Trajectories in Intensity of Medical Interventions at the End of Life: Clustering Analysis in a Pediatric, Single-Center Retrospective Cohort, 2013-2021.

OBJECTIVE: Pediatric deaths often occur within hospitals and involve balancing aggressive treatment with minimization of suffering. This study first investigated associations between clinical/demographic features and the level of intensity of various therapies these patients undergo at the end of life (EOL). Second, the work used these data to develop a new, broader spectrum for classifying pediatric EOL trajectories. DESIGN: Retrospective, single-center study, 2013-2021. SETTING: Four hundred sixty-one bed tertiary, stand-alone children's hospital with 112 ICU beds. PATIENTS: Patients of age 0-26 years old at the time of death. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of 1111 included patients, 85.7% died in-hospital. Patients who died outside the hospital were older. Among the 952 in-hospital deaths, most occurred in ICUs (89.5%). Clustering analysis was used to distinguish EOL trajectories based on the presence of intensive therapies and/or an active resuscitation attempt at the EOL. We identified five simplified categories: 1) death during active resuscitation, 2) controlled withdrawal of life-sustaining technology, 3) natural progression to death despite maximal therapy, 4) discontinuation of nonsustaining therapies, and 5) withholding/noninitiation of future therapies. Patients with recent surgical procedures, a history of organ transplantation, or admission to the Cardiovascular ICU had more intense therapies at EOL than those who received palliative care consultations, had known genetic conditions, or were of older age. CONCLUSIONS: In this retrospective study of pediatric EOL trajectories based on the intensity of technology and/or resuscitation discontinued at the EOL, we have identified associations between these trajectories and patient characteristics. Further research is needed to investigate the impact of these trajectories on families, patients, and healthcare providers.

Opportunistic Infections, Mortality Risk, and Prevention Strategies in Patients With Vacuoles, E1 Enzyme, X-Linked, Autoinflammatory, Somatic (VEXAS) Syndrome.

Background: VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a genetic disorder characterized by bone marrow failure and systemic inflammation, putting patients at risk for infections. This study comprehensively examines the prevalence of opportunistic infections in patients with VEXAS, evaluating their impact on clinical outcomes and potential preventive measures. Methods: Patients with confirmed VEXAS were included. Survival analysis and logistic regression were used to identify associations between opportunistic infections and mortality. Infection rates (IRs) for Pneumocystis jirovecii pneumonia (PJP) and alphaherpesviruses were calculated over a prospective 8-month observation period in relationship to prophylaxis. Results: Of 94 patients with VEXAS, 6% developed PJP; 15% had alphaherpesvirus reactivation, with varicella zoster virus (VZV) being the most common herpesvirus; and 10% contracted a nontuberculous mycobacterial (NTM) infection. Risk of death was significantly increased per month following a diagnosis of PJP (hazard ratio [HR], 72.41 [95% confidence interval {CI}, 13.67-533.70]) or NTM (HR, 29.09 [95% CI, 9.51-88.79]). Increased odds for death were also observed in patients with a history of herpes simplex virus (HSV) reactivation (odds ratio [OR], 12.10 [95% CI, 1.29-114.80]) but not in patients with VZV (OR, 0.89 [95% CI, .30-2.59]). Prophylaxis for PJP (IR, 0.001 vs 0 per person-day, P < .01) and VZV (IR, 0.006 vs 0 per person-day, P = .04) markedly decreased infection rates with a number needed to treat of 4 and 7, respectively. Conclusions: Opportunistic infections are common in patients with VEXAS. Patients who develop PJP, HSV, or NTM are at increased risk for death. Prophylaxis against PJP and VZV is highly effective.

Altered transcriptomic immune responses of maintenance hemodialysis patients to the COVID-19 mRNA vaccine.

