Microfluidic Assays for Probing Neutrophil-Borrelia Interactions in Blood During Lyme Disease.

Human neutrophils are highly sensitive to the presence of Borrelia burgdorferi (Bb), the agent of Lyme disease (LD), in tissues. Although Bb is also found in the blood of LD patients, far less is known about how neutrophils respond to Bb in the presence of blood. In this study, we employed microfluidic tools to probe the interaction between human neutrophils and Bb and measured the activation of human neutrophils in blood samples from patients. We found that neutrophils migrate vigorously toward Bb in the presence of serum, and this process was complement-dependent. Preventing complement factor 5 cleavage or blocking complement receptors decreased neutrophil's ability to interact with Bb. We also found that spiking Bb directly into the blood from healthy donors induced spontaneous neutrophil motility. This response to Bb was also complement-dependent. Preventing complement factor 5 cleavage decreased spontaneous neutrophil motility in Bb-spiked blood. Moreover, we found that neutrophils in blood samples from acute LD patients displayed spontaneous motility patterns similar to those observed in Bb-spiked samples. Neutrophil motility was more robust in blood samples from LD patients than that measured in healthy and ill controls, validating the utility of the microfluidic assay for the study of neutrophil-Bb interactions in the presence of blood.

The ALT-70 cellulitis model maintains predictive value at 24 and 48 hours after presentation.

BACKGROUND: Cellulitis has many potential mimickers, and its misdiagnosis often leads to unnecessary hospitalizations and higher health care costs. The ALT-70 predictive model offers an objective tool to help differentiate between cellulitis and other clinically similar conditions at the time of initial emergency department (ED) presentation. OBJECTIVE: To evaluate the performance of the ALT-70 predictive model at 24 and 48 hours following ED presentation. METHODS: We performed a retrospective review of our prior cohort and expanded our data collection to include data at 24 and 48 hours after initial ED presentation. We compared classification measures for the ALT-70 at the time of initial ED presentation, 24 hours after presentation, and 48 hours after presentation. RESULTS: There was a statistically significant difference in median ALT-70 score between patients with true cellulitis and those with mimickers of cellulitis at all time points. Sensitivity, specificity, positive predictive value, and negative predictive value of the ALT-70 score was similar across all 3 time points. LIMITATIONS: Single-center design may reduce generalizability. CONCLUSION: At 24 and 48 hours, the ALT-70 performed similarly to the way it performed at the time of initial ED presentation, allowing for its use in a wider array of clinical settings.

Lidocaine-induced potentiation of thermal damage in skin and carcinoma cells.

OBJECTIVE: Lidocaine acts as a local anesthetic by blocking transmembrane sodium channel permeability, but also induces the synthesis of heat shock proteins and sensitizes cells to hyperthermia. A previous study reported two cases of deep focal skin ulceration at points corresponding to depot local lidocaine injection sites after treatment with non-ablative fractional resurfacing and it was hypothesized that lidocaine had focally sensitized keratinocytes to the thermal damage of laser treatment. The objective of this study was to investigate whether lidocaine potentiates hyperthermia damage to both normal and cancerous skin cells using an in vitro model. METHODS: Normal skin cell lines (fibroblasts, keratinocytes), skin cancer cell lines (melanoma, basal cell carcinoma), and a mucosal cancer cell line (cervical carcinoma) were exposed to various concentrations of lidocaine (0-0.3%) with or without hyperthermia (37°C, 42°C). RESULTS: Compared to normal skin cells, we demonstrate that cancer cell lines show significantly increased cell toxicity when a moderate temperature (42°C) and low lidocaine concentrations (0.1-0.2%) are combined. The toxicity directly correlates with a higher percentage of cells in S-phase (28-57%) in the cancer cell lines compared to normal skin cell lines (13-19%; R-square 0.6752). CONCLUSION: These results suggest that lidocaine potentiates thermal sensitivity of cell cycle active skin cells. The direct correlation between cell toxicity and S-phase cells could be harnessed to selectively treat skin and mucosal cancer cells while sparing the surrounding normal tissue. Additional research pre-clinically and clinically using several different heat sources (e.g., lasers, ultrasound, etc.) and lidocaine concentrations is needed to confirm and optimize these results. Lidocaine-enhanced hyperthermia may provide a non-invasive, alterative treatment option for highly proliferating, superficial skin, and mucosal lesions such as cancer or warts. Lasers Surg. Med. 51:88-94, 2019. © 2018 Wiley Periodicals, Inc.

