Methods: Tissue sections from the central nervous system of infected cases were examined by light microscopy, immunohistochemistry and in situ hybridization.

Results: All 13 cases of EV71 encephalomyelitis collected from Asia and France invariably showed stereotyped distribution of inflammation in the spinal cord, brainstem, hypothalamus, cerebellar dentate nucleus and, to a lesser extent, cerebral cortex and meninges. Anterior pons, corpus striatum, thalamus, temporal lobe, hippocampus and cerebellar cortex were always uninflamed. In contrast, the eight JE cases studied showed inflammation involving most neuronal areas of the central nervous system, including the areas that were uninflamed in EV71 encephalomyelitis. Lesions in both infections were nonspecific, consisting of perivascular Selleckchem PLX3397 and parenchymal infiltration by inflammatory cells, oedematous/necrolytic areas, microglial nodules and neuronophagia. Viral inclusions were absent. Conclusions: Immunohistochemistry and in situ hybridization assays were useful to identify the causative virus, localizing viral antigens and RNA, respectively, almost exclusively to neurones.

The stereotyped distribution of inflammatory lesions in EV71 encephalomyelitis appears to be very useful to help distinguish it from JE. “
“Multiple selleck antibody inhibitor sclerosis (MS) and neuromyelitis optica (NMO) are inflammatory autoimmune diseases that affect the central nervous system. Several genome-wide and candidate gene studies have identified genetic polymorphisms associated with the risk of MS or NMO. In particular, two recently published studies of meta-analysis in European-origin populations have suggested associations of single-nucleotide polymorphisms (SNPs) in CD6, TNFRSF1A and IRF8

with MS. The aim of our study was to assess the associations between SNPs in these three genes and the risk of inflammatory demyelinating disease (IDD) including MS and NMO. To the best of our knowledge, this is the first time such a study has been performed in an Asian population. A total of 21 SNPs of CD6, TNFRSF1A and IRF8 O-methylated flavonoid were genotyped in 178 IDD cases (79 MS and 99 NMO patients) and 237 normal controls in a Korean population. Logistic analyses revealed that one SNP in CD6 (rs12288280, P = 0.04) and three SNPs in TNFRSF1A (rs767455, rs4149577 and rs1800693, P = 0.01–0.03) were associated with NMO. However, there was no association of IRF8 polymorphisms with IDD, including MS and NMO. Using further information from the SNP Function Prediction website, two exonic splicing enhancers (ESEs), including the polymorphic site of rs767455, were predicted to be binding sites for splicing factors (SRp55, SF2/ASF2 and SF2/ASF1). Although additional studies are needed, our findings could provide information regarding the genetic aetiology of IDD in the Korean population.

The facts that the pain observed in patients with CRPS can result from multiple mechanisms and that patients with CRPS do not respond equally to the same medications may be due in

part to its evolution in time, but it also suggests that CRPS may result from multiple aetiologies. The results of this study demonstrating that a subset of CRPS patients show elevated numbers of the CD14+CD16+ monocyte subgroup may aid in elucidating some of the different mechanisms involved in its pathophysiology. A better understanding of these mechanisms may lead to novel treatments for this very severe, life-altering condition. This study has demonstrated an increase in the percentage of the CD14+CD16+ monocyte subgroup in individuals afflicted

with CRPS. In addition, other investigators have reported mast cell involvement [47], Y-27632 concentration leucocyte accumulation in the affected Crizotinib price extremity [48] and impaired neutrophil function [49] in patients with CRPS. Thus, further evaluation of the role the immune system plays in the pathogenesis of CRPS is warranted, and may aid in elucidating disease mechanisms as well as the development of novel therapies for its treatment. We wish to graciously thank Eric B. Wong MS and Jeffrey J. Gerbino for their technical assistance. This study was supported by grants from the Commonwealth of Pennsylvania Department of Health, Drexel University College of Medicine Pain Initiative and gifts from the Tilly Family Foundation and the Sunstein family. The authors certify that they have no commercial associations that might pose a conflict of interest in connection with this article. All funding sources for this study are listed in the Acknowledgements section. PatID Gender/ Age Initiating Event/Duration Signs/Symptoms/Overall

