Brucella suppress STING expression via miR-24 to enhance infection.

Brucellosis, caused by a number of Brucella species, remains the most prevalent zoonotic disease worldwide. Brucella establish chronic infections within host macrophages despite triggering cytosolic innate immune sensors, including Stimulator of Interferon Genes (STING), which potentially limit infection. In this study, STING was required for control of chronic Brucella infection in vivo. However, early during infection, Brucella down-regulated STING mRNA and protein. Down-regulation occurred post-transcriptionally, required live bacteria, the Brucella type IV secretion system, and was independent of host IRE1-RNase activity. STING suppression occurred in MyD88-/- macrophages and was not induced by Toll-like receptor agonists or purified Brucella lipopolysaccharide (LPS). Rather, Brucella induced a STING-targeting microRNA, miR-24-2, in a type IV secretion system-dependent manner. Furthermore, STING downregulation was inhibited by miR-24 anti-miRs and in Mirn23a locus-deficient macrophages. Failure to suppress STING expression in Mirn23a-/- macrophages correlated with diminished Brucella replication, and was rescued by exogenous miR-24. Mirn23a-/- mice were also more resistant to splenic colonization one week post infection. Anti-miR-24 potently suppressed replication in wild type, but much less in STING-/- macrophages, suggesting most of the impact of miR-24 induction on replication occurred via STING suppression. In summary, Brucella sabotages cytosolic surveillance by miR-24-dependent suppression of STING expression; post-STING activation "damage control" via targeted STING destruction may enable establishment of chronic infection.

The distribution of covert microbial natural enemies of a globally invasive crop pest, fall armyworm, in Africa: Enemy release and spillover events.

Invasive species pose a significant threat to biodiversity and agriculture world-wide. Natural enemies play an important part in controlling pest populations, yet we understand very little about the presence and prevalence of natural enemies during the early invasion stages. Microbial natural enemies of fall armyworm Spodoptera frugiperda are known in its native region, however, they have not yet been identified in Africa where fall armyworm has been an invasive crop pest since 2016. Larval samples were screened from Malawi, Rwanda, Kenya, Zambia, Sudan and Ghana for the presence of four different microbial natural enemies; two nucleopolyhedroviruses, Spodoptera frugiperda NPV (SfMNPV) and Spodoptera exempta NPV (SpexNPV); the fungal pathogen Metarhizium rileyi; and the bacterium Wolbachia. This study aimed to identify which microbial pathogens are present in invasive fall armyworm, and determine the geographical, meteorological and temporal variables that influence prevalence. Within 3 years of arrival, fall armyworm was exposed to all four microbial natural enemies. SfMNPV probably arrived with fall armyworm from the Americas, but this is the first putative evidence of host spillover from Spodoptera exempta (African armyworm) to fall armyworm for the endemic pathogen SpexNPV and for Wolbachia. It is also the first confirmed incidence of M. rileyi infecting fall armyworm in Africa. Natural enemies were localised, with variation being observed both nationally and temporally. The prevalence of SfMNPV (the most common natural enemy) was predominantly explained by variables associated with the weather; declining with increasing rainfall and increasing with temperature. However, virus prevalence also increased as the growing season progressed. The infection of an invasive species with a natural enemy from its native range and novel pathogens specific to its new range has important consequences for understanding the population ecology of invasive species and insect-pathogen interactions. Additionally, while it is widely known that temporal and geographic factors affect insect populations, this study reveals that these are important in understanding the distribution of microbial natural enemies associated with invasive pests during the early stages of invasion, and provide baseline data for future studies.

A gene-based recessive diplotype exome scan discovers FGF6, a novel hepcidin-regulating iron-metabolism gene.

