From our previous study (Pokkali et al., 2009), an MOI of 3 was found optimum for infecting PMNs, and hence, same was kept as standard throughout this study. Because we aimed at observing the initial effect of mycobacterial vaccine strains on neutrophils, early time point

of 4 h was chosen. Uninfected neutrophils (Control) served as negative BAY 80-6946 mouse control, and 10 nm phorbol myristate acetate (PMA) (Sigma Chemicals)–stimulated cells were used as positive control. After 4 h, the neutrophil culture supernatants (Nu sups) were collected, centrifuged, and used to stimulate peripheral blood mononuclear cells (PBMCs), and the remaining was stored in aliquots at −70 °C until use. The cells were washed with PBS twice and used for fluorescence-activated cell sorting (FACS) staining protocol as given in the section ‘cell phenotyping https://www.selleckchem.com/products/BAY-73-4506.html by flow cytometry’. The buffy coat containing PBMCs was collected after Ficoll-Hypaque density gradient centrifugation. The cells were washed once with Hanks’ balanced salt solution (HBSS) and suspended in RPMI 1640 medium supplemented with 1% FBS. The cell viability was always found to be > 95% through trypan

blue exclusion test, and the cell density was adjusted to 1 × 106 mL−1. The cells were stimulated with 200 μL of infected Nu sups and cultured in 12 Well Clear TC-Treated Multiple Well Plates (Corning

Life Sciences) for 18 h at 37 °C in a humidified 5% CO2 incubator. After 18 h, the cells were harvested and stained for FACS as given in the section ‘cell phenotyping by flow cytometry’. Cell Fluorouracil manufacturer surface expression of CD32, CD64, TLR-4, and CXCR3 on neutrophils (CD16+ve); CD69 and CXCR3 on T helper cells (CD4+ve); and CCR5 and CCR7 on monocytes (CD14+ve) was determined by staining the cells using the monoclonal mouse anti-human conjugated antibodies, i.e. CD16 (clone 3G8)–fluorescein isothiocyanate (FITC), TLR-4 (clone HTA125)–phycoerythrin (PE), CD32 (clone FL18.26), CD64 (clone 10.1), CD4 (clone RPA T4), CD14 (clone M5E2)–allophycocyanin (APC), CD69 (clone FN50)–phycoerythrin-cyanine5 (PE-Cy5) (BD Pharmingen), and CCR5 (clone 45549)–FITC, CCR7 (clone 150503), CXCR3 (clone 49801)–PE (R & D Systems), and their fluorescence emission was detected in FL-1 (FITC), FL-2 (PE), FL-3 (PE-Cy5), and FL-4 (APC) channels. The above specified clones were used throughout the study. Briefly, cells were incubated with PBS containing the combinations of antibodies at saturation for 20 min at 4 °C. Cells were washed and fixed with 1% paraformaldehyde (Sigma Chemicals) in PBS and analyzed on a FACSCalibur flow cytometer (Becton Dickinson).

72 Also similar to IBD, patients suffering from untreated coeliac disease have increased numbers of FoxP3+ Tregs and IL-10-producing Tr1 cells in the intestine,73–77 the latter known to be

gliadin specific.78 The failure Fulvestrant cost of Tregs to control inflammation in this disease may therefore be a consequence of their functional impairment or target resistance. Circulating FoxP3+ CD4+ T cells from patients with active coeliac disease do not efficiently inhibit autologous effector T cells, but they are functional when co-cultured with T cells from healthy donors.77 Moreover, Tregs from healthy adults fail to suppress effector T cells isolated from coeliac patients.77 Analogous to the data from IBD studies, these data suggest that in coeliac disease the immune defect is not intrinsic to the Tregs, but rather is related to the resistance of effector T cells to suppression. Coeliac disease therefore represents an ideal setting in which to test whether antigen-specific Treg cell therapy can reverse established mucosal disease. Not only is the antigen well-defined, but it could also be administered

and removed as necessary. The availability of tetramers to track gliadin-specific T-cell responses would also allow quantitative Selleckchem Compound Library monitoring of crucial components of the response to therapy in these patients.79 Inflammatory bowel disease is thought to be a multi-step process involving an initial barrier injury, leading to a shift in the normal intestinal microbiota,20,80 increasing numbers of Enterobacteriaceae and reducing the species thought to protect from IBD, such as Faecalibacterium and Roseburia.20 The microbiota facilitate post-thymic education of the immune system and are important for tolerance to microbial antigens,81 so changes in the

