Absence Of Leukocytes Research Articles

Predicting antibiotic susceptibility in urinary tract infection with artificial intelligence-model performance in a multi-centre cohort.

To develop an artificial intelligence model to predict an antimicrobial susceptibility pattern in patients with urinary tract infection (UTI). 26 087 adult patients with culture-proven UTI during 2015-2020 from a university teaching hospital and three community hospitals in Hong Kong were included. Cases with asymptomatic bacteriuria (absence of diagnosis code of UTI, or absence of leucocytes in urine microscopy) were excluded. Patients from 2015 to 2019 were included in the training set, while patients from the year 2020 were included as the test set.Three first-line antibiotics were chosen for prediction of susceptibility in the bacterial isolates causing UTI: namely nitrofurantoin, ciprofloxacin and amoxicillin-clavulanate. Baseline epidemiological factors, previous antimicrobial consumption, medical history and previous culture results were included as features. Logistic regression and random forest were applied to the dataset. Models were evaluated by F1-score and area under the curve-receiver operating characteristic (AUC-ROC). Random forest was the best algorithm in predicting susceptibility of the three antibiotics (nitrofurantoin, amoxicillin-clavulanate and ciprofloxacin). The AUC-ROC values were 0.941, 0.939 and 0.937, respectively. The F1 scores were 0.938, 0.928 and 0.906 respectively. Random forest model may aid judicious empirical antibiotics use in UTI. Given the reasonable performance and accuracy, these accurate models may aid clinicians in choosing between different first-line antibiotics for UTI.

Comparison of cytokine levels in prostatic secretion between the IIIa and IIIb subtypes of prostatitis.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), also known as National Institutes of Health (NIH) type III prostatitis, is a common disorder with an unclear etiology and no known curative treatments. Based on the presence or absence of leukocytes in expressed prostatic secretion (EPS), CP/CPPS is classified further into IIIa (inflammatory) and IIIb (noninflammatory) subtypes. However, the severity of symptoms is not entirely consistent with the white blood cell (WBC) count. Following the preliminary finding of a link between inflammatory cytokines and CP/CPPS, we performed this clinical study with the aim of identifying cytokines that are differentially expressed according to whether the prostatitis subtype is IIIa or IIIb. We found that granulocyte colony-stimulating factor (G-CSF), interleukin-18 (IL-18), and monocyte chemoattractant protein-1 (MCP-1) levels were significantly elevated and interferon-inducible protein-10 (IP-10) and platelet-derived growth factor-BB (PDGF-BB) levels were downregulated in the EPS of patients with type IIIa prostatitis. In a word, it is a meaningful study in which we investigate the levels of various cytokines in EPS according to whether prostatitis is the IIIa or IIIb subtype. The combination of G-CSF, IL-18, MCP-1, IP-10, and PDGF-BB expression levels could form a basis for classification, diagnosis, and therapeutic targets in clinical CP/CPPS.

Aspirin modulates production of pro-inflammatory and pro-resolving mediators in endothelial cells.

Endothelial cells synthesize biochemical signals to coordinate a response to insults, resolve inflammation and restore barrier integrity. Vascular cells release a variety of vasoactive bioactive lipid metabolites during the inflammatory response and produce pro-resolving mediators (e.g., Lipoxin A4, LXA4) in cooperation with leukocytes and platelets to bring a halt to inflammation. Aspirin, used in a variety of cardiovascular and pro-thrombotic disorders (e.g., atherosclerosis, angina, preeclampsia), potently inhibits proinflammatory eicosanoid formation. Moreover, aspirin stimulates the synthesis of pro-resolving lipid mediators (SPM), so-called Aspirin-Triggered Lipoxins (ATL). We demonstrate that cytokines stimulated a time- and dose-dependent increase in PGI2 (6-ketoPGF1α) and PGE2 formation that is blocked by aspirin. Eicosanoid production was caused by cytokine-induced expression of cyclooxygenase-2 (COX-2). We also detected increased production of pro-resolving LXA4 in cytokine-stimulated endothelial cells. The R-enantiomer of LXA4, 15-epi-LXA4, was enhanced by aspirin, but only in the presence of cytokine challenge, indicating dependence on COX-2 expression. In contrast to previous reports, we detected arachidonate 5-lipoxygenase (ALOX5) mRNA expression and its cognate protein (5-lipoxygenase, 5-LOX), suggesting that endothelial cells possess the enzymatic machinery necessary to synthesize both pro-inflammatory and pro-resolving lipid mediators independent of added leukocytes or platelets. Finally, we observed that, endothelial cells produced LTB4 in the absence of leukocytes. These results indicate that endothelial cells produce both pro-inflammatory and pro-resolving lipid mediators in the absence of other cell types and aspirin exerts pleiotropic actions influencing both COX and LOX pathways.