Standard analyses applied to genome-wide association data are well designed to detect additive effects of moderate strength. However, the power for standard genome-wide association study (GWAS) analyses to identify effects from recessive diplotypes is not typically high. We proposed and conducted a gene-based compound heterozygosity test to reveal additional genes underlying complex diseases. With this approach applied to iron overload, a strong association signal was identified between the fibroblast growth factor-encoding gene, FGF6, and hemochromatosis in the central Wisconsin population. Functional validation showed that fibroblast growth factor 6 protein (FGF-6) regulates iron homeostasis and induces transcriptional regulation of hepcidin. Moreover, specific identified FGF6 variants differentially impact iron metabolism. In addition, FGF6 downregulation correlated with iron-metabolism dysfunction in systemic sclerosis and cancer cells. Using the recessive diplotype approach revealed a novel susceptibility hemochromatosis gene and has extended our understanding of the mechanisms involved in iron metabolism.

Brucella abortus Cyclic Dinucleotides Trigger STING-Dependent Unfolded Protein Response That Favors Bacterial Replication.

Brucella abortus is a facultative intracellular bacterium that causes brucellosis, a prevalent zoonosis that leads to abortion and infertility in cattle, and undulant fever, debilitating arthritis, endocarditis, and meningitis in humans. Signaling pathways triggered by B. abortus involves stimulator of IFN genes (STING), which leads to production of type I IFNs. In this study, we evaluated the pathway linking the unfolded protein response (UPR) and the endoplasmic reticulum-resident transmembrane molecule STING, during B. abortus infection. We demonstrated that B. abortus infection induces the expression of the UPR target gene BiP and XBP1 in murine macrophages through a STING-dependent pathway. Additionally, we also observed that STING activation was dependent on the bacterial second messenger cyclic dimeric GMP. Furthermore, the Brucella-induced UPR is crucial for induction of multiple molecules linked to type I IFN signaling pathway, such as IFN-ß, IFN regulatory factor 1, and guanylate-binding proteins. Furthermore, IFN-ß is also important for the UPR induction during B. abortus infection. Indeed, IFN-ß shows a synergistic effect in inducing the IRE1 axis of the UPR. In addition, priming cells with IFN-ß favors B. abortus survival in macrophages. Moreover, Brucella-induced UPR facilitates bacterial replication in vitro and in vivo. Finally, these results suggest that B. abortus-induced UPR is triggered by bacterial cyclic dimeric GMP, in a STING-dependent manner, and that this response supports bacterial replication. In summary, association of STING and IFN-ß signaling pathways with Brucella-induced UPR unravels a novel link between innate immunity and endoplasmic reticulum stress that is crucial for bacterial infection outcome.

Orosomucoid-like 3 Supports Rhinovirus Replication in Human Epithelial Cells.

Polymorphism at the 17q21 gene locus and wheezing responses to rhinovirus (RV) early in childhood conspire to increase the risk of developing asthma. However, the mechanisms mediating this gene-environment interaction remain unclear. In this study, we investigated the impact of one of the 17q21-encoded genes, ORMDL3 (orosomucoid-like 3), on RV replication in human epithelial cells. ORMDL3 knockdown inhibited RV-A16 replication in HeLa, BEAS-2B, A549, and NCI-H358 epithelial cell lines and primary nasal and bronchial epithelial cells. Inhibition varied by RV species, as both minor and major group RV-A subtypes RV-B52 and RV-C2 were inhibited but not RV-C15 or RV-C41. ORMDL3 siRNA did not affect expression of the major group RV-A receptor ICAM-1 or initial internalization of RV-A16. The two major outcomes of ORMDL3 activity, SPT (serine palmitoyl-CoA transferase) inhibition and endoplasmic reticulum (ER) stress induction, were further examined: silencing ORMDL3 decreased RV-induced ER stress and IFN-ß mRNA expression. However, pharmacologic induction of ER stress and concomitant increased IFN-ß inhibited RV-A16 replication. Conversely, blockade of ER stress with tauroursodeoxycholic acid augmented replication, pointing to an alternative mechanism for the effect of ORMDL3 knockdown on RV replication. In comparison, the SPT inhibitor myriocin increased RV-A16 but not RV-C15 replication and negated the inhibitory effect of ORMDL3 knockdown. Furthermore, lipidomics analysis revealed opposing regulation of specific sphingolipid species (downstream of SPT) by myriocin and ORMDL3 siRNA, correlating with the effect of these treatments on RV replication. Together, these data revealed a requirement for ORMDL3 in supporting RV replication in epithelial cells via SPT inhibition.