Amino acid Pain Score NRS(0-10) Pain Medications Other Conditions CRPS01 F/68 Kyphoscoliosis; disc disease at L5-S1/22 years L5-S1 sensory loss; spontaneous burning pain in both legs; weakness; inability to move toes; severe dystrophic changes. Pain (NRS) 8 NSAIDs; anti-epileptic drugs (AED), antidepressants; intermittent narcotics; spasmolytics. L4-L5 bilateral radiculopathy; arthrosclerosis; GERD; osteoporosis; osteoarthritis; IBS; headaches CRPS02 F/44 Fall; brachial plexus traction injury (BPTI)/4·5 years Paresthesias; deep ache; deep muscle joint pain; dynamic and static allodynia; generalized from BPTI; weakness; poor initiation of movement. Pain (NRS) 8 Intravenous ketamine; intravenous lidocaine; narcotics; AED; antidepressants, lenalidomide. C5-C6 disk herniation; L4-L5-S1 radiculopathy; mitral valve prolapse; Asthma; headaches. CRPS03 F/46 Fall; repetitive strain of right brachial plexus/9 years Dynamic and static mechano allodynia; cold allodynia right upper quadrant; autonomic dysregulation; neurogenic oedema; dystonia of trunk; weakness.

Urinary Emmprin, MMP-9 and TIMP-1 may be noninvasive potential biomarkers that could be used for long-term follow-up of children with UPJ narrowing on conservative X-396 in vivo treatment to determine those who might develop

obstruction. “
“151 CLASS II EXPRESSING RENAL TUBULAR CELLS LEAD TO RECONSTITUTION OF CD4 T CELLS IN CLASS II DEFICIENT MICE Y M WANG1, GY ZHANG1, A SAWYER1, JH ZHOU1, M HU2, G ZHENG2, Y WANG2, DC HARRIS2, SI ALEXANDER1 1Centre for Kidney Research, Children’s Hospital at Westmead, Sydney, NSW; 2Centre for Transplantation and Renal Research, University of Sydney, Westmead Millennium Institute, Sydney, NSW, Australia Aims: To identify whether reconstitution of Class II expression in thymus by Class II expressing renal tubular cells may lead Nivolumab manufacturer to reconstitution of kidney specific CD4 T cells in Class II deficient mice. Background: Regulatory T cells (Tregs) are generated

in thymus and are of the CD4 subset. Tregs require MHC Class II to be selected in the thymus. MHC Class II knockout (Class II−/−) mice are deficient in CD4 T cells. Studies have shown that renal tubular cells can express MHC class II. This study identifies the induction of CD4 T cells and Tregs by reconstitution of Class II expressing tubular cells into thymus. Methods: Renal tubular cells were isolated from C57BL/6 Ly5.1 mice and were cultured with IFN-γ. The cultured tubular cells were assessed for Class II expression and

then injected into the thymus of Class II−/− mice. CD4, CD8 and Tregs were assessed by flow cytometry prior and after tubular cell injection. Two months after thymus injection, CD4 T cells and Tregs were assessed Cediranib (AZD2171) in kidney and spleen by immunohistochemical staining. Results: 30% of tubular cells expressed MHC Class II after ten-day co-culture with IFN-γ. CD4+ T cells in Class II−/− mice increased from less than 1% of total CD3+ T cells before tubular cell injection to 1.4% at week four and 7% at two months after tubular cell thymic injection. Immunohistochemical staining showed that there were increased CD4+ T cells and Tregs in spleen and kidney for these class II deficient mice. Conclusions: Reconstitution of Class II expression in thymus by class II expressing renal tubular cells lead to reconstitution of CD4 T cells including Tregs in Class II deficient mice.

Our results demonstrate the neuroprotective effects of –Cu, −Cu+Mn and +Mn diets in a murine model of scrapie. However, neuronal death induced by infection with prions seems to be independent of apoptosis marker signalling. Moreover, copper-modified diets were neuroprotective against the possible toxicity of the prion transgene in Tga20 control and infected mice even though manganese supplementation could not counteract this toxicity. “
“We report a clinical case report AZD2281 of the MV2K+C subtype of sporadic Creutzfeldt-Jakob disease (sCJD). The patient was a 72-year-old woman who exhibited progressive dementia over the course of 22 months. Diffusion-weighted

