Epidermal Infection Research Articles

Endoreduplication-mediated initiation of symbiotic organ development in Lotus japonicus.

Many leguminous plants have a unique ability to reset and alter the fate of differentiated root cortical cells to form new organs of nitrogen-fixing root nodules during legume-Rhizobium symbiosis. Recent genetic studies on the role of cytokinin signaling reveal that activation of cytokinin signaling is crucial to the nodule organogenesis process. However, the genetic mechanism underlying the initiation of nodule organogenesis is poorly understood due to the low number of genes that have been identified. Here, we have identified a novel nodulation-deficient mutant named vagrant infection thread 1 (vag1) after suppressor mutant screening of spontaneous nodule formation 2, a cytokinin receptor gain-of-function mutant in Lotus japonicus. The VAG1 gene encodes a protein that is putatively orthologous to Arabidopsis ROOT HAIRLESS 1/HYPOCOTYL 7, a component of the plant DNA topoisomerase VI that is involved in the control of endoreduplication. Nodule phenotype of the vag1 mutant shows that VAG1 is required for the ploidy-dependent cell growth of rhizobial-infected cells. Furthermore, VAG1 mediates the onset of endoreduplication in cortical cells during early nodule development, which may be essential for the initiation of cortical cell proliferation that leads to nodule primordium formation. In addition, cortical infection is severely impaired in the vag1 mutants, whereas the epidermal infection threads formation is normal. This suggests that the VAG1-mediated endoreduplication of cortical cells may be required for the guidance of symbiotic bacteria to host meristematic cells.

Lotus japonicus SUNERGOS1 encodes a predicted subunit A of a DNA topoisomerase VI that is required for nodule differentiation and accommodation of rhizobial infection

A symbiotic mutant of Lotus japonicus, called sunergos1-1 (suner1-1), originated from a har1-1 suppressor screen. suner1-1 supports epidermal infection by Mesorhizobium loti and initiates cell divisions for organogenesis of nodule primordia. However, these processes appear to be temporarily stalled early during symbiotic interaction, leading to a low nodule number phenotype. This defect is ephemeral and near wild-type nodule numbers are reached by suner1-1 at a later point after infection. Using an approach that combined map-based cloning and next-generation sequencing we have identified the causative mutation and show that the suner1-1 phenotype is determined by a weak recessive allele, with the corresponding wild-type SUNER1 locus encoding a predicted subunit A of a DNA topoisomerase VI. Our data suggest that at least one function of SUNER1 during symbiosis is to participate in endoreduplication, which is an essential step during normal differentiation of functional, nitrogen-fixing nodules.

Interleukin-1α released from HSV-1 infected skin keratinocytes initiates protective immunity against systemic infection (VIR1P.964)

Abstract Herpes simplex virus-1 (HSV-1) is a common human pathogen that typically causes self-limiting disease. However, neonates and patients with compromised immune systems are at risk of developing potentially fatal systemic disease. Previously we reported that IL-1α is released by keratinocytes treated with poly(I:C), a double stranded RNA analogue often utilized to simulate virus infections. The objective of this study was to examine whether IL-1 signaling plays a role in initiating protective immunity against HSV-1 infections. Extracellular release of IL-1α protein could be detected 6-24 hours after HSV-1 infection of primary human keratinocytes (4-5-fold increase, p<0.01). This release was not associated with a correlating change in mRNA levels suggesting that the cells released a pre-formed pool of protein. HSV-1 triggered activation of inflammatory caspases-1/4 as determined using fluorescence labeled peptides (6-fold, p<0.001); however, the cellular release of IL-1α was independent of caspases-1/4. IL-1R knockout (KO) mice exhibited an increased mortality following cutaneous HSV-1 infection (60 versus 25%, p<0.01). Caspase-1/4 deficiency did not affect survival rates; hence, the protective function of IL-1R must be exclusively due to IL-1α signaling. Recruitment of leukocytes to the infected epidermis was delayed in IL-1R KO mice. Hence, HSV-1 triggered IL-1α release from infected cells appears important for initiating protective immunity against systemic HSV-1 infections.

