Cell cycle-dependent centrosome clustering precedes proplatelet formation.

Platelet-producing megakaryocytes (MKs) primarily reside in the bone marrow, where they duplicate their DNA content with each cell cycle resulting in polyploid cells with an intricate demarcation membrane system. While key elements of the cytoskeletal reorganizations during proplatelet formation have been identified, what initiates the release of platelets into vessel sinusoids remains largely elusive. Using a cell cycle indicator, we observed a unique phenomenon, during which amplified centrosomes in MKs underwent clustering following mitosis, closely followed by proplatelet formation, which exclusively occurred in G1 of interphase. Forced cell cycle arrest in G1 increased proplatelet formation not only in vitro but also in vivo following short-term starvation of mice. We identified that inhibition of the centrosomal protein kinesin family member C1 (KIFC1) impaired clustering and subsequent proplatelet formation, while KIFC1-deficient mice exhibited reduced platelet counts. In summary, we identified KIFC1- and cell cycle-mediated centrosome clustering as an important initiator of proplatelet formation from MKs.

Platelet Dysfunction and Thrombosis in JAK2V617F-Mutated Primary Myelofibrotic Mice.

OBJECTIVE: The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2V617F (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2V617F transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl3-induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation. CONCLUSIONS: Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.

High-content, label-free analysis of proplatelet production from megakaryocytes.

BACKGROUND: The mechanisms that regulate platelet biogenesis remain unclear; factors that trigger megakaryocytes (MKs) to initiate platelet production are poorly understood. Platelet formation begins with proplatelets, which are cellular extensions originating from the MK cell body. OBJECTIVES: Proplatelet formation is an asynchronous and dynamic process that poses unique challenges for researchers to accurately capture and analyze. We have designed an open-source, high-content, high-throughput, label-free analysis platform. METHODS: Phase-contrast images of live, primary MKs are captured over a 24-hour period. Pixel-based machine-learning classification done by ilastik generates probability maps of key cellular features (circular MKs and branching proplatelets), which are processed by a customized CellProfiler pipeline to identify and filter structures of interest based on morphology. A subsequent reinforcement classification, by CellProfiler Analyst, improves the detection of cellular structures. RESULTS: This workflow yields the percent of proplatelet production, area, count of proplatelets and MKs, and other statistics including skeletonization information for measuring proplatelet branching and length. We propose using a combination of these analyzed metrics, in particular the area measurements of MKs and proplatelets, when assessing in vitro proplatelet production. Accuracy was validated against manually counted images and an existing algorithm. We then used the new platform to test compounds known to cause thrombocytopenia, including bromodomain inhibitors, and uncovered previously unrecognized effects of drugs on proplatelet formation, thus demonstrating the utility of our analysis platform. CONCLUSION: This advance in creating unbiased data analysis will increase the scale and scope of proplatelet production studies and potentially serve as a valuable resource for investigating molecular mechanisms of thrombocytopenia.

Protective effect of fungal extracellular vesicles against murine candidiasis.

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterised. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from Candida albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here, we investigated whether immunisation with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunisation, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunised with EVs when compared with EVs + Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs + ADJ, while IL-12p70, TGFß, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, -20 or -80°C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations.

Bensal HP Attenuates the Inflammatory Response in Hair Shaving Associated Dermatitis.

Shaving is an ubiquitous practice, and cutaneous irritation and inflammation are common sequelae, which may be worsened by underlying skin conditions or poor hair removal techniques. Moisturizing shaving creams and aftershaves are available to help maintain or restore the epidermal barrier; however, many continue to suffer from post-shave redness, itching, and pain. To reduce post-shave inflammation, some products have included botanical and other natural ingredients, which are often favored by consumers. We evaluated Bensal HP, an ointment containing 3% oak bark extract, 3% salicylic acid, and 6% benzoic acid, which has documented anti-inflammatory and antimicrobial properties, in a murine model of shave irritation to determine whether it would be useful in this clinical setting. Shaving dermatitis was simulated using a depilatory agent and electric clippers, and the shaved area was photographed and treated with Bensal HP daily for four days. Compared to untreated controls, mice treated with Bensal HP experienced a visible reduction in skin irritation and inflammation. These findings were mirrored on histology, as Bensal HP-treated areas demonstrated increased epidermal integrity and decreased dermal inflammatory infiltrate compared to untreated skin. Using immunohistochemistry, fewer neutrophils and macrophages were noted, and cytokine analysis also revealed decreased IL-6 in Bensal HP-treated skin at 24 and 96 hours after shaving. These results highlight the potential of Bensal HP as an anti-inflammatory treatment for shave irritation. Given the product's application against a variety of inflammatory and infectious skin disorders, its use against shave irritation may also improve comorbid skin conditions, such as pseudofolliculitis barbae.