Virtually there: An innovative approach for training oncology pharmacy residents in gynecologic oncology.

OBJECTIVE: To develop an alternative approach to provide oncology pharmacy practice residents' education and training in the management of gynecologic malignancies in the absence of a specialist in this area at their institution. SETTING: Gynecologic oncology is a unique specialty in oncology. There is a need for more oncology clinical pharmacy specialists to participate in the care of patients with gynecologic malignancies as many do not have specific education in this area. PRACTICE DESCRIPTION: A virtual learning experience was developed that included all aspects of a typical experience with the exception of direct patient care. Postgraduate year 2 oncology pharmacy residents from 3 different programs were included. PRACTICE INNOVATION: Although the number of oncology clinical pharmacy specialists who are subspecialized in gynecologic oncology has grown, it is difficult to find experienced preceptors in gynecology oncology. We set to offer a virtual learning environment for programs that did not have a dedicated or highly specialized pharmacist in this area. EVALUATION: A pre- and postlearning assessment of the resident's knowledge of gynecologic malignancies was administered. Each trainee independently completed a validated 20-question gynecologic oncology knowledge assessment tool before and again after completion of all sessions. Midpoint and end-of-experience evaluations were completed via the phone with each resident. All evaluations were documented in PharmAcademic (McCreadie Group, Ann Arbor, MI), a required software program for postgraduate residency training programs. RESULTS: To date, 7 oncology pharmacy practice residents completed the virtual experience. A 42% improvement in scores pertaining to gynecologic oncology knowledge was identified. Residents were also satisfied with the overall virtual experience. Based on the assessment tool, all the residents gave positive evaluations with "always true" for 6 of the 7 questions. CONCLUSIONS: This pilot of a virtual experience was a successful platform to provide clinical knowledge and skills for oncology pharmacy residents in gynecologic oncology.

Tunicamycin-induced photoreceptor atrophy precedes degeneration of retinal capillaries with minimal effects on retinal ganglion and pigment epithelium cells.

Endoplasmic reticulum (ER) stress is recognized as a contributing factor to various ocular neurovascular pathologies including retinitis pigmentosa, glaucoma, and diabetic retinopathy (DR). ER stress in particular is implicated in the development of DR, which is significantly influenced by inflammation driven retinal vascular degeneration and dysfunction. Ultimately, loss of vision occurs if left untreated. However, the identity of the target cells and their temporal involvement in diabetes-mediated dysfunction need further investigation. Early diabetes-induced stress in photoreceptor cells is proposed as the driver of inflammatory mediated neurovascular changes during diabetes. Although tunicamycin induced ER stress results in photoreceptor loss, its consequences for retinal vascular degeneration and retinal ganglion (RGC) and pigment epithelium (RPE) cell loss remains unclear. Here we show intravitreal delivery of tunicamycin primarily induced ER stress in photoreceptor cells resulting in their loss by apoptosis. This was concomitant with induced expression of the unfolded protein response marker CHOP in these cells. We also demonstrated significant degeneration of retinal capillaries following the loss of photoreceptor cells with minimal impact on loss of RGC and RPE cells. However, activation of retinal microglial and Muller cells were noticeable. Thus, our data support the notion that ER stress mediated dysfunction and/or loss of photoreceptor cells in response to inflammation and oxidative stress could precede retinal vascular and neuronal dysfunction and degeneration.

Identifying the need to refine the potential patient risk factors for niraparib-induced thrombocytopenia.