RNA-seq Analysis of Host and Viral Gene Expression Highlights Interaction between Varicella Zoster Virus and Keratinocyte Differentiation

Varicella zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread.

Rhizobial infection does not require cortical expression of upstream common symbiosis genes responsible for the induction of Ca(2+) spiking.

For the establishment of an effective root nodule symbiosis, a coordinated regulation of the infection processes between the epidermis and cortex is required. However, it remains unclear whether the symbiotic genes identified so far are involved in epidermal and/or cortical infection, e.g. epidermal and cortical infection thread formation or cortical cell division. To analyze the symbiotic gene requirements of the infection process, we have developed an epidermis-specific expression system (pEpi expression system) and examined the symbiotic genes NFR1, NFR5, NUP85, NUP133, CASTOR, POLLUX, CCaMK, CYCLOPS, NSP1 and NSP2 for involvement in the infection process in the epidermis and cortex. Our study shows that expression of the upstream common symbiosis genes CASTOR, POLLUX, NUP85 and NUP133 in the epidermis is sufficient to induce formation of infection threads and cortical cell division, leading to the development of fully effective nodules. Our system also shows a requirement of CCaMK, CYCLOPS, NSP1 and NSP2 for the entire nodulation process, and the different contributions of NFR1 and NFR5 to cortical infection thread formation. Based on these analyses using the pEpi expression system, we propose a functional model of symbiotic genes for epidermal and cortical infection.

Use of Antibiotics for Epidermal Wounds in Emergency Room: Correct Prophylaxis and Right Choice of the Treatment

Background: antibiotic therapy and prophylaxis of epidermal wounds in emergency room are often made em- pirically in case of clinics due to infective complications, although there are no scientific evidences supporting their valid- ity yet. Furthermore, resistance to many antibiotics is a constantly growing problem, especially in Lombardy, Italy. When needed, better prefer beta-lactams or macrolides for their large-spectrum activity. Methods: We identified all patients (pts) treated to our emergency room for epidermal wounds in a period between 1st and 31st july 2012. Then, we performed a retrospective, observational analysis of epidemiological, clinical and therapeutic features of those pts. <12 years old individuals were exluded. We considered at high risk of infections pts with at least one of the following comorbidities: diabetes, neoplastic disease, therapy with corticosteroids, immunodepression. Results: A total of 201 pts were evaluated, 143 males (71.1%) and 58 females (28.9%). The median age was 46,3 yrs. Among the pts 101 (50.2%) needed treatment with suture. 13 (6.5%) pts came after at least 12 hrs after the accident. 107 (53,2%) pts did not need therapy, while in 82 (40.8%) cases antibiotic prophylaxis (89% beta-lactams, 9.8% macrolides, 1.2% others) has been prescribed; 4 (2%) were adviced to start antibiotic therapy only in case of acute epidermal infection development at home; we don't have this information about 8 pts (4%). In 185 (93.5%) pts clinical signs of acute epider- mal infection were missing. 15 (7.5%) pts had an high risk of developing infections in history taking; 3 of them received antibiotic prophylaxis, 8 didn't, 4 not known. In 143 individuals (71,1%) the history taking has been insufficient. Conclusions: Even if acute local infection is possible in wounded pts, only subjects at high risk should be closely screened and monitored to detect early infection, in order to evaluate the necessity of specific antibiotic therapy. Clinical evaluation should also include comorbilities as valvulopathy or immunosuppression.

Abstract LB-61: Vemurafenib promotes RAS wild-type tumor formation in a mouse model of HPV-driven cutaneous squamous cell carcinoma.