O-032 Primary human endothelial and stromal cells from the uterine endometrium co-cultured in vitro in a 3D-system as a model to study the physiopathology of endometriosis

Abstract Study question Is an uterine endothelial-stromal cell 3D-system able to respond to inflammatory/immune factors presented in patient’s endometriotic serum and can this be reversible by hormonal treatment? Summary answer The endothelial-stromal cells system is responsive to the serum from women with endometriosis and its cytokine profile may be reverse with hormonal treatment. What is known already Endometriosis’s declined fertility is mainly attributed to poor oocyte quality, inhibition of ovulation, an anatomical commitment of tubes and uterus, and loss of endometrial receptivity during the implantation window. Changes in the inflammatory/immune profile in pelvic and peripheral blood also suggest a possible interference of several cytokines playing a role in women's reduced fertility. Three-dimensional (3D) cell cultures open up new study possibilities, by guaranteeing interactions between spatially organized tissues mimicking the natural microenvironment and can significantly contribute to obtaining essential data for understanding endometrial physiology and its associated diseases. Study design, size, duration This is a prospective cohort study with oocyte donation women from a private IVF center and patients under endometriosis treatment in an University-affiliated gynecology service. Endometrium biopsy (n = 9) and non-endometriotic serum blood (n = 15) were collected from oocyte donors in the same day of oocyte picked (antagonist protocol), before ovary puncture and serum blood samples were collected from patients diagnostic with endometriosis (n = 15). Samples were collected between Jan/2016 and May/2017 after signing the informed consent form. Participants/materials, setting, methods Endometriotic serum samples were obtained from patients with (n = 10) or without (n = 5) estrogen/progestin therapy. Tissue biopsies were digested and submitted to magnetic microbeads. Endothelial and stromal cells layers were added one-by-one to a mixture of extracellular matrix components. The 3D-system received endometriotic or control serum for additional 48h. Supernatants were excluded and the cells homogenized for cytokine evaluation through cytometric bead array. The ANOVA-Tukey’s test were used for statistical analysis, p < 0.05 were considered significant. Main results and the role of chance Oocyte donors were 24,2 ± 3,41 years old and had a body mass index (BMI) of 22,7 ± 1,38. Patients with endometriosis were 34,0 ± 3,93 years old (p = 0,21) and had a BMI of 25,4 ± 4,13 (p = 0.02). The morphology assessed by ultrastructural analysis of the 3D-system showed the presence of cells embedded in an abundant extracellular matrix, with no directional organization and with morphology compatible with fibrocytes and fibroblasts. The cells showed characteristics of viable cells with loose chromatin nuclei, evident nucleoli, and well-defined organelles. Eventually, mitosis were observed. Specific presence or absence of stromal and endothelial cells markers (cytokeratin, vimentin, IGFBP1, von Willebrand factor) were confirmed by immunofluorescence and flow cytometry. Cell viability were assessed by Fixable Cell Viability 510 dye staining. Changes in the expression profile of the cytokines TNF-alpha (p < 0.0001), IL-10 (p < 0.001) and IL-2 (p < 0.001) were detected in the endothelial-stromal cells treated with the serum of endometriotic patients in comparison with hormonally-treated and non-endometriotic groups. These results suggest amelioration of the immune response by endometrial cells when submitted to a serum environment under hormonal treatment. Serum of endometriotic patients (hormonally treated and non-treated) also increased the levels of IL-6 and IL-8 in the co-cultured cells. Limitations, reasons for caution Endometrial biopsies were collected in the initial secretory phase of young oocyte donor women, which may not reflect to the eutopic endometrial response in patients with endometriosis. The reduced size of patients cohort may also increase the risks of bias in cytokine analysis. Wider implications of the findings The possibility of endometrial cells, even in the absence of leukocytes, to become a protagonist in the expression of cytokines and the ability of uterine cells to respond and contribute to a systemic inflammatory/immunomodulating environment with cytokine production may reflect on the pathophysiology of endometriosis and uterine reproductive functions. Trial registration number Not Applicable