gut flora in IBD may be a driving force for effector T-cell responses through against commensal bacteria and must therefore be considered in the context of cellular therapy. Indeed, in mice, colitis does not occur unless microbial antigens are present to drive activation and differentiation of T cells.82 The intestinal microbiota also plays an important role in modulating Tregs. For example, certain species of commensal bacteria specifically promote FoxP3+ Tregs in the colon,83,84 and some species of bacteria induce tolerance by signalling through TLR2 on Tregs.85 Hence, depending on the balance of species, microbial communities may either drive pathogenic T-cell responses or induce Tregs in a normal homeostatic environment. It follows that for Treg cellular therapy to be effective in IBD, microbial communities may need to be shifted towards a balance of species that is more permissive of tolerance. One way that the microbiome could be manipulated is by administration of probiotics.

Regarding Tregs, numerous studies reported decreased levels of Tregs and/or suppressed Treg function in patients with myocarditis or idiopathic cardiomyopathies [25-29]. In the present study, Vemurafenib clinical trial similar blood levels of Tregs (defined as CD4+CD25+CD127low and expressed as% CD3+ T cells) were observed in patients with iDCM and age-matched patients with stable and chronic ischaemic cardiomyopathy. A novel finding is that iDCM patients with low levels of Tregs (<4%) showed a significant of improvement of systolic LV function after IA therapy, whereas patients with higher levels (≥4%) did not respond to this treatment.

The number of Tregs increased in responders in the observation period and shows

no difference to other groups 6 months after IA. In addition to these results, we found that another subset of helper T cells is influenced by IA + IgG substitution. These Th17 cells play an important role in the induction of autoimmune tissue injury. They are distinct from Th1 or Th2 cells because they do not produce classical Th1 or Th2 cytokines such as IFN-γ or IL-4. There is a functional antagonism between Th17 and Treg cells. Both populations are regulated by variable levels of TGF-ß and IL-6. At a steady-state level or in absence of inflammatory stimuli, TGF-ß Tyrosine Kinase Inhibitor Library cell line suppresses the generation of T effector cells and induces FoxP3 regulatory T cells and thereby maintain self-tolerance. In state of inflammation, IL-6 suppresses the generation of TGF-ß-induced Treg cells and induces a pro-inflammatory T cell response predominated by Th17 cells [30, 31]. In our study, IA-responding patients had higher levels of Th17 cells compared to non-responders and control patients with ischaemic heart failure. These observations have to be confirmed in larger trials. But this observation may be a first step to characterize a subgroup of patients with iDCM who do best benefit from IA therapy. It is not known Edoxaban how IA therapy can affect cell-mediated immune responses.

Particularly, it is not known whether non-specific removal of IgG antibodies and/or non-specific ‘immune-modulatory’ effects secondary to plasmapheresis and/or IgG substitution after IA are responsible for this phenomenon. Autoantibody-induced inflammation can be separated into two components, autoantibody production and local inflammatory response. Tregs suppress both components, thereby controlling autoimmune inflammation. Follicular Tregs may suppress follicular T helper cell–mediated antibody production. CD4+CD25+FoxP3+ Tregs have the capacity to control inflammation by suppressing cytokine production in T helper cells. Furthermore Tregs are able to suppress innate cells via IL-10 production. These IL-10 producing cells may also play a pivotal role in regulating Th17 cells [32].

mirabilis. Orf9 belongs to the group 1 family of glycosyltransferases (Pfam00534, E value = 9 × e−28) and shares 33% identity to glycosyltransferase of Herpetosiphon aurantiacus. Therefore, orf7, orf9, and orf12 were proposed to encode the three glycosyltransferases and were named wpaA, wpaB, and wpaD, respectively. Among four known pathways for synthesis and translocation www.selleckchem.com/products/MK-2206.html of O-antigen (Hug et al., 2010; Valvano, 2011), the Wzx/Wzy-depending pathway occurs in the synthesis of the majority of O-antigens, especially heteropolymeric O-antigens. Both Wzx (flippase)

and Wzy (O-antigen polymerase) are highly hydrophobic inner membrane proteins, usually sharing little sequence identities with their homologues. In the O40-antigen gene cluster, orf6 and orf8 are the only two genes encoding predicted membrane proteins. Orf6 has 12 predicted transmembrane segments, which is a typical topology for Wzx proteins, and shares 46% identity or 63% similarity with putative flippase of E. coli O91. It was proposed that orf6 encodes the O-antigen flippase and was named wzx. Orf8 exhibited no sequence identity to any protein in GenBank. However, the transmembrane region search indicated that it had 10 predicted transmembrane segments with a large selleck products periplasmic loop of 34 amino acid residues. One or two such loops have

been reported for a number of O-antigen polymerases (Islam et al., 2010; Islam et al., 2011; Daniels et al., 1998) and seemed to be important in the recognition of the O-unit or/and for the catalytic activity (Valvano,