OBJECTIVE: Niraparib is a poly (ADP-ribose) polymerase inhibitor (PARP) approved for use in maintenance therapy for ovarian cancer that is associated with the unpredictable grade 3/4 thrombocytopenia. This study was conducted to refine patient dosing recommendations for niraparib based upon clinical practice observations of grade 3/4 thrombocytopenia. METHODS AND MATERIALS: Six patient cases were reviewed to identify similarities in patient factors. An in vitro study was conducted using healthy volunteer blood spiked with Niraparib concentrations ranging from 0 ng/mL to 5000 ng/mL. Manual platelet counts were evaluated at different time intervals for each concentration and compared to untreated controls. Data was then analyzed based on percent change in platelet count versus untreated control for each concentration/time point. RESULTS: In three patients with body weight > 80 kg and platelet count >200 × 109/L, decreased creatinine clearance (CrCl) <60 mL/min was identified as potential signal. An additional three patients with weights below 77 kg and/or baseline platelet counts <150 × 109/L were re-evaluated, and it was observed that all had decreased CrCl of <60 mL/min. Albumin <3.5 g/dL was also observed in some patients with thrombocytopenia. The in vitro study, observed a direct concentration-dependent relationship between niraparib and thrombocytopenia. CONCLUSION: The data suggests that renal insufficiency and hypoalbuminemia may be associated with the development of niraparib-induced thrombocytopenia. Moreover, the preliminary in vitro studies also demonstrated a concentration-dependent relationship between niraparib and direct toxicity to platelets.

Space: A Final Frontier for Vacuolar Pathogens.

There is a fundamental gap in our understanding of how a eukaryotic cell apportions the limited space within its cell membrane. Upon infection, a cell competes with intracellular pathogens for control of this same precious resource. The struggle between pathogen and host provides us with an opportunity to uncover the mechanisms regulating subcellular space by understanding how pathogens modulate vesicular traffic and membrane fusion events to create a specialized compartment for replication. By comparing several important intracellular pathogens, we review the molecular mechanisms and trafficking pathways that drive two space allocation strategies, the formation of tight and spacious pathogen-containing vacuoles. Additionally, we discuss the potential advantages of each pathogenic lifestyle, the broader implications these lifestyles might have for cellular biology and outline exciting opportunities for future investigation.

Brucella Peptide Cross-Reactive Major Histocompatibility Complex Class I Presentation Activates SIINFEKL-Specific T Cell Receptor-Expressing T Cells.

Brucella spp. are intracellular pathogenic bacteria remarkable in their ability to escape immune surveillance and therefore inflict a state of chronic disease within the host. To enable further immune response studies, Brucella was engineered to express the well-characterized chicken ovalbumin (OVA). Surprisingly, we found that CD8 T cells bearing T cell receptors (TCR) nominally specific for the OVA peptide SIINFEKL (OT-1) reacted to parental Brucella-infected targets as well as OVA-expressing Brucella variants in cytotoxicity assays. Furthermore, splenocytes from Brucella-immunized mice produced gamma interferon (IFN-γ) and exhibited cytotoxicity in response to SIINFEKL-pulsed target cells.To determine if the SIINFEKL-reactive OT-1 TCR could be cross-reacting to Brucella peptides, we searched the Brucella proteome using an algorithm to generate a list of near-neighbor nonamer peptides that would bind to H2Kb Selecting five Brucella peptide candidates, along with controls, we verified that several of these peptides mimicked SIINFEKL, resulting in T cell activation through the "SIINFEKL-specific" TCR. Activation was dependent on peptide concentration as well as sequence. Our results underscore the complexity and ubiquity of cross-reactivity in T cell recognition. This cross-reactivity may enable microbes such as Brucella to escape immune surveillance by presenting peptides similar to those of the host and may also lead to the activation of autoreactive T cells.

Defining the impact of the use of granulocyte colony stimulating factors on the incidence of chemotherapy-induced neutropenia in patients with gynecologic malignancies.