Background: End-stage renal disease (ESRD) patients experience immune compromise characterized by complex alterations of both innate and adaptive immunity, and results in higher susceptibility to infection and lower response to vaccination. This immune compromise, coupled with greater risk of exposure to infectious disease at hemodialysis (HD) centers, underscores the need for examination of the immune response to the COVID-19 mRNA-based vaccines. Methods: The immune response to the COVID-19 BNT162b2 mRNA vaccine was assessed in 20 HD patients and cohort-matched controls. RNA sequencing of peripheral blood mononuclear cells was performed longitudinally before and after each vaccination dose for a total of six time points per subject. Anti-spike antibody levels were quantified prior to the first vaccination dose (V1D0) and 7 d after the second dose (V2D7) using anti-spike IgG titers and antibody neutralization assays. Anti-spike IgG titers were additionally quantified 6 mo after initial vaccination. Clinical history and lab values in HD patients were obtained to identify predictors of vaccination response. Results: Transcriptomic analyses demonstrated differing time courses of immune responses, with prolonged myeloid cell activity in HD at 1 wk after the first vaccination dose. HD also demonstrated decreased metabolic activity and decreased antigen presentation compared to controls after the second vaccination dose. Anti-spike IgG titers and neutralizing function were substantially elevated in both controls and HD at V2D7, with a small but significant reduction in titers in HD groups (p<0.05). Anti-spike IgG remained elevated above baseline at 6 mo in both subject groups. Anti-spike IgG titers at V2D7 were highly predictive of 6-month titer levels. Transcriptomic biomarkers after the second vaccination dose and clinical biomarkers including ferritin levels were found to be predictive of antibody development. Conclusions: Overall, we demonstrate differing time courses of immune responses to the BTN162b2 mRNA COVID-19 vaccination in maintenance HD subjects comparable to healthy controls and identify transcriptomic and clinical predictors of anti-spike IgG titers in HD. Analyzing vaccination as an in vivo perturbation, our results warrant further characterization of the immune dysregulation of ESRD. Funding: F30HD102093, F30HL151182, T32HL144909, R01HL138628. This research has been funded by the University of Illinois at Chicago Center for Clinical and Translational Science (CCTS) award UL1TR002003.

Immune response to the mRNA COVID-19 vaccine in hemodialysis patients: cohort study.

Background: End-stage renal disease (ESRD) patients experience immune compromise characterized by complex alterations of both innate and adaptive immunity, and results in higher susceptibility to infection and lower response to vaccination. This immune compromise, coupled with greater risk of exposure to infectious disease at hemodialysis (HD) centers, underscores the need for examination of the immune response to the COVID-19 mRNA-based vaccines. Methods: A transcriptomic analysis of the immune response to the Covid-19 BNT162b2 mRNA vaccine was assessed in 20 HD patients and cohort-matched controls. RNA sequencing of peripheral blood mononuclear cells (PBMCs) was performed longitudinally before and after each vaccination dose for a total of six time points per subject. Anti-spike antibody levels were quantified prior to the first vaccination dose (V1D0) and seven days after the second dose (V2D7) using anti-Spike IgG titers and antibody neutralization assays. Anti-spike IgG titers were additionally quantified six months after initial vaccination. Clinical history and lab values in HD patients were obtained to identify predictors of vaccination response. Results: Transcriptomic analyses demonstrated differing time courses of immune responses, with predominant T cell activity in controls one week after the first vaccination dose, compared to predominant myeloid cell activity in HD at this time point. HD demonstrated decreased metabolic activity and decreased antigen presentation compared to controls after the second vaccination dose. Anti-spike IgG titers and neutralizing function were substantially elevated in both controls and HD at V2D7, with a small but significant reduction in titers in HD groups (p < 0.05). Anti-spike IgG remained elevated above baseline at six months in both subject groups. Anti-spike IgG titers at V2D7 were highly predictive of 6-month titer levels. Transcriptomic biomarkers after the second vaccination dose and clinical biomarkers including ferritin levels were found to be predictive of antibody development. Conclusion: Overall, we demonstrate differing time courses of immune responses to the BTN162b2 mRNA COVID-19 vaccination in maintenance hemodialysis subjects (HD) comparable to healthy controls (HC) and identify transcriptomic and clinical predictors of anti-Spike IgG titers in HD. Analyzing vaccination as an in vivo perturbation, our results warrant further characterization of the immune dysregulation of end stage renal disease (ESRD). Funding: F30HD102093, F30HL151182, T32HL144909, R01HL138628This research has been funded by the University of Illinois at Chicago Center for Clinical and Translational Science (CCTS) award UL1TR002003.