MRI during this period showed abnormal hyperintensity in the cerebral cortex in the early stage. The clinical course was similar to that of previously reported patients with the MV2K or MV2K+C subtype of sCJD. However, histopathological examination revealed unique features: severe extensive spongiform changes with perivacuolar deposits in the cerebrum and basal ganglia, plaque-like PrP deposits in the cerebrum, and only mild changes in the cerebellum with small amyloid plaques (∼20 μm in diameter), smaller than those in the MV2K subtype or variant CJD (40–50 μm in diameter). Molecular analysis showed a methionine/valine heterozygosity Ixazomib at codon 129 and no pathogenic mutation in

the PrP gene (PRNP). Western blot analysis of the protease-resistant PrP (PrPSc) in the right temporal pole revealed the type 2 pattern, which is characterized by a single unglycosylated band, in contrast to the doublet described for the typical MV2 subtype of sCJD. The other intermediate band might exist in the cerebellum with kuru plaques. Therefore, small amyloid plaques in the cerebellum can be crucial for MV2K+C subtype. “
“Frequencies of typical myohistological changes such as ragged red fibers (RRF) and cytochrome c oxidase (COX)-deficient fibers have been suggested

to be dependent on underlying mitochondrial DNA (mtDNA) defect. However, there are no systematic studies comparing frequencies of myohistological changes and underlying genotypes. others The histopathological changes were analysed in 29 patients with genetically confirmed mitochondrial myopathies. Genotypes included multiple mtDNA deletions due to POLG1 mutations (n = 11), single mtDNA deletion (n = 10) and mtDNA point mutation m.3243A>G (n = 8). Histochemical reactions, including Gomori-trichome, COX/SDH (succinate dehydrogenase) and SDH as well as immunohistological reaction with COX-antibody against subunit I (COI) were carried out in muscle biopsy sections of all patients. The COX-deficient fibers were observed most frequently in all three patient groups. The frequencies of myopathological changes were not significantly different in the different genotypes in all three histochemical stains.

Cells from spinal cord were restimulated in vitro with MOG peptide and stained intracellularly for IL-17 and IFN-γ. As shown in Fig. 4, MOG-specific T cells from inflamed click here spinal cords belonged to Th1, Th17, and Th17/Th1 subsets. However, the percentage of CD4+ T cells from LFA-1+/+ and LFA-1−/− mice producing these cytokines was absolutely comparable on the level of antigen-specific cells. In addition, the amount of cytokines

produced did not differ (MFI for IL-17: 20 020±1457 (LFA-1+/+) versus 21 460±1080 (LFA-1−/−), p=0.436; MFI for IFN-γ: 15 436±2127 (LFA-1+/+) versus 14 940±804 (LFA-1−/−), p=0.832). The same results were obtained for IL-2 and TNF-α. However, it is important to note that the increased total number of antigen-specific cells finally results in a higher absolute number of cytokine-producing CD4+ T cells. Interestingly, there was also no correlation between EAE score of an individual animal and cytokine production on the single cell level. Again, only the number of infiltrating CD4+ T cells correlates with disease severity (see above). Polyfunctional Th1 cells producing multiple effector cytokines at the same time are believed to be particularly

destructive in inflammation 12. Therefore, we also analyzed whether the frequency of IL-2, IL-17, IFN-γ double JNK inhibitor or triple producers was altered between WT and KO mice, but did not find any significant differences (data not shown). Alternatively, a change in Th2 or anti-inflammatory cytokines could influence the severity of disease. Therefore, we tested for the production of IL-4 and IL-10. Only

very few (<2%) antigen-specific CD4+ T cells in the spinal cord produced these two cytokines. However, for we did not observe any significant differences between LFA-1+/+ and LFA-1−/− T cells (data not shown). To analyze the general capacity of T cells to produce certain cytokines, we additionally used an antigen-independent stimulation with PMA and ionomycin. Also, with this kind of stimulation, none of the analyzed cytokines differed between KO and WT mice (data not shown). Taken together, these results clearly show that loss of LFA-1 does not alter the cytokine pattern of autoreactive CD4+ T cells. Therefore, only the increased total number of antigen-specific, cytokine-producing cells in LFA-1−/− mice can be accounted for the increased severity of EAE. Treg play an important role for the suppression of chronic inflammation 8, 13. They control the expansion as well as the function of autoreactive effector T cells. Utilizing intracellular staining for the lineage-specific transcription factor FoxP3, we analyzed Treg in the spinal cord of LFA-1−/− and LFA-1+/+ mice after EAE induction. The absolute number of Treg was the same in both groups (Fig. 5).

