1b). Using multiple regression analysis, we evaluated independent effects of genetic and non-genetic factors on the development of thyroid autoantibodies. The reference categories for the analysis were CT60 CTLA-4 genotype, age, family history of AITD and cigarette smoking. In the case of thyroid peroxidase antibodies, selleck chemicals we confirmed a significant contribution of CT60 CTLA-4 genotype (P < 0·007) and younger age (P < 0·05), while family history and cigarette smoking did not prove to have any effect. In thyroglobulin antibodies, no contribution of either genotype or non-genetic factors

was confirmed. The genotyping in the group of 75 PPT patients revealed the AA genotype in 17 (22·7%) patients, the AG genotype in 36 (48%) and the GG genotype in 22 (29·3%) patients, showing no deviation from HWE (χ2 0·096, P = 0·757). As presented in Table 2, the patients with different genotypes did not differ in age, number of pregnancies, family history of AITD and smoking status. However, females with the G-allele carrying genotypes presented significantly more often with positive values of thyroid peroxidase antibodies (P < 0·04), while

the proportion of thyroglobulin antibody-positive patients did not differ significantly between the three genotypes. Similarly, more patients with the G-allele carrying genotypes had at least one type of thyroid autoantibody elevated compared

to the AA genotype (P < 0·04) (Table 2). Furthermore, the median value of thyroid peroxidase antibodies was Y-27632 chemical structure significantly lower in the AA genotype compared to the AG and GG genotypes (median, 12, 130 and 423 U/ml, respectively, P < 0·006) (Fig. 2a). In contrast to thyroid peroxidase antibodies, the median values of thyroglobulin antibodies did not differ significantly between the three genotypes (Fig. 2b). For the evaluation of thyroid autoantibody Montelukast Sodium development with multiple regression analysis, the reference categories were CT60 CTLA-4 genotype, age, number of pregnancies, family history of AITD and cigarette smoking. For thyroid peroxidase antibodies, we established a significant contribution of CT60 CTLA-4 genotype (P < 0·04), while the effect of other factors was not confirmed. In thyroglobulin antibodies, no significant contribution of genetic or non-genetic factors was found. In PPT patients, 41 (54·7%) were hyperthyroid at presentation, while hypothyroidism was established in 34 (45·3%) patients. As presented in Table 3, the median value of thyroid peroxidase antibodies was significantly higher in the hypothyroid form of disease (P < 0·0001). Similarly, the median value of thyroglobulin antibodies was higher, although the difference was statistically insignificant.

Because infusion of haploidentical male mouse splenocytes

was found previously to prevent diabetes in NOD mice we looked for, but found no evidence of, persistent chimeric lymphocytes from haploidentical paternal origin within the dams’ splenocytes. Gestation per se appears to have no aggravating or ameliorating effects on pre-existent autoimmune beta cell destruction, but pregnancy from MHC partially DAPT nmr mismatched males delays diabetes onset in female NOD mice. In type 1 diabetes, autoimmune mechanisms are involved in the destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans leading to the eventual need for insulin replacement therapy in patients [1,2]. Pregnancy has the capacity to alter both immune EPZ-6438 clinical trial response and beta cell function, but its effects on the development of autoimmune diabetes are largely unknown. Pregnancy is reported to ameliorate autoimmune diseases [3–5] through establishing a privileged state of tolerance potentially by shifting immune responses towards a less inflammatory state (reviewed in Piccinni [6]). However, this may not be the case for type 1 diabetes. Pregnancy also increases insulin demand with an expansion of beta cell mass [7–9], and a

number of islet autoantibody-positive women develop diabetes during gestation [10]. Evidence in humans indicates that increasing insulin demand aggravates autoimmune diabetes, and that increasing insulin demand PD184352 (CI-1040) at a late stage of preclinical disease will anticipate the onset of clinical diabetes [11–13]. We reasoned that examining the effect of gestation in the non-obese diabetic (NOD) mouse may be informative with respect to accelerating or delaying the onset of autoimmune diabetes. Because it is reported that the relative matching of the fetus may be important in the maternal tolerance state, we further reasoned that partially or

