Home HD in Australia is practiced without remote monitoring, although most units will maintain an on-call service for patients via both nursing and medical staff, as well as machine technicians if needed. Some centres internationally mandate remote monitoring for home HD with the benefits of documenting Imatinib chemical structure adherence to treatment regimens, providing patient reassurance and allowing for data collection to study physiological effects of NHD. Additional safety precautions for patients undertaking alternative HD regimes, especially NHD at home, include securing of blood lines, floor moisture sensors that may aid in detection of blood or dialysate

leaks and the taping of a moisture sensor (such as an enuresis alarm or newly developed sensor patch) close to the AVF needle sites may allow the patient to recognize early needle dislodgement. There is limited literature

comparing parameters for patients undertaking different NHD schedules, either alternate-night (3.5 nights per week) or more frequent NHD (5–7 nights selleck kinase inhibitor per week). One Australian study (n = 34) compared biochemical and volume parameters between these regimens and reported significantly lower urea and creatinine levels (pre- and post-HD), higher calcium levels, reduced ultrafiltration rates and intradialytic weight gains in those undertaking the more frequent NHD regimen.41 In this study, 38% of patients doing alternate-night NHD still required phosphate binders compared with none in the more frequent group. The study concluded Thiamet G that NHD performed 5–7 nights per week offered optimum biochemical and volume outcomes, but alternate-night NHD may have additional appeal related to cost advantages with reduced consumable expenditure. A flexible dialysis programme should therefore offer varying time and frequency options for home HD patients to be sympathetic to the clinical rehabilitation and lifestyle aspirations of the individuals on dialysis. One further Australian study also assessing the control of biochemical

parameters in NHD patients receiving alternate-night HD (n = 26) showed that after conversion from conventional HD there was improvement in parameters of bone and mineral metabolism as well as reduction in vascular calcification.49 Alternate-night NHD is therefore effective and offers lifestyle advantages for patients compared with more frequent NHD and, although not as efficient as 5–7 nights per week, it may be that alternate-night is potentially more cost-effective. Alternative HD regimens like SDHD and NHD allow for increased flexibility in dialysis treatments and are associated with significant physiological and quality of life improvements when compared with conventional HD, although survival benefits are as yet unproven. Although larger studies are required to confirm benefits, there is an increasing interest in using these schedules.

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 Venetoclax in vivo 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 Dabrafenib 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 Abiraterone cost 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 salvage of hardware and reconstruction selleck of soft tissue defect remain challenging. In this report, we presented our experience on the use of the distally based saphenous neurocutaneous perforator flap combined with vacuum-assisted closure (VAC) therapy for the coverage of the soft tissue defect and the exposed hardware in the lower extremity with fracture. Between January 2008 and July 2010, seven patients underwent the VAC therapy followed by transferring a reversed saphenous neurocutaneous perforator flap for reconstruction of the wound with exposed hardware around the distal tibia. The sizes of the flaps ranged

from 6 × 3 cm to 15 × 6 cm. Six flaps survived completely. Partial necrosis occurred in one patient. There were no other complications of repair and donor sites. Bone healing was achieved in all patients. In conclusion, the reversed saphenous neurocutaneous perfortor flaps combined with the VAC therapy might be one of the options to PXD101 molecular weight cover the complex wound with exposed hardware in the lower extremities. © 2013 Wiley Periodicals, Inc. Microsurgery 33:625–630, 2013. “
“Postoperative flap

monitoring is a key component for successful free tissue transfer. Tissue oxygen saturation measurement (TOx) with near-infrared spectrophotometry (NIRS) is a method used for this purpose. The aim of this study was to identify external variables that can affect TOx. Patients who had breast reconstruction with free flaps were monitored Tideglusib prospectively and intra-operative details were recorded. Flap TOx was recorded with NIRS pre-extubation, postextubation, and then every four hours for 36 hours. At each of these time points, blood oxygen saturation (SO2), amount of supplemental oxygen, and blood pressure were recorded. Thirty flaps were monitored. Initially, a significant trend over time was detected such that for every increase of 24 hours, TOx decreased on average by 2.1% (P = 0.025). However, when accounting for SO2 levels, this decrease was no longer significant

