To reveal bound antibodies we used horseradish peroxidase (HRP)-c

To reveal bound antibodies we used horseradish peroxidase (HRP)-conjugated secondary antibodies. Blots were developed with enhanced chemiluminescence (ECL) reagent (Pierce; Thermo Scientific, Rockford, IL, USA). To obtain semi-quantitative

estimates for the total tyrosine phosphorylation, it was quantified and densitometry analysis was performed using Tina 2·0 software (Raytest, Straubenhardt, Germany). Values were normalized to the intensity of actin bands. For comparisons of quantitative values we used the unpaired Student’s t-test. The frequency of autoantibodies in HAE patients and control group was compared using Fisher’s exact test. Two-tailed P-values of 0·05 or less were considered statistically significant. Data are expressed as mean values of MFI ± s.d. In 29 of the 61 (47·5%) patients, at least one of the tested autoantibodies was found in the serum, as detailed

in Table 1. We did Selleck Tigecycline not find any difference in gender ratio when HAE patients with autoantibodies were compared with those without autoantibodies [male (12 of 25), female (17 of 36)]. Additionally, we did not find a difference in the average mean of the complement 4 (C4) levels between these two groups of HAE patients [0·095 ± 0·05 versus 0·088 ± 0·05, P = not significant (n.s.)]. In the healthy control group, five of 50 (10%) had serum autoantibodies. This frequency is statistically lower compared to HAE patients [five of 50 (10%) versus 29 of 61 (47·5%), P = 0·0001]. Two had positive anti-nuclear antibodies (4%), two of 50 Phosphoglycerate kinase (4%) had anti-cardiolipin antibodies and in one serum we found positive anti-S. cerevisiae antibodies. Seven of 61 HAE patients (11·4%) suffered from the following selleck inhibitor immunoregulatory disorders; one patient had systemic lupus erythematosus (SLE), two patients had coeliac disease,

one patient had mixed connective tissue disease, one patient had systemic sclerosis, one patient had Crohn’s disease and one patient multiple sclerosis-like syndrome. Expression of CD69 and CD5 was found to be statistically higher on memory B cells (CD19+CD27+) from HAE patients compared to healthy controls (4·59 ± 4·41 versus 2·06 ± 1·81, P = 0·04, 8·22 ± 7·17 versus 3·65 ± 3·78, P = 0·05, respectively). Expression of CD21 on memory B cells was also significantly higher when compared to that on memory B cells from healthy controls (2·43 ± 0·54 versus 1·92 ± 0·41, P = 0·01). In contrast, we did not find any statistical difference in the expression of MHC-II, CD40 and CD86 on the memory B cells of the two groups. Results are summarized in Table 2. Memory B cells isolated from the HAE group expressed a significantly higher amount of TLR-9 (8·17 ± 4·1 versus 4·56 ± 1·6, P = 0·0027). Furthermore, the expression of TLR-9 in B cells from HAE patients who had autoantibodies was much higher than that of memory B cells from both the control group (10 ± 4·7 versus 4·56 ± 1·6, P = 0·0002) and from HAE patients without autoantibodies (10 ± 4·7 versus 5·8 ± 0·9, P = 0·036).

Fibrinolysis is an important defence mechanism against thrombosis

Fibrinolysis is an important defence mechanism against thrombosis, but has only been studied locally in BP and no systemic data are available. The aim of this observational study was to evaluate systemic fibrinolysis and coagulation activation in patients with BP. We measured parameters of fibrinolysis and coagulation by immunoenzymatic methods in plasma from 20 patients with BP in an active phase and during remission after

corticosteroid treatment. The controls were 20 age- and sex-matched healthy subjects. Plasma levels of plasminogen activator inhibitor type 1 (PAI-1) antigen, PAI-1 activity and tissue plasminogen activator (t-PA) antigen were significantly higher in the BP patients with active disease than in healthy controls (P = 0·0001 for all), as were the plasma levels of the fibrin fragment d-dimer and prothrombin fragment