Abstract A subset of melanoma patients (18-30%) treated with Vemurafenib (and other RAF inhibitors) develop spontaneous cutaneous squamous-cell carcinoma (cSCC) and/or keratoacanthoma (KA). Preclinical studies demonstrate that RAF inhibitors paradoxically activate the MAPK pathway and stimulate growth of RAS mutated cells. Based on these observations it has been proposed that drug induced MAPK activation in pre-cancerous tissues may account for the high incidence of cSCC and KA in treated patients. Because human cSCC is frequently positive for human papillomavirus (HPV) DNA, we hypothesized that RAF inhibitors promote tumorigenesis in HPV infected epidermis. To investigate this possibility, we used a transgenic murine model (K14-HPV16) of cSCC, driven by basal keratinocyte-specific expression (K14 promoter) of HPV type 16 early genes including E6 and E7, which inactivates p53 and pRB tumor suppressors. Exposure to Vemurafenib elevated MAPK markers in epidermis and increased cSCC incidence from 22% to 70%. Exome sequencing revealed that 100% of cSCCs from untreated mice harbored activating RAS mutations, yet 55% of cSCCs from Vemurafenib treated mice were RAS wild-type. Concomitant treatment with a MEK inhibitor reduced cSCC frequency, and MEK inhibitor alone was sufficient to completely regress established cSCCs. Together, these results suggest that cSCC is dependent upon MAPK activation induced either by a RAS oncogene or alternatively by exposure to RAF inhibitors in HPV infected keratinocytes. Citation Format: Matthew Holderfield, Edward Lorenzana, Ben Weisburd, Lisa Lomovasky, Majid Ghoddusi, Dylan Daniel, Nancy Pryer, Frank McCormick, Darrin Stuart. Vemurafenib promotes RAS wild-type tumor formation in a mouse model of HPV-driven cutaneous squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-61. doi:10.1158/1538-7445.AM2013-LB-61

Rhizobial Infection Is Associated with the Development of Peripheral Vasculature in Nodules ofMedicago truncatula

Nodulation in legumes involves the coordination of epidermal infection by rhizobia with cell divisions in the underlying cortex. During nodulation, rhizobia are entrapped within curled root hairs to form an infection pocket. Transcellular tubes called infection threads then develop from the pocket and become colonized by rhizobia. The infection thread grows toward the developing nodule primordia and rhizobia are taken up into the nodule cells, where they eventually fix nitrogen. The epidermal and cortical developmental programs are synchronized by a yet-to-be-identified signal that is transmitted from the outer to the inner cell layers of the root. Using a new allele of the Medicago truncatula mutant Lumpy Infections, lin-4, which forms normal infection pockets but cannot initiate infection threads, we show that infection thread initiation is required for normal nodule development. lin-4 forms nodules with centrally located vascular bundles similar to that found in lateral roots rather than the peripheral vasculature characteristic of legume nodules. The same phenomenon was observed in M. truncatula plants inoculated with the Sinorhizobium meliloti exoY mutant, and the M. truncatula vapyrin-2 mutant, all cases where infections arrest. Nodules on lin-4 have reduced expression of the nodule meristem marker MtCRE1 and do not express root-tip markers. In addition, these mutant nodules have altered patterns of gene expression for the cytokinin and auxin markers CRE1 and DR5. Our work highlights the coordinating role that bacterial infection exerts on the developing nodule and allows us to draw comparisons with primitive actinorhizal nodules and rhizobia-induced nodules on the nonlegume Parasponia andersonii.

First Isolation, In Egypt, of a granulovirus (AgseGVEG) from Agrotis segetum Schiffermuller (Lepidoptera: Noctuidae)

A new Granulovirus was isolated from cutworm Agrotis segetum larvae collected from the field in Qalubaya governorate. Transverse section of larvae showed infected fat body, epidermis and mid-gut cells. Transmition electromicroscope evealed ovoid occlusion bodies (granules), each granule contains a single rod-shaped virion with one nucleocapsid, typical of granuolosis virus. Also, the scanning electromicroscopy of apurified viral suspension confirmed a baculovirus morphology. The size of virus particles in the present isolation ranged between 1.9 m to 2.5m. Preliminary bioassay indicated that A. ipsilon neonate larvae are susceptible to AgseGV EG and the calculated LC50 value was 3.79x10 6 capsules/ml.

