Electrophysiology, muscle this website weight, peroneal nerve length, and histomorphometry were also analyzed. Only the peroneal nerve length and the ratio of

highest muscle force/muscle weight demonstrated the equivalence between the sides. A small variability of TA muscle force and TA muscle weight was observed between the sides suggesting dominance. Optimization of electrical stimulation and preload as well as the use of correct anesthesia were fundamental to acquire the highest muscle force. © 2011 Wiley Periodicals, Inc. Microsurgery, 2012 “
“Bone nonunion in the pediatric population usually occurs in the context of highly unfavorable biological conditions. Recently, the vascularized fibular periosteal flap has been reported as a very effective procedure for treating this condition. Even though a vascularized tibial periosteal graft (VTPG) was described long ago and has been successfully employed in one adult case, there has been no other report published on the use of this technique. We report on the use of VTPG, pedicled in the anterior tibial vessels, for the treatment of two complex pediatric bone nonunion case: a recalcitrant supracondylar femoral pseudarthrosis secondary to an infection in an 11-year-old girl, and a tibial nonunion secondary to a failed bone defect reconstruction in a 12-year-old girl. Rapid healing was obtained in both cases. find more In the light of the data presented,

we consider VTPG as a valuable surgical option for the treatment of complex bone nonunions in children. © 2014 Wiley Periodicals, Inc. Microsurgery, 2014. “
“Despite increasing

use of lateral lower leg perforator flaps, comprehensive anatomical data are still lacking. The aim of this article was to comprehensively document the pattern of usable lateral lower leg perforators. Systematic mapping of 16 cadaver leg perforators in a well-defined area was performed to elucidate location, course, length, 5-Fluoracil diameter, and origin. Overall, 197 perforators were found in 16 lateral lower legs. The mean number of perforators per leg with a diameter ≥ 0.3 mm was 13.4 ± 3.6. Most perforators were found in the distal third (39.0%), followed by the middle third (32.0%), and proximal third (29.0%). A musculocutaneous course was found in 26.9% of the perforators, whereas 73.1% revealed a septocutaneous course. Most septocutaneous perforators (50.0%) were found in the distal third and most musculocutaneous perforators (58.5%) in the proximal third (P < 0.001). The majority of perforators originated from the anterior tibial artery (53.0%), followed by the peroneal artery (41.6%), and the popliteal artery (5.1%). Popliteal artery perforators (1.64 mm) were significantly larger than anterior tibial artery (0.91 mm) and peroneal artery perforators (1.02 mm; P < 0.001). These results may facilitate tissue transfer around the lateral lower leg. © 2014 Wiley Periodicals, Inc. Microsurgery, 2014.

For the 0.1-μg dose, lymphocyte and eosinophil numbers were significantly higher in 20- compared with 1-week-old mice (* in Fig. 3A, B). For the 10-μg dose, this was opposite; the cell numbers decreased with age (Fig. 3A, B). In a separate study, mice were sensitized by i.n. instillation of OVA in Al(OH)3 and challenged i.n. with OVA. The main and interaction effects are reported above the figures. When a significant effect of age or a significant sex and age interaction

was found, the result of the post hoc test is given on the figure. Fig. 4A shows the OVA-specific IgE response in 1-, 6- and 20-week-old female and male mice. Significant main effects of both sex and age were found. Sensitized females produced higher levels of OVA-specific IgE compared with males (Fig. 4A). Small molecule library manufacturer Further, the IgE response increased with age as 20-week-old mice had significantly higher levels than 1-week-old mice. The same pattern was observed for OVA-specific IgG1 production; females had significantly higher antibody production than males, and the response in both sexes increased with age (Fig. 4B). Cells from both SLNs and MLNs were stimulated with OVA ex

vivo. In MLNs, IL-4 was undetectable. Only IL-10 secretion was influenced by the sex of the mice, with females releasing significantly more IL-10 than males (Fig. 5A). IL-5 and IL-13 secretion was higher in 1-week-old mice compared with Y-27632 datasheet older mice (Fig. 5B, C). INFγ was affected by age in the same manner as IL-17A secretion (Fig. 5D, E); 6-week-old mice had significantly lower IFNγ and IL-17A secretion than oxyclozanide 20-week-old mice and for IFNγ also significantly