F1+2 (P = 0·0001 for both). During remission after treatment, levels of PAI-1 VX 770 antigen and PAI-1 activity decreased significantly (P = 0·008 and P = 0·006, respectively), and there was also a significant decrease in plasma levels of d-dimer (P = 0·0001) and F1+2 (P = 0·0001). Fibrinolysis is inhibited in patients with active BP, due mainly to an increase in plasma levels of PAI-1. Corticosteroids not only induce the regression of BP lesions, but also reduce the inhibition of fibrinolysis, which may contribute Ceritinib supplier to decreasing thrombotic risk. Bullous pemphigoid (BP) is an autoimmune blistering disease that occurs typically in the elderly [1] and is burdened with a high risk of death, due mainly to sepsis and cardiovascular events [2]. It involves the skin and rarely the mucous membranes, and is characterized by the presence of blisters usually surrounded by erythematous–oedematous lesions. The diagnosis is supported by histology showing

a subepidermal blister with a dermal mixed inflammatory cell infiltrate usually rich in eosinophils, and a direct immunofluorescence examination of perilesional skin revealing the linear deposition of immunoglobulin (Ig)G and/or C3 in the basement membrane zone (BMZ). Circulating Vorinostat anti-BMZ autoantibodies can be detected by means of an enzyme-linked immunosorbent assay (ELISA) for two hemidesmosomal antigens, BP180 and BP230 [3, 4]. Autoantibodies against these antigens play an important role in the pathogenesis of BP, as well as complement activation and leucocyte infiltration [1, 5]. Inflammatory cells (particularly autoreactive T cells and eosinophils) participate in blister formation by producing and releasing a number of cytokines and soluble factors that amplify and maintain tissue damage [6-8]. The inflammatory response induces an activation of blood coagulation which is involved both locally, by amplifying the inflammatory network in lesional skin, and systemically, by leading to a prothrombotic state [4, 9-12].

Representative Th17 cell clones from ovarian and colon cancers ar

Representative Th17 cell clones from ovarian and colon cancers are shown in Fig. 1A. To further investigate whether these tumor-infiltrating Th17 clones were homogeneously expanded from a single cell or were comprised of heterogeneous cell populations, TCR-Vβ gene expression was determined using RT-PCR with TCR-Vβ-specific Pictilisib in vitro primers 29, 30. As shown in Fig. 1B, two Th17 clones (CTh17-18 and CTh17-20) derived from the colon cancer TILs of different patients shared the same TCR-Vβ6A gene, and the OTh17-8 clone derived from an ovarian

cancer TILs expressed TCR-Vβ13B gene. We analyzed TCR-Vβ gene expression in primary (E0) Th17 clones and Th17 clones following different rounds of expansion (E1–E3) and obtained the same expression patterns (data not shown). Thus, the results of TCR profiling analyses confirmed that each of these Th17 clones had been expanded from a single cell. We next sought to determine gene expression levels of the lineage-specific transcriptional factors learn more in these Th17 clones using real-time PCR. As expected, we found that all primary Th17 clones (E0) markedly expressed RORγt and IRF-4 when compared with naïve CD4+ T cells (Fig. 1C). In contrast, Th17 clones had minimal or

no expression of T-bet, GATA3 and FOXP3, which are critical transcriptional regulators for Th1, Th2 and Treg development, respectively 6. Recent studies have suggested that Th17 cells exhibit distinct cytokine and chemokine receptor expression profiles which are involved in their regulation and biological functions 31–34. Thus, we next evaluated the mRNA expression of cytokines elaborated by the tumor-infiltrating Th17 clones after stimulation with OKT3, using real-time PCR. Representative second data from three primary Th17 clones (E0) are shown in Fig. 1D. Th17 clones expressed high levels of IL-17A and IL-22, and moderate levels of IL-21, but

not IL-4 and IFN-γ, all consistent with previous reports characterizing Th17 cells from other tissue sites 19, 33, 35, 36. These results were further confirmed by ELISA analysis of secreted cytokines in Th17 clone culture supernatants (data not shown). Unexpectedly, we found that these primary Th17 clones minimally expressed IL-23 receptor (IL-23R), although recent studies have suggested that Th17 cells highly express IL-23R, and that IL-23 plays a critical role as a growth/stabilization and development factor for late-stage Th17 cells 12, 19, 37. We then analyzed chemokine receptor expression on Th17 clones by FACS analysis. We observed that all Th17 clones expressed CCR2, CCR4, CCR5, CCR6, CCR7 and CXCR3, similar to the expression pattern in other T-cell lineages, including Tregs 27, 38.