Basic concepts of autologous platelet concentrate preparation

The application of PRP products has gained vast popularity in a number of clinical fields of medicine—dermatology, orthopedics, sports traumatology, aesthetic and plastic reconstructive surgery, neurosurgery, maxillofacial surgery, cardiac surgery and others. As their use in practice increases, many existing techniques for preparation become obvious. A basic difference in the methods of extraction of autologous platelet concentrates is the use of standard laboratory centrifuges and consumables or the extraction through variety of commercial systems with specific kits. There are a lot of factors that affect the quality of PRP products—type of anticoagulant, number of centrifugations (spins), time of rotation and centrifugal force and the activator that is used. Differences in these factors lead to products with variations in platelet counts and the presence or absence of leukocytes. This in turn is essential for the levels of growth factors, which are a key element in the recovery process. The main growth factors released by platelet α-granules after activation are: platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), insulin-like growth factor (IGF), and epidermal growth factor (EGF). An important characteristic of PRP is that it represents an autologous product, which is prepared from the patient’s own blood. Therefore, the use of autologous PRP eliminates any concerns about the risk of cross-contamination, disease transmission or immune reactions. By using different methods, it is possible to get various PRP types in regard to the content of bioactive molecules. Beside platelets as a dominant PRP element, other bioactive factors may also be involved in the immune response modulation process.

Commercial blood cell separation systems versus tube centrifugation methods for the preparation of platelet-rich plasma: a preliminary cross-sectional study.

Clinical studies claim that platelet-rich plasma (PRP) accelerates tissue healing due to its high concentration of growth factors and that the combination with leukocytes improves the antimicrobial effect of the concentrate. Most of these studies obtained PRP using different separation systems, and few analyzed the content of the PRP used for treatment. This study characterized the composition of PRP and white blood cells (WBC) from a single donor produced by three commercially available PRP separation systems and two anticoagulated general analytical tubes. Five patients donated 50 mL of blood, which was processed to produce PRP and WBC using three PRP concentration systems (i.e., Easy PRP Kit, GloPRP, and Wego) and two tubes for general analysis anticoagulated with ethylenediaminetetraacetic acid (EDTA) and citrate. Platelets and WBC in combination with their concentrates were analyzed by automated systems in a clinical laboratory. There were no significant differences in the average concentrations of PRP platelets and WBC between GloPRP and the tubes for general analysis with EDTA and citrate; however, the Easy PRP Kit gave results much superior to the rest of the methods, especially comparing it with the Wego Kit, whose concentrates were especially low, even nonexistent for WBC. The Easy PRP Kit concentrates WBC-rich PRP, resulting in increased WBC concentrations, compared with low WBC-low PRP of GloPRP and general tube methods for EDTA and citrate analysis and the even lower concentration of PRP from the Wego Kit, with the absence of leukocytes.

Optimizing Platelet-Rich Plasma (PRP) Injections: A Narrative Review

Platelet-rich plasma (PRP) is an orthobiologic treatment that has gained popularity as a potential alternative treatment for various musculoskeletal conditions. The physiologic role of platelets in the healing cascade provides clarity regarding its potential as it releases various growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta-1 (TGF-β1), and vascular endothelial growth factor (VEGF). However, there are various characteristics of PRP treatments including platelet count, presence or absence of leukocytes and red blood cells, as well as the use of an activating agent that introduces heterogeneity among preparations. This aim of this article is to provide clarity, where available, regarding the optimal characteristics for PRP treatments regarding tendon and ligament injuries as well as articular and muscular pathology.

Detection of Reproductive Status in Ongole Crossbred (PO) Cow Based On Vaginal Epithel Morphology and Profile Hormone