Purpose The objectives of this study were to characterize the incidence of chemotherapy-induced neutropenia (CIN) and febrile neutropenia (FN) with specific chemotherapy agents commonly used in the treatment of gynecologic malignancies, as well as defining the impact of granulocyte colony stimulating factors (G-CSF) on the prevention of CIN and FN in this patient population. Methods This retrospective analysis was conducted from a database of 635 gynecologic cancer patients who received chemotherapy between 1 September 2007 and 31 August 2008. A logistic regression analysis was conducted to determine the impact of potential covariates on the overall incidence of CIN. Results Overall, 28.3% of patients experienced CIN with one or more cycles chemotherapy, and 13.1% had treatment delays or dose reduction associated with CIN. The use of G-CSF prior to administration of chemotherapy resulted in a decrease in the incidence of CIN from 29.8% to 19.6% compared to no G-CSF use. No difference was observed in number of treatment delays or dose reductions in the 46 (7.2%) of gynecologic cancer patients that received G-CSF prophylaxis. Multivariate analysis found that both age and the number of current cycles jointly may predict risk of CIN. Conclusions Patients with gynecologic malignancies appear to be at a higher risk of development of neutropenia when treated with chemotherapy. The proactive use of G-CSF did decrease the risk of CIN by over 30%. Prospective study is warranted to determine the impact of G-CSF to reduce CIN in patients with gynecologic malignancies receiving chemotherapy.

Observational Case Series Evaluation of the Granisetron Transdermal Patch System (Sancuso) for the Management of Nausea/Vomiting of Pregnancy.

Objective The objective of this study was to observe the efficacy of antiemetic therapy (no emesis/retching episodes and no rescue medication use) when granisetron is administered via a transdermal patch system (TDS) in women who are 6 to 14 weeks pregnant when compared with oral ondansetron by evaluating the frequency of the use of rescue medications for control of nausea/vomiting of pregnancy (NVP). Methods This was an observational case series study to observe the potential benefits of granisetron TDS compared with oral ondansetron for management of NVP in pregnant patients during the first trimester. Dates of data collection were September 1, 2014, through December 31, 2015. There was no direct contact with patient. The oral ondansetron and granisetron TDS patients were matched by age, 4:1. The proportion of patients who received rescue antiemetics was calculated from those patients who continued to experience NVP. Risk factors for NVP were identified and compared between groups. Descriptive statistics were used to describe study results. Results Patients were prescribed rescue antiemetics in 0/3 patients in the granisetron TDS group compared with 2/12 patients in the oral ondansetron group. Conclusion Prospective efficacy studies on the use of granisetron TDS for management of NVP are needed to confirm this clinical observation.

The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response.

Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ΔbpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ΔbpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ΔcgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ΔbpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection.

Equivalency challenge: Evaluation of Lipodox® as the generic equivalent for Doxil® in a human ovarian cancer orthotropic mouse model.

BACKGROUND: The objective of this study was to evaluate the in vivo growth inhibition activity and tumor distribution of Doxil® compared to Lipodox® as its generic (GLD) in human ovarian cancer orthotopic mouse model. METHODS: In the efficacy study 50 mice were randomized to: vehicle, Doxil® 5mg/kg or 10mg/kg, or GLD 5mg/kg or 10mg/kg for a total of three cycles with monitoring for response and toxicity with 10 mice in each arm. In the microdialysis(MD) study, 60 mice were randomized to: Doxil® 5mg/kg or 10mg/kg, or GLD 5mg/kg or 10mg/kg single dose (n=15 mice/arm). MD sample time points included total of 29 samples from baseline through 100h and were evaluated with a validated PaperSpray LC/MS assay. RESULTS: There was 15.7% decrease (p<0.0001) in efficacy of GLD the 5mg/kg and 21.3% decrease (p<0.0001) in efficacy of the 10mg/kg dose of GLD when compared to equivalent doses of Doxil®. The intratumoral concentration for the GLD ranged from 1.0 to 25.5ng/mL (5mg/kg) and 2.9-35.6ng/mL (10mg/kg) compared to 2.7-42.2ng/mL (p<0.04, 5mg/kg) and 2.0-76ng/mL (p<0.02, 10mg/kg) for the Doxil®, respectively. CONCLUSION: Significant differences in preclinical efficacy were observed between Doxil® and GLD. These may be due to significant pharmacodynamic effects of drug distribution and decrease uptake of GLD in tumor tissue. A prospective clinical comparison of these two products is warranted to determine equivalency.

