Dual Roles of CD40 on Microbial Containment and the Development of Immunopathology in Response to Persistent Fungal Infection in the Lung

Persistent pulmonary infection with Cryptococcus neoformans in C57BL/6 mice results in chronic inflammation that is characterized by an injurious Th2 immune response. In this study, we performed a comparative analysis of cryptococcal infection in wild-type versus CD40-deficient mice (in a C57BL/6 genetic background) to define two important roles of CD40 in the modulation of fungal clearance as well as Th2-mediated immunopathology. First, CD40 promoted microanatomic containment of the organism within the lung tissue. This protective effect was associated with: i) a late reduction in fungal burden within the lung; ii) a late accumulation of lung leukocytes, including macrophages, CD4⁺ T cells, and CD8+ T cells; iii) both early and late production of tumor necrosis factor-α and interferon-γ by lung leukocytes; and iv) early IFN-γ production at the site of T cell priming in the regional lymph nodes. In the absence of CD40, systemic cryptococcal dissemination was increased, and mice died of central nervous system infection. Second, CD40 promoted pathological changes in the airways, including intraluminal mucus production and subepithelial collagen deposition, but did not alter eosinophil recruitment or the alternative activation of lung macrophages. Collectively, these results demonstrate that CD40 helps limit progressive cryptococcal growth in the lung and protects against lethal central nervous system dissemination. CD40 also promotes some, but not all, elements of Th2-mediated immunopathology in response to persistent fungal infection in the lung.CD40, a 48-kDa type I transmembrane protein and member of the tumor necrosis factor receptor family, is a well-described costimulatory molecule expressed on B cells, dendritic cells (DC), macrophages, basophils, and platelets as well as nonhematopoietic cells including fibroblasts, epithelial, and endothelial cells. The ligand for CD40, known as CD154 or CD40L, is a type II transmembrane protein member of the tumor necrosis factor (TNF) superfamily expressed primarily by activated T cells, B cells, and platelets.^1,2,3 CD40 can be induced on DC, monocytes, and macrophages under inflammatory conditions.^4,5 Signaling via the CD40/CD40L pathway exerts numerous biological effects including: i) increased cytokine expression (especially TNF-α and Th1 cytokines interleukin (IL)-12 and interferon (IFN)-α) and nitric oxide production; ii) upregulation of additional costimulatory molecules (CD80 and CD86) on antigen-presenting cells (APC); iii) enhanced cell survival (particularly of B and T cells, DC, and endothelial cells); iv) Ig isotype switching; and v) somatic hypermutation of Ig.^1,4,5The CD40/CD40L signaling pathway contributes to adaptive Th1 immune responses required to clear Leishmanisa spp.,^6,7,8 Trypanosoma spp.,^6,7,8,9 Shistosoma mansoini,¹⁰ and the fungi Candida albicans¹¹ and Pneumocystis spp.¹² The enhanced production of IFN-γ, TNF-α, and nitric oxide associated with CD40/CD40L signaling is thought to be responsible for this protective effect. However, other studies have suggest that CD40/CD40L signaling is not required for successful host defense against Listeria monocytogenes,^13,14 Toxoplasma gondi,¹⁵ lymphocytic choriomeningitis virus,^16,17 or the fungus Hisoplasma capsulatum.^18,19 In models of Mycobacterium spp. infection, CD40 appears dispensable for clearance of an i.v. infection,^20,21 but essential for clearing the organism in response to aerosolized infection in the lungs.^22,23 Thus, the role of CD40 in antimicrobial host defense varies and depends not only on the specific pathogen but also on the primary site of infection.Cryptococcus neoformans, an opportunistic fungal pathogen acquired through inhalation, causes significant morbidity and mortality primarily in patients with AIDS, lymphoid or hematological malignancies, or patients receiving immunosuppressive therapy secondary to autoimmune disease or organ transplantation.^24,25 Infection in non-immunocompromised patients has been reported.^26,27,28 Murine models of cryptococcal infection in CBA/J or BALB/c mice demonstrate that development of a Th1 antigen-specific immune response characterized by IFN-γ production and classical activation of macrophages is required to eradicate the organism.^{29,30,31,32,33,34,35,36,37,38,39,40} In contrast, a model of persistent cryptococcal infection has been developed using C57BL/6 mice;^{41,42,43,44,45,46,47} this model reflects many features observed in humans diagnosed with allergic bronchopulmonary mycosis.⁴⁸ Specifically, these mice fail to clear the organism from the lung and develop characteristic Th2-mediated immunopathology including: i) tissue eosinophilia; ii) airway hyperreactivity, mucus production, and fibrosis; and iii) alternative macrophage activation associated with YM1 crystal deposition.The molecular mechanisms responsible for the immunopathologic response associated with persistent cryptococcal infection are not clearly defined. These features are abrogated in the absence of IL-4,⁴⁵ whereas more severe Th2-mediated lung injury occurs in the absence of IFN-γ.^29,41 TNF-α exerts a protective effect by enhancing IFN- γ production and the subsequent classical activation of lung macrophages.^31,35,49,50 Lymphocytes are critical mediators of this Th2 response as the pathological features of chronic cryptococcal infection are substantially diminished in CD4 T cell-depleted mice despite no change in fungal clearance.⁴²Although interactions between CD4 T cells and APC are critical determinants of T cell polarization in response to cryptococcal lung infection,^{49,51,52,53,54,55} the contribution of specific costimulatory molecules including the CD40/CD40L signaling pathway has not been fully elucidated. In vitro studies suggest that activation of the CD40/CD40L pathway in response to Cryptococcus promotes IFN-γ production by T cells and TNF-α, and nitric oxide (NO) production by monocytes.⁵⁶ In the absence of CD40L, primary pulmonary infection with a weakly virulent strain of C. neoformans was associated with impaired fungal clearance; however, measurements of immune function at the site of infection in the lung or evidence of systemic fungal dissemination were not evaluated.⁵⁷ The potential to target CD40 therapeutically is highlighted by studies showing that treatment of mice with disseminated or intracerebral cryptococcal infection with an agonist antibody to CD40 in combination with IL-2 improves survival.^58,59 In this study, we used gene-targeted CD40-deficient mice (on a C57BL/6 genetic background), a clinically relevant model, and assessments of immune function and histopathology in the lung to identify two unique roles for the CD40-signaling pathway in response to persistent cryptococcal lung infection.     