fully mismatched fetuses may provide advantage in controlling maternal autoimmunity. We therefore mated NOD female mice with male NOD mice, major histocompatibility complex (MHC) haploidentical mice and fully MHC mismatched mice and followed the female mice for diabetes development during and after pregnancy. The findings of our study are inconsistent with the notion that pregnancy accelerates the development of autoimmune diabetes, but support amelioration when mating is with haploidentical males. NOD mice were obtained originally from Taconics (Germantown, NY, USA) and C57BL/6J mice from The Jackson Laboratory (Bar Harbor, ME, USA), and the colonies established in the animal facilities at the Diabetes Research Institute Munich. The frequency of diabetes within untreated female NOD mice at the time of the study was 89% at age 36 weeks. Four male CByB6F1/J mice, F1 hybrids of female BALB/cByJ and male C57BL/6J mice were purchased at age 8 weeks from The Jackson Laboratory.

3B). These experiments confirmed that the reduced response to FO-1 was dependent on the defective expression (Fig. 2A) of specific activating NK receptors. NVP-BEZ235 As expected, all the receptors analyzed (with the exception of CD16) also displayed lower capability of inducing target cell killing in “hypoxic” NK cells (see redirected killing assay in Supporting Information Fig. 2). In addition, “hypoxic” NK cells displayed a reduced ability to kill different

targets including MeCoP and FO-1 melanoma cell lines and the EBV+ 721.221 B-cell line (Fig. 4A). These results are in line with the concept that the activating NK receptors targeted by hypoxia are involved in the recognition and killing of a wide panel of NK target cells. Since our data indicate that hypoxia does not affect CD16 expression and function, we further analyzed whether “hypoxic” NK cells maintained ADCC capability. NK cells were cultured under normoxic or hypoxic conditions and tested in a cytolytic assay against the 721.221 HLA-DR+ selleckchem target cell

line with or without an anti-HLA-DR mAb (to promote ADCC). As shown in Figure 4B, NK cells exposed to hypoxia were not cytolytic against this target (see also Fig. 4A, right panel). In contrast, they acquired a strong killing capability upon the addition of the anti-HLA-DR mAb (Fig. 4B) thus indicating that hypoxia did not prevent NK cells from performing ADCC. Due to the lack of basal killing (i.e. in the absence of mAbs), the overall lytic activity of “hypoxic” NK cells remained lower than that of “normoxic” NK cells; nevertheless, similar increases

above controls (i.e. mAb-induced cytotoxicity) were detected under hypoxic and normoxic conditions (Fig. 4B). The aim of the present study was to assess how NK cells could be influenced in their killing capability by the variation of pO2 in the surrounding microenvironment. Our experiments demonstrate that low levels of pO2, comparable to those hypoxic areas of certain solid tumors or infection sites or even normal lymphoid tissues, may greatly interfere with the expression Resminostat and function of major activating NK-cell receptors. The receptors affected by hypoxia play a major role in recognition and lysis of a wide panel of NK-cell targets. Accordingly, under hypoxia, NK cells strongly reduced their ability to kill both EBV-infected and tumor cells. The inhibitory effects of hypoxia, together with a series of recently identified suppressive mechanisms occurring at the tumor site, suggest that the efficacy of NK cells in clearing pathogens or tumor cells in vivo may have been overestimated. Indeed, the assays to evaluate NK-cell-mediated cytolysis are routinely performed under atmospheric O2 concentration. Our experiments indicate that hypoxia can both influence NK cells in their “resting status” and effectively counteract the stimulatory effect of the main cytokines activating the NK-cell function (including IL-2, IL-15, IL-12, and IL-21).

[29-31] GalNAc exposure may induce the injury of podocyte and PTECs by mesangial-podocyte crosstalk and glomerulotubular crosstalk, respectively. Recently, Roberta et al. found that oxidative stress and galactose deficient IgA1 were markers of progression in IgA nephropathy.[32] Moldoveanu et al. using HAA to detect the GalNAc of serum IgA1, the sensitivity as a diagnostic test of IgAN was 76.5%, with specificity 94%.[12] Furthermore, cells secreting antibodies specific for Gal-deficient IgA1 can be easily detected and enumerated in peripheral blood from IgAN patients.[33] It was also shown

in our data that serum IgG concentration was higher in the GalNAc exposure more than the 40% group. Using a lectin-binding assay to detect GalNAc exposure of IgA1 in serum might have potential as a non-invasive predictive test for IgAN prognosis. However, whether the immunosuppressive treatment will change the GalNAc exposure Kinase Inhibitor Library mouse level needs to be confirmed in further

