A Bacteriophage-Based, Highly Efficacious, Needle- and Adjuvant-Free, Mucosal COVID-19 Vaccine.

The U.S. Food and Drug Administration-authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected in two doses and effective in preventing COVID-19, but they do not induce efficient mucosal immunity or prevent viral transmission. Here, we report the first noninfectious, bacteriophage T4-based, multicomponent, needle- and adjuvant-free, mucosal vaccine harboring engineered Spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of two doses of this T4 SARS-CoV-2 vaccine 21 days apart induced robust mucosal immunity, in addition to strong systemic humoral and cellular immune responses. The intranasal vaccine induced broad virus neutralization antibody titers against multiple variants, Th1-biased cytokine responses, strong CD4+ and CD8+ T cell immunity, and high secretory IgA titers in sera and bronchoalveolar lavage specimens from vaccinated mice. All of these responses were much stronger in intranasally vaccinated mice than those induced by the injected vaccine. Furthermore, the nasal vaccine provided complete protection and sterilizing immunity against the mouse-adapted SARS-CoV-2 MA10 strain, the ancestral WA-1/2020 strain, and the most lethal Delta variant in both BALB/c and human angiotensin converting enzyme (hACE2) knock-in transgenic mouse models. In addition, the vaccine elicited virus-neutralizing antibodies against SARS-CoV-2 variants in bronchoalveolar lavage specimens, did not affect the gut microbiota, exhibited minimal lung lesions in vaccinated and challenged mice, and is completely stable at ambient temperature. This modular, needle-free, phage T4 mucosal vaccine delivery platform is therefore an excellent candidate for designing efficacious mucosal vaccines against other respiratory infections and for emergency preparedness against emerging epidemic and pandemic pathogens. IMPORTANCE According to the World Health Organization, COVID-19 may have caused ~15-million deaths across the globe and is still ravaging the world. Another wave of ~100 million infections is predicted in the United States due to the emergence of highly transmissible immune-escaped Omicron variants. The authorized vaccines would not prevent these transmissions since they do not trigger mucosal immunity. We circumvented this limitation by developing a needle-free, bacteriophage T4-based, mucosal vaccine. This intranasally administered vaccine generates superior mucosal immunity in mice, in addition to inducing robust humoral and cell-mediated immune responses, and provides complete protection and sterilizing immunity against SARS-CoV-2 variants. The vaccine is stable, adjuvant-free, and cost-effectively manufactured and distributed, making it a strategically important next-generation COVID vaccine for ending this pandemic.

Validación de Experimentos Cognitivos con PEBL y Wundts Lab / Validation of Cognitive Experiments with PEBL and WundtS Lab

Resumen Actualmente las herramientas tecnológicas están reemplazando los métodos tradicionales en diversos campos de la psicología, un fenómeno de alto impacto; por esta razón se realizó un análisis de las características de validez y confiabilidad de pruebas expérimentales computarizadas empleando un estudio psicométrico instrumental que contó con 267 participantes que realizaron 589 aplicaciones pareadas de pruebas, en tanto que la mayoría llevó a cabo más de dos tareas cognitivas. Mediante un análisis discriminante, se identificaron cuatro pruebas computarizadas PEBL y tres pruebas computarzadas Wundt's Lab, consistentes con medidas cognitivas validadas, a diferencia de tres pruebas PEBL y cuatro pruebas Wundt's Lab, que no mostraron tal evidencia. Adicionalmente, se encontraron convergencias entre los desempenos de pruebas Mental Rotation de PEBL y Wundt's Lab, así como asociaciones pareadas entre desempenos de pruebas de memoria operativa verbal y visoespacial, de atención y de percepción del movimiento. Finalmente, se evidenciaron asociaciones relevantes entre los resultados de los participantes en las pruebas y la variable "sexo". Los resultados apoyan la existencia de características psicométricas en siete pruebas computarizadas, haciendo relevante su uso en diversos campos de la evaluación cognitiva.