Altered transcription factor targeting is associated with differential peripheral blood mononuclear cell proportions in sarcoidosis.

Introduction: In sarcoidosis, peripheral lymphopenia and anergy have been associated with increased inflammation and maladaptive immune activity, likely promoting development of chronic and progressive disease. However, the molecular mechanisms that lead to reduced lymphocyte proportions, particularly CD4+ T-cells, have not been fully elucidated. We posit that paradoxical peripheral lymphopenia is characterized by a dysregulated transcriptomic network associated with cell function and fate that results from altered transcription factor targeting activity. Methods: Messenger RNA-sequencing (mRNA-seq) was performed on peripheral blood mononuclear cells (PBMCs) from ACCESS study subjects with sarcoidosis and matched controls and findings validated on a sarcoidosis case-control cohort and a sarcoidosis case series. Preserved PBMC transcriptomic networks between case-control cohorts were assessed to establish cellular associations with gene modules and define regulatory targeting involved in sarcoidosis immune dysregulation utilizing weighted gene co-expression network analysis and differential transcription factor involvement analysis. Network centrality measures identified master transcriptional regulators of subnetworks related to cell proliferation and death. Predictive models of differential PBMC proportions constructed from ACCESS target gene expression corroborated the relationship between aberrant transcription factor regulatory activity and imputed and clinical PBMC populations in the validation cohorts. Results: We identified two unique and preserved gene modules significantly associated with sarcoidosis immune dysregulation. Strikingly, increased expression of a monocyte-driven, and not a lymphocyte-driven, gene module related to innate immunity and cell death was the best predictor of peripheral CD4+ T-cell proportions. Within the gene network of this monocyte-driven module, TLE3 and CBX8 were determined to be master regulators of the cell death subnetwork. A core gene signature of differentially over-expressed target genes of TLE3 and CBX8 involved in cellular communication and immune response regulation accurately predicted imputed and clinical monocyte expansion and CD4+ T-cell depletion. Conclusions: Altered transcriptional regulation associated with aberrant gene expression of a monocyte-driven transcriptional network likely influences lymphocyte function and survival. Although further investigation is warranted, this indicates that crosstalk between hyperactive monocytes and lymphocytes may instigate peripheral lymphopenia and underlie sarcoidosis immune dysregulation and pathogenesis. Future therapies selectively targeting master regulators, or their targets, may mitigate dysregulated immune processes in sarcoidosis and disease progression.

Perinatal granulopoiesis and risk of pediatric asthma.

There are perinatal characteristics, such as gestational age, reproducibly associated with the risk for pediatric asthma. Identification of biologic processes influenced by these characteristics could facilitate risk stratification or new therapeutic targets. We hypothesized that transcriptional changes associated with multiple epidemiologic risk factors would be mediators of pediatric asthma risk. Using publicly available transcriptomic data from cord blood mononuclear cells, transcription of genes involved in myeloid differentiation was observed to be inversely associated with a pediatric asthma risk stratification based on multiple perinatal risk factors. This gene signature was validated in an independent prospective cohort and was specifically associated with genes localizing to neutrophil-specific granules. Further validation demonstrated that umbilical cord blood serum concentration of PGLYRP-1, a specific granule protein, was inversely associated with mid-childhood current asthma and early-teen FEV1/FVCx100. Thus, neutrophil-specific granule abundance at birth predicts risk for pediatric asthma and pulmonary function in adolescence.

Disruption of innate defense responses by endoglycosidase HPSE promotes cell survival.

The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we followed heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late-stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.

Immune network dysregulation precedes clinical diagnosis of asthma.

Allergic asthma is a chronic disease beginning in childhood that is characterized by dominant T-helper 2 cell activation without adequate counter-regulation by T-helper 1 cell and regulatory T cell activity. Prior transcriptomic studies of childhood asthma have primarily investigated subjects who already have a disease diagnosis, and have generally taken an approach of differential gene expression as opposed to differential gene interactions. The immune states that predispose towards allergic sensitization and disease development remain ill defined. We thus characterize immune networks of asthmatic predisposition in children at the age of 2, prior to the diagnosis of allergic asthma, who are subsequently diagnosed with asthma at the age of 7. We show extensive differences of gene expression networks and gene regulatory networks in children who develop asthma versus those who do not using transcriptomic data from stimulated peripheral blood mononuclear cells. Moreover, transcription factors that bind proximally to one another share patterns of dysregulation, suggesting that network differences prior to asthma diagnosis result from altered accessibility of gene targets. In summary, we demonstrate non-allergen-specific immune network dysregulation in individuals long before clinical asthma diagnosis.