prospective therapeutic trials. Proteinuria has a particularly strong association with poor kidney prognosis in IgA nephropathy.[3, 34-36] Remission of proteinuria is an important predictor of renal survival. The correlation of proteinuria with GalNAc exposure is not well established yet. Recently, Hastings et al. found that GalNAc exposure was not associated with the proteinuria at presentation of paediatric IgAN.[37] However, in a research carried Sorafenib mouse out by Camilla et al., it was suggested that some weak correlations were indeed found between proteinuria and IgA galactose deficiency.[32] The proteinurias of both studies were detected once at the diagnosis of IgA nephropathy. Xie et al. demonstrated that proteinuria was strongly associated with the risk of end-stage renal disease in univariate analysis; however, it did not independently contribute to the risk in multivariate models.[35] Although

proteinuria at presentation is an important consideration, increasing evidence suggests that proteinuria overtime more closely correlates with disease outcome. Several studies suggest that regardless of the peak level of proteinuria, partial remission to protein Tryptophan synthase excretion <1/g will improve the renal progression.[38, 39] Repeated measurements of proteinuria averaged over time have been shown to predict GFR loss better than proteinuria at presentation in several studies. Expanded proteinuria evaluation beyond 1-time cross-sectional assessments at the time of diagnosis to include longitudinal measurements of proteinuria for improved quantification of disease activity and risks of progression are very important.[40, 41] The therapy of steroid and angiotensin converting enzyme inhibitor/ angiotensin receptor blocker (antagonist) (ACEI/ARB) could drastically improve the clinical parameters but could not affect the HAA-IgA levels.

Ubiquitin-positive NCIs, which are evident in the degenerating lower motor neurons, have long been recognized as a characteristic feature of the cellular pathology. However, TDP-43 immunostaining may reveal positive neuronal and glial cytoplasmic inclusions (NCIs and GCIs) not only in the affected lower motor neuron nuclei but also in the other apparently normal non-motor neuron nuclei, indicating that SALS is a multisystem neuronal-glial proteinopathy of TDP-43 affecting a wide range of both neurons and glial cells in the CNS.[20] TDP-43

pathology is also evident in many patients with superoxide dismutase p38 MAPK assay 1 (SOD1)-unrelated familial ALS, in whom mutations within the TDP-43 gene (TARDBP) have been identified; interestingly, although

rare, TARDBP mutations have also been identified in patients with SALS.[21, 22] Based on these pathological and genetic findings, TDP-43 has been implicated as an important contributor to the pathogenesis of ALS. PolyQ diseases are inherited neurodegenerative disorders caused by expanded CAG repeats that encode abnormally long polyQ stretches in the disease proteins. The polyQ-positive NCIs and neuronal intranuclear inclusions (NIIs) are widespread in the CNS beyond the degenerative areas, indicating that the diseases are also multisystem neuronal proteinopathies.[23] TDP-43 pathology in the polyQ diseases was first reported in HD.[15] Unlike the neurodegenerative diseases showing TDP pathology mainly in the Seliciclib in vivo limbic system, patients with HD have TDP-43-positive NCIs in the neocortex, where many polyQ-positive inclusions are also observed. More recently, intermediate-length polyQ expansions

(27–33 Qs) in ataxin 2 (ATX2), the causative gene of SCA2, were reported to be a genetic risk factor for SALS.[16] In cases of HD, Schwab et al. have reported that TDP-43 was frequently colocalized with huntingtin in dystrophic neuritis Tangeritin and various intracellular inclusions, but not in intranuclear inclusions.[15] Recently, Tada et al. examined autopsied patients with genetically confirmed HD with SALS, and found that two different proteinaceous inclusions coexisted in a small number of neurons in the affected brain. However, the two disease proteins, huntingtin and TDP-43, were not co-localized within the inclusions, although the regional distributions of TDP-43-positive inclusions and expanded polyQ (1C2)-positive inclusions partly overlapped.[19] Biochemically, TDP-43 isoforms similar to those seen in SALS were observed in one of the patients examined.[19] In these cases of HD with SALS, it seems that two distinct pathological pathways may each affect the brain. It is tempting to speculate that “aging” may promote the deleterious effect of mutant huntingtin on motor neurons and on TDP-43. We have previously reported the occurrence of TDP-43 pathology in SCA3/MJD.