Lotus japonicus ARPC1 Is Required for Rhizobial Infection

Remodeling of the plant cell cytoskeleton precedes symbiotic entry of nitrogen-fixing bacteria within the host plant roots. Here we identify a Lotus japonicus gene encoding a predicted ACTIN-RELATED PROTEIN COMPONENT1 (ARPC1) as essential for rhizobial infection but not for arbuscular mycorrhiza symbiosis. In other organisms ARPC1 constitutes a subunit of the ARP2/3 complex, the major nucleator of Y-branched actin filaments. The L. japonicus arpc1 mutant showed a distorted trichome phenotype and was defective in epidermal infection thread formation, producing mostly empty nodules. A few partially colonized nodules that did form in arpc1 contained abnormal infections. Together with previously described L. japonicus Nck-associated protein1 and 121F-specific p53 inducible RNA mutants, which are also impaired in the accommodation of rhizobia, our data indicate that ARPC1 and, by inference a suppressor of cAMP receptor/WASP-family verpolin homologous protein-ARP2/3 pathway, must have been coopted during evolution of nitrogen-fixing symbiosis to specifically mediate bacterial entry.

Complement Inhibitors from Scabies Mites Promote Streptococcal Growth – A Novel Mechanism in Infected Epidermis?

BackgroundScabies is highly prevalent in socially disadvantaged communities such as indigenous populations and in developing countries. Generalized itching causes discomfort to the patient; however, serious complications can occur as a result of secondary bacterial pyoderma, commonly caused by Streptococcus pyogenes (GAS) or Staphylococcus aureus. In the tropics, skin damage due to scabies mite infestations has been postulated to be an important link in the pathogenesis of disease associated with acute rheumatic fever and heart disease, poststreptococcal glomerulonephritis and systemic sepsis. Treatment of scabies decreases the prevalence of infections by bacteria. This study aims to identify the molecular mechanisms underlying the link between scabies and GAS infections.Methodology/Principal FindingsGAS bacteria were pre-incubated with blood containing active complement, phagocytes and antibodies against the bacteria, and subsequently tested for viability by plate counts. Initial experiments were done with serum from an individual previously exposed to GAS with naturally acquired anti-GAS antibodies. The protocol was optimized for large-scale testing of low-opsonic whole blood from non-exposed human donors by supplementing with a standard dose of heat inactivated human sera previously exposed to GAS. This allowed an extension of the dataset to two additional donors and four proteins tested at a range of concentrations. Shown first is the effect of scabies mite complement inhibitors on human complement using ELISA-based complement activation assays. Six purified recombinant mite proteins tested at a concentration of 50 µg/ml blocked all three complement activation pathways. Further we demonstrate in human whole blood assays that each of four scabies mite complement inhibitors tested increased GAS survival rates by 2–15 fold.Conclusions/SignificanceWe propose that local complement inhibition plays an important role in the development of pyoderma in scabies infested skin. This molecular link between scabies and bacterial infections may provide new avenues to develop alternative treatment options against this neglected disease.

Phase II trials of cetuximab (CX) plus cisplatin (CDDP), 5-fluorouracil (5-FU) and radiation (RT) in immunocompetent (ECOG 3205) and HIV-positive (AMC045) patients with squamous cell carcinoma of the anal canal (SCAC): Safety and preliminary efficacy results.