Therefore, higher absolute IDWG needs to be strictly controlled d

Therefore, higher absolute IDWG needs to be strictly controlled despite the corresponding IDWG% possibly being relatively small in heavy haemodialysis patients. “
“Podocytes (glomerular epithelial cells) lie on the urinary aspect of the glomerular capillary and play a key role in the selective filter that underlies click here kidney function. They are injured in various forms of renal disease: the extents of this injury and its reversibility have major implications for treatment and prognosis. Until recently, podocytes were difficult

to study in vitro because of a previous lack of techniques for obtaining differentiated cells in quantities adequate for research. In recent years, this problem has been solved for rodent and human podocytes and there has been an explosion of research using cultured cells. These authors have led the development and characterization of human podocyte cell lines and in this article describe the INK-128 methods that have allowed them to do this. In recent years, one of the fastest moving areas of research progress in nephrology has been the appreciation of the importance

of the visceral glomerular epithelial cell, hereinafter referred to as the podocyte, in health and disease. Podocytes play a key role in the prevention of proteinuria in the healthy situation, are important targets of injury in a variety of renal diseases and are important determinants of outcome.1,2 Improved understanding of podocyte biology has however come from two main arenas: first, molecular genetics of single gene disorders which lead to rare forms of congenital nephrotic syndrome; and second, focused study of this specialized cell type in vivo and in vitro. The purpose of this article is to review the current state of knowledge in relation to the in vitro study of podocytes. The authors have most experience of human podocyte culture, but where relevant we will also discuss study of podocytes from

other species. Our aim is to help new investigators to join this exciting field. When cells are directly separated from tissue and propagated in vitro they are referred to as ‘primary culture cells’. For podocytes, this typically requires isolation of glomeruli by differential sieving, plating of glomeruli onto a collagen surface (use of collagen surface is optional, currently we use tissue culture treated surface instead) and outgrowth of cobblestone-like cells (further details will be given later). Some of the early work on rat3 and human4 podocytes used primary culture podocytes, but the problem was that these cells did not develop the features of differentiated cells and they continued to proliferate, whereas differentiated podocytes are quiescent cells that do not proliferate. When specific markers of differentiated podocytes (such as nephrin and podocin) became known in the early 1990s, it was clear that podocytes suitable for in vitro study needed to demonstrate expression of these markers.

It is applicable for direct detection in stained sputum smear pre

It is applicable for direct detection in stained sputum smear preparations, which help in reducing the time needed for bacterial growth

and should facilitate the adequate choice of antituberculosis therapy (Johnson et al., 2006) limiting the extent and severity of MDR-TB transmission and infection. Our data suggest that Jordan may soon face a rapid increase in the number of new cases of drug-resistant tuberculosis, and therefore the application of a simple PCR method for easy detection of drug resistance in such a resource-limited area for regular monitoring of drug resistance patterns is essential. The authors Vincristine concentration thank Drs Yusra Rehani and Saied Abu Nadi in the TB section in the Directorate of Chest Diseases and Foreigners Health for providing the drug-resistant M. tuberculosis isolates. This study was supported by grant 133/2007 from the Deanship of research at Jordan University of Science and Technology, Irbid, Jordan. “
“The mammalian target of rapamycin (mTOR) pathway is an important integrator of Src inhibitor nutrient-sensing signals in all mammalian

cells, and acts to coordinate the cell proliferation with the availability of nutrients such as glucose, amino acids and energy (oxygen and ATP). A large part of the immune response depends on the proliferation and clonal expansion of antigen-specific T cells, which depends on mTOR activation, and the pharmacological inhibition

of this pathway by rapamycin is therefore potently immunosuppressive. It is only recently, however, that we have started to understand the more subtle details of how the mTOR pathway is involved in controlling the differentiation of effector versus memory CD8+ T cells and the decision to generate different CD4+ helper T-cell subsets. In particular, this review will focus on how nutrient sensing via mTOR controls the expression of the master transcription factor for regulatory T cells in order to maintain the balance between tolerance and Chloroambucil inflammation. All cells need to be able to coordinate their proliferation and differentiation with their metabolic demands and the availability of essential nutrients. The mammalian target of rapamycin (mTOR) signalling pathway acts as an important integrator of nutrient-sensing pathways, which in turn control and coordinate the metabolism of the cell according to its need to proliferate or functionally differentiate.[1] T-cell activation is intimately coupled to metabolism and energy generation, with a switch from primarily oxidative phosphorylation in resting T cells to an aerobic form of glycolysis, known as the ‘Warburg effect’,[2] during activation and proliferation.