Hormonal fluctuations in livestock will affect vaginal cytology good overview on the condition of estrous until pregnancy. The purpose of this study was to determine the physiological condition of Ongole crossbred (PO) cow during estrous and determine pregnancy by the description of vaginal epithelial cells, progesterone, and estrogen hormone profiles. The materials were used 35 cows with physiological status (estrous, 5th pregnancy period, 16th pregnancy period, 22nd pregnancy period, and 60th pregnancy period). Samples of Vaginal smear were stained with Giemsa, then it was observed using a microscope, with 40 times magnification. The progesterone and estrogen were analyzed by the ELISA method. The parameters measured were the percentage of vaginal epithelial cells, such as (parabasal, intermediate, and superficial) started estrous phase until the time of pregnancy in cows (5, 16, 22, and 60 days), hormone concentration, as well as the presence or absence of leukocytes. The result showed the Ongole crossbred cow estrous phase percentage of superficial cells 56.27%±6.49 higher than 26.23%±7.98 intermediate cells, followed by parabasal cells 17.50%±4.74. While in Ongole crossbred that were 5th pregnancy period until the 60th predominantly intermediate cell 80.43%±1.31, then the superficial cells 18.09%±1.30 and 1.48%±0.04 parabasal cells. Progesterone concentration was 63.74±1.07 ng.mL-1 in estrus cows, and steadily increased 93.71±0.94 ng.mL-1 to 149.5±0.71 ng.mL-1 in pregnant cows (5-60 days). The concentration of high estrogen levels were 122.38 ± 0.63 ng.mL-1 in the estrous phase, then decreased 81.54±0.44 ng.mL-1 in the pregnancy phase. In conclusion, the concentration of hormone showed a diagnosis of pregnancy, which done by looking at changes in vaginal epithelial cells at the Ongole crossbred cow, and the cow estrous phase showed greater superficial cells compared by pregnant cows (5-60 days).

Platelet-Rich Plasma and its Use for Cicatricial and Non-Cicatricial Alopecias: A Narrative Review.

The concept and description of platelet-rich plasma (PRP) started in the field of hematology and is being extensively used in other fields of medicine. Interest in the application of PRP has been increasing in dermatology, such as in tissue regeneration, wound healing, scar revision, skin-rejuvenating effects, and alopecia. PRP is an autologous blood product containing high concentrations of platelets in a small volume of plasma. Different preparations of PRP may lead to different volumes of PRP, platelet concentration, and presence or absence of leukocytes. PRP is being used as a new therapy for some types of non-cicatricial alopecia such as androgenetic alopecia (AGA) and alopecia areata (AA) and, recently, new insights refer to the possibility of action in the field of cicatricial alopecia, like lichen planopillaris (LPP) and frontal fibrosing alopecia (FFA). This article aims to identify the major indications for the application of PRP in the field of hair disorders, including non-cicatricial and cicatricial alopecia.

ENVIRONMENTAL AND CLINICAL CONTEXT IN MENINGOCOCCEMIA: BIOCHEMICAL MODIFICATION OF STIEHM-DAMROSCH/NIKLASSON SEVERITY SCORE - PROGNOSTIC FACTOR

The study considers meningococcemia which is one of the most severe form of meningococcal disease and represents a significant worldwide health problem. The occurrence of meningococcemia was investigated based on a case study developed in a Romanian hospital using data taken from the medical records of patients with the diagnosis of meningococcemia. The pathogen can produce a fulminant form of the disease that can lead to death within a few hours of onset. The signs and symptoms of meningococcemia can include an early upper respiratory tract infection with coryza, pharyngitis, tonsillitis, and/or laryngitis which can rapidly be followed by a collapse of the immune system and can ultimately cause multisystem organ failure. We considered the Stiehm-Damrosch/ Niklasson severity score a useful instrument which includes biochemical and clinical factors. Regarding the biochemical factors of the score, the absence of leukocytes in cerebrospinal fluid together with the low level of glucose can be considered important predictors of a severe prognosis.

PLASMA RICH IN GROWTH FACTORS FOR PERSISTENT MACULAR HOLE: A PILOT STUDY.

Persistent macular hole (PMH) is a challenge for retina surgeons. There are limited surgical techniques described achieving PMH closure. For more than a decade, techniques with blood derivatives have been described without optimal outcomes. The aim of this article is to report the usefulness of the plasma rich in growth factors (PRGF) as a new way to improve PMH closure. This is an observational study. Two patients with PMH were chosen at the FOSCAL Internacional, Floridablanca, Colombia. For each patient, a complete ophthalmological examination before and after treatment was performed. It included best-corrected visual acuity and fundus examination. Features of swept-source optical coherence tomographic angiography (Topcon) at baseline and after treatment were included. Preoperative evaluation included the following: previous vitreoretinal surgery for macular hole, macular hole measurement and classification, and dissociated optic nerve fiber layer in en-face map, confirming a prior internal limiting membrane peeling. Plasma rich in growth factors-Endoret technology is characterized by moderate platelet concentration and absence of leukocytes, which requires calcium chloride for platelet activation. It is obtained from each patient's blood sample, submitted to a series of processes to acquire a three-dimensional fibrin scaffold, rich in hundreds of proteins, which seems to have therapeutic potential. This PRGF membrane is placed into the macular hole with forceps through a pars plana vitrectomy after complete air-fluid exchange. Supernatant was placed on the macula and left it for 2 minutes. It is then aspirated, and gas or silicone oil was left as a tamponade. Follow-up period was of 12 months for each patient. Complete macular hole closure was achieved in both cases. Best-corrected visual acuity changed from 20/200 preoperatively to 20/100 postoperatively in Patient #1 and from 20/1,500 to 20/100 in Patient #2. Plasma rich in growth factors has been primarily investigated for ocular surface diseases. This is a pilot study using this technology for PMH treatment. The use of PRGF may be an effective and safe surgical technique with satisfactory anatomical and functional results for PMH. Because of its own biomaterials and proteins, PRGF not only allows anatomical closure but also seems to stimulate tissue regeneration.