Heme oxygenase protects hippocampal neurons from ethanol-induced neurotoxicity

Ethanol has deleterious effects on neuronal cells both in vivo and in vitro, but the mechanisms are unknown. Here, treatment with increasing doses of ethanol (from 20 up to 600mM) decreased the viability of a mouse hippocampal neuroblastoma cell line, HT22. The glutathione concentration decreased and intracellular reactive oxygen species (ROS) increased in a dose-and time-dependent manner, suggesting that the neurotoxicity was due to oxidative stress. Expression of heme oxygenase (HO)-1, a redox regulator and heat shock protein, increased with time after ethanol treatment, but HO-2 was expressed constitutively. The addition of 5microM zinc protoporphyrin IX (ZnPP IX), a competitive HO inhibitor, with the ethanol further reduced cell viability and increased intracellular ROS, but these effects were reversed by co-treatment with 50nM bilirubin, a well-known antioxidant and a product of HO catalysis. These results suggest that HO has a protective role in hippocampal neurons as an intrinsic factor against ethanol-induced oxidative stress and the protection depends on the degree of oxidative stress.     

Effects of exercise training on pathological cardiac hypertrophy related gene expression and apoptosis

This study determined whether exercise training prevents pathological hypertrophy in the left ventricle by modulation of myocardial and apoptosis-associated genes. We used spontaneously hypertensive rats (n=15, non-exercise SHR), exercise-trained SHR (n=15, treadmill exercise for 12 weeks), and sedentary Wistar-Kyoto (WKY) rats (n=15). Exercise-trained SHR expressed adaptive changes such as reduced body weight, heart rate, blood pressures, left ventricle wall thickness, lipid profiles, and homocysteine level. The mRNA expression of angiotensin converting enzyme, endothelin-1, and brain natriuretic peptides in the heart was lower in the exercise-trained SHR and in the WKY than in the non-exercise SHR, whereas mRNA expression of caveolin-3 and eNOS in the heart was higher. Bcl-2 protein was higher in the exercise-trained SHR than in the WKY and the non-exercise SHR. In contrast, Bax protein levels were lower in the exercise-trained SHR and in the WKY than in the non-exercise SHR. Furthermore, the levels of the active forms of caspase-3 (20 kDa) were lower in the exercise-trained SHR and in the WKY than in the non-exercise SHR. These findings suggest that exercise training prevents pathological hypertrophy in the left ventricle by modulation of myocardial genes and that it interferes with a signal transduction pathway of apoptosis secondary to the pathological cardiac hypertrophy.     