The Bench-to-Bedside Story of IL-17 and the Therapeutic Efficacy of its Targeting in Spondyloarthritis.

TNF-blocking biologics have revolutionized the care of patients with spondyloarthritis, a group of clinically overlapping conditions that includes ankylosing spondylitis and psoriatic arthritis. However, incomplete response rates speak to the need for alternative therapeutic approaches. Over the last decade, animal models, genetics, and translational studies have implicated the excessive production of a pro-inflammatory cytokine interleukin-17 (IL-17) along with another IL-17-promoting cytokine IL-23 in the pathogenesis of spondyloarthritis. Genome-wide studies identified disease associations with multiple genes regulating IL-23/IL-17 immune pathway activity. Direct examination of the patient blood and tissues revealed excessive IL-17 and IL-23 production by diverse cell types. Murine models both underscored the sufficiency of excess IL-23 in driving disease phenotype and predicted utility in IL-23/IL-17 pathway blockade. However, the clinical efficacy of agents such as secukinumab and ustekinumab, which block IL-17 and IL-23/IL-12 respectively, provided exciting proof of concept.

Is it equivalent? Evaluation of the clinical activity of single agent Lipodox® compared to single agent Doxil® in ovarian cancer treatment.

BACKGROUND: In response to the critical shortage of liposomal doxorubicin (Doxil®) in the United States, the Food and Drug Administration (FDA) approved temporary importation of doxorubicin hydrochloride liposome (Lipodox®). The objective was to compare toxicity and clinical activity of Lipodox® with Doxil®. METHODS: Recurrent ovarian cancer patients who received Lipodox® were compared 3:1 to matched control Doxil® patients who had received Doxil®. Patients were matched based on age, stage, dose, platinum sensitivity, and prior treatments from an existing de-identified database. Patients receiving combination regimens were excluded. RESULTS: The data from 40 Lipodox® patients was compared to 120 matched control Doxil® patients. In this study, 17 (42.5%) of the Lipodox® patients were switched to Doxil®. The overall response rate Lipodox® was 4.3% (1/23) compared to 18% (20/111) in matched control Doxil® patients. In the platinum-sensitive patients, 100% progressed in the Lipodox® group compared to 78.4% in matched control Doxil® patients. The mean time to progression was 4.1 ± 2.8 months for Lipodox® and 6.2 ± 7.2 months in control Doxil®s (p = 0·25). Toxicity was similar in the Lipodox® group and control Doxil® group. CONCLUSION: Lipodox® for treatment of recurrent ovarian cancer did not appear to have equivalent efficacy compared to Doxil®. A prospective clinical study is warranted before Lipodox® can be deemed equivalent substitution for Doxil®.

Stability study of carboplatin infusion solutions in 0.9% sodium chloride in polyvinyl chloride bags.

BACKGROUND AND PURPOSE: Carboplatin is a platinum-containing compound with efficacy against various malignancies. The physico-chemical stability of carboplatin in dextrose 5% water (D5W) has been thoroughly studied; however, there is a paucity of stability data in clinically relevant 0.9% sodium chloride infusion solutions. The manufacturer's limited stability data in sodium chloride solutions hampers the flexibility of carboplatin usage in oncology patients. Hence, the purpose of this study is to determine the physical and chemical stability of carboplatin-sodium chloride intravenous solutions under different storage conditions. METHODS: The physico-chemical stability of 0.5 mg/mL, 2.0 mg/mL, and 4.0 mg/mL carboplatin-sodium chloride solutions prepared in polyvinyl chloride bags was determined following storage at room temperature under ambient fluorescent light and under refrigeration in the dark. Concentrations of carboplatin were measured at predetermined time points up to seven days using a stability-indicating high-performance liquid chromatography method. RESULTS: All tested solutions were found physically stable for at least seven days. The greatest chemical stability was observed under refrigerated storage conditions. At 4℃, all tested solutions were found chemically stable for at least seven days, with nominal losses of ≤6%. Following storage at room temperature exposed to normal fluorescent light, the chemical stability of 0.5 mg/mL, 2.0 mg/mL, and 4.0 mg/mL solutions was three days, five days, and seven days, respectively. CONCLUSION: The extended physico-chemical stability of carboplatin prepared in sodium chloride reported herein permits advance preparation of these admixtures, facilitating pharmacy utility and operations. Since no antibacterial preservative is contained within these carboplatin solutions, we recommend storage, when prepared under specified aseptic conditions, no greater than 24 h at room temperature or three days under refrigeration.

