David A. Norris

Presence of IgE antibodies to staphylococcal exotoxins on the skin of patients with atopic dermatitis. Evidence for a new group of allergens.

In the current study, we investigated whether Staphylococcus aureus grown from affected skin of atopic dermatitis (AD) patients secreted identifiable toxins that could act as allergens to induce IgE-mediated basophil histamine release. The secreted toxins of S. aureus grown from AD patients were identified by ELISA using antibodies specific for staphylococcal enterotoxin (SE) exfoliative toxin (ET), or toxic shock syndrome toxin (TSST-1). S. aureus isolates from 24 of 42 AD patients secreted identifiable toxins with SEA, SEB, and TSST accounting for 92% of the isolates. 32 of 56 AD sera (57%) tested contained significant levels of IgE primarily to SEA, SEB, and/or TSST. In contrast, although SEA, SEB, or TSST secreting S. aureus could be recovered from the skin of psoriasis patients, their sera did not contain IgE antitoxins. Freshly isolated basophils from 10 AD patients released 5-59% of total histamine in response to SEA, SEB, or TSST-1 but only with toxins to which patients had specific IgE. Basophils from eight other AD patients and six normal controls who had no IgE antitoxin failed to demonstrate toxin-induced basophil histamine release. Stripped basophils sensitized with three AD sera containing IgE to toxin released 15-41% of total basophil histamine only when exposed to the relevant toxin, but not to other toxins. Sensitization of basophils with AD sera lacking IgE antitoxin did not result in release of histamine to any of the toxins tested. These data indicate that a subset of patients with AD mount an IgE response to SEs that can be grown from their skin. These toxins may exacerbate AD by activating mast cells, basophils, and/or other Fc epsilon-receptor bearing cells armed with the relevant IgE antitoxin.

Genome-wide association study in alopecia areata implicates both innate and adaptive immunity

Alopecia areata (AA) is among the most highly prevalent human autoimmune diseases, leading to disfiguring hair loss due to the collapse of immune privilege of the hair follicle and subsequent autoimmune attack. The genetic basis of AA is largely unknown. We undertook a genome-wide association study (GWAS) in a sample of 1,054 cases and 3,278 controls and identified 139 single nucleotide polymorphisms that are significantly associated with AA (P ≤ 5 × 10−7). Here we show an association with genomic regions containing several genes controlling the activation and proliferation of regulatory T cells (Treg cells), cytotoxic T lymphocyte-associated antigen 4 (CTLA4), interleukin (IL)-2/IL-21, IL-2 receptor A (IL-2RA; CD25) and Eos (also known as Ikaros family zinc finger 4; IKZF4), as well as the human leukocyte antigen (HLA) region. We also find association evidence for regions containing genes expressed in the hair follicle itself (PRDX5 and STX17). A region of strong association resides within the ULBP (cytomegalovirus UL16-binding protein) gene cluster on chromosome 6q25.1, encoding activating ligands of the natural killer cell receptor NKG2D that have not previously been implicated in an autoimmune disease. By probing the role of ULBP3 in disease pathogenesis, we also show that its expression in lesional scalp from patients with AA is markedly upregulated in the hair follicle dermal sheath during active disease. This study provides evidence for the involvement of both innate and acquired immunity in the pathogenesis of AA. We have defined the genetic underpinnings of AA, placing it within the context of shared pathways among autoimmune diseases, and implicating a novel disease mechanism, the upregulation of ULBP ligands, in triggering autoimmunity.

Ultraviolet light induces binding of antibodies to selected nuclear antigens on cultured human keratinocytes.