Implementation of a novel population panel management curriculum among interprofessional health care trainees

Background

Gaps in chronic disease management have led to calls for novel methods of interprofessional, team-based care. Population panel management (PPM), the process of continuous quality improvement across groups of patients, is rarely included in health professions training for physicians, nurses, or pharmacists. The feasibility and acceptance of such training across different healthcare professions is unknown. We developed and implemented a novel, interprofessional PPM curriculum targeted to diverse health professions trainees.

Methods

The curriculum was implemented annually among internal medicine residents, nurse practitioner students and residents, and pharmacy residents co-located in a large, academic primary care site. Small groups of interprofessional trainees participated in supervised quarterly seminars focusing on chronic disease management (e.g., diabetes mellitus, hypertension, or chronic obstructive pulmonary disease) or processes of care (e.g., emergency department utilization for nonacute conditions or chronic opioid management). Following brief didactic presentations, trainees self-assessed their clinic performance using patient-level chart review, presented individual cases to interprofessional staff and faculty, and implemented subsequent feedback with their clinic team. We report data from 2011 to 2015. Program evaluation included post-session participant surveys regarding attitudes, knowledge and confidence towards PPM, ability to identify patients for referral to interprofessional team members, and major learning points from the session. Directed content analysis was performed on an open-ended survey question.

Results

Trainees (n?=?168) completed 122 evaluation assessments. Trainees overwhelmingly reported increased confidence in using PPM and increased knowledge about managing their patient panel. Trainees reported improved ability to identify patients who would benefit from multidisciplinary care or referral to another team member. Directed content analysis revealed that trainees viewed team members as important system resources (n?=?82).

Conclusions

Structured interprofessional training in PPM is both feasible and acceptable to trainees across multiple professions. Curriculum participants reported improved panel management skills, increased confidence in using PPM, and increased confidence in identifying candidates for interprofessional care. The curriculum could be readily exported to other programs and contexts.

     

Correlation between the presence of anti HPV33 VLP antibodies and HPV DNA in cervical neoplasia patients

Summary. The L1 capsid proteins derived from human papillomavirus (HPV) type 33 were expressed in a recombinant baculovirus system using Sf9 insect cells. Selected sera originating in women from case-control study carried out in Spain and Colombia found negative and positive for HPV16, 18, 31, 33 and 35 DNA were tested in ELISA for the presence of IgG antibodies to purified virus-like particles (VLP). The reactivity was type-restricted with the possible exception of HPV31. Received July 4, 1996 Accepted September 17, 1996     

The protective effect of allopurinol on cholestatic liver injury induced by bile duct ligation.

To determine whether oxygen free radicals are responsible for the pathogenesis of the cholestasis induced by ligation of common bile duct (CBD) variables which reflect the hepatic function in the serum, the amount of superoxide radical production, and xanthine oxidase(XO) activity were studied. The activity of serum alanine aminotransferase, bilirubin level in the serum and the amount of superoxide radical production were lower in a CBD ligation with allopurinol treated group than in a CBD ligation without allopurinol treated group. Abnormalities of the microscopic structures were reduced in a CBD ligation with allopurinol treated group than in a CBD ligation without allopurinol treated group. Allopurinol, an inhibitor of XO, prevented the hepatic damage induced by CBD ligation through the inhibition of XO. These experiments demonstrate that oxygen free radicals are responsible for the pathogenesis of the cholestatic liver.     