(P = 0.19). An increase by 1% in SO2 produced an increase in TOx reading of 0.36 (P = 0.007). The amount of supplemental O2, systolic blood pressure, and diastolic blood pressure did not have a significant impact on TOx (P > 0.05). The TOx values were highest in the free TRAM flaps and were lower in decreasing order in the muscle-sparing TRAM, DIEP, and SIEA flaps (P > 0.05). The TOx values did not significantly correlate with vessel size, perforator number, or perforator row. Postoperative flap TOx was found to correlate with SO2 and was not significantly dependent on blood pressure, supplemental O2, or surgical variables. Careful interpretation of oximetry values is essential in decision making during postoperative flap monitoring. © 2014 Wiley Periodicals, Inc. Microsurgery, 2014.

Survival was not prolonged when IL-4Rα−/− donors were paired with WT hosts, or when IL-4 was blocked in WT controls (WT into WT) (Fig. 3A). To gauge the immunological impact of IL-4Rα deficiency, we measured donor T-cell cytokine production. We found that, in contrast to all other donor/host pairings, WT donor T cells did not produce large amounts

of IFN-γ and IL-17 when transferred into IL-4Rα−/− hosts (Fig. 3B). This donor/host pairing was also unique in the production of IL-10, a cytokine known to suppress both Th1 and Th17 responses (Fig. 3D). Given the improved survival of IL-4Rα-deficient hosts (WT into IL-4Rα−/−), we next asked whether STAT6-deficient sOva Rag2−/− STA-9090 datasheet hosts exhibit a similar phenotype. Surprisingly, we found that survival was not prolonged when WT donors

were transferred into STAT6−/− host and, in stark contrast to IL-4Rα-deficient hosts, that donor T cells produced large amounts of IFN-γ and IL-17 but little IL-10 (Fig. 3C). Survival was also unaffected when STAT6−/− donors were transferred into WT or STAT6−/− hosts, consistent with our finding that IL-4Rα−/− donors are pathogenic in both IL-4Rα-sufficient and deficient settings (Fig. 3A). Thus, Lenvatinib in vivo in the context of systemic autoimmune disease, IL-4Rα can promote lethal pathology by delivering STAT6-independent signals to innate lymphocytes and nonimmune cells. Although IL-4Rα-deficient Terminal deoxynucleotidyl transferase hosts survived longer than WT counterparts, they did eventually succumb to lethal autoimmune disease, typically culminating between

15 and 30 days posttransfer. However, in contrast to WT hosts, which exhibit massive weight loss and disseminated alopecia [14], moribund IL-4Rα−/− hosts were not emaciated and had a more localized alopecia characterized by patches of complete hair loss (Supporting Information Fig. 5 and data not shown). Also unlike WT hosts, IL-4Rα−/− hosts harbored large numbers of IL-4/IL-13 double-positive donor T cells at day 30, which suggests a shift toward a more Th2-type inflammatory response. The percentage of IL-10+ donor T cells was also increased at this later time point, as was the percentage of IFN-γ+ and IL-17+ cells, though it should be noted that these emerging Th1 and Th17 responses were lesser in magnitude than those seen in WT hosts at day 7 (Fig. 3E and Supporting Information Fig. 5). Thus, IL-4Rα-deficient hosts develop a systemic pathology that is different from that of WT hosts, one that is not only delayed, but also clinically and immunologically distinct.