Antibodies which bind to different nuclear antigens in tissue sections or in permeabilized cell cultures are useful markers of subsets of connective tissue disease, especially of lupus erythematosus (LE), but whether these antibodies are able to react with these intracellular sequestered antigens in vivo and cause immunologic tissue damage has been a matter of much debate. We report experiments which show that ultraviolet light-irradiated, cultured human keratinocytes bind IgG antibodies from the sera of LE patients with either monospecific anti-SSA/Ro, anti-RNP, or anti-Sm activity, which implies that these antigens have been made accessible on the cell surface by ultraviolet irradiation. Normal human sera or LE patients sera with anti-double-stranded DNA, anti-single-stranded DNA, or antihistone activity do not bind to the surface of irradiated human keratinocytes. In control experiments, all antisera produced the expected patterns of nuclear and cytoplasmic staining of fixed permeabilized, unirradiated keratinocytes. Careful study of the viability and permeability of irradiated keratinocytes by several techniques showed that this apparent cell membrane expression of extractable nuclear antigens (SSA/Ro, RNP, and Sm) following irradiation was seen on injured keratinocytes whose cell membranes were intact, but not on dead cells. It is particularly significant that all six monospecific anti-SSA/Ro sera bound to irradiated keratinocytes, since this antibody antigen system is highly associated with photosensitive cutaneous LE.

Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis.

Recent studies have suggested that T cells play a critical role in the pathogenesis of psoriasis. Guttate psoriasis is a well-defined form of psoriasis frequently associated with streptococcal throat infection. This study tested the hypothesis that T cells in acute guttate psoriasis skin lesions may be activated by streptococcal superantigens. Peripheral blood as well as lesional and perilesional skin biopsies were analyzed for T cell receptor V beta repertoire using monoclonal antibodies against 10 different V beta families. Skin biopsies from all patients with acute guttate psoriasis, but not skin biopsies from patients with acute atopic dermatitis or inflammatory skin lesions induced in normal subjects with sodium lauryl sulfate, demonstrated selective accumulation of V beta 2+ T cells (P < 0.05). The expansion of V beta 2+ T cells occurred in both the CD4+ and the CD8+ T cell subsets. Sequence analysis of T cell receptor beta chain genes of V beta 2-expressing T cells from skin biopsies of patients with guttate psoriasis showed extensive junctional region diversity that is more compatible with a superantigen rather than a conventional (nominal) antigen-driven T cell response. All streptococcal isolates from patients with guttate psoriasis secreted streptococcal pyrogenic exotoxin C, a superantigen known to stimulate marked V beta 2+ T cell expansion. These data support the concept that acute guttate psoriasis is associated with superantigenic stimulation of T cells triggered by streptococcal superantigen(s).

Subacute Cutaneous Lupus Erythematosus Associated with Hydrochlorothiazide Therapy

Photosensitive eruptions with clinical and histologic features of subacute cutaneous lupus erythematosus and antibodies to SS-A(Ro) antigen occurred in five patients taking hydrochlorothiazide. After drug therapy was discontinued, the eruptions cleared. In one patient anti-SS-A antibodies disappeared after discontinuation of thiazide, and in another rechallenge with hydrochlorothiazide produced an acute dermatitis with a photodistribution. These eruptions may represent a new type of photosensitive drug reaction in which the photoactive drug may be synergistic with anti-SS-A antibody in producing cutaneous lesions of photosensitive subacute cutaneous lupus erythematosus.

Constitutively Active Inflammasome in Human Melanoma Cells Mediating Autoinflammation via Caspase-1 Processing and Secretion of Interleukin-1β

Interleukin-1β (IL-1β) is a pleiotropic cytokine promoting inflammation, angiogenesis, and tissue remodeling as well as regulation of immune responses. Although IL-1β contributes to growth and metastatic spread in experimental and human cancers, the molecular mechanisms regulating the conversion of the inactive IL-1β precursor to a secreted and active cytokine remains unclear. Here we demonstrate that NALP3 inflammasome is constitutively assembled and activated with cleavage of caspase-1 in human melanoma cells. Late stage human melanoma cells spontaneously secrete active IL-1β via constitutive activation of the NALP3 inflammasome and IL-1 receptor signaling, exhibiting a feature of autoinflammatory diseases. Unlike human blood monocytes, these melanoma cells require no exogenous stimulation. In contrast, NALP3 functionality in intermediate stage melanoma cells requires activation of the IL-1 receptor to secrete active IL-1β; cells from an early stage of melanoma require stimulation of the IL-1 receptor plus the co-stimulant muramyl dipeptide. The spontaneous secretion of IL-1β from melanoma cells was reduced by inhibition of caspase-1 or the use of small interfering RNA directed against ASC. Supernatants from melanoma cell cultures enhanced macrophage chemotaxis and promoted in vitro angiogenesis, both prevented by pretreating melanoma cells with inhibitors of caspases-1 and -5 or IL-1 receptor blockade. These findings implicate IL-1-mediated autoinflammation as contributing to the development and progression of human melanoma and suggest that inhibiting the inflammasome pathway or reducing IL-1 activity can be a therapeutic option for melanoma patients.