Abstract Currently, technological tools are replacing traditional methods in many fields of psychology, a phenomenon of high impact. For this reason, validity and reliability characteristics analysis of computerized experimental tests were carried out, using an instrumental psychometric study, which had 267 participants taking part in 589 paired applications of tests. Through a discriminant analysis, four computerized PEBL tests and three computerized Wundt's Lab tests were identified, consistent with validated cognitive measures, as opposed to three PEBL tests and four Wundt's Lab tests, which did not show such evidence. Additionally, convergences were found between PEBL and Wundt's Lab's Mental Rotation test performances, as well as paired associations between verbal and visuospatial working memory, and attention and movement perception test performances. Finally, relevant associations were shown between the results of the participants in the tests and the sex variable. Results support the existence of psychometric characteristics in seven computerized tests, making their use relevant in various fields of cognitive evaluation.

David Versus Goliath: The Clinical Consequences of an Influenza A Viral Infection in an Unvaccinated Patient.

Acute influenza virus (AIV) infection can manifest as a severe life-threating illness in patients who are not vaccinated, and furthermore, have comorbidities that place them at risk for rapid respiratory decompensation. Each year influenza causes death in individuals with high risk for contracting this infection, although the illness is preventable by vaccination. Complications of AIV infection, such as bacterial pneumonia are treatable, but other severe complications such as acute respiratory distress syndrome (ARDS) leading to diffuse alveolar damage (DAD) are limited to supportive therapy and self-resolution. In most cases, ARDS leading to DAD is fatal, due to the insidious severity of symptoms which lead to rapid oxygen desaturation without correction, and despite supportive therapy. Regardless of a poor prognosis, the clinical signs and symptoms are congruent with imaging and attest to the importance of vaccination, which protect against high mortality rates.

Diferencias en las decisiones utilitarias y morales entre hombres y mujeres / Differences on Utilitarian and Moral Decision Between Male and Female / Diferencias nas decisões utilitárias e morais entre homens e mulheres

Resumen Objetivo. Los juicios morales se basan en decisiones que toman en cuenta la representación de las normas y la ley, los valores, la funcionalidad y la situación en sí. La moral se ha estudiado con "dilemas morales hipotéticos" para identificar el tipo de resultado y el proceso detrás del razonamiento moral. No obstante, los juicios en sí mismos no son suficientes para establecer diferencias en el tipo de resolución o la relación con otros procesos cognitivos. Por lo anterior, el presente estudio buscó comparar el desempeño en tareas de maximización de la utilidad, control cognitivo y juicios morales, teniendo en cuenta el sexo y otras variables sociodemográficas. Método. Participaron 73 estudiantes universitarios (50 mujeres, 20 hombres y 3 con sexo no reportado, la edad promedio fue 19.53 años (DE = 1.68 años). El Iowa Gambling Task (IGT) se utilizó para identificar comportamientos de maximización de la utilidad. Además, el estudio empleó la prueba de cambios de tarea y la aplicación web Máquina Moral. Resultados. Se encontró una diferencia entre las variables de IGT, sin observar diferencias en la prueba de cambios de tarea. Conclusión. En cuanto al juicio moral, los hombres dieron más valor al cumplimiento de normas que las mujeres. Algunas variables de la tarea IGT apoyan las diferencias entre los sexos. Los resultados son congruentes con las diferencias mostradas por la literatura.

Abstract Objective. Moral judgments are based on decisions that take into account the representation of norms and law, values, functionality and situations themselves. Morality has been studied with "hypothetic moral dilemmas", in order to identify the type of outcome and the process behind moral reasoning. But judgments by themselves are not enough to establish differences in the type of resolution or the relationship with other cognitive processes. The present paper aimed to compare performance in tasks of utility maximization, cognitive control, and moral judgments, taking into account sex and other sociodemographic variables. Method. Seventy-three university students participated (50 women, 20 men and 3 with unreported gender, the average age was 19.53 years (SD = 1.68 years). The Iowa Gambling Task (IGT) was used to identify behaviors of utility maximization. In addition, we used the switch costs and the web application of moral machine tasks. Results. A difference between variables of the IGT, but no differences in the switch costs task were found. Conclusion. Regarding moral judgment, males gave more value to respect norms than females. Some variables of the IGT task support outcomes related to differences between sexes. Results are congruent with differences shown in existing literature.