lower than the 1-week-old mice. A significant age and sex interaction was found for the total number of cells in MLNs (Fig. 5F). The post hoc test revealed that only in the oldest age group did females have significantly higher number of cells compared with males (bracket in Fig. 5F). In SLNs, IL-4, -5, -10, -13 and IFNγ were undetectable and IL-17A produced at very low levels (data not shown and Fig. 5G). IL-17A production increased with age but was not affected by sex. The total number of cells in SLNs was unaffected by both sex and age (Fig. 5H). Control groups of mice were immunized i.n. with OVA alone. When comparing the OVA and OVA + Al(OH)3 treatments, MLN cell numbers, but not SLN cell numbers, were highly increased after using the adjuvant for sensitization, and this was observed for all age groups (data not shown). In contrast to the control groups (data not shown), a pronounced airway inflammatory cell influx dominated by macrophages, lymphocytes, some epithelial cell shedding and in particular by eosinophils was found in BALF of the mice. However, only lymphocytes, epithelial cells and eosinophils were significantly affected by the investigated experimental factors. The number of lymphocytes, eosinophils and epithelial cells in BALF was significantly higher in female mice compared with male mice (Fig. 6A, B, C).

Intracellular cytokine staining for IFN-γ, TNF and IL-4 confirmed Th0 (IFN-γ+, TNF+ and IL-4+) and Th1 (IFN-γ+, TNF+ and IL-4low) cytokine profiles for CD4+ and CD4− NKT cells, respectively (Fig. 3). More extensive cytokine analysis

was conducted using CBA to analyse supernatants from cultures of FACS-sorted NKT cell populations stimulated with PMA and ionomycin for 16 h MI-503 to maximize cytokine output (Fig. 4). A striking finding was that CD4+ NKT cells produced higher cytokine concentrations of IFN-γ, TNF, IL-4, IL-13, GM-CSF and IL-2, despite intracellular flow cytometry analysis showing similar proportions of IFN-γ+ and TNF+ cells in CD4+ and CD4− NKT cell cultures after 4 h stimulation. We did not detect NKT cell production of IL-17 or selleck compound IL-10 (data not shown). Our data suggest that CD4+ NKT cells exhibit a more prolonged cytokine production than CD4− NKT cells. Having identified differential cell surface

antigen expression within the CD4+ and CD4− NKT cell subsets, we examined whether this reflected unreported functional heterogeneity. We focused on two antigens (CD161 and CD62L) known to be significant for classifying conventional T cell subsets. Analysis of FACS-sorted subpopulations showed that more CD161+ NKT cells were IFN-γ+ or TNF+ after 4 h stimulation than CD161− cells (Fig. 5a). This was broadly consistent those with CBA analysis of supernatants after 16 h of in-vitro stimulation (Fig. 5b). Differences were seen in cytokine production of sorted CD4+ and CD4− NKT cell subsets separated on the basis of CD161; however, these were inconsistent and the trend varied between cytokine types (Fig. 5b). NKT cell subsets defined by CD62L and CD4 expression provided more consistent trends. CD62L expression is lost transiently after stimulation, which prevented intracellular flow cytometry of unsorted NKT cell cultures, but CBA analysis of supernatants from

sorted cells revealed striking differences in the cytokine profiles at 16 h (Fig. 6). As expected, cultures of CD4+ NKT cells had the highest cytokine concentrations, but differential CD62L expression correlated well with cytokine production within each subset. For example, CD62L−CD4+ NKT cells were the most potent producers of IL-4 and IL-13 (with a similar trend for many other cytokines (Fig. 6), whereas the lower cytokine production by CD62L+ NKT cells was similar to CD4− NKT cells (CD4−CD62L+ and CD4−CD62L−). IFN-γ was an exception, with a similar concentration of IFN-γ detected in cultures of all four subsets defined by CD62L and CD4 expression. Most human NKT cell studies have involved cells derived exclusively from peripheral blood.

1 and 18.5% positive cells respectively (Fig. 5A and B). Furthermore, 23.3% of the memory B cells expressed the type I receptor activin receptor-like kinase (Alk) 2. In naive B cells, none of the three type I receptors were detected. Since a hetero-oligomeric receptor complex consisting of type I and type II receptors are needed to bind BMP and induce signaling, the functional effects observed in naive B cells were surprising, unless the stimulation conditions used (CD40L/IL-21) could upregulate BMP receptor expression. To test this hypothesis, we cultured

mononuclear cells from peripheral blood in the presence of CD40L/IL-21 for 24 h and then stained with anti-BMP receptor Abs, anti-CD19 or anti-CD20 Abs. buy GDC-0449 Interestingly, stimulation with CD40L/IL-21 doubled the MFI values of Alk-2 expression in CD19+ B cells, whereas only minor differences were seen for the other receptors (Fig. 5C). Specific analysis of naive and memory B cells by anti-CD27 Ab was not possible in stimulated mononuclear cells as CD40L/IL-21-induced downregulation of CD27 (Supporting check details Information Fig. 5) as shown previously 39. Stimulation of FACS-sorted naive B cells for 48 h confirmed that Alk-2 expression could be induced in naive