A predictive model for diagnosis of lower extremity cellulitis: A cross-sectional study.

BACKGROUND: Cellulitis has many clinical mimickers (pseudocellulitis), which leads to frequent misdiagnosis. OBJECTIVE: To create a model for predicting the likelihood of lower extremity cellulitis. METHODS: A cross-sectional review was performed of all patients admitted with a diagnosis of lower extremity cellulitis through the emergency department at a large hospital between 2010 and 2012. Patients discharged with diagnosis of cellulitis were categorized as having cellulitis, while those given an alternative diagnosis were considered to have pseudocellulitis. Bivariate associations between predictor variables and final diagnosis were assessed to develop a 4-variable model. RESULTS: In total, 79 (30.5%) of 259 patients were misdiagnosed with lower extremity cellulitis. Of the variables associated with true cellulitis, the 4 in the final model were asymmetry (unilateral involvement), leukocytosis (white blood cell count ≥10,000/uL), tachycardia (heart rate ≥90 bpm), and age ≥70 years. We converted these variables into a points system to create the ALT-70 cellulitis score as follows: Asymmetry (3 points), Leukocytosis (1 point), Tachycardia (1 point), and age ≥70 (2 points). With this score, 0-2 points indicate ≥83.3% likelihood of pseudocellulitis, and ≥5 points indicate ≥82.2% likelihood of true cellulitis. LIMITATIONS: Prospective validation of this model is needed before widespread clinical use. CONCLUSION: Asymmetry, leukocytosis, tachycardia, and age ≥70 are predictive of lower extremity cellulitis. This model might facilitate more accurate diagnosis and improve patient care.

Successful Use of Cyclosporin A for Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis in Three Children.

BACKGROUND/OBJECTIVES: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are medical emergencies. Mainstays of treatment include removal of the offending agent, supportive care, and wound care. The use of immunosuppressive agents such as corticosteroids and intravenous immunoglobulin (IVIg) is controversial. Some case reports and small studies report the successful use of cyclosporin A (CsA) for SJS/TEN in halting disease progression, fostering reepithelialization, and reducing mortality. OBJECTIVE: To report on the efficacy of cyclosporine A in the treatment of SJS/TEN in three pediatric patients. METHODS: We describe three pediatric patients seen at a tertiary care hospital in Boston, Massachusetts, diagnosed with SJS/TEN confirmed by skin biopsy who were successfully treated with CsA with improvements seen in time to cessation of disease progression or new lesion formation, reepithelialization, and duration of hospital stay. RESULTS: The average time cessation of disease progression or new lesion formation after CsA administration was 2.2 days (range 1.5-3 days) and average time to remission or reepithelialization was 13 days (range 10-15 days). The average length of hospital stay was 11.7 days (range 4-19 days). CONCLUSIONS: We describe three pediatric patients treated successfully with CsA and provide evidence for the use of cyclosporine in children with SJS/TEN. These results further support previous observations that CsA use for SJS/TEN produces consistently favorable outcomes. The results in this case series are limited by their observational nature. Additional trials are needed to evaluate the safety and efficacy of CsA use in children.

Inhibition of Langerhans cell maturation by human papillomavirus type 16: a novel role for the annexin A2 heterotetramer in immune suppression.