In summary, our data suggest that RWE-stimulated enhancement of IL-1β production in LPS-treated THP-1 cells is mainly the consequence of the substantially increased pro-IL-1β expression and elevated caspase-1 activation. The induced gene transcription and expression

of pro-IL-1β together with key inflammasome components (caspase-1 and NLRP3) is dependent on the ROS production by the RWE-associated NADPH oxidases. Nevertheless, it is important to note that pollen grains and sub-pollen particles are complex EPZ-6438 biological packages composed of many components that can alter the functions of human cells. However, the observed interplay of RWE and LPS suggests a critical role of bacterial endotoxin in the pollen-induced allergic reactions that should be taken into account in designing treatments for allergic airway https://www.selleckchem.com/products/cobimetinib-gdc-0973-rg7420.html inflammations. The work was supported in part by the TÁMOP 4.2.1/B-09/1/KONV-2010-0007 project (to J.T. and A.B.), the TÁMOP-4.2.2.A-11/1/KONV-2012-0023 project (to S.B., J.T. and A.V.) the TÁMOP-4.2.2/B-10/1-2010-0024 project (to A.V.), the UD Faculty of Medicine Research Fund – Bridging Fund (to S.B.) and the Hungarian Science and Research Fund (K-73347 to A.B.). The project is co-financed by the European Union and the European Social Fund. S.B. is

a receiver of Lajos Szodoray Post-doctoral Fellowship and Janos Bolyai Post-doctoral Fellowship. The authors declare no competing interests. “
“Twelve Vibrio cholerae isolates with genes for a type III secretion system (T3SS) were detected among 110 environmental and 14 clinical isolates. T3SS-related genes were distributed among the various serogroups and pulsed-field gel electrophoresis

of NotI-digested genomes showed genetic diversity in these strains. However, the restriction fragment length polymorphism profiles of the T3SS-related genes had similar patterns. Additionally, naturally competent T3SS-negative V. cholerae incorporated the ca. 47 kb gene cluster of T3SS, which had been integrated into a site on the chromosome by recombination. Therefore, it is suggested that horizontal gene transfer of T3SS-related genes occurs among V. cholerae in natural ecosystems. Vibrio cholerae live ubiquitously in natural aquatic environments, such as rivers, estuaries and coastal Phospholipase D1 waters. There more than 200 recognized serogroups, among which serogroup O1 and O139 strains are known to produce CT and cause epidemic cholera [1]. Many serogroups of non-O1, non-O139 V. cholerae can also cause mild or severe diarrhea; certain of these strains possess the ctxAB gene encoding CT [2-5], whereas others do not produce CT. The virulence determinants of non-O1, non-O139 V. cholerae without ctxAB have not been well characterized. Gram-negative pathogenic bacteria have a T3SS that plays an important role in their pathogenesis [6]. Among Vibrio species, the genes for T3SS were first identified in V.

In addition, treatment with the dltD mutant Ku-0059436 clinical trial correlated with a significant down-regulation of Toll-like receptor-2 expression and of downstream proinflammatory cytokine expression in the colitic mice. These results show that molecular cell

surface characteristics of probiotics are crucial when probiotics are considered for use as supporting therapy in IBD. Inflammatory bowel diseases (IBD), such as Crohn’s disease (CD) and ulcerative colitis (UC), are chronic illnesses that involve inflammation of the intestinal tract [1]. An increased prevalence of these diseases has been documented in developed countries. It is estimated that more than 3 million people are affected in North America and Europe [2,3]. The pathogenesis of these diseases is not fully understood, but besides genetic, environmental and immunoregulatory factors, the enteric microbiota seem to play an important role. It is thought that the inflammation results from an aberrant mucosal immune response against the indigenous microbiota in genetically susceptible hosts [4]. Additionally, it has been found that IBD is linked to an altered microbiota composition (dysbiosis) [5]. Among the mechanisms by which

bacteria may promote inflammatory signalling, recent evidence suggests that microbe-associated molecular patterns (MAMPs) derived from intestinal bacteria may modulate IBD via stimulation of their respective innate immune receptors, including Toll-like receptors (TLRs) [6]. This is reflected, for example, by the dysregulation of several TLRs and susceptibility genes, such as nucleotide-binding oligomerization