We also look forward to OAB assessment with universal acceptance

We also look forward to OAB assessment with universal acceptance of in the future. “
“Objectives: We studied the influence of preoperative detrusor underactivity in patients with stress urinary incontinence on the postoperative continence rates and patient satisfaction. Methods: Medical records of 41 female patients who had detrusor underactivity and had undergone a midurethral sling procedure with a follow up of at least 12 months were reviewed. The preoperative evaluation included a history taking, physical examination, voiding diary for 3 days and an urodynamic study. Detrusor underactivity was defined at pressure flow study

by a maximal flow rate (Qmax) less than 15 mL/sec and a detrusor pressure at maximal flow rate (PdetQmax) less than PS-341 price 20 cmH2O. The postoperative evaluation included a continence state, questionnaire regarding patient satisfaction (5: very satisfied, 1: check details very unsatisfied), uroflowmetry and residual urine volume. Results: The mean patient age was 52.9 (range 39–68) years. Preoperatively, mean Qmax was 12.6 ± 2.1 mL/sec, mean residual urine volume was 16.1 ± 32.3

mL and mean PdetQmax was 13.1 ± 4.7 cmH2O. Postoperative continence rate was 88% (36/41). Five patients experienced minimal incontinence when they coughed violently. The amount of patients satisfied with postoperative status was 71%. Postoperatively, three patients needed medication with alpha blocker because of voiding difficulty. There was significant differences between preoperative and postoperative Qmax (13.1 ± 0.9 mL/sec vs 17.1 ± 0.9 mL/sec, P < 0.05). In addition postoperative residual urine volume (26.1 ± 27.9 mL) was significantly increased compared to the preoperative residual urine volume (16.1 ± 32.3 mL) (P < Adenosine triphosphate 0.05). Conclusion: Midurethral sling

can be done safely for the patients with stress urinary incontinence and detrusor underactivity. However, the evaluation of preoperative detrusor function is important since the therapeutic outcome and postoperative voiding pattern may be affected by detrusor underactivity. “
“Objectives: The possible relationship between urological disease and inferior vena cava (IVC) reflux was examined. Methods: Transabdominal color Doppler ultrasonography of the IVC was performed. The patient was placed supine and the convex probe was positioned in vertical to the upper abdominal wall. Then the extent of reflux in the IVC accompanying each heart beat was examined near the diaphragm. A total of 403 patients (202 males and 201 females aged 12–90 years) were studied. The relationship between the existence of IVC reflux or its severity and urological disease was examined. Results: The 202 males included 104 and 98 subjects without and with IVC reflux, respectively, while the 201 females included 64 and 137 subjects without and with IVC reflux, respectively. The prevalence of IVC reflux was significantly higher in females than males.

T helper type 2 development can be influenced by such cytokines a

T helper type 2 development can be influenced by such cytokines as IL-33 and thymic stromal lymphopoietin,54,55 but IL-4 remains the primary signal that drives Th2 commitment from naive precursors.55,56 The Th2 differentiation involves the integration of signals both from

the T-cell receptor and from IL-4 signalling via STAT6, which culminates in the induction of the GATA3 transcription factor. GATA3 subsequently promotes transcription at the Th2 cytokine locus containing the IL-4, IL-5 and IL-13 Stem Cell Compound Library clinical trial genes. This pathway also acts acutely to inhibit expression of the IL-12Rβ2 subunit.57 Consequently, induction of GATA3 serves to block Th1 development while positively regulating Th2 commitment. Moreover, while there seems to be some level

of plasticity in Th2 cells,58 GATA3 is involved in an autoregulatory feedback loop check details that maintains Th2 commitment even in the absence of further IL-4 signalling.59,60 Hence, autoregulation by GATA3 represents an important stabilizing mechanism for Th2 commitment. However, early reports demonstrated that IFN-α/β could inhibit IL-5 secretion and eosinophil migration during allergic responses.61,62 Furthermore, IFN-α/β treatment of bulk CD4+ T cells during acute stimulation seemed to inhibit IL-5, but not IL-4 or IL-13. This was somewhat curious considering the dominant role played by IL-4 and GATA3 in Th2 effector function. Yet, despite these and other similar studies, one central question remained: can IFN-α/β regulate the ability of IL-4 to drive Th2 differentiation? Recently, Huber et al.63 found that unlike the Th1-promoting cytokines IL-12 and IFN-γ, IFN-α/β potently and specifically inhibited the ability of IL-4 to drive Th2 differentiation of human cells but not murine cells. Moreover, IFN-α/β destabilized pre-committed Th2 cells and blocked Th2 cytokine expression. Interferon-α/β also reduced expression of the Th2 marker, CRTH2. It appears to do this, at least in part, by suppressing mRNA and protein levels of GATA3, Amisulpride which is critical for expression of CRTH2 as well as Th2-associated cytokines. While the underlying mechanism of GATA3 suppression is not yet clear, there are a few clues. First, as neither IL-12 nor