After the simultaneous vaccination (Day 42), the frequency of fatigue was higher in Group 2. While information regarding simultaneous vaccinations is scarce, Vajo et al. have reported finding no significant differences in systemic reactions between single and simultaneous vaccinations (18). Although the seasonal influenza vaccine is recommended only for the elderly and other high risk people, healthy adults were enrolled

in this study. In the case of a pandemic, all age groups would be naïve against a pandemic virus. Because the participants in this study work in facilities which produce influenza vaccines, they appear to be an appropriate target population for both the pandemic and seasonal vaccines. Should a pandemic occur, the present study would provide useful information because healthy adults (including police officers, firefighters, and healthcare professionals) will have high 3-Methyladenine concentration priority for pandemic Talazoparib nmr vaccination. However, it is important that the elderly and children also be evaluated, because their response to vaccination may be different from the participants in this study due to differences in basic immunity.

Because the pandemic H1N1 virus is no longer the pandemic virus and the vaccine has become one of the components of the seasonal vaccine, it would be difficult to repeat the current study in a high-risk population. Although the results of Phosphoprotein phosphatase the present study would not be directly applicable in a future pandemic, interaction between pandemic and seasonal vaccines is a very important factor to be evaluated in any pandemic situation, especially in high-risk groups. Shingo Uno, Kazuhiko Kimachi, Junko Kei, Keiichiro Miyazaki, Ayano Oohama, Tomohiro Nishimura, Kayo Ibaragi, Koichi Odoh, Yasuhiro Kudo and Yoichiro Kino are employees of Kaketsuken. Kaketsuken designed and implemented this study, as well as evaluating the study results. Data analysis

for this study was performed by Statcom. Kaketsuken was the sole funding source of this study. We thank Fujio Matsuo of Statcom for his valuable advice on the design of this study. We also thank the following Kaketsuken staff members: Shigemi Yamamoto, Keiko Shindo, Mariko Miyata, Emiko Sato, Akiko Saeki, Takayuki Masaki, Seiichi Harada and Nobuo Mon’nai for their great contribution in the preparation of study vaccinations and blood sample collections for this study. “
“In Africa, adolescent girls have high HIV risk. Early sexual debut may be a risk factor, although evidence has not been systematically compiled. A systematic review was conducted. Quantitative studies from sub-Saharan Africa with biologically confirmed HIV infection measures were included. A total of 128 full texts were screened. Twenty-five met the inclusion criteria, most cross-sectional. Half of studies, and all with large sample sizes, reported significant bivariate associations.

This work was supported by the Roche Research Fund for Biology, the Bonizzi-Theler Stiftung, the GEBERT-RÜF-STIFTUNG, the Swiss National Science Foundation, the Vontobel Foundation, and UBS AG on behalf of a client. Conflict of interest: The authors declare no financial

or commercial conflict of interest. “
“Mast cells are proposed to be one of the targets for mucosal vaccine adjuvants. We previously demonstrated that mucosal adjuvants containing IgG immune complexes could activate connective tissue mast cells enhancing immune responses. Here we suggest that mucosal mast cells (MMC) may also contribute to augmentation of antigen-specific selleckchem immune responses following treatment with antigens complexed with IgG. We demonstrated that both bone marrow (BM)-derived cultured MMC and tissue resident MMC incorporated ovalbumin (OVA) at a greater level in the presence of anti-OVA IgG. Co-culture of OVA/IgG-pulsed BM-derived

MMC with splenocytes from OT-II mice promoted OVA-specific activation and proliferation of T cells, a process known as cross-presentation. Furthermore, BM-derived cultured MMC underwent apoptosis following treatment with IgG immune complexes, a feature that has been described to favour phagocytosis of mast cells by professional antigen-presenting cells. This article is protected by copyright. All rights reserved. “
“Infections caused Selleck Fostamatinib by the Racecadotril leading nosocomial pathogen Staphylococcus epidermidis are characterized by biofilm formation on implanted medical devices. In a previous study, we found that ClpP protease plays an essential role in biofilm formation of S. epidermidis. However, the mechanism by which ClpP impacts S. epidermidis biofilms has remained unknown. Here, we show that the Spx protein accumulates in the clpP mutant strain of S. epidermidis and controls biofilm formation of S. epidermidis via a pronounced effect on the transcription of the icaADBC operon coding