Resumo Escopo. Os juízos morais estão baseados em decisões que levam em conta a representação das normas e a lei, os valores, a funcionalidade e a situação concreta. A moral tem se estudado com "dilemas morais hipotéticos" para identificar o tipo de resultado e o processo detrás do razoamento moral. Mas os juízos em si não são suficientes para estabelecer diferencias no tipo de resolução ou a relação com outros processos cognitivos. Por tanto, o escopo de este estudo foi comparar o desempenho em tarefas de maximização da utilidade, controle cognitivo e juízos morais, levando em conta o sexo e outras variáveis sócio demográficas. Metodologia. Participaram 73 estudantes universitários (50 mulheres, 20 homens e 3 com sexo não reportado, a idade de média foi de19.3 anos, (DE = 1.68 anos). O Iowa Gambling Task (IGT) foi utilizado para identificar comportamentos de maximização da utilidade. Além, o estudo empregou a prova de mudança de tarefa e a aplicação web Máquina Moral. Resultados. Foi achada uma diferencia entre as variáveis de IGT, mas não há diferencias na prova de mudança de tarefa. Em quanto ao juízo moral, os homens deram mais valor ao cumprimento de normas que as mulheres. Algumas variáveis da tarefa IGT apoiam as diferencias entre os sexos. Conclusão. Os resultados são congruentes com as diferencias mostradas na literatura.

ZIKV Demonstrates Minimal Pathologic Effects and Mosquito Infectivity in Viremic Cynomolgus Macaques.

To evaluate the effects of ZIKV infection on non-human primates (NHPs), as well as to investigate whether these NHPs develop sufficient viremia to infect the major urban vector mosquito, Aedes aegypti, four cynomolgus macaques (Macaca fascicularis) were subcutaneously infected with 5.0 log10 focus-forming units (FFU) of DNA clone-derived ZIKV strain FSS13025 (Asian lineage, Cambodia, 2010). Following infection, the animals were sampled (blood, urine, tears, and saliva), underwent daily health monitoring, and were exposed to Ae. aegypti at specified time points. All four animals developed viremia, which peaked 3⁻4 days post-infection at a maximum value of 6.9 log10 genome copies/mL. No virus was detected in urine, tears, or saliva. Infection by ZIKV caused minimal overt disease: serum biochemistry and CBC values largely fell within the normal ranges, and cytokine elevations were minimal. Strikingly, the minimally colonized population of Ae. aegypti exposed to viremic animals demonstrated a maximum infection rate of 26% during peak viremia, with two of the four macaques failing to infect a single mosquito at any time point. These data indicate that cynomolgus macaques may be an effective model for ZIKV infection of humans and highlights the relative refractoriness of Ae. aegypti for ZIKV infection at the levels of viremia observed.

Effects of goal clarification on impulsivity and academic procrastination of college students / Efectos de la clarificación de metas sobre la impulsividad y la procrastinación académica de los estudiantes universitarios

Abstract Nearly 50% of the college population struggles with academic procrastination, which is an impulsivity problem that often leads to emotional difficulties and college dropout. This study aimed to assess whether an online intervention on clarification of academic goals could reduce impulsivity and academic procrastination in college students. Forty-eight participants were assigned to three different types of interventions: (a) SMART-type goal clarification treatment (setting specific, measurable, agreed upon, realistic and time-based goals); (b) instructional intervention for the abandonment of procrastination (conventional self-help type intervention); and (c) a waiting list. Only SMART intervention produced a statistically signif icant decrease in impulsivity (measured in terms of a hyperbolic discounting test; Whelan & McHugh, 2009), and academic procrastination (measured with the Procrastination Assessment Scale-Student --- PASS), in both cases with small-to-moderate treatment effects. In conclusion, the study showed that online SMART-type goal clarification led to positive changes in impulsive ness and academic procrastination of college students, whereas a self-help protocol failed to produce similar effects. Potential reasons for reduced treatment effects of the SMART interven tion are examined (e.g., experimental control). Also, prospective lines of research are discussed in view of the scarcity of experimental studies in this area.