B cells (Fig. 5D). Taken together, naive and memory B cells expressed the type II receptor ACTR-IIB and the BMP type I receptor Alk-2 after stimulation with CD40L/IL-21. To investigate how the various BMPs mediate their functional effects in naive and memory B cells, we next investigated BMP-induced signaling. We stimulated peripheral blood CD19+ B cells with BMP-6 for various periods of time and examined activation of Smad1/5/8. BMP-6 induced phosphorylation of Smad1/5/8 after 30 min and reached maximum at 3 h of stimulation (Fig. 6A). The phosphorylation was still enhanced after 24 h. Furthermore, we tested the effects of BMP-2, -4, -6 however and -7, and all BMPs induced activation of Smad1/5 (Fig. 6B). The BMPs also induced phosphorylation of pSmad1/5 in the presence of CD40L/IL-21 (Fig. 6B), although weaker as CD40L/IL-21 reduced the phosphorylation level of Smad1/5/8 (Supporting Information

Fig. 6). As BMP-6 potently suppressed plasma cell differentiation and Ig production, we used this BMP to investigate the expression of key regulators of plasma cell differentiation, in addition to the BMP target genes ID1, ID2 and ID3. Real-time RT-PCR was performed on IgD-depleted memory B cells cultured for 2 or 4 days in the presence of CD40L/IL-21, with or without BMP-6. The expression of ID1 was increased 7.2- and 4.5-fold by BMP-6 after 2 and 4 days respectively (Fig. 7A). ID3 expression was increased 3.4-fold at day 4 in the presence of BMP-6, whereas ID2 was increased less than 2-fold. Furthermore, CD40L/IL-21 significantly increased the expression of IRF4, PRDM1 (gene encoding Blimp-1) and XBP1 at day 4 compared with day 2 (Fig. 7B).

© 2012 Wiley Periodicals, Inc. Microsurgery, 2012. “
“Reconstruction of the radial head can be complicated in cases of wide resection, particularly in those cases including the proximal radial shaft. In such cases, radial head replacement may not be possible because of lack of adequate bone stock. Here, we report the use of a radial head prosthesis incorporated with a vascularized fibula for immediate anatomic restoration of the forearm and elbow. We present a case of a pathologic fracture

non-union in the proximal radius in a 57-year-old female with a history of multiple myeloma. Non-operative management of the fracture was unsuccessful after chemotherapy and radiation. The proximal radius and radial head were resected

and reconstructed with vascularized fibula graft in conjunction with immediate radial head prosthesis. The osteotomy site healed at 6-weeks and follow-up at 1 year showed good functional outcome. We PI3K Inhibitor Library feel that the use of this find more construct has definite promise and may be considered for reconstruction following resection of the proximal radius. © 2014 Wiley Periodicals, Inc. Microsurgery 34:475–480, 2014. “
“The distally based sural flap has become popular for reconstruction of the foot and leg. However, this flap often fails due to venous congestion. In this report, we developed a new modification of the distally based sural flap. The procedure comprised three stages. In the first stage, the flap was raised cephalad to the midpoint of the posterior aspect of the leg, involving

reanastomosis of the short saphenous vein (SSV) at the proximal end of the flap. In the second stage, ligature of the SSV was performed. In the third stage, the entire flap was raised. We treated eight patients with the flap. All flaps survived completely. Duplex scanning indicated that venous drainage of the flap was provided by the tenuous venae comitantes (VCs) surrounding the SSV. Reanastomosis of the SSV may prevent rapid venous overloading of the VCs. Our new modification may be useful to avoid venous congestion. check © 2013 Wiley Periodicals, Inc. Microsurgery 33:534–538, 2013. “
“Background: Acute postoperative pain following craniofacial or esthetic surgery, or trauma is readily treated with medicinal regimens. Facial pain persisting for more than six months is defined as chronic and must be distinguished from nontraumatic atypical facial pain or “tic-douloureaux.” Our surgical experience managing chronic facial (trigeminal) pain is reviewed to provide insight into the success of our current algorithm for managing patients with chronic facial pain. Methods: We performed a retrospective review of nine consecutive patients operated for post-traumatic chronic trigeminal nerve pain. Most patients were women (mean age 41 years). Data evaluated included mechanism of nerve injury, physical exam, CT scans, computer-aided neurosensory testing, and diagnostic nerve blocks.

