An Analysis of Skin Test Responses to Spherulin-Based Coccidioidin (Spherusol®) Among a Group of Subjects with Various Forms of Active Coccidioidomycosis.

BACKGROUND: A reformulated skin test for coccidioidomycosis, Spherusol®, was recently approved for use in the USA. We hypothesized that it could be useful in predicting severity of illness and outcome in various types of coccidioidomycosis. METHODS: Subjects with non-meningeal coccidioidomycosis attending a clinic in the coccidioidal endemic region were skin tested with Spherusol® and clinical data were collected at the time of testing and at follow-up. RESULTS: Twenty-seven subjects were studied, eight of whom had extrathoracic dissemination. A total of 15 subjects had positive tests, including 11 of 19 subjects with non-disseminated pulmonary disease and four with extrathoracic disseminated coccidioidomycosis. Among those with non-disseminated pulmonary disease, age ≥ 65 years, female sex, and antifungal therapy were significantly associated with a negative test on univariate but not multivariate analysis. For 23 subjects, there was a trend for those not on antifungal therapy at the time of follow-up to have a positive test but no association with coccidioidal complement-fixation titer or overall outcome. CONCLUSIONS: Not all subjects with non-disseminated pulmonary coccidioidomycosis were found to be skin test positive and half of those with extrathoracic disseminated disease manifested dermal hypersensitivity. In this small study, the results of the skin test were not clinically predictive of disease severity or outcome.

Longitudinal Changes in Retinotopic Rod Function in Intermediate Age-Related Macular Degeneration.

Purpose: Although impairment of rod function in the early stages of age-related macular degeneration (AMD) has been well recognized, data on longitudinal changes in rod function at multiple retinal locations remain limited. This study investigated the longitudinal changes in retinotopic rod function in eyes with intermediate AMD (iAMD). Methods: Complete ophthalmic examination, multimodal imaging, and scotopic perimetry were performed at baseline and at 12-month follow-up. Perimetric scotopic retinal sensitivities for the 505-nm stimulus were repeatedly measured for 20 minutes after exposing to a single photobleach (∼30%). The rod intercept time (RIT) and retinal sensitivity at seven retinal loci within the central 12° were ascertained. Using the 95% limit of measurement variability derived from the control eyes as a reference, the proportion of test points with a significant change in retinal sensitivity or RIT at follow-up was determined. Results: Twenty iAMD and 6 control eyes were included. Decline in rod function was detected at 12-month follow-up in eyes with iAMD, but not in control eyes. Approximately 25% of test points in iAMD eyes showed a significant increase in RIT compared to 6% of test points with a decrease in RIT over the 12-month period (P < 0.001). Similarly, 40% of test points demonstrated a reduction in retinal sensitivity compared to the 7% of test points with an increase in retinal sensitivity at follow-up (P < 0.001). Conclusions: There are detectable retinotopic changes in rod function over 12 months in iAMD eyes, indicating an ongoing disease progression in rod impairment or loss with time.

Ex Vivo Cytokine Release, Determined by a Multiplex Cytokine Assay, in Response to Coccidioidal Antigen Stimulation of Whole Blood among Subjects with Recently Diagnosed Primary Pulmonary Coccidioidomycosis.