Resumen Cerca del 50% de la población universitaria experimenta procrastinación académica, un problema asociado con impulsividad, dificultades emocionales y deserción. El estudio evaluó si una intervención en línea en clarificación de metas académicas reduce la impulsividad y la procrastinación académica de estudiantes universitarios. Cuarenta y ocho estudiantes fueron distribuidos en tres tipos de intervención: (a) clarificación de metas tipo SMART (estable cer metas específicas, acordadas en colaboración, medibles, realistas, y basadas en criterios temporales); (b) seguimiento de instrucciones para abandonar la procrastinación (protocolo convencional de tipo autoayuda), y (c) lista de espera. La intervención SMART fue la única que produjo una disminución estadísticamente significativa en impulsividad -medida en términos de descuento hiperbólico (Whelan & McHugh, 2009)- y procrastinación académica -medida a través del Procrastination Assessment Scale-Student (PASS)---- , en ambos casos con efectos de tratamiento de pequeños a moderados. En conclusión, el estudio demostró la efectividad de un protocolo en línea de clarificación de metas tipo SMART para reducir la impulsividad y la procrastinación académica de estudiantes universitarios, efectos que no fueron encontrados con la implementación de un protocolo de tipo autoayuda. Se discuten posibles razones por las cuales los efectos del tratamiento SMART no fueron mayores (e.g., control experimental), y líneas potenciales de investigación a futuro, esto especialmente considerando los escasos estudios experimentales en esta área.

Attitudes and Beliefs of Pathology Residents Regarding the Subspecialty of Clinical Chemistry: Results of a Survey.

CONTEXT: -Previous studies suggest that training in pathology residency programs does not adequately prepare pathology residents to become competent in clinical chemistry. OBJECTIVES: -To define the beliefs of pathology residents in the United States regarding their preparation for practicing clinical chemistry in their career, their attitude toward the discipline, and the attractiveness of clinical chemistry as a career. DESIGN: -The residents of all pathology residency programs in the United States were given the opportunity to participate in an online survey. RESULTS: -Three hundred thirty-six pathology residents responded to the survey. Analysis of the survey results indicates that pathology residents are more likely to believe that their income may be lower if they select a career that has a clinical chemistry focus and that their faculty do not value clinical chemistry as much as the anatomic pathology part of the residency. Residents also report that clinical chemistry is not as enjoyable as anatomic pathology rotations during residency or preferable as a sole career path. A large proportion of residents also believe that they will be slightly prepared or not prepared to practice clinical chemistry by the end of their residency and that they do not have enough background and/or time to learn clinical chemistry during their residency programs to be able to practice this specialty effectively post graduation. CONCLUSIONS: -Our survey results suggest that many pathology residents do not have a positive attitude toward clinical chemistry and do not experience a supportive learning environment with an expectation that they will become competent in clinical chemistry with a residency alone.

Early diagnosis of Ehrlichia ewingii infection in a lung transplant recipient by peripheral blood smear.

Ehrlichiosis in lung transplant (LT) recipients is associated with severe outcomes. Ehrlichia ewingii is a less frequent cause of symptomatic ehrlichiosis, characterized by cytoplasmic inclusions (morulae) within circulating neutrophils. We report a case of E. ewingii infection in an LT recipient diagnosed promptly by blood smear exam and confirmed with molecular studies.

An Intradermal Inoculation Mouse Model for Immunological Investigations of Acute Scrub Typhus and Persistent Infection.

Scrub typhus is a neglected tropical disease, caused by Orientia tsutsugamushi, a Gram-negative bacterium that is transmitted to mammalian hosts during feeding by Leptotrombidium mites and replicates predominantly within endothelial cells. Most studies of scrub typhus in animal models have utilized either intraperitoneal or intravenous inoculation; however, there is limited information on infection by the natural route in murine model skin or its related early host responses. Here, we developed an intradermal (i.d.) inoculation model of scrub typhus and focused on the kinetics of the host responses in the blood and major infected organs. Following ear inoculation with 6 x 104 O. tsutsugamushi, mice developed fever at 11-12 days post-infection (dpi), followed by marked hypothermia and body weight loss at 14-19 dpi. Bacteria in blood and tissues and histopathological changes were detected around 9 dpi and peaked around 14 dpi. Serum cytokine analyses revealed a mixed Th1/Th2 response, with marked elevations of MCP-1/CCL2, MIP-1α/CCL3 and IL-10 at 9 dpi, followed by increased concentrations of pro-inflammatory markers (IL-6, IL-12, IFN-γ, G-CSF, RANTES/CCL5, KC/CCL11, IL-1α/ß, IL-2, TNF-α, GM-CSF), as well as modulatory cytokines (IL-9, IL-13). Cytokine levels in lungs had similar elevation patterns, except for a marked reduction of IL-9. The Orientia 47-kDa gene and infectious bacteria were detected in several organs for up to 84 dpi, indicating persistent infection. This is the first comprehensive report of acute scrub typhus and persistent infection in i.d.-inoculated C57BL/6 mice. This is a significant improvement over current murine models for Orientia infection and will permit detailed studies of host immune responses and infection control interventions.