SUN-229 The Role of Testosterone in Spermatogenesis: Lessons from Proteome Profiling of Human Spermatozoa in Testosterone Deficiency

Testosterone is required during spermatogenesis to mantain the blood-testis barrier and for meiosis, Sertoli-spermatid adhesion and sperm release. Aim of the study was to better understand the effect of hypothalamic-pituitary-testicular axis on sperm protein composition and function.12 male patients with low testosterone and LH levels were consecutively enrolled for this study. Five normogonadic men were enrolled as control group in the protocol. If spermatozoa were detectable in seminal samples, the the ejaculates were washed and the sperm cells were selected using 50% PercollTM. The recovered sperm cells were then suspended in PBS and subjected to a residual leukocyte depletion using CD45 Dynabeads® magnetic cell sorting. The absence of leukocytes was further confirmed both by microscopic observation after staining and by performing a RT-PCR for CD45. Protein solubilization was independently performed on each sperm sample. A comparative proteomic analysis was performed comparing two pools: controls vs hypogonadic patients. The TMTduplexTM Isobaric Mass Tagging Kit was used for the peptide labeling. MS/MS analysis was performed using a LTQ Orbitrap Velos. Data were processed using Proteome Discoverer 1.4.1.14 software, using very stringent filtering criteria. Proteins identified by SEQUEST were analyzed using the PANTHER classification system. Proteomic results have been confirmed by Western Blot. The following primary antibody were used: anti-Lactoferrin polyclonal antibody and anti-PIP polyclonal antibodyWe included in our study 5 oligozoospermic hypogonadic patients with a sperm count >10 x 106/ml and without leukocyte contamination. 42 proteins were under-expressed in the pool of hypogonadic patients; 12 of these 42 increased proteins have been previously reported to be related with spermatogenesis, sperm motility, sperm-oocyte interaction and acrosomal reaction. The distribution of proteins for molecular function revealed that the protein pattern impaired by low blood testosterone is involved in protein binding and hydrolase. Twelve proteins have been over-expressed in the pool of hypogonadic patients, including markers of defective spermatogenesis. Western-blot analysis confirmed proteomic data. This is the first application of high resolution mass spectrometry -based proteomics aimed to reveal an array of proteins in spermatozoa reflecting an impairment of spermatogenesis and sperm function in male secundary hypogonadism.

Leukocyte Distribution in the Open Eye Tears of Normal and Dry Eye Subjects

ABSTRACTPurpose: Leukocytes accumulate in the eye with sleep, but little is known about the presence or absence of leukocytes in awake, open eye tears. This study sought to compare normal and dry eye subjects for daily variation in open eye leukocyte composition.Materials and Methods: Ten normal subjects and nine dry eye subjects were enrolled. Subjects were trained for self-collection of tear samples using an ocular surface wash with 5 mL of phosphate buffered saline per eye. Subjects performed washes at awakening, between 8 and 9 am, between 11 am and 12 pm, and between 4 pm and 5 pm on four separate days. Leukocytes were isolated from the wash and were counted with a cell counter before staining with an anti-CD45 antibody and viability stain. Stained leukocytes were then analyzed via flow cytometry. Side scatter characteristics were used to distinguish granulocytes from lymphocytes. Results were interpreted both by time of wash as well as time from awakening.Results: At awakening, dry eye subjects had approximately twice as many recovered leukocytes and had a statistically significantly higher granulocyte-to-lymphocyte ratio as compared to normals. Leukocytes were rapidly cleared from the eye with a significant decrease in leukocyte counts at the 8 am time point as compared to awakening. Leukocyte counts across all open eye time points appeared to be consistent, with no differences between normal and dry eye subjects.Conclusions: There is a low level, constitutively expressed population of leukocytes in the open eye tears of normal and dry eye subjects. Higher levels of granulocytes in dry eye disease subjects warrants further investigation into this population of cells, and their role in homeostasis and dysregulation.