Pattern of cutaneous immunoglobulin G deposition in subacute cutaneous lupus erythematosus is reproduced by infusing purified anti-Ro (SSA) autoantibodies into human skin-grafted mice.

Subacute cutaneous lupus and neonatal lupus are closely associated with the presence of anti-Ro (SSA) autoantibodies, but there is no direct evidence establishing a role for anti-Ro (SSA) in these diseases. After parental injection into mice, IgG from sera containing anti-Ro (SSA) will bind human skin grafted onto the mice. To determine whether the antibody binding is due to anti-Ro (SSA), affinity-purified anti-Ro (SSA) and serum depleted of anti-Ro (SSA) were prepared. After injection into human skin-grafted mice, purified anti-Ro (SSA) antibodies bound an antigen in the human skin graft, while preabsorbing anti-Ro (SSA) serum with Ro (SSA) virtually abolished binding to the human skin graft. Moreover, the pattern of IgG deposition was primarily epidermal and was identical in the human skin-grafted mice injected with purified anti-Ro (SSA) when compared with that found in five patients with subacute lupus (four adults, one neonate). These data directly show that anti-Ro (SSA) antibodies bind to the skin, and support the hypothesis that anti-Ro (SSA) autoantibodies are involved in the disease process that produces subacute cutaneous lupus and neonatal lupus.

ALDH1A isozymes are markers of human melanoma stem cells and potential therapeutic targets.

Although the concept of cancer stem cells (CSCs) is well‐accepted for many tumors, the existence of such cells in human melanoma has been the subject of debate. In this study, we demonstrate the existence of human melanoma cells that fulfill the criteria for CSCs (self‐renewal and differentiation) by serially xenotransplanting cells into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. These cells possess high aldehyde dehydrogenase (ALDH) activity with ALDH1A1 and ALDH1A3 being the predominant ALDH isozymes. ALDH‐positive melanoma cells are more tumorigenic than ALDH‐negative cells in both NOD/SCID mice and NSG mice. Biological analyses of the ALDH‐positive melanoma cells reveal the ALDH isozymes to be key molecules regulating the function of these cells. Silencing ALDH1A by siRNA or shRNA leads to cell cycle arrest, apoptosis, decreased cell viability in vitro, and reduced tumorigenesis in vivo. ALDH‐positive melanoma cells are more resistant to chemotherapeutic agents and silencing ALDH1A by siRNA sensitizes melanoma cells to drug‐induced cell death. Furthermore, we, for the first time, examined the molecular signatures of ALDH‐positive CSCs from patient‐derived tumor specimens. The signatures of melanoma CSCs include retinoic acid (RA)‐driven target genes with RA response elements and genes associated with stem cell function. These findings implicate that ALDH isozymes are not only biomarkers of CSCs but also attractive therapeutic targets for human melanoma. Further investigation of these isozymes and genes will enhance our understanding of the molecular mechanisms governing CSCs and reveal new molecular targets for therapeutic intervention of cancer. STEM Cells2012;30:2100–2113

A serological marker for neonatal lupus erythematosus

Precipitating antibodies to cellular antigens, designated SS‐A(Ro), were found in the sera of seven of eight infants with lupus erythematosus, but they were not found in seventy‐one control

Immunogenetics of the Neonatal Lupus Syndrome

Abstract Infants with neonatal lupus erythematosus have congenital heart block, transient cutaneous lesions, or both. Mothers of these infants have SSA/Ro autoantibodies that are passed across the ...