High-risk human papillomaviruses (HPVs) are sexually transmitted viruses causally associated with several cancers. During its natural life cycle, HPV16, the most common high-risk genotype, infects the epithelial basal cells in a process facilitated through a recently identified receptor, the annexin A2 heterotetramer (A2t). During infection, HPV16 also interacts with Langerhans cells (LC), the APC of the epithelium, inducing immune suppression, which is mediated by the HPV16 L2 minor capsid protein. Despite the importance of these virus-immune cell interactions, the specific mechanisms of HPV16 entry into LC and HPV16-induced immune suppression remain undefined. An N-terminal peptide of HPV16 L2 (aa 108-126) has been shown to specifically interact with A2t. In this study, we show that incubation of human LC with this peptide blocks binding of HPV16. Inhibiting this interaction with an A2t ligand or by small interfering RNA downregulation of A2t significantly decreases HPV16 internalization into LC in an L2-dependent manner. A2t is associated with suppression of LC maturation as demonstrated through attenuated secretion of Th1-associated cytokines and decreased surface expression of MHC class II on LC exposed to A2t. Conversely, small molecule inhibition of A2t prevents HPV16-induced suppression of LC immune function as indicated by significantly increased secretion of inflammatory cytokines and surface expression of CD86 in HPV16 treated LC pre-exposed to A2t inhibitors. These results demonstrate that HPV16 suppresses LC maturation through an interaction with A2t, revealing a novel role for this protein.

Cellulitis: A Review.

IMPORTANCE: Cellulitis is an infection of the deep dermis and subcutaneous tissue, presenting with expanding erythema, warmth, tenderness, and swelling. Cellulitis is a common global health burden, with more than 650,000 admissions per year in the United States alone. OBSERVATIONS: In the United States, an estimated 14.5 million cases annually of cellulitis account for $3.7 billion in ambulatory care costs alone. The majority of cases of cellulitis are nonculturable and therefore the causative bacteria are unknown. In the 15% of cellulitis cases in which organisms are identified, most are due to ß-hemolytic Streptococcus and Staphylococcus aureus. There are no effective diagnostic modalities, and many clinical conditions appear similar. Treatment of primary and recurrent cellulitis should initially cover Streptococcus and methicillin-sensitive S. aureus, with expansion for methicillin-resistant S. aureus (MRSA) in cases of cellulitis associated with specific risk factors, such as athletes, children, men who have sex with men, prisoners, military recruits, residents of long-term care facilities, those with prior MRSA exposure, and intravenous drug users. Five days of treatment is sufficient with extension if symptoms are not improved. Addressing predisposing factors can minimize risk of recurrence. CONCLUSIONS AND RELEVANCE: The diagnosis of cellulitis is based primarily on history and physical examination. Treatment of uncomplicated cellulitis should be directed against Streptococcus and methicillin-sensitive S. aureus. Failure to improve with appropriate first-line antibiotics should prompt consideration for resistant organisms, secondary conditions that mimic cellulitis, or underlying complicating conditions such as immunosuppression, chronic liver disease, or chronic kidney disease.

The evolving field of human papillomavirus receptor research: a review of binding and entry.

Human papillomaviruses (HPVs) infect epithelia and can lead to the development of lesions, some of which have malignant potential. HPV type 16 (HPV16) is the most oncogenic genotype and causes various types of cancer, including cervical, anal, and head and neck cancers. However, despite significant research, our understanding of the mechanism by which HPV16 binds to and enters host cells remains fragmented. Over several decades, many HPV receptors and entry pathways have been described. This review puts those studies into context and offers a model of HPV16 binding and entry as a framework for future research. Our model suggests that HPV16 binds to heparin sulfate proteoglycans (HSPGs) on either the epithelial cell surface or basement membrane through interactions with the L1 major capsid protein. Growth factor receptors may also become activated through HSPG/growth factor/HPV16 complexes that initiate signaling cascades during early virion-host cell interactions. After binding to HSPGs, the virion undergoes conformational changes, leading to isomerization by cyclophilin B and proprotein convertase-mediated L2 minor capsid protein cleavage that increases L2 N terminus exposure. Along with binding to HSPGs, HPV16 binds to α6 integrins, which initiate further intracellular signaling events. Following these primary binding events, HPV16 binds to a newly identified L2-specific receptor, the annexin A2 heterotetramer. Subsequently, clathrin-, caveolin-, lipid raft-, flotillin-, cholesterol-, and dynamin-independent endocytosis of HPV16 occurs.

A paradigm shift in therapeutic vaccination of cancer patients: the need to apply therapeutic vaccination strategies in the preventive setting.