All 325 patients who had AKI and required dialysis during one years study period were enrolled. Baseline characteristic data and clinical

outcomes between IHD and APD were colleted and compared. Results: Only 194 patients were analyzed. 51.6% received IHD and 48.4 % received APD. There were similar in mean age and sex of patients in both groups. Percentage of patients who had respiratory support and required inotropic drug at the beginning of dialysis were much more Selleck Alectinib in APD group (90.4% vs 67%, P. Conclusion: Overall mortality rate of AKI patients was still high despite dialysis support. Patients who had received APD were more critically ill, leading to higher mortality than IHD patients. However, APD could be used in AKI in resource-limited

setting. VERNAWATI SRI A, NAINGGOLAN GINOVA Division of Nephrology and Hypertension, Dept. of Internal Medicine, Dr. Cipto Mangunkusumo Hospital, University of Indonesia Introduction: Rhabdomyolysis is the liberation of components of injured skeletal muscle including electrolytes, myoglobin, and other sarcoplasmic proteins into the circulation that can cause Acute Kidney Injury (AKI). We measured buy LY294002 kidney function (eGFR) after recovered from AKI using serum Creatinine and compared the result with several methods.1,2 Methods: This is a case of 4 injured patiens suffered from AKI caused by Rhabdomyolysis. In recovery phase, we examined eGFR using several methods: CKD-EPI, Cystatin C and 24 hours urine collection Creatinine Clearance. (figure 1) Results: The case is taken from an accident Clomifene of a collapsed tunnel in Papua, Indonesia, May 2013. Five workers trapped

more than 19 hours had rhabdomyolysis and four of them developed AKI. All patients are male 29–50 years old. Laboratorium findings showed high Creatinine Kinase ranged from 53.102 U/L to 181.414 U/L, hyperphosphatemia, hyperkalemia, hyperuricemia and hypocalsemia. Three patients with AKI received haemodialysis for 2 to 4 weeks duration. Improvement of urine output was noted in the recovery phase, followed by polyuria phase on day 8 to 26. Improving level of serum Creatinine started on day 8 then decreased to the level of 1 mg/dL on day 48. Microscopic haematuria became undetected on day 32. The result of eGFR in recovery phase using several methods are listed in table 1. (table 1) Three patients with normal eGFR by CKD-EPI showed higher Cystatin C level and lower Creatinine Clearance Test. This discrepancy suggests that eGFR by CKD-EPI cannot be used independently to measure kidney function in Rhabdomyolysis. We hypothesized that muscle damage in rhabdomyolysis have led to low production of creatinine. Conclusion: Determination of eGFR using serum creatinine and CKD-EPI method is not accurate and cannot be used independently in the case of rhabdomyolysis. We suggest several methods, such as Cystatin C or Creatinine Clearance Test, should be used.2,3 1. Raymond V, Mehmed SS, Ekrem E, Norbert L.

In IgAN, complement system has attracted great attention. In patients with IgAN, except for the characteristic IgA deposition, C3 Selleck Palbociclib is the most commonly co-deposited molecule, approximately affects 90% patients. Serological complement activation was also found in IgAN. Additionally, the elevated urine factor H level in patients

with IgAN was reported in recent study. Accumulating evidences from plasma, urine and renal biopsy samples suggested the involvement of factor H and complement activation in IgAN pathogenesis. CFH, CFHR3 and CFHR1 are regulators of complement system, which is a key system for immune surveillance and homeostasis. In

systemic autoimmune diseases, such as SLE, activation of the complement system is involved in pathogenesis. In recent years, following the identification of aberrant glycosylated IgA1 and anti-glycan antibodies in patients with IgAN, it have been convinced that IgAN is an autoimmune disease, in which IgA1-containing immune complexes were the initiator MAPK Inhibitor Library for glomerular injury. In our recent study, we enrolled two populations, Beijing Discovery Cohort of IgAN-GWAS and Beijing Follow-up Cohort, to explore the genetic mechanism of variants in CFH, CFHR3 and CFHR1 on IgAN. In the Beijing Discovery Cohort, we found the top

SNP rs6677604 was associated with glomerular mesangial C3 deposition by genotype-phenotype correlation analysis. In the Beijing Follow-up Cohort, after the confirmation of tight linkage between rs6677604-A and CFHR3-1Δ, we found rs6677604-A was associated with higher factor H levels and lower complement activation split product C3a, which implied less system complement activation. Furthermore, factor H levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition, indicated the important role of factor H in controlling complement activation in IgAN. Besides rs6677604, serum IgA levels and galactose deficient IgA1 levels, GPX6 which were pathogenic initiator of IgA nephropathy, were also found to be associated with mesangial C3 deposition in IgAN. Our findings, together with our present understanding of IgAN pathogenesis, suggested that variants in CFH, CFHR3 and CFHR1 regulated pathogenic IgA1 induced system complement activation due to its effect on factor H levels, which might influence circulating IgA1-containing immune complex formation and the following mesangium deposition, and at last contributed to IgAN susceptibility.

