seen after complex hernia repair.

Biological alternatives to synthetic meshes are increasingly utilised in complex abdominal wall reconstruction. There is a lack of evidence demonstrating that non-cross-linked porcine acellular dermal matrix vascularizes and integrates with human tissue in suboptimal wound conditions. We aimed to evaluate these properties in Strattice™ (Life Cell Inc., Branchburg, NJ) following ventral hernia repair. A retrospective review of patients with high-risk ventral hernia repair utilising Strattice™ as an onlay after open component separation was conducted. Patients with postoperative wound exploration and exposure of the onlay were included in this review. One patient underwent punch biopsy for histological analysis. Eleven patients with wound complications necessitating postoperative debridement and exposure of Strattice™ onlay were identified. The onlay was partially debrided in two cases, and one case required complete excision. Vascularisation was clinically evident in 10 of 11 cases (91%) as demonstrated by the presence of granulation tissue and/or the ability to support a skin graft. Histological analysis of one onlay 3 months postoperatively showed neovascularisation and collagen remodelling with minimal inflammatory response. Strattice™ demonstrated resistance to rejection, ability to undergo vascularisation and incorporation into host tissues in sub-optimal wound conditions following ventral hernia repair.

MHC-restricted phosphopeptides from insulin receptor substrate-2 and CDC25b offer broad-based immunotherapeutic agents for cancer.

Cancer cells display novel phosphopeptides in association with MHC class I and II molecules. In this study, we evaluated two HLA-A2-restricted phosphopeptides derived from the insulin receptor substrate (IRS)-2 and the cell-cycle regulator CDC25b. These proteins are both broadly expressed in multiple malignancies and linked to cancer cell survival. Two phosphopeptides, termed pIRS-21097-1105 and pCDC25b38-46, served as targets of strong and specific CD8 T-cell memory responses in normal human donors. We cloned T-cell receptor (TCR) cDNAs from murine CD8 T-cell lines specific for either pIRS-21097-1105 or pCDC25b38-46. Expression of these TCRs in human CD8 T cells imparted high-avidity phosphopeptide-specific recognition and cytotoxic and cytokine-secreting effector activities. Using these cells, we found that endogenously processed pIRS-21097-1105 was presented on HLA-A2(+) melanomas and breast, ovarian, and colorectal carcinomas. Presentation was correlated with the level of the Ser(1100)-phosphorylated IRS-2 protein in metastatic melanoma tissues. The highest expression of this protein was evident on dividing malignant cells. Presentation of endogenously processed pCDC25b38-46 was narrower, but still evident on HLA-A2(+) melanoma, breast carcinoma, and lymphoblastoid cells. Notably, pIRS-21097-1105-specific and pCDC25b38-46-specific TCR-expressing human CD8 T cells markedly slowed tumor outgrowth in vivo. Our results define two new antigens that may be developed as immunotherapeutic agents for a broad range of HLA-A2(+) cancers.

Something to Reed about: fibroids, cutaneous leiomyomas, and renal cell carcinoma.

A 48 year-old woman with a history of fibroids presents with asymptomatic skin lesions. Biopsy reveals cutaneous leiomyomas and subsequent genetic evaluation confirms the diagnosis of hereditary leiomyomatosis and renal cell cancer. In this report, we review the typical presentation of the syndrome as well as recommendations for surveillance.

Symptomatic squamous papilloma of the uvula: report of a case and review of the literature.

Background. Oral squamous papillomas are benign pedunculated masses that grow most commonly on the palate. These benign lesions rarely cause symptoms. Methods. Here we present the case of a large, elongated squamous papilloma of the uvula causing dysphagia. We also review pertinent literature related to these lesions. Results. This patient underwent surgical excision of her atypically symptomatic oral lesion, with complete resolution of symptoms. Conclusion. Oral squamous papillomas are benign lesions which are usually asymptomatic. Dysphagia due to a squamous papilloma of the uvula has only been reported once in the literature previously. The development of symptoms such as dysphagia due to squamous papilloma of the uvula is uncommon; however this may be more likely in the presence of particularly large lesions.

Aberrant VEGF expression associated with neoplasm-induced extramedullary hematopoiesis in an epithelioid hemangioendothelioma: a case report.

The authors present the first report of extramedullary hematopoiesis occurring in association with an epithelioid hemangioendothelioma. A 64-year-old man sought medical attention for upper chest pain. CT evaluation identified a 2.8-cm mass involving the right hemithorax in the area of the right upper lobe and superior vena cava. A biopsy revealed the presence of an epithelioid malignant vascular neoplasm, which on subsequent resection was found to be an epithelioid hemangioendothelioma. The neoplasm was closely associated with an area of extramedullary hematopoiesis. VEGF immunohistochemical staining of the neoplasm revealed a ring of intensely positive staining cells around the periphery of the area of extramedullary hematopoiesis. This finding provides evidence for the role of aberrant VEGF expression in neoplasm-induced extramedullary hematopoiesis. This report discusses possible mechanisms by which extramedullary hematopoiesis may occur in vascular neoplasms.

