The differential expression of toll like receptors and RIG-1 correlates to the severity of infectious diseases.

The toll like receptors (TLRs) and RIG-1 are proteins involved in the initial reaction of the innate immune system to infectious diseases and, thus, can provide much information to the surgical pathologist in terms of the molecular dynamics of the infection. The TLRs (TLR1, 2, 3, 4, 7, 8) and RIG-1 distribution as determined by immunohistochemistry was examined in the following diseases: human papillomavirus (n = 30 including 15 squamous intraepithelial lesions (SIL), 5 cancers, and 10 controls); molluscum contagiosum (n = 8 including 4 controls), SARS-CoV2 (n = 52 including 20 mild, 5 fatal, and 27 controls) and reovirus infection as oncolytic therapy. Mild, regressing infection (molluscum contagiosum, mild SARS-CoV2 and low grade SIL) each showed the same pattern: marked up regulation of at least three of the TLRs/RIG-1 with decreased expression of none compared to the controls. Severe infection (fatal SARS-CoV2, and cervical cancer) each showed marked decrease expression in at least three of the TLRs/RIG-1. We recently documented an equivalent marked decrease expression of the TLRs/RIG-1 in the placenta in fatal in utero infections. The reoviral infected tissues showed an overall pattern of marked increase expression of TLRs/RIG-1, consistent with a strong anti-viral response. Thus, the in situ testing of infectious diseases by a panel of these early infectious disease recognition proteins may allow the surgical pathologist to predict the outcome of the disease which, in turn, may assist in the understanding of the role of the TLRs/RIG-1 in determining the fate of a given infectious process.

The molluscum contagiosum virus death effector domain containing protein MC160 RxDL motifs are not required for its known viral immune evasion functions.

The molluscum contagiosum virus (MCV) uses a variety of immune evasion strategies to antagonize host immune responses. Two MCV proteins, MC159 and MC160, contain tandem death effector domains (DEDs). They are reported to inhibit innate immune signaling events such as NF-κB and IRF3 activation, and apoptosis. The RxDL motif of MC159 is required for inhibition of both apoptosis and NF-κB activation. However, the role of the conserved RxDL motif in the MC160 DEDs remained unknown. To answer this question, we performed alanine mutations to neutralize the arginine and aspartate residues present in the MC160 RxDL in both DED1 and DED2. These mutations were further modeled against the structure of the MC159 protein. Surprisingly, the RxDL motif was not required for MC160's ability to inhibit MAVS-induced IFNß activation. Further, unlike previous results with the MC159 protein, mutations within the RxDL motif of MC160 had no effect on the ability of MC160 to dampen TNF-α-induced NF-κB activation. Molecular modeling predictions revealed no overall changes to the structure in the MC160 protein when the amino acids of both RxDL motifs were mutated to alanine (DED1 = R67A D69A; DED2 = R160A D162A). Taken together, our results demonstrate that the RxDL motifs present in the MC160 DEDs are not required for known functions of the viral protein.

Compound heterozygous CORO1A mutations in siblings with a mucocutaneous-immunodeficiency syndrome of epidermodysplasia verruciformis-HPV, molluscum contagiosum and granulomatous tuberculoid leprosy.

PURPOSE: Coronin-1A deficiency is a recently recognized autosomal recessive primary immunodeficiency caused by mutations in CORO1A (OMIM 605000) that results in T-cell lymphopenia and is classified as T(-)B(+)NK(+)severe combined immunodeficiency (SCID). Only two other CORO1A-kindred are known to date, thus the defining characteristics are not well delineated. We identified a unique CORO1A-kindred. METHODS: We captured a 10-year analysis of the immune-clinical phenotypes in two affected siblings from disease debut of age 7 years. Target-specific genetic studies were pursued but unrevealing. Telomere lengths were also assessed. Whole exome sequencing (WES) uncovered the molecular diagnosis and Western blot validated findings. RESULTS: We found the compound heterozygous CORO1A variants: c.248_249delCT (p.P83RfsX10) and a novel mutation c.1077delC (p.Q360RfsX44) (NM_007074.3) in two affected non-consanguineous siblings that manifested as absent CD4CD45RA(+) (naïve) T and memory B cells, low NK cells and abnormally increased double-negative (DN) ϒδ T-cells. Distinguishing characteristics were late clinical debut with an unusual mucocutaneous syndrome of epidermodysplasia verruciformis-human papilloma virus (EV-HPV), molluscum contagiosum and oral-cutaneous herpetic ulcers; the older female sibling also had a disfiguring granulomatous tuberculoid leprosy. Both had bilateral bronchiectasis and the female died of EBV+ lymphomas at age 16 years. The younger surviving male, without malignancy, had reproducibly very short telomere lengths, not before appreciated in CORO1A mutations. CONCLUSION: We reveal the third CORO1A-mutated kindred, with the immune phenotype of abnormal naïve CD4 and DN T-cells and newfound characteristics of a late/hypomorphic-like SCID of an EV-HPV mucocutaneous syndrome with also B and NK defects and shortened telomeres. Our findings contribute to the elucidation of the CORO1A-SCID-CID spectrum.

Aberrant humoral immune reactivity in DOCK8 deficiency with follicular hyperplasia and nodal plasmacytosis.