IFN-γ inhibits Th2 commitment, the effect is not likely to be mediated by STAT4 or STAT1. Furthermore, the inhibition of Th2 cells by IFN-α/β paralleled recent studies demonstrating that type-III interferon (IFN-λ) can also suppress Th2 responses.64 Since both IFN-α/β and IFN-λ activate STAT2 and drive ISGF3 complex formation,65 STAT2 may play a crucial role in suppressing human Th2 development. In addition to Th2 cells, there is increasing evidence that Th17 cells contribute to a variety of inflammatory processes involved in autoimmunity and allergic diseases.66 The Th17 cells are regulated by combined signalling via transforming growth factor-β, IL-6, IL-23 and IL-1β, culminating in the induction of the transcription factor retinoic acid-related orphan receptor γT.

30 Next to the standardization element, this NIH GU mucosal immun

30 Next to the standardization element, this NIH GU mucosal immunology working group will study number and functional phenotypes of cells obtained from selleck compound non-invasive specimens with brushes and see if they are equivalent to those isolated from biopsies. This will then inform the field to what extent endocervical

sampling is representative for the local cellular immunology. Good results have been obtained in measuring total cell number and their phenotype but it remains to be seen if cell yield is sufficiently high enough to follow antigen-specific responses to an HIV vaccine. Ulcerative and non-ulcerative pathogens that infect the vagina have been shown to affect the local immunity.31,32 The presence of these pathogens may confound the relationship between an experimental medication

or vaccine and the local immune response. Therefore it is important to test for sexually transmitted infections such as C. trachomatis, N. gonorrhea, T. vaginalis, Human papilloma virus selleck as well as shedding for HSV. In addition, serum samples should be taken to test for active syphilis, HIV and HSV. Menstrual cycle phase have been shown to be a powerful determinant for levels of cytokines and numbers of immune cells.24,33 Serum or urine samples for endogenous hormones should be collected and menstrual phase applied to the analysis of results. Finally, vitamin D is an important immune modulator, can be tested in serum and considered as a confounder.34 Planning a sampling strategy for a clinical trial requires balancing of study objectives and endpoints, participant acceptability, available infrastructure and study budget. Extra samples may be needed to account for local confounding factors

as mentioned above, other vaginal infections (yeast, bacterial vaginosis) and to perform diagnostic tests (Amsel versus Nugent, InPouch versus wet mount) for the participant. The use of antiretroviral drugs for the prevention of heterosexual transmission introduced concerns about the development of HIV drug resistance and has heightened the interest in product PK/PD. As a result cervical, endocervical and uterine biopsies are now more commonly collected in Phase I safety studies. Figure 2 selleck screening library proposes a basic set of samples to study mucosal immunology and confounding factors without additional biopsies. The figure incorporates sampling methods, volumes of samples, markers, storage issues and volumes for assays. The sampling strategy is specific to each individual trial, since each trial has its own objectives and endpoints. Nevertheless, we believe that it is feasible and desirable to establish standardized sampling methods in conjunction with standardized assays, so that results are comparable between trials.

While podocyte depletion has been linked to the development of gl

While podocyte depletion has been linked to the development of glomerulosclerosis, there is very limited information in human pre-disease stages. Methods: Kidneys from 14 adult male Caucasian Americans without renal disease were collected at autopsy in Mississippi, USA. Age and history of hypertension were obtained from medical records. Nglom, podocyte number and density were estimated using unbiased stereology. Age was dichotomized into younger and older (cut-off: 40 years), and Nglom as normal and low (cut-off: 0.6 million). Data is presented as median and inter quartile range (IQR). Results: Median age was

39 (IQR: 21–50 years) with 31% of subjects categorized as hypertensive. Median Nglom was SCH772984 mouse 0.95 (IQR: 0.61–1.3 million nephrons). Podocyte number

in younger (433; IQR: 386–512), normotensive (424; IQR: 358–506) and normal Nglom subjects (424; IQR: 356–493) was higher than in older (357; IQR: 317–425; P < 0.001), hypertensive (359; IQR: 315–433; P < 0.05) and low Nglom subjects (358; IQR: 301–409; P < 0.05). Similarly, podocyte density (podocytes per 106 μm3 of glomerular Tyrosine Kinase Inhibitor Library tuft) was lower in subjects who were older (195; IQR: 139–241), hypertensive (194; IQR: 94–241) and with low Nglom (121; IQR: 71–266) compared to subjects who were younger (275; IQR: 216–318; P < 0.0001), normotensive (260; IQR: 194–295; P < 0.001) and with normal Nglom (240; IQR: 194–289; P < 0.01). Discussion: This preliminary report suggests that older age, hypertension and low Nglom are associated with podocyte depletion in adults without kidney disease, raising questions about the limit for podocyte depletion before the