4030 Background: Epidermal growth factor receptor (EGFR) expression and HPV infection are common in SCAC. We therefore initiated 2 phase II studies to evaluate the safety and efficacy of the EGFR inhibitor CX given concurrently with CDDP/5-FU/RT in HIV-positive (AMC045) and immunocompetent (E3205) patients with SCAC. Methods: All patients received CX (400 mg/m2 loading, then 250 mg/m2/wk IV x 6-8 wks) plus CDDP (75 mg/m2 IV q28 days x 2) and 5-FU (1000 mg/m2/day IV infusion days 1-4 q 28 days x 2) concurrently with RT (45-54 Gy) beginning with CX dose 2. Patients in E3205 also received 2 cycles of CDDP/5-FU alone prior to CX/CDDP/5-FU/RT; this was discontinued on recommendation of the NCI Anorectal Task Force after 28 patients. Both trials were powered to detect a reduction in 3-year local-regional failure (LRF) rate from 35% to 17.5% (alpha=0.10, beta=0.10), the primary end point. Other endpoints included progression free survival (PFS) and overall survival (OS). The results below include complete toxicity and preliminary efficacy data (including only the first 28 patients from E3205). Results: Expedited reporting was required for type I (any grade 5, grade 4 cardiac) or II (grade 4 RT skin, diarrhea) adverse events, with prespecified rates of &gt;5% or &gt;20%, respectively defined as unacceptable. Early stopping rules were not invoked for either trial. LRF rates data will be presented after more detailed case review is completed. Conclusions: CX plus CDDP/5-FU/RT is feasible in patients with SCAC, including patients with HIV infection. Preliminary safety and efficacy data appear encouraging, but accrual without neoadjuvant CDDP/5-FU continues in E3205, and additional followup of both study cohorts is required in order to determine whether pre-specified efficacy endpoints were met. [Table: see text]

Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti–Medicago symbiosis

Legumes and soil bacteria called rhizobia have coevolved a facultative nitrogen-fixing symbiosis. Establishment of the symbiosis requires bacterial entry via root hair infection threads and, in parallel, organogenesis of nodules that subsequently are invaded by bacteria. Tight control of nodulation and infection is required to maintain the mutualistic character of the interaction. Available evidence supports a passive bacterial role in nodulation and infection after the microsymbiont has triggered the symbiotic plant developmental program. Here we identify in Sinorhizobium meliloti, the Medicago symbiont, a cAMP-signaling regulatory cascade consisting of three receptor-like adenylate cyclases, a Crp-like regulator, and a target gene of unknown function. The cascade is activated specifically by a plant signal during nodule organogenesis. Cascade inactivation results in a hyperinfection phenotype consisting of abortive epidermal infection events uncoupled from nodulation. These findings show that, in response to a plant signal, rhizobia play an active role in the control of infection. We suggest that rhizobia may modulate the plant's susceptibility to infection. This regulatory loop likely aims at optimizing legume infection.

Screening of drugs to counteract human papillomavirus 16 E6 repression of E-cadherin expression.

Persistent infections with certain high-risk human papillomavirus (HPV) types such as 16 and 18 can result in the development of cervical cancer. Neither of the two prophylactic vaccines against HPV16 and 18 that are in current use have any therapeutic efficacy for prevalent HPV infections. Ablative therapy is widely used for the treatment of HPV cervical dysplasia however disease recurrence is a widely recognized problem. Thus there is a continuing need for therapeutic approaches for the treatment of HPV infections. The HPV16 E6 viral oncoprotein represses surface expression of the cellular adhesion molecule, E-cadherin. Reduced E-cadherin expression on HPV-infected keratinocytes is associated with lowered numbers of antigen-presenting Langerhans cells in the infected epidermis, potentially reducing immune surveillance for HPV. Four chemicals reported to up-regulate E-cadherin were screened for their ability to counteract E6 repression of surface E-cadherin. 5-Aza-2'-deoxycytidine (AzaDC), a DNA methyltransferase inhibitor, and Indole-3-carbinol (I3C), reported to increase E-cadherin through a p21(Waf1/Cip1)-dependent mechanism, had low cytotoxicity and increased or restored E-cadherin expression and adhesive function in HPV16 E6 expressing HCT116 cells. Doxorubicin, also known to induce p21(Waf1/Cip1), increased E-cadherin in E6 expressing cells but had some associated cytotoxicity. Tamoxifen, which can restore adhesive function of surface E-cadherin, was ineffective in counteracting E6 repression of E-cadherin. AzaDC and I3C both show potential to restore antigen-presenting cells to HPV infected skin by antagonizing E6 repression of E-cadherin, thereby counteracting an important immune evasion mechanism of HPV16 and reinstating immune function at the infected site.