The potential role of vitamin D in the progression of benign and malignant melanocytic neoplasms.

Hormonally active vitamin D3, 1,25-(OH)2D3, is believed to have a role in the prevention of cancer formation and in limiting the aggressiveness of cancers that do arise. Therefore,much interest is presently being focused on 1,25-(OH)2D3 and its analogues as potential treatments for various cancers including melanoma. This article discusses the evidence in favour of a role for 1,25-(OH)2D3 in protection against the progression of melanocytic lesions and also summarizes the mechanisms by which 1,25-(OH)2D3 may act to protect against melanoma development and progression.

A multiplex real-time PCR method for quantification of BK and JC polyomaviruses in renal transplant patients.

BACKGROUND: Infection of BK or JC human polyomavirus can lead to polyomavirus-associated nephropathy in renal transplant patients. Thus effective management of these patients requires early detection and quantification of these viruses in urine and blood. DESIGN: The aim of this study was to evaluate and validate a multiplex real-time PCR-based method for monitoring BK and/or JC viral loads in renal transplant patients. Analytic parameters such as limit of quantification, linear dynamic range, sensitivity and specificity, as well as reliability of the assays were determined. Seventy-six plasma or urine samples spiked with variable amounts of BK and JC viral DNA ranging from low (7.0 x 10(3) or 1.5 x 10(4)), to medium (1.0 x 10(6) to high (1.0 x 10(8) or 1.0109 copies/mL) levels of viruses were tested. In addition, 45 clinical urine or plasma samples with known copy numbers of BK or JC viruses, which were isolated from the renal transplant patients from 4 US medical centers, were also tested. RESULTS: BK and/or JC viruses can be detected with distinguishable melting temperature of 64 degrees C or 68 degrees C, respectively. On the basis of the need for clinical monitoring of different types of specimens, the low limit of quantification for plasma or urine was set at 7.0 x 10(3) or 1.5 x 10(4) copies/mL, respectively with the linear dynamic range Z6 logs. The assay exhibits 100% specificity, 97.9% sensitivity with low intra-assay and interassay variability (coefficient of variation <4%). CONCLUSIONS: This clinically validated method has the necessary utility to monitor BK and JC viremia and viruria in renal transplant patients.

Enhanced immunity and protective efficacy against SIVmac251 intrarectal challenge following ad-SIV priming by multiple mucosal routes and gp120 boosting in MPL-SE.

Previously, 39% of rhesus macaques primed orally, intranasally, and intratracheally with adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinants and boosted with gp120 in monophosphoryl lipid A-stable emulsion (MPL-SE) remained aviremic or cleared or controlled viremia at the threshold of detection following SIV(mac251) intrarectal challenge (Study B). In contrast, no macaques primed orally and intranasally with Ad-SIV recombinants and boosted with gp120 in Quillaja Saponaria-21 exhibited undetectable viremia post-challenge (Study A). We conducted a detailed comparison of the studies to elucidate the effect of different vaccine regimens on induced immunity associated with the different challenge outcomes. Quantitative viral load comparisons were statistically analyzed. All immune responses were assessed at identical timepoints post-immunization, and cellular immunity was re-evaluated on cryopreserved cells from Study B macaques to match Study A data acquired with frozen cells. Study B exhibited greater protective efficacy, increased levels of p11C and p54m tetramer positive cells and a trend toward enhanced interferon-gamma secreting cells in response to Env and Gag peptides, modestly enhanced serum neutralizing antibodies, and greater positivity in anti-gp120 rectal IgA and IgG antibodies. Study A macaques exhibited greater positivity in salivary IgA anti-gp120 antibodies. Thus, the vaccine regimen using oral-intranasal-intratracheal priming and protein boosting in MPL-SE was superior, eliciting greater protective efficacy against pathogenic SIV(mac251) and enhanced SIV-specific immunity, systemically and at rectal sites. The mechanism(s) by which binding antibodies, lacking neutralizing activity against the primary challenge virus, may contribute to protection requires further study.

Evaluation of combination DNA/replication-competent Ad-SIV recombinant immunization regimens in rhesus macaques.

Combination vaccine regimens in which priming with recombinant DNA is followed by boosting with recombinant viral vectors have been shown in previous studies to effectively enhance cellular immunity. However, no information exists concerning possible synergy of the cellular immune response when DNA immunization is followed by administration of a recombinant vector able to replicate. As our approach makes use of replication-competent Ad HIV and SIV recombinants, we performed a pilot experiment in six rhesus macaques in which we compared immunogenicity resulting from priming with one or two DNA recombinants encoding the SIVsmH4 env and rev genes with that elicited by a single replication-competent Ad5hr-SIV env/rev priming immunization. All macaques were subsequently administered an Ad5hr-SIV env/rev booster immunization followed by two immunizations with SIV gp120 protein. The choice of the env gene as target immunogen allowed comparison of induced cellular immune responses as well as binding and neutralizing antibodies elicited in serum and mucosal secretions. We report here that all immunized monkeys developed strong cellular immunity to the SIV envelope as shown by secretion of interferon-gamma, lysis of envelope-expressing target cells, and/or proliferation in response to gp120 or inactivated SIV. Similarly, all macaques developed anti-gp120 binding antibodies and neutralizing antibodies in serum and IgG and IgA binding antibodies in mucosal secretions. We did not observe consistently enhanced immune responses in any immunization group. We conclude that two sequential immunizations with the same replication-competent Ad5hr-SIV recombinant is as effective as priming with one or two recombinant DNA vaccines followed by a single Ad5hrSIV recombinant immunization.