Pediatric lung transplantation: Dynamics of the microbiome and bronchiolitis obliterans in cystic fibrosis.

BACKGROUND: Compositional changes in the microbiome are associated with the development of bronchiolitis obliterans (BO) after lung transplantation (LTx) in adults with cystic fibrosis (CF). The association between the lower airway bacterial community and BO after LTx in children with CF remains largely unexplored and is possibly influenced by frequent antibiotic therapy. The objectives of this study were to examine the relationship between bacterial community dynamics and the development of BO and analyze antibiotic resistance trends in children after LTx for CF. METHODS: For 3 years from the time of transplant, 12 LTx recipients were followed longitudinally, with 5 subjects developing BO during the study period. A total of 82 longitudinal bronchoalveolar lavage samples were collected during standard of care bronchoscopies. Metagenomic shotgun sequencing was performed on the extracted microbial DNA from bronchoalveolar lavage specimens. Taxonomic profiling was constructed using WEVOTE pipeline. The longitudinal association between development of BO and temporal changes in bacterial diversity and abundance were evaluated with MetaLonDA. The analysis of antibiotic resistance genes was performed with the ARGs-OAP v2.0 pipeline. RESULTS: All recipients demonstrated a Proteobacteria-predominant lower airways community. Temporal reduction in bacterial diversity was significantly associated with the development of BO and associated with neutrophilia and antibiotic therapy. Conversely, an increasing abundance of the phylum Actinobacteria and the orders Neisseriales and Pseudonocardiales in the lower airways was significantly associated with resilience to BO. A more diverse bacterial community was related to a higher expression of multidrug resistance genes and increased proteobacterial abundance. CONCLUSIONS: Decreased diversity within bacterial communities may suggest a contribution to pediatric lung allograft rejection in CF.

Serotonin Transporter Deficiency is Associated with Dysbiosis and Changes in Metabolic Function of the Mouse Intestinal Microbiome.

Serotonin transporter (SERT) plays a critical role in regulating extracellular availability of serotonin (5-HT) in the gut and brain. Mice with deletion of SERT develop metabolic syndrome as they age. Changes in the gut microbiota are being increasingly implicated in Metabolic Syndrome and Diabetes. To investigate the relationship between the gut microbiome and SERT, this study assessed the fecal and cecal microbiome profile of 11 to 12 week-old SERT+/+ and SERT-/- mice. Microbial DNA was isolated, processed for metagenomics shotgun sequencing, and taxonomic and functional profiles were assessed. 34 differentially abundant bacterial species were identified between SERT+/+ and SERT-/-. SERT-/- mice displayed higher abundances of Bacilli species including genera Lactobacillus, Streptococcus, Enterococcus, and Listeria. Furthermore, SERT-/- mice exhibited significantly lower abundances of Bifidobacterium species and Akkermansia muciniphilia. Bacterial community structure was altered in SERT-/- mice. Differential abundance of bacteria was correlated with changes in host gene expression. Bifidobacterium and Bacilli species exhibited significant associations with host genes involved in lipid metabolism pathways. Our results show that SERT deletion is associated with dysbiosis similar to that observed in obesity. This study contributes to the understanding as to how changes in gut microbiota are associated with metabolic phenotype seen in SERT deficiency.

Bronchiolitis obliterans syndrome susceptibility and the pulmonary microbiome.