J Drugs Dermatol. 2016;15(7):836-840.

Bensal HP Treatment for Burn and Excision Wounds: An In-Vivo Assessment of Wound Healing Efficacy and Immunological Impact.

Natural ingredients are of increasing interest within the field of dermatology. Bensal HP, an ointment containing 3% oak bark extract, 3% salicylic acid, and 6% benzoic acid, is believed to be efficacious against a variety of inflammatory and infectious dermatidites. Here we evaluate Bensal HP's ability to influence wound healing, which has yet to be studied in this setting. Bensal HP applied to burn wounds on the dorsal surface of BALB/c mice significantly attenuated wound expansion in the first few days post-injury as compared to controls. Histological analysis mirrored these findings with accelerated maturation of the wound bed and increased collagen deposition by the end of the study period. Cytokine analysis revealed decreased IL-6 and TNFα secretion in the Bensal HP-treated burns as compared to controls. Similarly, excisional wounds treated with Bensal HP demonstrated comparable wound healing as compared to controls with positive histologic features and increased collagen deposition. Furthermore, IL-6 production was attenuated in the Bensal-HP treated wounds at day 3, with no differences appreciated in IL-6 at day 7 or in TNFα at either time point. While Bensal-HP represents a therapeutic strategy to enhance the histologic and immunologic milieu in burn and excisional wounds, further study is needed to fully elucidate the full potential of this treatment.

N-acetylcysteine S-nitrosothiol Nanoparticles Prevent Wound Expansion and Accelerate Wound Closure in a Murine Burn Model.

BACKGROUND: The treatment of cutaneous wounds in the clinical setting continues to be a clinical challenge and economic burden, with burn wounds being especially formidable. Direct mechanical injury coupled with the transfer of thermal energy leads to tissue necrosis, pro-inflammatory cytokine release and the eventual expansion of an initial wound. Our current therapeutic armamentarium falls short of options to help prevent wound expansion, and therefore new modalities are required. Nitrosating substances such as RSNOs have been proven to be effective in promoting wound closure due to their ability to modulate inflammation, cytokine production and vascular function. OBJECTIVE: We aim to evaluate the efficacy of n-actetylcysteine s-nitrosothiol nanoparticles (NAC-SNO-np) on thermal burn wounds and associated expansion. METHODS: A multi-burn model was utilized to induce three burn wounds on the dorsal surface of BALB/c mice, allowing for evaluation of the burn itself and peripheral tissue. Wounds were excised and processed for histology and immunohistochemistry on day 7 following wounding. RESULTS: Following treatment with NAC-SNO-np, burn wound expansion was attenuated and wound healing was accelerated. Histological analysis revealed increased collagen deposition as well as increased macrophage and decreased neutrophil infiltration into the wound bed. CONCLUSION: NAC-SNO-np represents a platform that harnesses the nitrosative properties of NAC-SNO in order to accelerate the transition from inflammatory to proliferative wound healing. Further studies are needed in order to translate to the clinical setting.

Antimicrobial photodynamic therapy: an effective alternative approach to control fungal infections.

Skin mycoses are caused mainly by dermatophytes, which are fungal species that primarily infect areas rich in keratin such as hair, nails, and skin. Significantly, there are increasing rates of antimicrobial resistance among dermatophytes, especially for Trichophyton rubrum, the most frequent etiologic agent worldwide. Hence, investigators have been developing new therapeutic approaches, including photodynamic treatment. Photodynamic therapy (PDT) utilizes a photosensitive substance activated by a light source of a specific wavelength. The photoactivation induces cascades of photochemicals and photobiological events that cause irreversible changes in the exposed cells. Although photodynamic approaches are well established experimentally for the treatment of certain cutaneous infections, there is limited information about its mechanism of action for specific pathogens as well as the risks to healthy tissues. In this work, we have conducted a comprehensive review of the current knowledge of PDT as it specifically applies to fungal diseases. The data to date suggests that photodynamic treatment approaches hold great promise for combating certain fungal pathogens, particularly dermatophytes.

HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function.