Inducible Lung Epithelial Resistance Requires Multisource Reactive Oxygen Species Generation To Protect against Viral Infections.

ABSTRACTViral pneumonias cause profound worldwide morbidity, necessitating novel strategies to prevent and treat these potentially lethal infections. Stimulation of intrinsic lung defenses via inhalation of synergistically acting Toll-like receptor (TLR) agonists protects mice broadly against pneumonia, including otherwise-lethal viral infections, providing a potential opportunity to mitigate infectious threats. As intact lung epithelial TLR signaling is required for the inducible resistance and as these cells are the principal targets of many respiratory viruses, the capacity of lung epithelial cells to be therapeutically manipulated to function as autonomous antiviral effectors was investigated. Our work revealed that mouse and human lung epithelial cells could be stimulated to generate robust antiviral responses that both reduce viral burden and enhance survival of isolated cells and intact animals. The antiviral protection required concurrent induction of epithelial reactive oxygen species (ROS) from both mitochondrial and dual oxidase sources, although neither type I interferon enrichment nor type I interferon signaling was required for the inducible protection. Taken together, these findings establish the sufficiency of lung epithelial cells to generate therapeutically inducible antiviral responses, reveal novel antiviral roles for ROS, provide mechanistic insights into inducible resistance, and may provide an opportunity to protect patients from viral pneumonia during periods of peak vulnerability.

Nanoscale Imaging Reveals a Tetraspanin-CD9 Coordinated Elevation of Endothelial ICAM-1 Clusters.

Endothelial barriers have a central role in inflammation as they allow or deny the passage of leukocytes from the vasculature into the tissue. To bind leukocytes, endothelial cells form adhesive clusters containing tetraspanins and ICAM-1, so-called endothelial adhesive platforms (EAPs). Upon leukocyte binding, EAPs evolve into docking structures that emanate from the endothelial surface while engulfing the leukocyte. Here, we show that TNF-α is sufficient to induce apical protrusions in the absence of leukocytes. Using advanced quantitation of atomic force microscopy (AFM) recordings, we found these structures to protrude by 160 ± 80 nm above endothelial surface level. Confocal immunofluorescence microscopy proved them positive for ICAM-1, JAM-A, tetraspanin CD9 and f-actin. Microvilli formation was inhibited in the absence of CD9. Our findings indicate that stimulation with TNF-α induces nanoscale changes in endothelial surface architecture and that—via a tetraspanin CD9 depending mechanism—the EAPs rise above the surface to facilitate leukocyte capture.

Abstract 422: The role of CCL2 in the breast cancer tumor microenvironment and metastasis

Abstract Background: Although many effective breast cancer treatments exist, metastasis remains a major problem. Increasing evidence suggests that leukocyte-associated chemokines may play an important role in tumor growth and metastasis. For instance, recent data shows that reduction of CCL2 diminishes the recruitment of inflammatory monocytes to the tumor microenvironment (TME) and inhibits metastatic seeding to lung and bone marrow. Therefore, our goal is to examine the impact of CCL2 on the TME and pre-metastatic niche (i.e., lung). Methods: We are investigating how delivery of this chemokine affects breast tumor growth and metastasis by treating mice possessing highly metastatic and poorly metastatic tumors with CCL2, a CCL2 receptor (CCR2) antagonist, or vehicle for two weeks. Treatment will start 7 days after tumor implantation. On days 7, 14 or 21, tumors, lungs, and regional lymph nodes will be harvested and tumor size, number of metastases, and leukocyte infiltrate in both the tumor and the metastatic niche will be analyzed by FACS. This will allow us to fully characterize how intranasal delivery of CCL2 will affect the presence of mesenchymal stem cells (CD44+, CD73+, CD90+, CD146+/NOTCH3+, CD19-, CD45-), subsets of T cells, and myeloid cells in the TME or pre-metastatic niches. We will evaluate the intracellular cytokine and enzymatic profile (IL-4, IL-13, IL-17, ARG1 or IFNγ, IL-12, iNOS2) of Brefeldin-treated leukocytes to reveal whether they are pro- or anti-tumorigenic, respectively. Results: Our preliminary studies demonstrate that CCL2 enhances the release of hydrogen peroxide from neutrophils, a mechanism utilized by these cells to kill tumors cells. Furthermore, the addition of CCL2 to co-cultures of neutrophils and tumor cells increases the ability of neutrophils to kill tumor cells. Conclusions & Future Directions: Our data shows that delivery of CCL2 in vitro enhances the activity of neutrophils and their ability to kill tumor cells. Ongoing studies are examining the effects of in vivo and ex-vivo delivery of CCL2 or a CCR2 antagonist on primary tumor growth and metastasis. We will also investigate how administration of CCL2 or a receptor antagonist will affect the presence or absence of leukocytes in the TME. These findings will determine whether the addition of CCL2 during treatment of the primary tumor will provide a mechanism for reducing the potential of future breast cancer metastases. Citation Format: Nicole Lavender, Jiqing Sai, Jinming Yang, Sergey V. Novitskiy, Ann Richmond. The role of CCL2 in the breast cancer tumor microenvironment and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 422. doi:10.1158/1538-7445.AM2015-422