CD8+ T cell exhaustion, suppressed gamma interferon production, and delayed memory response induced by chronic Brucella melitensis infection.

Brucella melitensis is a well-adapted zoonotic pathogen considered a scourge of mankind since recorded history. In some cases, initial infection leads to chronic and reactivating brucellosis, incurring significant morbidity and economic loss. The mechanism by which B. melitensis subverts adaptive immunological memory is poorly understood. Previous work has shown that Brucella-specific CD8(+) T cells express gamma interferon (IFN-γ) and can transition to long-lived memory cells but are not polyfunctional. In this study, chronic infection of mice with B. melitensis led to CD8(+) T cell exhaustion, manifested by programmed cell death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) expression and a lack of IFN-γ production. The B. melitensis-specific CD8(+) T cells that produced IFN-γ expressed less IFN-γ per cell than did CD8(+) cells from uninfected mice. Both memory precursor (CD8(+) LFA1(HI) CD127(HI) KLRG1(LO)) and long-lived memory (CD8(+) CD27(HI) CD127(HI) KLRG1(LO)) cells were identified during chronic infection. Interestingly, after adoptive transfer, mice receiving cells from chronically infected animals were able to contain infection more rapidly than recipients of cells from acutely infected or uninfected donors, although the proportions of exhausted CD8(+) T cells increased after adoptive transfer in both challenged and unchallenged recipients. CD8(+) T cells of challenged recipients initially retained the stunted IFN-γ production found prior to transfer, and cells from acutely infected mice were never seen to transition to either memory subset at all time points tested, up to 30 days post-primary infection, suggesting a delay in the generation of memory. Here we have identified defects in Brucella-responsive CD8(+) T cells that allow chronic persistence of infection.

Neutrophils in pediatric autoimmune disease.

PURPOSE OF REVIEW: As first immune responders, neutrophils are essential mediators of host defense, and also contribute to chronic pathologic inflammation at the crossroads of innate and adaptive immunity. In this review, we will highlight the current understanding of the role of neutrophils in pediatric rheumatology, with a focus on juvenile idiopathic arthritis (JIA) and lupus. RECENT FINDINGS: In inflamed tissues, neutrophils extrude neutrophil extracellular traps containing autoantigen that potentially drives lupus and rheumatoid factor-positive JIA. However, the contribution of neutrophil extracellular traps to pathogenesis remains an area of intense investigation. In JIA joints, neutrophils are activated to such an extent that associated circulating levels of S100A proteins may serve as biomarkers, correlating with disease activity, predicting response to treatment and heralding flares. Beyond the effects of 'normal' activation, neutrophils in JIA and lupus display dysregulation in gene expression, subset activation, and apoptosis. SUMMARY: The role of neutrophils in pediatric rheumatology is an understudied area, but garnering increasing attention. Although clearly implicated in JIA and lupus, the specific contributions of neutrophils to pathogenesis and the use of neutrophil activity surrogates as biomarkers require further study. Clarification of these outstanding issues will have implications for diagnosis and treatment of pediatric rheumatologic conditions.

Brucella induces an unfolded protein response via TcpB that supports intracellular replication in macrophages.

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.