High serum CXCL10 in Rickettsia conorii infection is endothelial cell mediated subsequent to whole blood activation.

BACKGROUND: The pathophysiological hallmark of Rickettsia conorii (R. conorii) infection comprises infection of endothelial cells with perivascular infiltration of T-cells and macrophages. Although interferon (IFN)-γ-induced protein 10 (IP-10)/CXCL10 is induced during vascular inflammation, data on CXCL10 in R. conorii infection is scarce. METHODS: Serum CXCL10 was analyzed in two cohorts of southern European patients with R. conorii infection using multiplex cytokine assays. The mechanism of R. conorii-induced CXCL10 release was examined ex vivo using human whole blood interacting with endothelial cells. RESULTS: (i) At admission, R. conorii infected patients had excessively increased CXCL10 levels, similar in the Italian (n=32, ∼56-fold increase vs controls) and the Spanish cohort (n=38, ∼68-fold increase vs controls), followed by a marked decrease after recovery. The massive CXCL10 increase was selective since it was not accompanied with similar changes in other cytokines. (ii) Heat-inactivated R. conorii induced a marked CXCL10 increase when whole blood and endothelial cells were co-cultured. Even plasma obtained from R. conorii-exposed whole blood induced a marked CXCL10 release from endothelial cells, comparable to the levels found in serum of R. conorii-infected patients. Bacteria alone did not induce CXCL10 production in endothelial cells, macrophages or smooth muscle cells. CONCLUSIONS: We show a massive and selective serum CXCL10 response in R. conorii-infected patients, likely reflecting release from infected endothelial cells characterized by infiltrating T cells and monocytes. The CXCL10 response could contribute to T-cell infiltration within the infected organ, but the pathologic consequences of CXCL10 in clinical R. conorii infection remain to be defined.

An embedded microretroreflector-based microfluidic immunoassay platform.

We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light. Fluidic force discrimination is used to increase specificity of the assay, following which a difference imaging algorithm that can see single 3 µm magnetic particles without optical calibration is used to detect and quantify signal intensity from each sub-array of retroreflectors. We demonstrate the utility of embedded microretroreflectors as a new sensing modality through a proof-of-concept immunoassay for a small, obligate intracellular bacterial pathogen, Rickettsia conorii, the causative agent of Mediterranean Spotted Fever. The combination of large sensing area, optimized surface chemistry and microfluidic protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in a sensitive immunoassay with a limit of detection of roughly 4000 R. conorii per mL.

Increased expression of the homeostatic chemokines CCL19 and CCL21 in clinical and experimental Rickettsia conorii infection.

BACKGROUND: Based on their essential role in concerting immunological and inflammatory responses we hypothesized that the homeostatic chemokines CCL19 and CCL21 may play a pathogenic role in rickettsiae infection. METHODS: Serum levels of CCL19 and CCL21 in patients with R. africae and R. conorii infection were analyzed by enzyme immunoassays. Lungs from R. conorii infected mice were examined for CCL19, CCL21 and CCR7 expression by immunohistochemistry. RESULTS: We found that patients with R. africae infection (n = 15) and in particular those with R. conorii infection (n = 16) had elevated serum levels of CCL19 on admission, with a decline during follow-up. While a similar pattern was seen for CCL21 in R. africae infection, patients with R. conorii infection showed persistently increased CCL21 levels during follow-up. In experimental R. conorii infection, we found strong immunostaining of CCL19 and CCL21 in the lungs, particularly in individuals that had received lethal doses. Immunofluorescence showed co-localization of CCR7 to endothelial cells, macrophages and fibroblasts within the lung tissue of R. conorii infected mice. CONCLUSIONS: Our findings suggest that the CCL19/CCL21/CCR7 axis is up-regulated during R. africae and in particular during R. conorii infection, which may potentially contribute to the pathogenesis of these disorders.