An extraordinary variety of potential therapeutic vaccine strategies directed against a wide variety of tumor antigens has been explored in clinical trials. To date, none of these cancer immunotherapies have been approved by the Food and Drug Administration for use in humans. A significant problem is that the vast majority of such clinical trials are carried out in patients with advanced or metastatic cancer. The immune systems of these patients are considerably compromised as a result of tumor- and treatment-mediated immunosuppression. Even in cases where patients are immunized in the adjuvant setting, where there is minimal residual disease, vaccines directed against tumor-associated antigens have failed to mediate eradication of tumors in the overwhelming majority of cases. Recently, we and others have experimented with administering therapeutic cancer vaccines in the preventive setting. This is achieved by vaccinating at the earliest possible stage of carcinogenesis. These studies have demonstrated that early vaccination is extremely effective in eliciting an anti-tumor immune response that leads to unprecedented improvements in the survival of mice that spontaneously develop cancer. Certain human cancers, notably prostate adenocarcinoma and cervical cancer, can currently be detected at very early stages of carcinogenesis. Therapeutic vaccines are available for these diseases, opening up the possibility of administering vaccinations early to patients diagnosed with pre-malignant lesions to halt disease progression. In addition, new technologies have become available in the past decade that will soon yield very sensitive and specific diagnostic tests for a plethora of other cancers. Earlier detection of these cancers, combined with existing vaccines directed against them, will soon make them targets for therapeutic vaccination in the preventive setting. The ability to immunize patients at the very earliest stages of carcinogenesis, when they have fully competent immune systems, has the potential to cause a paradigm shift in how therapeutic cancer vaccines are tested and used clinically.

Reversal of human papillomavirus-specific T cell immune suppression through TLR agonist treatment of Langerhans cells exposed to human papillomavirus type 16.

Human papillomavirus (HPV) type 16 infects the epithelial layer of cervical mucosa and is causally associated with the generation of cervical cancer. Langerhans cells (LC) are the resident APCs at the site of infection and therefore are responsible for initiating an immune response against HPV16. On the contrary, LC exposed to HPV16 do not induce a specific T cell immune response, which leads to the immune evasion of HPV16. Demonstrating that TLR7 and TLR8 are expressed on human LC, we hypothesized that imidazoquinolines would activate LC exposed to HPV16, leading to the induction of an HPV16-specific cell-mediated immune response. Surprisingly, both phenotypic and functional hallmarks of activation are not observed when LC are exposed to HPV16 virus-like particles and treated with imiquimod (TLR7 agonist). However, we found that LC are activated by 3M-002 (TLR8 agonist) and resiquimod (TLR8/7 agonist). LC exposed to HPV16 virus-like particles and subsequently treated with 3M-002 or resiquimod highly up-regulate surface activation markers, secrete proinflammatory cytokines and chemokines, induce CCL21-directed migration, and initiate an HPV16-specific CD8(+) T cell response. These data strongly indicate that 3M-002 and resiquimod are promising therapeutics for treatment of HPV infections and HPV-induced cervical lesions.

A major role for the minor capsid protein of human papillomavirus type 16 in immune escape.

High-risk human papillomavirus (HPV) infection of the cervical epithelium is causally linked with the generation of cervical cancer. HPV does not activate Langerhans cells (LC), the APC at the site of infection, leading to immune evasion. The HPV protein responsible for inducing this immune escape has not been determined. We demonstrate that LC exposed to the minor capsid protein L2 in HPV16L1L2 virus-like particles do not phenotypically or functionally mature. However, HPV16L1 virus-like particles significantly induce activation of LC. Our data suggest that the L2 protein plays a specific role in the induction of this immune escape of HPV16 through the manipulation of LC. This novel function is the first immune modulating action attributed to the L2 protein and adds significantly to our understanding of the mechanism of HPV immune escape.

Diffuse Reflectance Spectroscopy with Infrared Thermography for Accurate Prediction of Cellulitis.