HSV Neutralization by the Microbicidal Candidate C5A

Genital herpes is a major risk factor in acquiring human immunodeficiency virus type-1 (HIV-1) infection and is caused by both Herpes Simplex virus type 1 (HSV-1) and HSV-2. The amphipathic peptide C5A, derived from the non-structural hepatitis C virus (HCV) protein 5A, was shown to prevent HIV-1 infection but neither influenza nor vesicular stomatitis virus infections. Here we investigated the antiviral function of C5A on HSV infections. C5A efficiently inhibited both HSV-1 and HSV-2 infection in epithelial cells in vitro as well as in an ex vivo epidermal infection model. C5A destabilized the integrity of the viral HSV membrane. Furthermore, drug resistant HSV strains were inhibited by this peptide. Notably, C5A-mediated neutralization of HSV-1 prevented HIV-1 transmission. An in vitro HIV-1 transmigration assay was developed using primary genital epithelial cells and HSV infection increased HIV-1 transmigration. Treatment with C5A abolished HIV-1 transmigration by preventing HSV infection and by preserving the integrity of the genital epithelium that was severely compromised by HSV infection. In conclusion, this study demonstrates that C5A represents a multipurpose microbicide candidate, which neutralizes both HIV-1 and HSV, and which may interfere with HIV-1 transmission through the genital epithelium.

Characterization of Trichoderma polysporum from Spitsbergen, Svalbard archipelago, Norway, with species identity, pathogenicity to moss, and polygalacturonase activity

Abstract Six isolates of Trichoderma polysporum-like species isolated from a moss (Sanionia uncinata) in high arctic wetlands at Spitsbergen Island, Svalbard, Norway, were examined for species identification, effects of temperature on growth, pathogenicity to the moss, and polygalacturonase (PGase) activity. All isolates from Spitsbergen were identified as T. polysporum based on morphology, sequences of the rDNA-ITS regions, and growth response to temperature. In addition, two isolates of T. polysporum from Germany and Japan were used. All isolates from Spitsbergen, Germany and Japan infected epidermal tissues of the moss, but did not cause any symptoms in in vitro inoculation tests at 0 °C. One of the isolates from Spitsbergen and the German isolate showed high activity of PGase, an enzyme produced by many saprotrophic and pathogenic fungi, in a pectin liquid medium. The enzyme activity was evident for both isolates even at 0 °C. The present results document the presence of T. polysporum in the high arctic wetlands. The epidermal infection to the moss and PGase production at 0 °C of the T. polysporum isolates suggest that the species likely inhabits a niche as a saprotroph or an endophyte in the arctic environments.

Gr-1 + cells, but not neutrophils, limit virus replication and lesion development following flank infection of mice with herpes simplex virus type-1

Neutrophils are prominent in epidermal and dermal layers of human herpetic lesions and are rapidly recruited into the skin follow epidermal abrasion and infection of mice with herpes simplex virus type-1 (HSV-1). Herein, we demonstrate that early production of neutrophil-attracting chemokines KC/MIP-2 is associated with transient recruitment of neutrophils into the skin of HSV-1-infected mice in temporal association with the development of herpetic lesions. Treatment of HSV-1-infected mice with a Ly6G-specific mAb induced systemic neutropenia, but surprisingly did not alter virus replication or lesion development. In contrast, depletion of Gr-1 + cells with mAb RB6-8C5 led to enhanced virus growth and lesion severity. Thus, while neutrophils are prominent in zosteriform lesions of HSV-1-infected mice, they do not appear to play a major role in controlling virus replication or lesion development and/or healing. In contrast, Gr-1 + cells limit both virus replication and lesion development in the zosteriform model.