Clinical and epidemiological differences between septic arthritis of the knee and hip caused by oxacillin-sensitive and -resistant s. aureus

OBJECTIVE:To establish the risk factors for joint infection by oxacillin-resistant Staphylococcus aureus (MRSA) using clinical and epidemiological data.METHODS:All septic arthritis cases of the knee and hip diagnosed and treated in our institution from 2006 to 2012 were evaluated retrospectively. Only patients with cultures identified as microbial agents were included in the study. The clinical and epidemiological characteristics of the patients were analyzed, seeking the differences between populations affected by MRSA and oxacillin-sensitive Staphylococcus aureus (MSSA).RESULTS:S. aureus was isolated in thirty-five patients (46.0%) in our total sample, 25 in the knee and 10 in the hip. Of these 35 patients, 22 presented with MSSA and 13 presented with MRSA. Provenance from a health service-related environment, as described by the Centers for Disease Control and Prevention, was the only variable associated with oxacillin-resistant strains of this bacterium (p = 0.001).CONCLUSION:Provenance from a health service-related environment was associated with a higher incidence of MRSA-related septic arthritis, suggesting that this agent should be considered in the initial choice of antibiotic treatment. Previous surgeries of the knee or affected limb and the absence of leukocytes might also be related to infection with this agent.

Complement-Mediated Cellular Injury

Complement activation and recruitment of inflammatory leukocytes is an important defense mechanism against bacterial infection. However, complement also can mediate cellular injury and contribute to the pathogenesis of various diseases. With the appreciation that the C5b-9 membrane attack complex can injure cells in the absence of leukocytes, a role for the terminal complement pathway in inducing cell injury and kidney disease was shown in several experimental models, including the rat passive Heymann nephritis model of human membranous nephropathy. In podocytes, sublytic C5b-9 activates a variety of downstream pathways including protein kinases, lipid metabolism, reactive oxygen species, growth factors/gene transcription, endoplasmic reticulum stress, and the ubiquitin-proteasome system, and it impacts the integrity of the cytoskeleton and slit diaphragm proteins. C5b-9 also injures other kidney cells, including mesangial, glomerular endothelial, and tubular epithelial cells, and it contributes to the pathogenesis of mesangial-proliferative glomerulonephritis, thrombotic microangiopathy, and acute kidney injury. Conversely, certain C5b-9 signals limit complement-induced injury, or promote recovery of cells. In addition to C5b-9, complement cleavage products, such as C5a and C1q, can injure kidney cells. Thus, the complement system contributes to various kidney pathologies by causing cellular damage in both an inflammation-dependent and inflammation-independent manner.