Rapid detection of trace bacteria in biofluids using porous monoliths in microchannels.

We present advancements in microfluidic technology for rapid detection of as few as 10 rickettsial organisms in complex biological samples. An immuno-reactive filter, macroporous polyacrylamide monolith (PAM), fabricated within a microfluidic channel enhances solid-phase immuno-capture, staining and detection of targeted bacteria. Bacterial cells in samples flowing through the channel are forced to interact with the PAM filter surface due to size exclusion, overcoming common transport and kinetic limitations for rapid (min), high-efficiency (~100%) capture. In the process, targeted cells in sample volumes of 10 µl to >100 µl are concentrated within a sub-50 nl region at the PAM filter edge in the microchannel, thus concentrating them over 1000-fold. This significantly increases sensitivity, as the hydrophilic PAM also yields low non-specific immuno-fluorescence backgrounds with samples including serum, blood and non-targeted bacteria. The concentrated target cells are detected using fluorescently-labeled antibodies. With a single 2.0×2.0×0.3 mm PAM filter, as few as 10 rickettsial organisms per 100 µl of lysed blood sample can be analyzed within 60 min, as compared to hours or even days needed for conventional detection methods. This method is highly relevant to rapid, multiplexed, low-cost point of care diagnostics at early stages of infection where diagnostics providing more immediate and actionable test results are needed to improve patient outcomes and mitigate potential natural and non-natural outbreaks or epidemics of rickettsial diseases.

How Cytokines Co-occur across Rickettsioses Patients: From Bipartite Visual Analytics to Mechanistic Inferences of a Cytokine Storm.

Several intersecting host, vector, and environmental factors have led to a re-emergence of rickettsial diseases such as Mediterranean Spotted Fever (MSF), and Dermacentor spp.-borne necrosis-erythema lymphadenopathy (DEBONEL). Some rickettsiae produce diffuse endothelial infection and systemic microvascular leakage leading in some cases to high morbidity and mortality. Unfortunately, little is known about the molecular pathways triggered by these diseases in humans. We therefore analyzed how candidate cytokines co-occur across acutely-ill patients with either a localized (DEBONEL), or a systemic (MSF) form of rickettsiosis, using bipartite visual analytics. The results revealed a network core consisting of a small set of MSF patients exhibiting high expressions of cytokines implicated in microvascular leakage, endothelial repair, and pro-inflammatory immune responses, and a network periphery consisting of a mixture of MSF and DEBONEL patients with relatively lower overall cytokine expressions. These results provide evidence of pathways triggered by rickettsiae in humans, and a testable hypothesis for the mechanisms in a rickettsia-induced cytokine storm with the translational goal of identifying therapeutic targets.

A complex interaction between Rickettsia conorii and Dickkopf-1--potential role in immune evasion mechanisms in endothelial cells.

The pathophysiological hallmark of spotted fever group rickettsioses comprises vascular inflammation. Based on the emerging importance of the wingless (Wnt) pathways in inflammation and vascular biology, we hypothesized that Dickkopf-1 (DKK-1), as a major modulator of Wnt signaling, could be involved in the pathogenesis in rickettsial infections. Our major findings were: (i) While baseline concentration of DKK-1 in patients with R. conorii infection (n = 32) were not different from levels in controls (n = 24), DKK-1 rose significantly from presentation to first follow-up sample (median 7 days after baseline). (ii) In vitro experiments in human umbilical vein endothelial cells (HUVECs) showed that while heat-inactivated R. conorii enhanced the release of interleukin-6 (IL-6) and IL-8, it down-regulated the release of endothelial-derived DKK-1 in a time- and dose-dependent manner. (iii) Silencing of DKK-1 attenuated the release of IL-6, IL-8 and growth-related oncogene (GRO)α in R. conorii-exposed HUVECs, suggesting inflammatory effects of DKK-1. (iv) Silencing of DKK-1 attenuated the expression of tissue factor and enhanced the expression of thrombomodulin in R. conorii-exposed HUVECs suggesting pro-thrombotic effects of DKK-1. The capacity of R. conorii to down-regulate endothelial-derived DKK-1 and the ability of silencing DKK-1 to attenuate R. conorii-induced inflammation in endothelial cells could potentially reflect a novel mechanism by which R. conorii escapes the immune response at the site of infection.