development of glomerulosclerosis. 187 SAFETY AND EFFICACY OF RAPID IRON POLYMALTOSE INFUSION IN NON DIALYSIS DEPENDENT CHRONIC KIDNEY DISEASE STAGE III A – STAGE Glycogen branching enzyme V PATIENTS M GUPTA, G HARRIS, C HOLMES Bendigo Hospital, Bendigo, Australia Aim: Assess safety and efficacy of a rapid iron polymaltose infusion in Non Dialysis dependent Chronic Kidney Disease patients stage IIIA-V. Background: Hypo-responsiveness to erythropoiesis stimulating agents ESAs and Iron deficiency is a common cause of anaemia in Dialysis and Non Dialysis dependent Chronic Kidney Disease patients stage IIIA-V (ND-CKD SIIIA-V). Across many Australian hospitals Iron polymaltose. (1 gram) IP infused slowly over 4 hours and 50 minutes. In last 4 months experience gained with rapid IP infusion over 73 minutes. Data is lacking on rapid IP infusion in ND-CKD SIIIA-V patients. Methods: We studied 63 (39 Male, 24 Female) ND-CKD SIIIA-V patients from January 2013 to Mid-March 2014, 34 patients mean age 73.

, 2010) GeneChip® data for biological replicates were normalized

, 2010). GeneChip® data for biological replicates were normalized, averaged, and analyzed using GeneSpring GX 7.3 Analysis Platform software (Agilent Technologies, Redwood City, CA), as previously described (Anderson et al., 2006). Genes that exhibited ≥ twofold increase in transcript titer in response to growth phase or growth in human serum in comparison with cells grown in control conditions were determined to be ‘present’ by Affymetrix algorithms during the induced condition and that demonstrated a significant change in expression (t-test P cutoff of ≤ 0.05) where considered differentially expressed. At least two biological replicates

were included in each analysis. To confirm GeneChip® results, primer sets (Table 1) were designed for selected ORFs to measure RNA expression by RT-PCR. RNA was isolated and purified from LB cultures of A. baumannii ATCC 17978 and 98-37-09 cells Ibrutinib mouse Y-27632 clinical trial at exponential or stationary phase of growth, as described above. Forty nanograms of purified RNA from each sample was serially diluted (twofold) and subjected to RT-PCR using the AccessQuick™ RT-PCR System (Promega, Madison, WI) in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Austin, TX) using the following parameters:

reverse transcription at 45 °C for 45 min, amplification of cDNA at 94 °C for 2 min, then 30 cycles of (94 °C for 30 s, 56 °C for 30 s, and 68 °C for 1 min), ending with a final extension at 68 °C for 7 min. For primer sets requiring lower stringency 45 or 52 °C was substituted for the Cyclic nucleotide phosphodiesterase 56 °C during PCR amplification. RT-PCR products were visualized by electrophoresis in a 2% agarose gel (UltraPure Agarose; Invitrogen, Carlsbad, CA) and ethidium

bromide staining (Thermo Scientific). Acinetobacter baumannii strains were cultured overnight in LB medium and then used to inoculate a 96-well round bottom plate with 100 μL per well of LB or 100% serum containing various concentrations of minocycline (0.25–2 μg mL−1) to a final bacterial concentration of 105 colony-forming units (CFUs) mL−1. Cultures were grown at 37 °C for 48 h. After 48 h, cultures were serially diluted in PBS and plated to enumerate CFUs mL−1 on LB agar. Assays including the efflux inhibitor phenylalanine arginine beta-naphthylamide (PAβN; Sigma) were performed as described above, except that each well also contained 60 μg mL−1 PAβN. It is well established that the expression patterns of bacterial genes, including many virulence factors, dramatically change as cells transition from exponential to stationary phase of growth. Despite its importance as an emerging bacterial pathogen, no studies have comprehensively assessed the growth phase-dependent changes in A. baumannii gene expression. Thus, we initially set out to define and compare the expression profiles of two genetically diverse A. baumannii strains, ATCC 17978 and 98-37-09, during exponential and stationary phases of growth in laboratory medium.