BACKGROUND: Lung transplantation outcomes remain complicated by bronchiolitis obliterans syndrome (BOS), a major cause of mortality and retransplantation for patients. A variety of factors linking inflammation and BOS have emerged, meriting further exploration of the microbiome as a source of inflammation. In this analysis, we determined features of the pulmonary microbiome associated with BOS susceptibility. METHODS: Bronchoalveolar lavage (BAL) samples were collected from 25 patients during standard of care bronchoscopies before BOS onset. Microbial DNA was isolated from BAL fluid and prepared for metagenomics shotgun sequencing. Patient microbiomes were phenotyped using k-means clustering and compared to determine effects on BOS-free survival. RESULTS: Clustering identified 3 microbiome phenotypes: Actinobacteria dominant (AD), mixed, and Proteobacteria dominant. AD microbiomes, distinguished by enrichment with Gram-positive organisms, conferred reduced odds and risks for patients to develop acute rejection and BOS compared with non-AD microbiomes. These findings were independent of treatment models. Microbiome findings were correlated with BAL cell counts and polymorphonuclear cell percentages. CONCLUSIONS: In some populations, features of the microbiome may be used to assess BOS susceptibility. Namely, a Gram-positive enriched pulmonary microbiome may predict resilience to BOS.

A Circulating MicroRNA Signature Serves as a Diagnostic and Prognostic Indicator in Sarcoidosis.

MicroRNAs (miRNAs) act as post-transcriptional regulators of gene expression. In sarcoidosis, aberrant miRNA expression may enhance immune responses mounted against an unknown antigenic agent. We tested whether a distinct miRNA signature functions as a diagnostic biomarker and explored its role as an immune modulator in sarcoidosis. The expression of miRNAs in peripheral blood mononuclear cells from subjects who met clinical and histopathologic criteria for sarcoidosis was compared with that observed in matched controls in the ACCESS (A Case Controlled Etiologic Study of Sarcoidosis) study. Signature miRNAs were determined by miRNA microarray analysis and validated by quantitative RT-PCR. Microarray analysis identified 54 mature, human feature miRNAs that were differentially expressed between the groups. Significant feature miRNAs that distinguished subjects with sarcoidosis from controls were selected by means of probabilistic models adjusted for clinical variables. Eight signature miRNAs were chosen to verify the diagnosis of sarcoidosis in a validation cohort, and distinguished subjects with sarcoidosis from controls with a positive predictive value of 88%. We identified both novel and previously described genes and molecular pathways associated with sarcoidosis as targets of these signature miRNAs. Additionally, we demonstrate that signature miRNAs (hsa-miR-150-3p and hsa-miR-342-5p) are significantly associated with reduced lymphocytes and airflow limitations, both of which are known markers of a poor prognosis. Together, these findings suggest that a circulating miRNA signature serves as a noninvasive biomarker that supports the diagnosis of sarcoidosis. Future studies will test the miRNA signature as a prognostication tool to identify unfavorable changes associated with poor clinical outcomes in sarcoidosis.

Atopic asthmatic immune phenotypes associated with airway microbiota and airway obstruction.

BACKGROUND: Differences in asthma severity may be related to inflammation in the airways. The lower airway microbiota has been associated with clinical features such as airway obstruction, symptom control, and response to corticosteroids. OBJECTIVE: To assess the relationship between local airway inflammation, severity of disease, and the lower airway microbiota in atopic asthmatics. METHODS: A cohort of young adult, atopic asthmatics with intermittent or mild/moderate persistent symptoms (n = 13) were assessed via bronchoscopy, lavage, and spirometry. These individuals were compared to age matched non-asthmatic controls (n = 6) and to themselves after six weeks of treatment with fluticasone propionate (FP). Inflammation of the airways was assessed via a cytokine and chemokine panel. Lower airway microbiota composition was determined by metagenomic shotgun sequencing. RESULTS: Unsupervised clustering of cytokines and chemokines prior to treatment with FP identified two asthmatic phenotypes (AP), termed AP1 and AP2, with distinct bronchoalveolar lavage inflammatory profiles. AP2 was associated with more obstruction, compared to AP1. After treatment with FP reduced MIP-1ß and TNF-α and increased IL-2 was observed. A module of highly correlated cytokines that include MIP-1ß and TNF-α was identified that negatively correlated with pulmonary function. Independently, IL-2 was positively correlated with pulmonary function. The airway microbiome composition correlated with asthmatic phenotypes. AP2, prior to FP treatment, was enriched with Streptococcus pneumoniae. Unique associations between IL-2 or the cytokine module and the microbiota composition of the airways were observed in asthmatics subjects prior to treatment but not after or in controls. CONCLUSION: The underlying inflammation in atopic asthma is related to the composition of microbiota and is associated with severity of airway obstruction. Treatment with inhaled corticosteroids was associated with changes in the airway inflammatory response to microbiota.