SSVEP and ERP measurement of cognitive fatigue caused by stereoscopic 3D

Characteristically within the resting brain there are slow fluctuations (around 0.1 Hz) of EEG and NIRS-(de)oxyhemoglobin ([deoxy-Hb], [oxy-Hb]) signals. An interesting question is whether such slow oscillations can be related to the intention to perform a motor act. To obtain an answer we analyzed continuous blood pressure (BP), heart rate (HR), prefrontal [oxy-Hb], [deoxy-Hb] and EEG signals over sensorimotor areas in 10 healthy subjects during 5 min of rest and during 10 min of voluntary finger movements. Analyses of prefrontal [oxy-Hb]/[deoxy-Hb] oscillations around 0.1 Hz and central EEG band power changes in the beta (alpha) band revealed that the positive [oxy-Hb] peaks preceded the central EEG beta (alpha) power peak by 3.6 ± 0.9 s in the majority of subjects. A similar relationship between prefrontal [oxy-Hb] and central EEG beta power was found during voluntary movements whereby the post movement beta power increase (beta rebound) is known to coexist with a decreased excitability of cortico-spinal neurons. Therefore, we speculate that the beta power increase ∼3 s after slow fluctuating [oxy-Hb] peaks during rest is indicative for a slow excitability change of central motor cortex neurons. This work provides the first evidence that initiation of finger movements at free will in relatively constant intervals around 10 s could be temporally related to slow oscillations of prefrontal [oxy-Hb] and autonomic blood pressure in the resting brain.     

Folate deficiency is associated with the formation of complex nuclear anomalies in the cytokinesis‐block micronucleus cytome assay

Chromosomal instability (CIN) is an important hallmark to oncogenesis and can be diagnosed morphologically by the presence of nuclear anomalies such as micronuclei (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBuds). We have identified additional nuclear anomalies formed under folate‐deficient conditions, defined as “fused” nuclei (FUS), “circular” nuclei (CIR), and “horse‐shoe” nuclei (HS) and investigated their suitability for inclusion as additional CIN biomarkers in the lymphocyte cytokinesis‐block micronucleus cytome (CBMN‐Cyt) assay. Although the morphological appearance of FUS, CIR, and HS suggested an origin from multiple NPB in the fusion region between the two nuclei, the very low frequency of dicentric chromosomes in metaphase spreads from these cultures did not support this model. Fluorescence in situ hybridization (FISH) analysis of cytokinesis‐blocked binucleated (BN) cells with peptide nucleic acid probes for telomeres and centromeres (PNA–FISH) revealed a high proportion of fusion regions contained both centromeric and telomeric DNA. This suggests that folate deficiency may disrupt the process of sister chromatid separation and chromosome segregation during mitosis. It was concluded that the FUS, CIR, and HS morphologies represent promising biomarkers of CIN that are sensitive to folate deficiency, and further validation and investigation of the mechanisms responsible for their formation is warranted. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Type and cause of liver disease in Korea: single-center experience, 2005-2010

Background/Aims

The aim of this study was to describe the types and causes of liver disease in patients from a single community hospital in Korea between April 2005 and May 2010.

Methods

A cohort of patients who visited the liver clinic of the hospital during the aforementioned time period were consecutively enrolled (n=6,307). Consistent diagnostic criteria for each liver disease were set by a single, experienced hepatologist, and the diagnosis of all of the enrolled patients was confirmed by retrospective review of their medical records.

Results

Among the 6,307 patients, 528 (8.4%) were classified as acute hepatitis, 3,957 (62.7%) as chronic hepatitis, 767 (12.2%) as liver cirrhosis, 509 (8.1%) as primary liver cancer, and 546 (8.7%) as a benign liver mass or other diseases. The etiologies in the acute hepatitis group in decreasing order of prevalence were hepatitis A (44.3%), toxic hepatitis (32.4%), other hepatitis viruses (13.8%), and cryptogenic hepatitis (9.1%). In the chronic hepatitis group, 51.2% of cases were attributed to viral hepatitis, 33.3% to nonalcoholic fatty liver disease, and 13.0% to alcoholic liver disease (ALD). Of the cirrhoses, 73.4% were attributable to viral causes and 18.1% to alcohol. Of the hepatocellular carcinoma cases, 86.6% were attributed to viral hepatitis and 11.6% to ALD. Among the benign tumors, hemangioma comprised 52.2% and cystic liver disease comprised 33.7%.

Conclusions

Knowledge of the current status of the type and cause of liver disease in Korea may be valuable as a basis for evaluating changing trends in liver disease in that country.