Mutations in the DOCK8 gene define the most common form of autosomal-recessive Hyper-IgE-syndrome (AR-HIES/OMIM#243700). In a patient with extensive molluscum contagiosum lesions, a homozygous DOCK8 gene deletion was demonstrated. In-vivo 18-FDG uptake showed multiple non-enlarged lymph nodes without uptake in the spleen. Lymph node biopsies for subsequent immunohistochemistry showed clear differences with the mouse model of DOCK8 deficiency in which these mice show no GCs. Unexpectedly, the patient's lymph nodes demonstrated lymphocyte polyclonality, follicular hyperplasia and an unusual IgE(+) plasma cell expansion. In contrast, the proliferative capacity of circulating B-cells was almost absent with little in-vitro Ig production or plasmablast formation. Also the T-cell proliferation indicated a partial defect. Hematopoietic stem cell transplantation (HSCT) was performed resulting in the disappearance of the molluscum contagiosum lesions. In sum, DOCK8 deficiency results in defective antibody responses and undirected plasma cell expansion in the lymph nodes, as part of a combined immunodeficiency cured by HSCT.

Soluble adenylyl cyclase defines a nuclear cAMP microdomain in keratinocyte hyperproliferative skin diseases.

Cyclic adenosine monophosphate (cAMP) is a nearly ubiquitous signaling molecule important for numerous signaling pathways in human skin. We studied a novel class of mammalian adenylyl cyclase, the soluble adenylyl cyclase (sAC). We examined sAC localization in normal human skin and found it to be present in keratinocytes, melanocytes, mononuclear cells, eccrine ducts, and nerves. In normal skin, sAC keratinocyte staining was evenly distributed throughout the cell. However, in certain hyperproliferative disorders of the skin, including psoriasis, verruca vulgaris, and SCCIS on sun-damaged skin, sAC keratinocyte staining was predominantly nuclear. In contrast, in other hyperproliferative disorders, such as basal cell carcinoma, sAC staining was similar to normal human skin. Using a model of epithelial differentiation, we established that sAC migrates into the nucleus when differentiated cells are induced to reenter the cell cycle. Previous work had determined that nuclear sAC activates the cAMP-response-element-binding (CREB) transcription factor, and we found that in psoriasis lesions, nuclear sAC occurs concomitantly with activation of CREB. Hence, sAC may play a role in the pathogenesis of certain hyperproliferative skin disorders via modulation of gene expression.

Molluscum contagiosum: recent advances in pathogenic mechanisms, and new therapies.

Two poxviruses, Molluscum contagiosum virus (MCV) and Variola virus are specific to humans. MCV is present worldwide and is directly passed by direct skin to skin contact to produce cutaneous and, rarely, mucosal lesions. It occurs predominantly in preadolescent children, sexually active adults, participants in sports with skin to skin contact, and in individuals with impaired cellular immunity. MCV characteristically proliferates within the follicular epithelium, and with routine fixation produces an area of retraction artifact separating layers 1 to 3 of CD34+ stromal cells that immediately surround the follicle from the surrounding dermis. This feature may be obscured when the lesions are inflamed, usually after rupture into the surrounding dermis. MCV is a cytoplasmically replicating virus. MCV-infected cells grow in size, while internal organelles are dislocated and eventually obliterated by a large intracytoplasmic inclusion. Rupture and discharge of the virus-packed cells occurs in a process similar to membrane debris and MCV accumulate in the crater-like ostium; MCV infection is spread by contact with infectious debris. In HIV-1-positive patients the histologic features, as well as the clinical features, may be atypical in patients with MCV infections. Not only are the lesions often large, but they may be verrucous and markedly hyperkeratotic. Recent sequencing of the MCV genome has increased our understanding and investigations into its mechanisms for avoiding host defense mechanisms. These include regions which encode for homologues of cellular chemokines and chemokine-binding proteins, a homolog of MHC1 and a viral FLICE-like inhibitory protein. Treatment, until recently, has depended upon tissue destruction including curettage, cryotherapy, CO(2) laser, electrodesiccation, trichloracetic acid and cantharadin. Recently, topical immune modulators have been used with some success. Understanding of the MCV genome is providing the basis for the development of drugs for therapy and prevention of MCV infections.

Two major antigenic polypeptides of molluscum contagiosum virus.

A library of molluscum contagiosum virus (MCV) transferred into the cowpox vector expression system was screened with 12 sera from molluscum patients. Two recombinant proteins of 70 and 34 kDa were detected by immunoblotting and mapped to the open-reading frames MC133L and MC084L, respectively. Consensus sites were found between the C-terminus of the 70-kDa MCV protein and the 14-kDa fusion protein of vaccinia and variola virus, and between the 34-kDa MCV protein and the 37.5-kDa viral membrane-associated protein of vaccinia and variola virus. Rabbit antisera against these two proteins were prepared. An immunofluorescence study demonstrated that the 70- and 34-kDa proteins were predominantly expressed on the surface of recombinant virus-infected HeLa cells, indicating the potential to be inserted into the membrane. On immunoelectron microscopy, antiserum against 70-kDa protein showed significant labeling of the MCV membrane, while the antiserum against 34-kDa protein failed to do so.