An unusual primary manifestation of systemic lupus erythematous in a Nepalese patient: The influence of epidemiological history

Abrupt and life-threatening presentations of multisystemic inflammatory diseases are rarely reported. A 31 year old Nepalese male patient, Buddhist, in Portugal since 2011, presented to the Emergency Department (ED) in September 2012 with fifteen days history of malar rash. He had been treated with an unknown drug 2 weeks ago. One week later, fever, diarrhea, sore throat, cough, chest pain, nonspecific abdominal pain, asthenia, marked muscle weakness, oral ulcers and unquantified weight loss appeared. Previously healthy. On examination, prostrate, afebrile; eupneic, BP: 102/70 mm Hg, HR: 99 bpm; vesicular, labial and gengival ulcers, eyelid and periorbital edema, malar rash with hyperpigmented and purpuric lesions, generalized exanthema also with hyperpigmented and purpuric macular lesions (affecting palms, trunk, extremities); left ear lobe lesion (erythematous base, slightly scaly and hyperpigmented); oropharynx with hyperaemia; normal heart sounds; crackles on the right low lung; painful abdomen, hepatomegaly and cervical ganglia were present. The laboratory exams revealed hemoglobin 12.9 g/dL, APTT 40.5 s (28), ferritin 1558 mg/dl, BUN 20 mg/dl, creatinine 1.39 mg/dl, proteins 5.1 mg/dl, albumin 2.1 mg/dl, AST 73 U/l, LDH 490 U/l, CK 337 U/l, myoglobin 334 ng/ml, VS 25 mm/1st hour. Ceftriaxone and azithromycin were empirically prescribed and switched to doxycycline (on day 10) because of persistent fever and progression of generalized rash. The remaining exams showed: proteinuria 7956 g/24 h, active urinary sediment, hypocomplementemia; chest X-ray and computed tomography angiography with bilateral pleural effusion, normal echocardiography; absence of leukocytes in feces; microbiological cultures (blood, urine, feces), serological research of Legionella, Streptococcus pneumoniae, Toxoplasma gondii, Rubella, HSV1/HSV2, CMV, Syphilis screening (CLIA), Chlamydia trachomatis, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Coxiella burnetii, Brucella (Rose Bengal) Borrelia burgdorferi, Leishmania, HIV1, 2, HBV and HCV, B19 Parvovirus and HH6 negative. Legionella 1/1000, Rickettsia conorii 1/80. Mantoux test anergic. ANA homogeneous pattern + + + +, anti-dsDNA Ab > 50; anti-dnDNA Ab, antiSSA60/SSB/Sm/RNP/ScL 70 Abs positives; myelogram without Leishmania's presence. The renal biopsy revealed diffuse proliferative lupus glomerulonephritis (LGN) with sclerosis lesions and extensive crescent formations (stage IV). This biopsy was traumatic with a consequent retroperitoneal hematoma with 15.7 cm in greatest axial axis submitted to catheterization. After SLE (systemic lupus erythematous) diagnosis was made, with bad prognostic factors, hydroxychloroquine and induction's therapy with methylprednisolone and cyclophosphamide (NIH protocol) was performed, in addition to atorvastatin, enalapril and furosemide, with an excellent clinical evolution. SLE can be a difficult to diagnose when severe forms are its first manifestation. Epidemiological history and the severity of presentation are extremely important on the differential diagnosis process (infection exclusion, the most), otherwise, the early recognition of SLE and specific management could change the course of the disease. When SLE first manifestations are severe, rapid diagnosis is important. In the case of a fever of unknown origin in a subject from a distant country, clinical suspicion and exclusion of infections caused by less frequent microorganisms in our geographical context are fundamental.

Lung epithelial cells are essential effectors of inducible resistance to pneumonia.

Infectious pneumonias are the leading cause of death worldwide, particularly among immunocompromised patients. Therapeutic stimulation of the lungs' intrinsic defenses with a unique combination of inhaled Toll-like receptor (TLR) agonists broadly protects mice against otherwise lethal pneumonias. As the survival benefit persists despite cytotoxic chemotherapy-related neutropenia, the cells required for protection were investigated. The inducibility of resistance was tested in mice with deficiencies of leukocyte lineages due to genetic deletions and in wild-type mice with leukocyte populations significantly reduced by antibodies or toxins. Surprisingly, these serial reductions in leukocyte lineages did not appreciably impair inducible resistance, but targeted disruption of TLR signaling in the lung epithelium resulted in complete abrogation of the protective effect. Isolated lung epithelial cells were also induced to kill pathogens in the absence of leukocytes. Proteomic and gene expression analyses of isolated epithelial cells and whole lungs revealed highly congruent antimicrobial responses. Taken together, these data indicate that lung epithelial cells are necessary and sufficient effectors of inducible resistance. These findings challenge conventional paradigms about the role of epithelia in antimicrobial defense and offer a novel potential intervention to protect patients with impaired leukocyte-mediated immunity from fatal pneumonias.