Diagnosing emerging and reemerging infectious diseases: the pivotal role of the pathologist.

CONTEXT: Molecular diagnostics continues to evolve very rapidly, and its impact in the diagnosis of infectious diseases is undeniable. Molecular tools have played a pivotal role in discovering and characterizing several emerging infectious agents and have now become the gold standard for the diagnosis of infectious diseases caused by fastidious or uncultivable agents. Multiple challenges still remain for the widespread use of cost-effective, validated, and commercially available molecular tools. Automated instruments capable of sample processing and multiplex nucleic acid amplification and postamplification analysis have already been approved by the US Food and Drug Administration (FDA) for use in the clinical setting. Nanobiotechnology is beginning to impact laboratory diagnostics in the clinical setting. OBJECTIVE: To address current nucleic acid techniques used in the clinical laboratory for diagnosis of infectious diseases. FDA-approved tests are listed, as well as molecular techniques (amplification and postamplification analysis). A comprehensive list of emerging pathogens during the last 4 decades is also presented. Biosurveillance systems are discussed in the context of molecular tools. The rapidly evolving field of nanobiotechnology is briefly addressed. DATA SOURCES: Original publications, major reviews, and book chapters were used to present a comprehensive, yet short, review of molecular diagnostics in infectious diseases. CONCLUSIONS: We will continue to witness an exponential growth of molecular techniques used for the initial diagnosis of infectious diseases. Molecular tools will also continue to have an impact on disease prognosis and response to therapeutic interventions. Automation, multiplexing, and miniaturization will continue to be driving forces in the development of new instruments.

The expanding spectrum of eschar-associated rickettsioses in the United States.

BACKGROUND: Until recently, Rickettsia rickettsii was the only substantiated cause of tick-borne spotted fever group (SFG) rickettsiosis in humans in the United States. Rickettsia parkeri, originally thought to be nonpathogenic in humans, was recently proved to be another cause of tick-borne SFG rickettsiosis. OBSERVATIONS: We report 3 cases of SFG rickettsiosis and discuss the epidemiology, clinical presentation, histopathologic features, and laboratory findings that support confirmed or probable diagnoses of R parkeri infection and describe the expanding list of eschar-associated SFG rickettsioses recognized in US patients. CONCLUSIONS: The SFG rickettsioses share many clinical manifestations and extensive antigenic cross-reactivity that may hamper specific confirmation of the causative agent.

Surface-expressed enolase contributes to the pathogenesis of clinical isolate SSU of Aeromonas hydrophila.

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound to human plasminogen and facilitated the latter's tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease alpha(2)-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT) A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues ((252)FYDAEKKEY(260)) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general.

The absence of Toll-like receptor 4 signaling in C3H/HeJ mice predisposes them to overwhelming rickettsial infection and decreased protective Th1 responses.

The importance of toll-like receptor 4 (TLR4) in immunity to rickettsiae remains elusive. To investigate the role of TLR4 in protection against rickettsioses, we utilized C3H/HeJ mice, which are naturally defective in TLR4 signaling, and compared the responses of C3H/HeN and C3H/HeJ mice following intravenous inoculation with Rickettsia conorii. Mice genetically defective in TLR4 signaling developed overwhelming, fatal rickettsial infections when given an inoculum that was nonfatal for TLR4-competent mice. In addition, mice lacking the ability to signal through TLR4 had significantly greater rickettsial burdens in vivo. Moreover, we observed greater concentrations of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha, IL-12p40, IL-12p70, and IL-17 in the sera of mice with intact TLR4 function as well as significantly greater quantities of activated CD4(+) and CD8(+) T lymphocytes. Additionally, we also observed that Th17 cells were present only in TLR4-competent mice, suggesting an important role for TLR4 ligation in the activation of this subset. In agreement with these data, we also observed significantly greater percentages of immunosuppressive regulatory T cells in the spleen during infection in TLR4-defective mice. Together, these data demonstrate that, while rickettsiae do not contain endotoxic lipopolysaccharide, they nevertheless initiate TLR4-specific immune responses, and these responses are important in protection.