The Potential for Emerging Microbiome-Mediated Therapeutics in Asthma.

PURPOSE OF REVIEW: In terms of immune regulating functions, analysis of the microbiome has led the development of therapeutic strategies that may be applicable to asthma management. This review summarizes the current literature on the gut and lung microbiota in asthma pathogenesis with a focus on the roles of innate molecules and new microbiome-mediated therapeutics. RECENT FINDINGS: Recent clinical and basic studies to date have identified several possible therapeutics that can target innate immunity and the microbiota in asthma. Some of these drugs have shown beneficial effects in the treatment of certain asthma phenotypes and for protection against asthma during early life. Current clinical evidence does not support the use of these therapies for effective treatment of asthma. The integration of the data regarding microbiota with technologic advances, such as next generation sequencing and omics offers promise. Combining comprehensive bioinformatics, new molecules and approaches may shape future asthma treatment.

Perinatal Bacterial Exposure Contributes to IL-13 Aeroallergen Response.

There is a high prevalence of aeroallergen sensitivity in asthmatic populations, and seroreactivity to aeroallergens early in infancy is associated with increased risk of developing asthma later in life. In addition to allergen sensitivity, asthma development has been associated with differential microbial exposure and infection in early life. We have previously shown that cord blood mononuclear cells respond to common aeroallergens (i.e., house dust mite [Der f1] and cockroach [Bla g2]) as assayed by lymphoproliferation and cytokine (IL-13 and IFN-γ) production. We hypothesized that there is a relationship between perinatal microbial exposure and response to specific aeroallergens. To test this hypothesis, we isolated DNA from cord blood serum samples with known lymphoproliferative and cytokine responses to Bla g2 and Der f1. Bacterial 16S ribosomal DNA amplicon libraries were generated and analyzed using high throughput sequencing of cord blood serum samples. In our analysis, we identified major compositional differences, including diversity and abundance of specific taxa, between groups whose IL-13 response to Der f1 and Bla g2 differed. We demonstrate a strong association between the ratio of Acinetobacter to Proteobacteria and IL-13 production and the probability of IL-13 production after allergen exposure. IL-13 concentrations in serum were also significantly correlated with the diversity of bacterial DNA. Together, these results underscore the relationship between immune responses to allergens and bacterial exposure during perinatal development.

Maduramicin Rapidly Eliminates Malaria Parasites and Potentiates the Gametocytocidal Activity of the Pyrazoleamide PA21A050.

New strategies targeting Plasmodium falciparum gametocytes, the sexual-stage parasites that are responsible for malaria transmission, are needed to eradicate this disease. Most commonly used antimalarials are ineffective against P. falciparum gametocytes, allowing patients to continue to be infectious for over a week after asexual parasite clearance. A recent screen for gametocytocidal compounds demonstrated that the carboxylic polyether ionophore maduramicin is active at low nanomolar concentrations against P. falciparum sexual stages. In this study, we showed that maduramicin has an EC50 (effective concentration that inhibits the signal by 50%) of 14.8 nM against late-stage gametocytes and significantly blocks in vivo transmission in a mouse model of malaria transmission. In contrast to other reported gametocytocidal agents, maduramicin acts rapidly in vitro, eliminating gametocytes and asexual schizonts in less than 12 h without affecting uninfected red blood cells (RBCs). Ring stage parasites are cleared by 24 h. Within an hour of drug treatment, 40% of the normally crescent-shaped gametocytes round up and become spherical. The number of round gametocytes increases to >60% by 2 h, even before a change in membrane potential as monitored by MitoProbe DiIC1 (5) is detectable. Maduramicin is not preferentially taken up by gametocyte-infected RBCs compared to uninfected RBCs, suggesting that gametocytes are more sensitive to alterations in cation concentration than RBCs. Moreover, the addition of 15.6 nM maduramicin enhanced the gametocytocidal activity of the pyrazoleamide PA21A050, which is a promising new antimalarial candidate associated with an increase in intracellular Na(+) concentration that is proposed to be due to inhibition of PfATP4, a putative Na(+) pump. These results underscore the importance of cation homeostasis in sexual as well as asexual intraerythrocytic-stage P. falciparum parasites and the potential of targeting this pathway for drug development.