Therefore, social play, far from being just a playful occasion for mothers and infants to have fun together, works as a special form of triadic interaction, suited to introducing infants to LBH589 the domains of cultural artifacts, such as conventional norms and symbolic language (Bruner, 1975, 1982; Tomasello, 1999). In that respect,

it requires the infants to make many adjustments in order to act as full participants as they must: (1) focus on the same object as their partner, which means directing attention in a way that is different from dyadic interaction, (2) use that object together with the partner, therefore acting according to the other’s actions, and (3) say something selleck chemicals in line with their partner’s comments and thus use language in a dialogic manner. Joint attention skills by the end of the first year of life are too immature to allow infants to satisfy those requirements, as the infants’ poor performance in Bakeman and Adamson’s (1984) study clearly

showed. Indeed, to perform better, those infants should have put their joint attention skills to work in a context of shared activity and used them as a means of collaborating—rather than simply “playing”—with another person. Becoming an effective partner in collaborative interaction is, Dichloromethane dehalogenase however, a gradual process. As we know from the literature on early cooperation, infants are relatively incompetent in that domain, whatever form the cooperation may take: 12-

to 18-month-olds involved in social games do not go beyond ritualized interactions (Ross & Lollis, 1987) and 14-month-old infants fail to coordinate their actions with those of another person in problem-solving tasks (Warnecken, Chen, & Tomasello, 2006; Warnecken & Tomasello, 2007). Moreover, the partner must be an adult as children can not collaborate with a peer before the age of two (e.g., Brownell & Carriger, 1990, 1991; Brownell, Ramani, & Zerwas, 2006; Eckerman, 1993; Eckerman & Peterman, 2001; Eckerman & Stein, 1990; Goldman & Ross, 1978; Hay, 1979). Recently, the emergence and early improvement of cooperative skills has been related to the development of infants’ social understanding (Brownell et al., 2006). According to the social cognitive perspective, infants approaching their first birthday recognize other people as intentional agents like themselves and therefore come to appreciate them as potential partners in collaborative interactions. Some months later, the achievement of the so-called “we intentionality” (Tomasello et al.

The cTECs are primarily responsible for the generation and survival of the positively selected CD4+ CD8+ immature T-cell pool with an immunocompetent TCR repertoire, whereas the main function of mTECs and medullary DCs is to secure the negative selection of self-reactive T cells. The two epithelial cell types are morphologically and functionally distinct, nevertheless, the evidence for their common bipotent progenitor cells has started to accumulate during recent years. A paper by Baik et al. published in this issue of the European Journal of Immunology AZD5363 order [1] adds new evidence and perspectives to our understanding of the bipotent thymic epithelial progenitor cell (TEPC)

differentiation and lineage marker expression. The early differentiation of TEPC depends on a transcriptional program activated by

the transcription factor FoxN1; in mice with Foxn1 mutations Talazoparib TECs do not develop and thymopoiesis is blocked [2]. The transcriptional regulation of the later dichotomy of cTECs and mTECs has remained thus far unknown. What is known is that the separation between cTECs and mTECs is associated with changes in their keratin expression patterns. Though not absolutely, keratin K8+ K5− cells are predominantly cTECs and K8−K5+ cells are mTECs, whereas K8+K5+ cells, as well as K14+ cells, are often considered as epithelial precursor cells at fetal stages [3, 4]. In the adult thymus, K8+K5+ cells are present at the cortico–medullary junction but their potency as progenitor cells is unknown. Other epithelial markers have proven to be informative tools in the identification of epithelial