domain-containing 2 Navitoclax chemical structure (NOD2), in colitis [7]. Some probiotics, which are defined as ‘live micro-organisms that when administered in adequate amounts can confer a health benefit on the host’[8], have been suggested to help in restoring the imbalances associated with IBD [9,10]. Therefore, probiotics might be useful as supporting therapeutic agents, although the results of clinical trials were not always unambiguous [10–12]. A crucial factor might be the choice of the probiotic strain. One of the best-documented and model probiotic strains is Lactobacillus rhamnosus GG (LGG) [13]. Well-substantiated health effects include prevention of acute diarrhoea Alectinib in children [14], prevention of antibiotic-associated diarrhoea [15–17], prevention of atopic disease [18] and treatment of recurrent Clostridium difficile-associated colitis [19]. In IBD patients, most promising clinical effects with LGG are in prevention of pouchitis [20] and maintenance of remission in UC [21], while clinical studies with LGG in patients with CD did not result in positive outcomes [22–24]. Some molecules of LGG have been suggested to be important for the probiotic effects based on in vitro studies. For example, two secreted proteins of LGG were demonstrated to prevent cytokine-induced apoptosis in intestinal epithelial cells [25].

Cryptosporidiosis has been also reported as a common serious primary cause of outbreaks of diarrhoea in newborn calves, goats and sheep. Presently, there is no effective therapeutic agent for the treatment of infection in immunodeficient individuals. Thus, there have been increasing efforts geared towards development of vaccines to control the disease. Cryptosporidium sp. infection is caused by ingestion of sporulated oocysts transmitted by the faecal-oral Ku-0059436 route. After being ingested, the oocysts excyst and release sporozoites that attach to and invade the microvilli of the epithelial cells

of the small intestine and cause pathology seen in the disease (2). In this process, the surface proteins of the sporozoites play an important role. Therefore, to develop the vaccine against the disease, many studies have focused on the analysis of the surface antigens of sporozoites. Among these antigens, the 15-kDa (Cp15) and 23-kDa (Cp23) are considered immunodominant and relevant to infection, and the most promising candidates for vaccine development (3,4). Cp23 is a glycoprotein, geographically conserved among C. parvum isolates and is present in both the sporozoite and merozoite stages. Cp23 was an immunogenic antigen in domestic isolates

of C. parvum (5). Colostrums from cattle hyperimmunized with recombinant (r) Cp23 provided protection against diarrhoea and significantly reduced oocyst shedding in calves. IgA-isotype Staurosporine supplier monoclonal antibodies to Cp23 orally administered to mice prior to inoculation with oocysts provide protection against C. parvum infection. Studies also have demonstrated cellular responses to Cp23 antigen by cells obtained from mice infected with C. parvum (6) and human peripheral blood mononuclear cells (PBMC)

(7). Wyatt et al. (8) demonstrated Cp23-specific T cell Adenosine triphosphate responses in calves after recovery from C. parvum infection. These observations suggest that the Cp23 antigen is involved in the generation of immune responses to C. parvum and may be a possible vaccine target antigen. The Cp15 protein is present on the surface of sporozoite of C. parvum (9). Studies have shown that Cp15 had strong immunogenicity to C. parvum. Tilley et al. found that this 15 kDa glycoprotein was among the most prominent antigen recognized by hyperimmune bovine colostrum (10). The oral administration of anti-Cp15 IgA monoclonal antibodies (McAbs) to suckling mice also provided protection against infection. Hill et al. noted that it was strongly recognized by both serum antibodies and faecal IgA in colostrum-deprived lambs (11). Spleen-derived McAbs against Cp15 have been shown to decrease infection levels in mouse models.