5 and E11.5 due to defects in placental vascularization, highlighting its role in placental vascular development [5]. Placentas of PPARγ-null mice are with an unsettled balance of pro- and anti-angiogenic factors, that is, increased proangiogenic factor proliferin and decreased anti-angiogenic factor proliferin-related protein.

This has been confirmed with “gain of function” studies because the PPARγ activator rosiglitazone Saracatinib manufacturer inhibits placental angiogenesis via regulating PRP and VEGF expression [90]. To this end, it is speculating that the critical PPARγ dimerization partner RXR may also have a role in placental angiogenesis because RXR-null mice show a similar phenotype to PPARγ [119]. Mammalian embryogenesis Mdm2 antagonist and placental development

are believed to take place under constant low-O2 relative to ambient O2 [54]. For example, in a human placenta the intervillous space O2 is as low as ~2% at ≤8–10 weeks of gestation at a time when placental vasculature forms; at the end of the first trimester this level rises threefold to ~8% when maternal blood is delivered into the placenta from the uterine spiral arteries; thereafter, O2 level gradually declines to ~6% at the end of the third trimester [102, 84], possibly due to the substantial increased demand of fetus. At the end of the third trimester, the O2 level in the human fetus is even lower, ~2.2% O2 (range 1.9–3.1%) and ~3.7% O2 (range 2.3–5.1%) in the umbilical artery and vein, respectively [102]. Low O2 or hypoxia is known to stimulate the expression of numerous hypoxia-responsive genes via HIF-1β, the also known as Arnt heterodimerization with

HIF-1α [32]. HIF-1β mediates hypoxia-induced transcription of many angiogenic genes in the placenta, including VEGF [41]. Thus, one would expect that HIF should play a critical role in placental angiogenesis. Surprisingly, vascular defect is likely to be secondary to the primary trophoblast defect in the Arnt-null mice [2]. This is because placentas of Arnt-null mice display greatly reduced size in the spongiotrophoblast and labyrinth layers but with increased numbers of giant trophoblast cells, suggesting that HIF-1β is critical for determining the fate of the trophoblasts [63]. The MAPK pathways are evolutionarily conserved signal transduction cascades that are implicated in control of different and even opposite cellular responses including proliferation, differentiation, and cell death. In vertebrates, multiple isoforms of MAPK have been identified and categorized into three subfamilies, that is, the ERKs, p38MAPK, and the JNKs or stress-activated protein kinases. The MAPK signaling is important for transmitting extracellular signals including growth factors, hormones, and chemokines into the intracellular targets for nearly all fundamental cellular processes. The p38MAPK comprises four members, including p38α/MAPK14, p38β/MAPK11, p38γ/MAPK12, and p38δ/MAPK13 [14].

These multifunctional lectins can hierarchically control a cascade of immunoregulatory events including the expansion, recruitment, and function of regulatory T cells, the promotion of tolerogenic

dendritic cells, and the execution of T-cell death programs. In addition, galectins can control cell adhesion and signaling events critical for implantation and are involved in fundamental processes linking tissue hypoxia to angiogenesis. In an attempt to integrate the regulatory roles of galectins to immunological and vascular programs operating during pregnancy. Here we outline the regulated expression and function of individual members of the galectin family within the fetoplacental unit and their biological implications for the development and preservation of successful pregnancies. “
“The binding of NKG2D to its ligands strengthens Ibrutinib chemical structure the cross-talk between natural killer (NK) cells and dendritic

cells, particularly at early stages, before the initiation of the adaptive immune response. We found that retinoic acid early transcript-1ε (RAE-1ε), one of the ligands of NKG2D, was persistently expressed on antigen-presenting cells in a transgenic mouse model (pCD86-RAE-1ε). By contrast, NKG2D expression on NK cells, NKG2D-dependent cytotoxicity and tumour rejection, and dextran sodium sulphate-induced colitis were all down-regulated in this mouse model. The down-regulation of learn more NKG2D on NK cells was reversed by stimulation with poly (I:C). The ectopic expression of RAE-1ε on dendritic cells maintained NKG2D expression levels and stimulated the activity of NK cells ex vivo, but the higher frequency of CD4+ NKG2D+ T cells in transgenic mice led to the down-regulation of NKG2D on NK cells in vivo. Hence, high levels of RAE-1ε expression on antigen-presenting cells would be expected to induce the down-regulation of NK cell activation by a regulatory T-cell subset.