Cellulitis is frequently misdiagnosed owing to its clinical mimickers, collectively known as pseudocellulitis. This study investigated diffuse reflectance spectroscopy (DRS) alone and in combination with infrared thermography (IRT) for the differentiation of cellulitis from pseudocellulitis. A prospective cohort study at an urban academic hospital was conducted from March 2017 to March 2018. Patients presenting to the emergency department with presumed cellulitis were screened for eligibility, and 30 adult patients were enrolled. Dermatology consultation conferred a final diagnosis of cellulitis or pseudocellulitis. DRS measurements yielded a spectral ratio between 556 nm (deoxyhemoglobin peak) and 542 nm (oxyhemoglobin peak), and IRT measurements yielded temperature differentials between the affected and unaffected skin. Of the 30 enrolled patients, 30% were diagnosed with pseudocellulitis. DRS revealed higher spectral ratios in patients with cellulitis (P = 0.005). A single parameter model using logistic regression on DRS measurements alone demonstrated a classification accuracy of 77.0%. A dual parameter model using linear discriminant analysis on DRS and IRT measurements combined demonstrated a 95.2% sensitivity, 77.8% specificity, and 90.0% accuracy for cellulitis prediction. DRS and IRT combined diagnoses cellulitis with an accuracy of 90%. DRS and IRT are inexpensive and noninvasive, and their use may reduce cellulitis misdiagnosis.

Point-of-care detection of neutrophils in live skin microsamples using chemiluminescence.

Many skin diseases are defined by the presence of neutrophils, which are among the first cells to respond to infection and inflammation. Currently, neutrophil identification in the skin is costly and slow. The objectives of the present work are to investigate the feasibility of detecting the presence of neutrophils in live skin microsamples using chemiluminescence and develop a device and procedures that will enable preclinical and clinical investigations. Our approach consists of collecting skin microsamples and exposing them to reagents that activate neutrophils and amplify the light emission produced by chemiluminescence. Experiments using live pig skin with and without inflammation show that it is feasible to detect the presence of neutrophils in the skin. The proposed method is minimally invasive, simple, fast, and does not require user specialization. The developed system is compact in size with a small footprint, which makes it portable and suitable for point-of-care diagnostics.

A microscale, full-thickness, human skin on a chip assay simulating neutrophil responses to skin infection and antibiotic treatments.

Human skin models are essential for understanding dermatological diseases and testing new treatment strategies. The use of skin biopsies ex vivo is the most accurate model. However, their use is expensive and exposes the donor to pain and scarring. While bioengineered skin samples provide a cheaper alternative, they have limitations due to their simple structure and functionality compared to human skin. Here, we present a skin-on-a-chip device designed to study neutrophil responses to Staphylococcus aureus skin infections. We integrate human skin microcolumns, which have a cross-section that is ∼100 times smaller than traditional skin biopsies, are full-thickness, and are collected using minimally invasive skin sampling techniques. We use human neutrophils directly from one drop of blood, without the need for blood separation. Using the skin-on-a-chip device with skin and blood samples from healthy donors, we show that the neutrophil responses correlate with the bacteria-load in the skin. A pre-incubation step increases the number of migrating neutrophils in response to a low concentration of bacteria. Antibiotic treatment of S. aureus-infected skin samples reduces the number of neutrophils migrating towards the skin. Overall, we validate a skin on a chip model that enables the study of neutrophil migration to the skin in the presence of microbes and following the administration of antibiotics, two situations relevant to clinical cases of human skin and soft tissue infections.

Microfluidic arenas for war games between neutrophils and microbes.

Measurements of neutrophil activities such as cell migration and phagocytosis are generally performed using low-content bulk assays, which provide little detail activity at the single cell level, or flow cytometry methods, which have the single cell resolution but lack perspective on the kinetics of the process. Here, we present a microfluidic assay for measuring the essential functions that contribute to the antimicrobial activity of neutrophils: migration towards the target, and killing of microbes. The assay interrogates the interactions between isolated human neutrophils and populations of live, proliferating microbes. The outcome is measured in a binary mode that is reflective of in vivo infections, which are either cleared or endure the host response. The outcome of the interactions is also characterized at single cell resolution for both the neutrophils and the microbes. We applied the assay to test the response of neutrophils from intensive care patients to live Staphylococcus aureus, and observed alterations of antimicrobial neutrophil activity in patients, including those with sepsis. By directly measuring neutrophil activity against live targets at high spatial and temporal resolution, this assay provides unique insights into the life-or-death contest shaping the outcome of interactions between populations of neutrophils and microbes.