cell phenotypes. For example, cTECs express proteosomal subunit beta-5t (encoded by Pmsb11), Ly-51/CD249 (Enpep), delta-like ligand 4 (Dll4), serine protease 16 (Prss16) and CD205 (DEC-205, Ly75) with the earliest cTEC-specific markers detectable at E12. In contrast, the markers associated with mTECs are tight junction proteins claudin-3 and -4 (Cldn3 and 4) and lectin UEA1 with commitment to mTEC lineage at E13. The differentiation and full maturation of mTECs critically Sitaxentan depends on RANK signaling that stimulates the expression of CD80, MHC class II, CD40 and Aire, all needed to promote tolerance towards self-antigens (reviewed in [5, 6]). The presence of a large pool of thymic epithelial cells in the early thymus expressing cTEC and mTEC markers has been considered as an indication that both epithelial cell types share a common bipotent progenitor cell [7]. The clonal progenitor activity was initially described for the mTEC lineage using chimeric mice [8]. The existence of bipotent TEPCs was first indirectly addressed by the transplantation of bulk reaggregated thymic organ cultures under the kidney capsule [9-11], the direct evidence came from using a clonal assay with single thymic epithelial cells expressing yellow fluorescent protein (YFP) [12].

4A). Expression of CD25 prior to activation may provide the CD95+CD25INT memory

population with an advantage in the absence of added costimulation by allowing them to respond to lower levels of IL-2. CD25 is known to be greatly upregulated on T cells after activation and would negate any benefit of CD25 expression prior to activation [40, 41]. However, we found that only the CD95+CD25INT population upregulated CD25 in response to anti-CD3 alone (Fig. 4B). Since IL-2 signaling is known to augment CD25 IWR 1 expression on activated T cells [42], we evaluated IL-2 responses by intracellular pSTAT5 levels and found that only the CD95+CD25INT memory population increased pSTAT5 levels (Fig. 4C). Stimulation in the presence of high concentrations of exogenous IL-2 demonstrated that both populations are capable of upregulating both CD25 and pSTAT5 levels (Fig. 4B and Supporting

Information Ribociclib cell line Fig. 3A). To test the function of CD25 expression within the CD95+CD25INT population, we tested their ability to activate in the absence of costimulation. We found that anti-CD25-blocking antibodies interfered with the ability of CD25INT cells to form aggregates, upregulate CD25, and phosphorylate STAT5 (Fig. 4A–C). The decrease in CD25 staining was not due to blocking of the anti-CD25 detection antibodies, since the anti-CD25-blocking antibodies do not interfere with the anti-CD25 detection antibody (Fig. 1C and Supporting Information Fig. 3A). To further compare differences between CD95+CD25NEG and CD95+CD25INT memory cells and the role of CD25 during activation in the absence of costimulation, proliferative responses were determined. When stimulated with anti-CD3 alone, the CD95+CD25INT but not the CD95+CD25NEG cells proliferated robustly

Montelukast Sodium (Fig. 4D). However, blocking CD25 on the CD95+CD25INT cells interfered with their ability to proliferate (Fig. 4D). Conversely, when stimulated in the presence of anti-CD28 or exogenous rhIL-2, the CD95+CD25NEG population proliferated robustly, demonstrating that the CD95+CD25NEG cells are capable of proliferation. The CD95+CD25INT memory population consistently proliferated as well or better than the CD95+CD25NEG memory population under all conditions (data not shown). Lastly, cytokine concentrations determined from supernatant showed that CD95+CD25INT cells produced more cytokines than the CD95+CD25NEG population and that blocking CD25 had a negative impact on these cytokine levels (Fig. 4E). Interestingly, blocking CD25 on the CD95+CD25INT population increased levels of detectable IL-2. This observation may be explained by a lack of IL-2 internalization and also a lack of negative feedback on IL-2 production. Collectively, these data suggest that CD95+CD25INT cells stimulated in the absence of costimulation are able to respond to lower concentrations of IL-2 due to their expression of CD25 prior to activation.

[1] Microvesicles have protein content similar to the plasma membrane of activated platelets and have procoagulant and inflammatory functions.[79, 80] In contrast, platelet exosomes only interact poorly with annexin-V and do not bind prothrombin and factor X. Platelet-derived exosomes are enriched in CD63, a tetraspanin protein also found on exosomes from other cell types.[81] Tetraspanin proteins have been implicated in adhesive as well as co-stimulatory and signalling functions. Platelet-derived exosomes may be released at

sites of vascular injury and could well learn more function in promotion of platelet and neutrophil adhesion.[1, 82] Endothelial dysfunction and vascular calcification is a significant risk factor for cardiovascular morbidity and mortality in patients with renal disease. In vitro, vesicles appear to be important in mediating vascular smooth muscle cell calcification.[83] In a recent study, it was found that phosphorylated fetuin-A is present in the calciprotein particles in serum of predialysis chronic kidney disease (CKD) patients. Increased calciprotein particle fetuin-A levels reflect an increasingly procalcific milieu and are associated with increased aortic stiffness.[84] Increased levels of circulating microparticles