T-cell costimulation and coinhibition generated by engagement of the B7 family and their receptor CD28 family are of central importance in regulating the T-cell response, making these pathways very attractive therapeutic targets. Here we describe HERV-H LTR-associating protein 2 (HHLA2) as a member of the B7 family that shares 10-18% amino acid identity and 23-33% similarity to other human B7 proteins and phylogenetically forms a subfamily with B7x and B7-H3 within the family. HHLA2 is expressed in humans but not in mice, which is unique within the B7 and CD28 families. HHLA2 protein is constitutively expressed on the surface of human monocytes and is induced on B cells after stimulation with LPS and IFN-γ. HHLA2 does not interact with other known members of the CD28 family or the B7 family, but does bind a putative receptor that is constitutively expressed not only on resting and activated CD4 and CD8 T cells but also on antigen-presenting cells. HHLA2 inhibits proliferation of both CD4 and CD8 T cells in the presence of T-cell receptor signaling. In addition, HHLA2 significantly reduces cytokine production by T cells including IFN-γ, TNF-α, IL-5, IL-10, IL-13, IL-17A, and IL-22. Thus, we have identified a unique B7 pathway that is able to inhibit human CD4 and CD8 T-cell proliferation and cytokine production. This unique human T-cell coinhibitory pathway may afford unique strategies for the treatment of human cancers, autoimmune disorders, infection, and transplant rejection and may help to design better vaccines.

Host b7x promotes pulmonary metastasis of breast cancer.

B7x (B7-H4 or B7S1) is an inhibitory member of the B7 family of T cell costimulation. It is expressed in low levels in healthy peripheral tissues, such as the lung epithelium, but is overexpressed in a variety of human cancers with negative clinical associations, including metastasis. However, the function of B7x in the context of cancer, whether expressed on cancer cells or on surrounding "host" tissues, has not been elucidated in vivo. We used the 4T1 metastatic breast cancer model and B7x knockout (B7x (-/-)) mice to investigate the effect of host tissue-expressed B7x on cancer. We found that 4T1 cells were B7x negative in vitro and in vivo, and B7x(-/-) mice had significantly fewer lung 4T1 tumor nodules than did wild-type mice. Furthermore, B7x(-/-) mice showed significantly enhanced survival and a memory response to tumor rechallenge. Mechanistic studies revealed that the presence of B7x correlated with reduced general and tumor-specific T cell cytokine responses, as well as with an increased infiltration of immunosuppressive cells, including tumor-associated neutrophils, macrophages, and regulatory T cells, into tumor-bearing lungs. Importantly, tumor-associated neutrophils strongly bound B7x protein and inhibited the proliferation of both CD4 and CD8 T cells. These results suggest that host B7x may enable metastasizing cancer cells to escape local antitumor immune responses through interactions with the innate and adaptive immune systems. Thus, targeting the B7x pathway holds much promise for improving the efficacy of immunotherapy for metastatic cancer.

B7x in the periphery abrogates pancreas-specific damage mediated by self-reactive CD8 T cells.

B7x (B7-H4 or B7S1) is the seventh member of the B7 family, and its in vivo function remains largely unknown. Despite new genetic data linking the B7x gene with autoimmune diseases, how exactly it contributes to peripheral tolerance and autoimmunity is unclear. In this study, we showed that B7x protein was not detected on APCs or T cells in both human and mice, which is unique in the B7 family. Because B7x protein is expressed in some peripheral cells such as pancreatic ß cells, we used a CD8 T cell-mediated diabetes model (AI4αß) in which CD8 T cells recognize an endogenous self-Ag, and found that mice lacking B7x developed more severe diabetes than control AI4αß mice. Conversely, mice overexpressing B7x in the ß cells (Rip-B7xAI4αß) were diabetes free. Furthermore, adoptive transfer of effector AI4αß CD8 T cells induced diabetes in control mice, but not in Rip-B7xAI4αß mice. Mechanistic studies revealed that pathogenic effector CD8 T cells were capable of migrating to the pancreas but failed to robustly destroy tissue when encountering local B7x in Rip-B7xAI4αß mice. Although AI4αß CD8 T cells in Rip-B7xAI4αß and AI4αß mice showed similar cytotoxic function, cell death, and global gene expression profiles, these cells had greater proliferation in AI4αß mice than in RIP-B7xAI4αß mice. These results suggest that B7x in nonlymphoid organs prevents peripheral autoimmunity partially through inhibiting proliferation of tissue-specific CD8 T cells, and that local overexpression of B7x on pancreatic ß cells is sufficient to abolish CD8 T cell-induced diabetes.