for the production of the biofilm exopolysaccharide polysaccharide intercellular adhesion (PIA). Notably, in contrast to Staphylococcus aureus, Spx controls PIA expression via an icaR-independent mechanism. Furthermore, Spx affected primary surface attachment, although not by regulating the production of the autolysin AtlE. Our results indicate that ClpP enhances the formation of S. epidermidis biofilms by degrading Spx, a negative regulator of biofilm formation. Staphylococcus epidermidis, previously regarded as an innocuous commensal bacterium of the human skin, has emerged as one of the most frequent causes of nosocomial infection in recent years. Staphylococcus epidermidis may cause persistent infections by forming biofilms on implanted medical devices, such as central venous catheters, urinary catheters, prosthetic heart valves and orthopedic devices.

These sperm exhibit altered

motility as well and an impairment in their ability to adhere to both the zona pellucida and to the oolemma proper in vitro, associated with impaired fertilization. Alteration in the sperm tail beating was noted, and fewer sperm were found within the oviducts of wild-type females mated with nectin-2 knockout males than wild-type males. Subsequent studies have shown that nectin-2 is expressed by Sertoli cells and nectin-3, its counter receptor, is present on spermatozoa.21 Knockout of either of these molecules is associated with alteration of sperm shape, motility, and male fertility. During sexual relations, semen is deposited in the vagina after ejaculation. Although the vaginal pH click here is approximately 4.5, due to the production of lactic acid by resident lactobacilli, during female sexual excitement, the vaginal pH rises toward neutral. Seminal fluid is slightly alkaline (pH 7.2 – 7.8) and has significant buffering capacity.22 In addition, the normal pH of cervical mucus in the absence of semen is approximately 7.0, in the late follicular

phase of the menstrual cycle. The characteristics of cervical mucus change at this time, allowing the entry of spermatozoa into the uterus and Fallopian tubes. Recent studies by Ceballo et al.23 suggest that HIV binds to human spermatozoa via heparin sulfate on the sperm surface, most likely involving syndecans 3 and 4, rather a mannose receptor. In addition, they showed selleck chemicals that spermatozoa were internalized and promoted the uptake of HIV by DC in culture, which subsequently

exhibited a marked increase in the expression of HLA-DR, CD40, CD83, and CCR7. The authors speculated that spermatozoa transmit the virus to mucosal DC’s within the reproductive tract and might alter the immune response against HIV by modulating their function. As sperm are foreign cells that enter the female reproductive tract at coitus, why Thiamine-diphosphate kinase is an immune response against them not mounted, as it is against microbes such as chlamydia and yeast.22,23 The female reproductive tract is capable of mounting an immune response to pathogens.24,25 There is increasing evidence that seminal plasma, which had conventionally been viewed solely as a transport medium for sperm, plays additional roles beyond this within the female reproductive tract (Table I). Seminal plasma has potent immunosuppressive activity, which can principally be attributed to its high content of TGF-beta26,27 and PGE prostaglandins.28 Emami et al.29 have provided evidence for the involvement of members of the seminal kallikrein-related peptidase (KLK) cascade in activation of latent TGF-beta in seminal plasma. Skibinski et al30 have shown that seminal plasma inhibits the function of both NK cell and T lymphocytes, and that the E series prostaglandins are responsible for the major portion of this suppression.

S2). However, we found no evidence of the presence of H-2Kb-positive CD4+ or CD8+ T cells in the spleens

of NOD mice mated with CByB6F1/J males. The majority of mice had insulin autoantibodies at 10 weeks confirming that they had ongoing autoimmunity (Fig. S3). However, we found no obvious effects on insulin autoantibody titres between unmated NOD dams (group A1) and NOD dams mated with haploidentical male CByB6F1/J mice (group C1) before breeding at age 10 weeks (P = 0·15) or after weaning at age 16 weeks (P = 0·8), and no difference between insulin autoantibodies at age 10 weeks and after weaning in dams mated with haploidentical male CByB6F1/J mice (P = 0·3). Finally, in a multivariate Cox proportional hazards model that included insulin autoantibody titre and mating group, mating with VX-809 concentration MHC haploidentical male CByB6F1/J mice was the only significant covariate (hazard ratio, 0·35, 95% confidence interval, 0·3–0·9; P = 0·04) in the model. The influence of gestation on the development of autoimmune diabetes Belinostat solubility dmso is discussed widely. Increased insulin demand accompanied by increased beta cell expansion [7–9], as well as tolerogenic