The elements of the cellular immune response in human coccidioidomycosis remain undefined. We examined the ex vivo release of an array of inflammatory proteins in response to incubation with a coccidioidal antigen preparation to ascertain which of these might be associated with diagnosis and outcome. Patients with a recent diagnosis of primary pulmonary coccidioidomycosis and a control group of healthy subjects were studied. Blood samples were incubated for 18 h with T27K, a soluble coccidioidal preparation containing multiple glycosylated antigens, and the supernatant was assayed for inflammatory proteins using the multiplex Luminex system. The presentation and course of illness were compared to the levels of the inflammatory proteins. Among the 31 subjects studied, the median time from diagnosis to assay was 15 days. Of the 30 inflammatory proteins measured, the levels of only 7 proteins, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 receptor alpha (IL-1RA), interleukin-1ß (IL-1ß), interferon gamma (IFN-γ), IL-2, IL-13, and tumor necrosis factor alpha (TNF-α), were more than 10-fold above the levels seen without antigen stimulation. The levels of IFN-γ and IL-2 were significantly elevated in those subjects not receiving triazole antifungal therapy compared to those who were receiving triazole antifungal therapy. While the levels of IL-1RA were nonspecifically elevated, elevated levels of IL-13 were seen only in those with active pulmonary coccidioidomycosis. Only six cytokines were specifically increased in subjects with recently diagnosed primary pulmonary coccidioidomycosis. While IFN-γ, IL-2, and TNF-α have been previously noted, the finding of elevated levels of the innate cytokines GM-CSF and IL-1ß could suggest that these, as well as IL-13, are early and specific markers for pulmonary coccidioidomycosis.IMPORTANCE Coccidioidomycosis, commonly known as Valley fever, is a common pneumonia in the southwestern United States. In this paper, we examined the release of 30 inflammatory proteins in whole-blood samples obtained from persons with coccidioidal pneumonia after the blood samples were incubated with a preparation made from the causative fungus, Coccidioides We found that six of these proteins, all cytokines, were specifically released in high concentrations in these patients. Three of the cytokines were seen very early in disease, and an assay for all six might serve as a marker for the early diagnosis of Valley fever.

Levator resection for congenital ptosis: Does pre-operative levator function or degree of ptosis affect successful outcome?

The aim of this study was to determine whether pre-operative levator function and degree of ptosis affect surgical outcomes in children with congenital ptosis undergoing anterior levator resection under general anaesthesia. Retrospective cohort study. Children with 'simple' congenital ptosis who underwent anterior levator resection under general anaesthesia. Consecutive cases were reviewed with regards to achievement of the desired lid height (surgical success), and the influence of preoperative levator function and degree of ptosis. The amount of pre-operative levator function and degree of ptosis, with corresponding surgical outcomes. Forty-two lids (37 patients) were included in the study: 36 primary and 6 revision cases (which were excluded from analysis due to small sample size). Mean age was 7 years (range 3-17 years), with similar male to female ratio (1.2:1). The overall success rate for primary was 86%. There were small to moderate trends towards greater amount of levator resection for lower levator function (r2 = -0.25, p < 0.05) and higher degree of ptosis (r2 = 0.38, p < 0.05). All successful primary cases (n = 31) had pre-operative levator function of at least 8 mm. Levator resection in children under general anaesthesia continues to be an imprecise science. Degree of ptosis and levator function were poorly correlated to each other; however, there was an expected small to moderate correlation between resection amount and levator function (negative correlation) or degree of ptosis (positive correlation). Patients with levator function of 8mm or more are likely to have a successful outcome.

Spontaneous Giant Pseudomeningocele in the Middle Cranial Fossa as a Cause of Pulsatile Proptosis.

The authors report a case of a 70-year-old man who presented with a long history of left pulsatile proptosis, irritation, and a pressure sensation behind the eye. Initial computed tomography brain demonstrated the presence of a suspected ethmoid sinus mucocele. Endoscopic sinus surgery was performed to drain the mucocele. On cautious opening of the nasal mucosa, cerebrospinal fluid was encountered requiring subsequent repair with local mucosal flap and fat graft. On further magnetic resonance imaging, the lesion was confirmed to be giant pseudomeningocele arising from the sphenoid sinus, with extensive invasion and erosion of the middle cranial fossa including the left lateral orbital wall. Here, the authors report, to the best of their knowledge, the first case of spontaneous giant pseudomeningocele in the middle cranial fossa causing pulsatile proptosis. In examining this case, the authors will also discuss the pathogenesis, diagnosis, and management of giant pseudomeningoceles, especially in the situation of a misdiagnosis.

Progress toward the Development of a NEAT Protein Vaccine for Anthrax Disease.