Bovine papillomavirus DNA can be detected in keratinocytes of equine sarcoid tumors

Bovine papillomavirus (BPV)-1 and -2 is linked to equine sarcoids, a commonly observed skin tumor in horses that is of considerable veterinary importance. Previous studies using in situ hybridization have detected BPV DNA only in fibroblasts and not in keratinocytes of sarcoids. In contrast, normal equine skin latently infected with BPV shows a dysplastic epithelium without dermal changes, similar to lesions induced by other papillomavirus types infecting the epithelium. The first goal of our study was to describe the epidermal and dermal characteristics of several stages in sarcoid development. Next, we explored whether BPV can infect epidermal cells in the horse using real-time PCR on laser-micro-dissected keratinocytes and fibroblasts. We found that latently infected normal skin samples and a subset of early stage sarcoids show dysplastic, koilocyte-like epithelial changes. BPV DNA was detected in keratinocytes in 40% of the samples with these particular epithelial properties, whereas advanced sarcoids only had BPV DNA in the fibroblasts. These data may indicate a novel and intriguing pathway of BPV infection in the horse composed of a first step of keratinocyte infection, followed by migration of viral material towards the dermis resulting in infection of sub-epidermal fibroblasts and their fully transformed phenotype. Additionally, an example of co-existence of a dermal BPV-1 and an epidermal BPV-2 infection in the same lesion is shown, indicating that horses can harbor infection with more than one BPV type at the same time.

The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus.

Bacterial infection of interior tissues of legume root nodules is controlled at the epidermal cell layer and is closely coordinated with progressing organ development. Using spontaneous nodulating Lotus japonicus plant mutants to uncouple nodule organogenesis from infection, we have determined the role of 16 genes in these two developmental processes. We show that host-encoded mechanisms control three alternative entry processes operating in the epidermis, the root cortex and at the single cell level. Single cell infection did not involve the formation of trans-cellular infection threads and was independent of host Nod-factor receptors and bacterial Nod-factor signals. In contrast, Nod-factor perception was required for epidermal root hair infection threads, whereas primary signal transduction genes preceding the secondary Ca2+ oscillations have an indirect role. We provide support for the origin of rhizobial infection through direct intercellular epidermal invasion and subsequent evolution of crack entry and root hair invasions observed in most extant legumes.

Epidermal injury and infection during poxvirus immunization is crucial for the generation of highly protective T cell-mediated immunity.

Introductory ParagraphVariola major infection (Smallpox) claimed hundreds of millions lives before it was eradicated by a simple vaccination strategy: epicutaneous application of the related orthopoxvirus Vaccinia virus (VACV) to superficially injured skin (skin scarification, s.s.)1. However, the remarkable success of this strategy was attributed to the immunogenicity of VACV rather than the unique vaccine delivery mode. We now demonstrate that VACV immunization via s.s., but not conventional injection routes, is essential to the generation of superior T cell-mediated immune responses that provide complete protection against subsequent challenges, independent of neutralizing antibodies. Skin-resident effector memory T cells (TEM) provide complete protection against cutaneous challenge, while protection against lethal respiratory challenge requires both respiratory mucosal TEM and central memory T cells (TCM). Vaccination with recombinant VACV (rVACV) expressing a tumor antigen was protective against tumor challenge only if delivered via s.s. route, but not by hypodermic injection. Finally, the clinically safer non-replicative Modified Vaccinia Ankara (MVA) also generated far superior protective immunity when delivered via s.s route compared to intramuscular injection used in MVA clinical trials. Thus, delivering rVACV -based vaccines, including MVA vaccines, through physically disrupted epidermis represents a uniquely powerful strategy with clear-cut advantages over conventional vaccination via hypodermic injection.