101,102 However, lymphokine-activated killer cells (LAKCs) lyse trophoblast, and activated NK cells cause abortion.18,103 This suggests that ‘tolerance’ can be broken by systemic activation. In this context, immunisation with OVA induces abortion in OVA transgenic mice,41 an occurrence not seen with classical transgenics. Thus, a ‘modified’ placenta can be seen/rejected as a transplant, but OVA at the trophoblast surface does

not necessarily have the high turnover of MHC class I.58 If this explanation is correct, OVA immunisation should 13 not affect an OVA–MHC recombinant protein transgenic foetus. This bears interest also, as in a Greek study, many patients with RSA were virus positive.104 The forced induction of class II alloantigen on the placenta to induce abortion, as reported CRM1 inhibitor by Athanassakis et al., is very controversial, as Mattson’s group did not reproduce

it. IDO blockade of abortions is mediated by CD4+, not by CD8+ T cells,69 pointing towards a crucial role of local macrophages and complement. Antigen-presenting cells, notably dendritic and CD11+ cells, are involved in the creation of a privileged local selleck products microenvironment,105 while also being crucial for decidualisation/implantation.106 In CBA × DBA/2 matings, syngeneic dendritic cell therapy increases local CD8+, γδ T cells, TGF-beta1, and PIBF, correlating with decreased abortion rates.105,107 A pivotal role was shown for galectin-1 (Gal-1), an immunoregulatory glycan-binding protein, synergising with progesterone. For the influence of stress in pregnancy, we direct readers to recent reviews.108,109 Maternal non-rejection of the foetus also necessitates local regulation /cohabitation with the local innate immune Tau-protein kinase system. Cytotoxic alloantibodies in many species call for complement regulation, and indeed activation of complement is abortifacient.110,111 Also, differential levels of MBL (mannan-binding lectin) are observed in CBA × DBA/2 versus CBA × BALB/c mice and in human patients.112 But complement is regulated at the fetal–placental interface by placental regulatory proteins. Mice made KO for crry destroy their embryos even in syngeneic pregnancy.113

MCP and DAF play this role in humans. Hence, prevastatin is to be tested for abortion and preeclampsia therapy. We will not detail uNK cells and angiogenesis, but according to the missing self-theory, MHC-negative trophoblast, while protected against T-cell effectors by lack of target molecules, should be destroyed by NK cells. The low lytic activity of uNK cells, per se, might seem to be a protection. In fact, while syncytia cannot be destroyed as easily by a single ‘hole’ and offers considerable capacity of self-repair, one should recall that activated NK cells are abortifacient as also seen in ‘natural’ CBA × DBA/2 matings.18 This activation is controlled by the NK-repressing activity of the already detailed HLA-G, placental factors, PIBF, and IL-10.

8c,d). In the present study, the serum levels of TNF-α, which is an inflammatory cytokine, were studied in the CLP model in the sera of rats (Fig. 9). Levels of TNF-α were found to be increased in

the CLP group when compared with the sham-operated animals, as seen in Fig. 9 (P < 0·01). In contrast to the CLP group, the serum levels of TNF-α were found to be decreased by the administration of SLD in septic rats (CLP + SLD groups) (P < 0·01). As shown in Fig. 9, administration of SLD alone in sham-operated rats did not affect the serum levels of TNF-α when compared with the non-treated sham group. In this present study, we determined that sildenafil has markedly protective effects against CLP, attenuating kidney and lung tissue injury, especially in the vascular bed, and decreasing oxidative stress, as confirmed RAD001 order by biochemical assays and histopathological study. This protection is due primarily

to the inhibition of oxidative stress, which is one of the important mechanisms of organ injury of polymicrobial sepsis, and inhibition of the degree of inflammation, as revealed clearly by our finding see more that pretreatment with sildenafil increased GSH and decreased the activation of MPO and LPO and levels of SOD. We observed a significant decrease in LPO and MPO and a decrease in SOD activity in the sildenafil-treated CLP rats compared with the vehicle-treated sham-operated rats, demonstrating the protective capacity of sildenafil Tenofovir in septic rats. Another result of our study is that sildenafil treatment improves inflammatory cells that accumulate