Bacillus anthracis is a sporulating Gram-positive bacterium that is the causative agent of anthrax and a potential weapon of bioterrorism. The U.S.-licensed anthrax vaccine is made from an incompletely characterized culture supernatant of a nonencapsulated, toxigenic strain (anthrax vaccine absorbed [AVA]) whose primary protective component is thought to be protective antigen (PA). AVA is effective in protecting animals and elicits toxin-neutralizing antibodies in humans, but enthusiasm is dampened by its undefined composition, multishot regimen, recommended boosters, and potential for adverse reactions. Improving next-generation anthrax vaccines is important to safeguard citizens and the military. Here, we report that vaccination with recombinant forms of a conserved domain (near-iron transporter [NEAT]), common in Gram-positive pathogens, elicits protection in a murine model of B. anthracis infection. Protection was observed with both Freund's and alum adjuvants, given subcutaneously and intramuscularly, respectively, with a mixed composite of NEATs. Protection correlated with an antibody response against the NEAT domains and a decrease in the numbers of bacteria in major organs. Anti-NEAT antibodies promote opsonophagocytosis of bacilli by alveolar macrophages. To guide the development of inactive and safe NEAT antigens, we also report the crystal structure of one of the NEAT domains (Hal) and identify critical residues mediating its heme-binding and acquisition activity. These results indicate that we should consider NEAT proteins in the development of an improved antianthrax vaccine.

Global metabolomic analysis of a mammalian host infected with Bacillus anthracis.

Whereas DNA provides the information to design life and proteins provide the materials to construct it, the metabolome can be viewed as the physiology that powers it. As such, metabolomics, the field charged with the study of the dynamic small-molecule fluctuations that occur in response to changing biology, is now being used to study the basis of disease. Here, we describe a comprehensive metabolomic analysis of a systemic bacterial infection using Bacillus anthracis, the etiological agent of anthrax disease, as the model pathogen. An organ and blood analysis identified approximately 400 metabolites, including several key classes of lipids involved in inflammation, as being suppressed by B. anthracis. Metabolite changes were detected as early as 1 day postinfection, well before the onset of disease or the spread of bacteria to organs, which testifies to the sensitivity of this methodology. Functional studies using pharmacologic inhibition of host phospholipases support the idea of a role of these key enzymes and lipid mediators in host survival during anthrax disease. Finally, the results are integrated to provide a comprehensive picture of how B. anthracis alters host physiology. Collectively, the results of this study provide a blueprint for using metabolomics as a platform to identify and study novel host-pathogen interactions that shape the outcome of an infection.

Cytokine Profiles from Antigen-Stimulated Whole-Blood Samples among Patients with Pulmonary or Nonmeningeal Disseminated Coccidioidomycosis.

The outcome of coccidioidomycosis depends on a robust specific cellular immune response. A T-helper type 1 (Th1) cellular immune response has been previously associated with resolution of clinical illness. However, the precise elements of this response and whether cytokines not involved with the Th1 response play a role in coccidioidomycosis are not known. Whole-blood samples were obtained from subjects with active coccidioidomycosis and controls and incubated for 18 h with T27K, a coccidioidal antigen preparation. The supernatant was then assayed for gamma interferon (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), IL-4, IL-6, IL-10, and IL-17A. A total of 43 subjects, 16 with acute pneumonia, 9 with pulmonary sequelae of nodules and cavities, and 18 with nonmeningeal disseminated coccidioidomycosis, were studied. Compared to concentrations in healthy immune and nonimmune donors, the median concentration of IL-17A was significantly higher in those with active coccidioidomycosis (for both, P < 0.01). In addition, IL-6 concentrations were higher while IL-2 and IFN-γ concentrations were significantly lower in those with nonmeningeal disseminated disease diagnosed within 12 months than in those with acute pneumonia (for all, P < 0.05). The cytokine profile among patients with active coccidioidomycosis is distinct in that IL-17A is persistently present. In addition, those with nonmeningeal disseminated disease have an increased inflammatory cytokine response and diminished Th1 responses that modulate over time.