“
“Bystander activation of T cells, i.e. the stimulation of unrelated (heterologous) T cells by cytokines during an Ag-specific T-cell response, has been best described for CD8+ T cells. In the CD8+ compartment, the release of IFN and IFN-inducers leads to the production of IL-15, which mediates the proliferation of CD8+ T cells, notably memory-phenotype CD8+ T cells. CD4+ T cells also undergo bystander activation, however, the signals inducing this BCKDHB Ag-nonspecific stimulation of CD4+ T cells are less well known. A study in this issue of the European Journal of Immunology sheds light on this aspect, suggesting that common γ-chain cytokines including IL-2 might be involved in bystander activation of CD4+ T cells. Bystander activation of T cells was first described by Tough and Sprent, showing that different viruses, virus-mimetics such as poly(I:C) or bacterial products such as LPS induced IFN-α/β secretion, which led to the proliferation and expansion of unrelated (heterologous) polyclonal T cells 1, 2.

We have recently reported the effects of C. trachomatis serovar D on endocervical epithelial

cells in vitro using novel techniques that allow more physiologic partial infection of exposed cells and discrete assessment of infected and noninfected bystander cells within a mixed culture (Ibana et al., 2011a). These experiments revealed that cell surface expression of MHC class I products is decreased on both infected and noninfected, bystander cells and suggest that soluble and nonsoluble factors are involved in this downregulation (Ibana et al., 2011a). In this study, we use similar techniques to assess the effects of C. trachomatis infection on endocervical epithelial cell expression of the host cell-expressed NK cell activating ligand, MHC class I-related protein A (MICA; Brunham & Rekart, 2008). Bcr-Abl inhibitor In all

infection analyses, a primary-like immortalized endocervical epithelial cell line (A2EN) was utilized. A2EN was derived from primary epithelial cells grown out from an endocervical explant and which were immortalized by transduction with PA317/LXSN-16E6E7-conditioned medium as described previously (Herbst-Kralovetz click here et al., 2008). These cells were propagated in antibiotic-free keratinocyte serum-free medium (KSFM) supplemented with 30 μg mL−1 recombinant epidermal growth factor (rEGF), 0.1 ng mL−1 bovine pituitary extract (Invitrogen, Carlsbad, CA), and 0.4 mM CaCl2 (Sigma, St. Louis, MO); referred to herein as cKSFM. A2EN cells were grown under 2% O2

and 5% CO2 at 37 °C (Ficarra et al., 2008). Cells were infected with C. trachomatis serovar D (D/UW-3/Cx) in SPG (10 mM sodium phosphate [pH 7.2], 0.25 M sucrose, 5 mM l-glutamic acid) at a multiplicity of infection (MOI) of 1–3 to achieve Exoribonuclease infection rates of ~40–60% (Ibana et al., 2011a, b) for mixed cell analyses. For cytolytic assays, an MOI of 15 was used to achieve infection rates of 80–85% (Kawana et al., 2007). A mock-infected control and infections with UV-inactivated elementary bodies (UVEB) were included for each infection condition. UVEB were prepared by exposing purified EBs to UV-light (mineralight UVSL-25, at 115 volts, 60 cycles, 0.12 Amps) for 2 h at a 10 mm distance. UVEB were confirmed free of infectious chlamydial particles by infecting HeLa cells at an MOI of up to 100. Immediately after infection, SPG was removed and replaced with cKSFM. Cells for immunofluorescent staining were cultured in 12-well culture plates on coverslips. Cells for flow cytometric analyses were cultured in six-well culture plates and harvested using a mild cell detaching agent, Accutase (Innovative Cell Technologies, San Diego, CA), at the indicated times post infection (hours postinfection or hpi). Mock-infected, UVEB-infected and C. trachomatis-infected cells grown on coverslips were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, then washed and permeabilized with 0.5% saponin.