in the lungs and result in lung injury in septic rats. According to our histopathological analysis, significant differences were found in terms of inflammation scores between the sepsis group and the other groups, except in the CLP + sildenafil 10 mg group. The CLP + sildenafil 20 mg/kg group had the lowest inflammation score in our study. Koksal et al. [50] reported that in caecal ligation and puncture (CLP)-induced sepsis, increased oxidative stress in tissue in parallel with plasma are important mechanisms due to the output of free radicals [50]. Moreover, according to Sakaguchi et al. [51], endotoxin injection resulted in lipid peroxide formation and membrane damage in experimental animals, causing a decreased level of free radical scavengers or quenchers [51]. ROS have been assumed to play a role in the induction of many proinflammatory cytokines and mediators important in producing the acute inflammatory responses associated with sepsis [12]. In our previous studies we determined that kidney, heart, lung and liver tissue exhibited oxidative stress in septic rats [40–42]. The proinflammatory effects of ROS include endothelial damage, formation of chemotactic factors, neutrophil reinforcement, cytokine release and mitochondrial injury [14–16], which all contribute to free radical overload and to oxidant–anti-oxidant imbalance.

3D). Finally, to confirm that LTi-like cell survival in vitro did not require IL-7, LTi prepared

from Rag−/−γc−/− mice (therefore unresponsive to IL-7) were cultured with CD45−podoplanin+ SSCL and in this assay survival was not affected (Fig. 3C). To examine whether adult LTi-like cells were able to survive without IL-7 or γc cytokine survival factors within selleck screening library the splenic T zone in vivo, tissue sections of spleen from IL-7−/− and Rag−/−γc−/− mice were stained for LTi-like cells and analyzed by immunofluorescent confocal microscopy. In both types of mice, LTi-like cells were distributed within the splenic T zones and these cells were in close proximity with VCAM-1+ stroma of T-cell areas suggesting interactions between LTi-like cells and surrounding T-zone stroma (Fig. 3E). Together, these data provide evidence that splenic adult LTi-like cells are able to survive in vivo in an IL-7-independent manner. In the embryo, IL-7 is important in controlling numbers of LTi. In transgenic Maraviroc price mice expressing high levels of IL-7, LTi numbers are greatly increased, resulting in increased numbers of Peyer’s patches and ectopic LN 20. Current data indicate that IL-7 improves embryonic LTi survival. Our previous studies have demonstrated that LTi persist in the adult spleen of IL-7−/− and γc−/− mice 18. In this study we identified IL-7Rα+ and IL-7Rα−

LTi-like cells in adult spleen. We found that CD45−podoplanin+ SSCL promote the survival of adult LTi-like cells in an IL-7 independent manner, and that LTi-like cells isolated from Rag−/−γc−/− next mice survived well in culture with CD45−podoplanin+ SSCL. Finally, splenic LTi-like cells exist in IL-7−/− and γc−/− mice in situ and these cells remain in the white pulp areas where they interact with VCAM-1+ T-zone stroma which express podoplanin. Taken together these data suggest that stromal-derived factors control the survival

of LTi in the adult. While IL-7 plays a key role in vivo, there appears to be redundancy in the signals supporting LTi-like cell survival in the adult spleen. All experiments were performed in accordance with UK laws and with the approval of the University of Birmingham ethics committee. C57Bl6, RAG2−/−, RAG2−/−/commonγchain−/− (Rag−/−γc−/−) and CD3ε-transgenic (CD3εtg) mice were all bred and maintained in the Biomedical Services Unit at the University of Birmingham. CD3εtg mice have a profound block in the early T-cell development at the CD4−CD8−CD25−CD44+ stage in the thymus and display a complete loss of T-lymphocytes 21. Mouse spleens were excised and digested in RPMI 1640 medium with 10% FCS plus 1 mg/mL collagenase/dispase (Roche) and 20 μg/mL DNase 1 (Sigma) at 37°C for 20 min. To aid digestion, tissues were agitated to disperse aggregates.