Reduced ethanol consumption and preference in cocaine- and amphetamine-regulated transcript (CART) knockout mice.

Cocaine- and amphetamine-regulated transcript (CART) is a neuropeptide implicated in addiction to drugs of abuse. Several studies have characterized the role of CART in addiction to psychostimulants, but few have examined the role of CART in alcohol use disorders including alcoholism. The current study utilized a CART knockout (KO) mouse model to investigate the role of CART in ethanol appetitive behaviors. A two-bottle choice, unlimited-access paradigm was used to compare ethanol appetitive behaviors between CART wild type (WT) and KO mice. The mice were presented with an ethanol solution (3%-21%) and water, each concentration for 4 days, and their consumption was measured daily. Consumption of quinine (bitter) and saccharin (sweet) solutions was measured following the ethanol preference tests. In addition, ethanol metabolism rates and ethanol sensitivity were compared between genotypes. CART KO mice consumed and preferred ethanol less than their WT counterparts in both sexes. This genotype effect could not be attributed to differences in bitter or sweet taste perception or ethanol metabolism rates. There was also no difference in ethanol sensitivity in male mice; however, CART KO female mice showed a greater ethanol sensitivity than the WT females. Taken together, these data demonstrate a role for CART in ethanol appetitive behaviors and as a possible therapeutic drug target for alcoholism and abstinence enhancement.

Immunological characterization of bronchoalveolar lavage fluid in patients with acute pulmonary coccidioidomycosis.

BACKGROUND: The specific cellular immunological characteristics of bronchoalveolar lavage (BAL) fluid in acute pulmonary coccidioidomycosis have not been defined. METHODS: BAL fluid from patients living in a coccidioidomycosis-endemic region of Arizona who were undergoing bronchoscopy because of pulmonary infiltrates was analyzed. Mononuclear cells from BAL fluid and peripheral blood mononuclear cells (PBMCs) were incubated with the coccidioidal antigen T27K in vitro, and cellular immunological assays were performed. RESULTS: Forty-six patients were studied. Twelve received a diagnosis of acute pulmonary coccidioidomycosis, 17 received other diagnoses, and 17 had no diagnosis established. There was an increased proportion of polyfunctional CD8(+) T cells after antigen stimulation from subjects with coccidioidomycosis as compared to those with another diagnosis (P = .025). In cells collected from BAL fluid and in PBMCs, the concentrations of interferon γ, tumor necrosis factor α, and interleukin 17 (IL-17) were all significantly increased in samples from those with acute pulmonary coccidioidomycosis, compared with the other 2 groups (for all, P<.05). CONCLUSIONS: When incubated in vitro with a coccidioidal antigen preparation, cells from both BAL fluid and peripheral blood obtained from patients with pulmonary coccidioidomycosis demonstrated specific cellular immune responses, including expression of IL-17.

Immunotherapeutic strategies to prevent and treat human herpesvirus 6 reactivation after allogeneic stem cell transplantation.

Human herpesvirus (HHV) 6 causes substantial morbidity and mortality in the immunocompromised host and has no approved therapy. Adoptive transfer of virus specific T cells has proven safe and apparently effective as prophylaxis and treatment of other virus infections in immunocompromised patients; however, extension to subjects with HHV6 has been hindered by the paucity of information on targets of cellular immunity. We now characterize the cellular immune response from 20 donors against 5 major HHV6B antigens predicted to be immunogenic and define a hierarchy of immunodominance of antigens based on the frequency of responding donors and the magnitude of the T-cell response. We identified specific epitopes within these antigens and expanded the HHV6 reactive T cells using a GMP-compliant protocol. The expanded population comprised both CD4(+) and CD8(+) T cells that were able to produce multiple effector cytokines and kill both peptide-loaded and HHV6B wild-type virus-infected target cells. Thus, we conclude that adoptive T-cell immunotherapy for HHV6 is a practical objective and that the peptide and epitope tools we describe will allow such cells to be prepared, administered, and monitored in human subjects.