(MP) or microvesicles MS-275 concentration have been detected in patients with CKD. Circulating levels of MP and microvesicles derived from endothelial cells correlate with arterial stiffness in haemodialysis

patients.[85-87] It is unclear whether exosomes and/or other circulating MP may play an important role in transporting or promoting vascular calcification in CKD or in other calcification-associated GPX6 diseases. Nephrolithiasis is associated with the formation of calcium oxalate, calcium phosphate, cystine, struvite or urate crystals in the kidneys. In vitro studies have demonstrated that renal brush border-derived exosomes/microvesicles of ∼100 nm in diameter can induce and promote calcium oxalate crystallization in nephrolithiasis.[88] In transplantation, it has been shown that the exchange of exosomes between dendritic cells may constitute a potential mechanism by which passenger leukocytes transfer alloantigens to recipient antigen-presenting cells, leading to an increased generation of donor-reactive T cells.[89] On the other hand, other studies have found that dendritic cell-derived exosomes may induce tolerance rather than immune stimulation.[90] Engineering of dendritic cells to release tolerogenic exosomes could be useful to prevent/ameliorate transplant rejection. Urine is the ideal biological sample for discovery of new biomarkers for kidney diseases because of the ease of non-invasive collection.

The concentration of peptide required to generate 50% of the maximal response was used as a measure of avidity. Mice were sacrificed 14 days after a single priming vaccination. Single-cell suspensions from individual spleens were cultured in complete medium in 25 cm2 upright flasks (3×106 cells/mL) supplemented with 10−8 M of the corresponding PSMA HLA-A*0201-binding peptide and 20 IU/mL IL-2 (R&D Systems, Abingdon, UK). Following 6 days stimulation in vitro, the cytolytic activity of the CTL cultures was assessed in Pirfenidone a standard

5-h 51Cr-release assay. Target cells were labeled with 51Cr with or without peptide for 1 h. Target (T) cells (5×103) were then cultured with effector (E) T cells at different E:T ratios.

Specific % lysis was calculated by the formula: (release by CTL−release by targets alone)/(release by 4% NP40−release by targets alone)×100. Splenocytes harvested from naïve HHD mice were pulsed with 1 mM PSMA27, PSMA663, or control HLA-A*0201-binding Panobinostat cell line peptide (VLHDDLLEA) at a concentration of 2×107/mL in PBS. The cells were then labeled with either 0.5 or 5 μM CFSE (Molecular Probes, Invitrogen) for 8 min at 37°C before adding FCS to a final concentration of 20% to quench the reaction. After washing, the cells were mixed at a 1:1 ratio such that each prevaccinated mouse received 1×107 cells pulsed with PSMA peptide and the same number pulsed with control peptide in 0.1 mL PBS by intravenous injection. Splenocytes were harvested from individual mice 20 h later, lymphocytes were isolated using density gradient centrifugation and CFSE staining was analyzed by FACS Canto (BD Pharmingen). Lymphocytes from the same mice were also used in an ELISpot assay as described. HHD mice were vaccinated with p.DOM-PSMA27, p.DOM-PSMA663, or p.DOM control vaccine 13 days prior to the assay. TRAMP-PSMA+ HHD+, and TRAMP-HHD+ cells were labeled with 10 and 1 μM CFSE, respectively, as described above and then mixed in a 1:1 ratio. The cell suspension was then mixed

with Matrigel® (BD Biosciences) at a ratio of 1:1 so that each mouse received 1×106 of each population in a total volume of 150 μL by subcutaneous injection. Matrigel® cell suspensions were kept on ice Nintedanib (BIBF 1120) until the time of injection, according to the manufacturer’s protocol. After 5 days, the Matrigel® plugs were harvested and digested with 1 mg/mL collagenase/dispase and 0.5 mg/mL DNAseI. CFSE staining of the cells released from the plug was analyzed using a FACS Calibur (BD). The spleens of the same mice were used in an ELISpot assay, as described, to identify those responding to vaccination; animals with an IFN-γ response of less than twice the background or <50 SFCs/106 cells were excluded from the analysis. Experimental groups were compared using a Mann–Whitney U-test. In vivo tumor lysis was analyzed using Fisher’s exact test.