Expression of cytosolic peroxiredoxins in Plasmodium berghei ookinetes is regulated by environmental factors in the mosquito bloodmeal.

The Plasmodium ookinete develops over several hours in the bloodmeal of its mosquito vector where it is exposed to exogenous stresses, including cytotoxic reactive oxygen species (ROS). How the parasite adapts to these challenging conditions is not well understood. We have systematically investigated the expression of three cytosolic antioxidant proteins, thioredoxin-1 (Trx-1), peroxiredoxin-1 (TPx-1), and 1-Cys peroxiredoxin (1-Cys Prx), in developing ookinetes of the rodent parasite Plasmodium berghei under various growth conditions. Transcriptional profiling showed that tpx-1 and 1-cys prx but not trx-1 are more strongly upregulated in ookinetes developing in the mosquito bloodmeal when compared to ookinetes growing under culture conditions. Confocal immunofluorescence imaging revealed comparable expression patterns on the corresponding proteins. 1-Cys Prx in particular exhibited strong expression in mosquito-derived ookinetes but was not detectable in cultured ookinetes. Furthermore, ookinetes growing in culture upregulated tpx-1 and 1-cys prx when challenged with exogenous ROS in a dose-dependent fashion. This suggests that environmental factors in the mosquito bloodmeal induce upregulation of cytosolic antioxidant proteins in Plasmodium ookinetes. We found that in a parasite line lacking TPx-1 (TPx-1KO), expression of 1-Cys Prx occurred significantly earlier in mosquito-derived TPx-1KO ookinetes when compared to wild type (WT) ookinetes. The protein was also readily detectable in cultured TPx-1KO ookinetes, indicating that 1-Cys Prx at least in part compensates for the loss of TPx-1 in vivo. We hypothesize that this dynamic expression of the cytosolic peroxiredoxins reflects the capacity of the developing Plasmodium ookinete to rapidly adapt to the changing conditions in the mosquito bloodmeal. This would significantly increase its chances of survival, maturation and subsequent escape. Our results also emphasize that environmental conditions must be taken into account when investigating Plasmodium-mosquito interactions.

Cardiac pressure overload initiates a systemic stem cell response.

BACKGROUND AIMS: Acute cardiac injury results in the activation and recruitment of resident and non-cardiac stem cells. In this study we sought to define the pattern of peripheral stem cells and resident cardiac stem cell (CSC) activation induced acutely by cardiac pressure overload (PO). METHODS: PO was induced in mice by transaortic constriction (TAC). CSC, endothelial progenitor cells (EPC), hematopoietic stem cells (HSC) and stage-specific embryonic antigen (SSEA)-1(+) cells were profiled in the heart, spleen and bone marrow after TAC by flow cytometry. RESULTS: The combination of a systemic and local stem cell response resulted in increases in SSEA-1 (+) cells and EPC in the heart 7 and 14 days post-TAC, respectively. Locally, modest SSEA-1(+) proliferation at 4 days preceded the elevated myocardial stem cell number. We observed no significant proliferation of EPC and CSC in the heart. The systemic stem cell response was characterized by a biphasic loss of splenic SSEA-1(+) cells at 2 and 7 days post-TAC and loss of bone marrow and spleen EPC at 4 and 7 days, respectively. Spleen size changed dynamically after TAC. A negligible response of HSC to TAC was observed. Significant EPC and SSEA-1(+) proliferation in the bone marrow and spleen occurred only after their local levels were decreased. CONCLUSIONS: Our results demonstrate that an orchestrated systemic stem cell response (EPC and SSEA-1 (+) ) takes place in response to TAC. The increase of SSEA-1(+) cells and EPC in the heart in response to pressure is likely to be because of a combination of local proliferation and stem cell recruitment.