Protection against mucosal simian immunodeficiency virus SIV(mac251) challenge by using replicating adenovirus-SIV multigene vaccine priming and subunit boosting.

Whereas several recent AIDS vaccine strategies have protected rhesus macaques against a pathogenic simian/human immunodeficiency virus (SHIV)(89.6P) challenge, similar approaches have provided only modest, transient reductions in viral burden after challenge with virulent, pathogenic SIV, which is more representative of HIV infection of people. We show here that priming with replicating adenovirus recombinants encoding SIV env/rev, gag, and/or nef genes, followed by boosting with SIV gp120 or an SIV polypeptide mimicking the CD4 binding region of the envelope, protects rhesus macaques from intrarectal infection with the highly pathogenic SIV(mac251). Using trend analysis, significant reductions in acute-phase and set point viremia were correlated with anti-gp120 antibody and cellular immune responses, respectively. Within immunization groups exhibiting significant protection, a subset (39%) of macaques have exhibited either no viremia, cleared viremia, or controlled viremia at the threshold of detection, now more than 40 weeks postchallenge. This combination prime-boost strategy, utilizing replication competent adenovirus, is a promising alternative for HIV vaccine development.

Boosting of SIV-specific immune responses in rhesus macaques by repeated administration of Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinants.

Previous non-human primate studies have shown replication competent adenovirus (Ad) HIVenv/rev and SIVenv/rev recombinants to be promising vaccine candidates. To broaden induced immunity in rhesus macaques, an Ad type 5 host range (Ad5hr) mutant vector with an inserted SIV gag gene was added to the vaccine regimen. Immunity to the encoded SIV Env, Rev, and Gag gene products was evaluated following two immunizations with the same recombinants. The vaccines were administered intranasally plus orally via stomach tube at weeks 0 and 12. The recombinants replicated well in the upper respiratory tract but poorly in the gut, suggesting enteric-coated capsules might improve oral delivery to the intestine. SIV-specific cellular immunity was induced in all 16 immunized macaques. Fourteen exhibited positive interferon-gamma (IFN-gamma) ELISPOT responses, and nine, including two lacking IFN-gamma responses, exhibited SIV-specific T-cell proliferative activity. IFN-gamma secreting peripheral blood mononuclear cells (PBMCs) in response to SIV Gag, Env, and Rev peptides were induced in 73, 53, and 27% of macaques, respectively, and were boosted two- to four-fold by the second immunization. A persistent response to Gag was evident at least 10 weeks thereafter. p11C tetramer staining confirmed elicitation of SIV Gag-specific CD8+ T-cells in Mamu-A*01 macaques. Proliferative responses were more frequent after the second immunization, and binding antibody titers to SIV gp120 were significantly boosted by the immunization regimen. We conclude that a second administration of recombinants based in the same Ad5hr vector can effectively boost immunity to inserted gene products, obviating development of several recombinants in different Ad serotypes for multiple immunizations.

Improved protection of rhesus macaques against intrarectal simian immunodeficiency virus SIV(mac251) challenge by a replication-competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regimen.

In this study we investigated the ability of a replication-competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regimen to induce protective immunity in rhesus macaques against pathogenic simian immunodeficiency virus(mac251). Immunization of macaques by two sequential administrations of the same recombinants by the same route resulted in boosting and persistence of SIV-specific cellular immune responses for 42 weeks past the initial immunization. Anti-SIV gp120 immunoglobulin G (IgG) and IgA antibodies were induced in secretory fluids, and all macaques exhibited serum neutralizing antibody activity. After intrarectal SIV(mac251) challenge, all of the macaques became infected. However, relative protection, as assessed by statistically significant lower SIV viral loads in plasma at both acute infection and set point, was observed in 8 out of 12 immunized non-Mamu-A(*)01 animals. Elevated mean cellular immune responses to Gag and Env, neutralizing antibody activity, and IgG and IgA binding antibody levels were observed in the eight protected macaques. Statistically significant correlations with protective outcome were observed for cellular immune responses to SIV Env and Gag and for SIV gp120-specific IgG antibodies in nasal and vaginal fluids. Two macaques that exhibited the greatest and most persistent viremia control also exhibited strong CD8(+) T-cell antiviral activity. The results suggest that a spectrum of immune responses may be necessary for adequate control of viral replication and disease progression and highlight a potential role for nonneutralizing antibodies at mucosal sites.