immune effects influenced by hormones and the fetus that is presenting paternal human leucocyte antigen (HLA) molecules affect the female immune system during pregnancy [6]. Here, we show that pregnancy per se has no accelerating impact on the development of autoimmune diabetes in NOD mice. We showed further that gestation via haploidentical male CByB6F1/J mates leads to a significantly delayed age at diabetes onset. Our findings in mice are of relevance to the hypothesis that increased insulin demand accelerates the development of autoimmune diabetes. It has been well described that pregnancy increases beta cell function Morin Hydrate requirements [16]. However, this did not accelerate diabetes in mice with pre-existing autoimmunity. This was true when female NOD mice were mated at 10 weeks or 13 weeks of age, when it is known that that pancreatic insulin content

is already compromised [17]. It is possible that there were transient effects on autoimmunity during gestation that were missed. It is also possible that beta cell mass was still sufficient to accommodate the extra demand of pregnancy. Consistent with the notion that pregnancy is a tolerogenic condition, we found that pregnancy delayed the onset of diabetes significantly in NOD females. This delay did not seem to be strictly due to changes associated with pregnancy, as the effect was not observed when syngeneic breeding partners were used, and insulin autoantibody titres were unaffected by pregnancy. Thus, we assumed that dams were conditioned specifically by MHC mismatching or other mismatching from the pups. Of note, the protective effect was most noticeable and only significant when male mates were partially mismatched at MHC.

Apparently, PMNs are attracted by the tumor cells via the chemokine-receptor axis CXCL16-CXCR6 [33]. In PDAC, PMN infiltration was associated with a distinct JQ1 clinical trial “micropapillary” growth

pattern, compatible with a PMN-mediated dispersal of the tumor cells [7]. Analyzing 112 pancreas biopsies, we found that prominent PMN infiltrates coincided with low E-cadherin expression, and since one single PMN contains about 1 pg elastase [34], it is possible that the infiltrating PMNs are responsible for the loss of E-cadherin. Loss of E-cadherin induces a more migratory phenotype, and an association between this migratory phenotype, evident as up-regulation of the pancreatic serine protease PRSS3 by pancreas tumor cells, and the occurrence of distant organ metastasis has been described by others [35], as has an epithelial-to-mesenchymal transition, which is also associated with enhanced migration and the generation of metastasis [36]. Although a cause-effect-chain cannot be established BGB324 conclusively, and some evidence is still correlative, our data support the concept that prominent PMN infiltrates favor the invasive growth and metastasis of PDAC cells. In our patients, we could not correlate the PMN infiltrate or the relative E-cadherin loss to any of the clinical

and pathological parameters. A study by Hong et al. with considerably more patients, however, showed that loss of E-cadherin was associated with poorer prognosis [37]. These authors also suggested that the microenvironment might affect the local E-cadherin expression, a presumption that perfectly fits into our concept that infiltrating PMNs degrade E-cadherin. In conclusion, we found that PMNs via elastase degrade E-cadherin on pancreatic tumor cells, resulting in an enhanced dyshesion, migration, and invasiveness of the tumor

cells, which — in turn — could contribute to tumor progression, metastasis, and poorer prognosis in PDAC. Peripheral blood from healthy human volunteers was obtained by puncture of peripheral veins and collected in heparin-NH4-coated check details tubes (Sarstedt, Nürnbrecht, Germany). PMNs were isolated by centrifugation on PolymorphPrep (Axis-Shield PoC AS, Oslo, Norway) which yielded an 85–95% pure PMN population. The PMNs were suspended in Hanks balanced salt solution and used within 1 h. Four human pancreatic cancer cell lines were used: MiaPaca-2, Su8686 (ATCC, Rockville, MD, USA), COLO-357, and T3M4 (provided by the European Pancreas Center, Heidelberg, Germany). Cells were grown in RPMI-1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL penicillin-streptomycin (Invitrogen, Karlsruhe, Germany) and were incubated at 37°C in a 5% CO2 humidified atmosphere. T3M4 (5 × 104) were plated in specialized cell culture dishes with a mobile insert in one compartment (ibidi, Martinsried, Germany) for 24 h.