2011). Therefore, orf8 was proposed to encode O-antigen polymerase and, accordingly, was designated wzy. These findings suggested that the biosynthesis of the P. alcalifaciens O40-antigen is mediated by the Wzx/Wzy-dependent process. orf15, orf16, and orf17 are homologues of wza, wzb, and wzc genes required for the biosynthesis and export of group 1 and 4��8C 4 capsular polysaccharides (CPS) (Whitfield, 2006). In particular, tyrosine–protein kinase Wzc and its cognate tyrosine phosphatase Wzb are essential for maintaining polymerization process, and Wza is involved in forming an outer membrane pore through which the CPS is translocated (Collins et al., 2007). Together with a nonessential gene named wzi, the wza, wzb, and wzc genes comprise a conserved locus within group 1 CPS biosynthesis clusters of E. coli (Whitfield, 2006). In contrast, in E. coli group 4 capsular producers, the wza, wzb, and wzc genes are accompanied by the ymcABCD genes and located outside the CPS gene cluster. Both group 1 and 4 capsules can be anchored to the cell surface by means of core-lipid A giving rise to the so-called KLPS. Some strains coexpress KLPS with a “normal” LPS, whereas others produce KLPS as the only serotype-specific polysaccharide (Whitfield, 2006). The latter seems to be the case of P.

The 24 h urine albumin excretion rate of diabetic db/db mice decreased after exposure to elevated miR-21. The same study also identified PTEN as a target of miR-21.38 Another study has reported overexpression of miR-377 in human and mouse mesangial cells when exposed to high glucose levels.39 MiR-377 has been demonstrated to reduce the expression of p21-activated kinase (PAK1) and manganese superoxide dismutase (mnSOD). This enhances fibronectin production, which is characteristic of mesangial cells in diabetic nephropathy. We anticipate that many other miRNAs check details expressed in podocytes, tubular and other renal cells will be deregulated under hyperglycaemic conditions. In diabetic nephropathy,

alteration of miRNA expression in response to several pathophysiological states is of interest, notably hypoxic-ischaemic and hyperglycaemic stimuli. The findings by Wang and colleagues have already provided the first glimpse of the effects of hyperglycaemia on miRNA expression in mesangial cells. In addition, hyperglycaemia has been found to affect endothelial dysfunction through miR-221.40 Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited renal diseases. Genetically, mutations in the polycystic kidney disease-1 gene (PKD1) account for 85%

of ADPKD; whereas mutations in the polycystic kidney disease-2 gene (PKD2) are responsible for the remainder.41 PKD2 encodes a protein termed polycystin-2. Aberrant expression of polycystin-2 causes abnormal proliferation of renal tubular and biliary epithelial cells, eventually leading to cystogenesis.42,43 Selleck MLN0128 The potential role of microRNAs in control of expression of PKD genes and in mediating functional effects has recently been explored. Two groups have demonstrated

that miR-17 directly targets the 3′UTR of PKD2 and post-transcriptionally represses the expression of PKD2.44,45 Moreover, they also showed that overexpression of miR-17 may promote cell proliferation via post-transcriptional repression of PKD2 in HEK293T cells. Finding new miRNAs that target PKD1 is an area of active research. Using a rat model of PKD, 30 differentially Erastin datasheet expressed miRNAs have been identified in diseased kidney tissues compared with healthy rat, 29 of which are downregulated.46 Two algorithms: TargetScan and miRanda, predicted targets for significantly deregulated miRNAs in PKD that were correlated with pathways affected in PKD as determined using KEGG, GeneOntology (GO), Biocarta and the Molecular Signature databases.47–50 The deregulated miRNAs in PKD were associated with genes in 24 functional categories, including several pathways important to cyst formation such as mTOR signalling, mitogen-activated protein kinase signalling, Wnt signalling and TGF-β pathway.46 However, these correlations require experimental validation. MiR-15a has been reported to modulate the expression of cell cycle regulator Cdc25A and affect hepatic cystogenesis in a rat model of PKD.

This result suggests that iNKT cell activation by microbes can lead to severe inflammation

in some cases. Recent studies have indicated that the iNKT cell response to Sphingomonas spp. is important in the pathogenesis of PBC, an autoimmune disease characterized by the destruction of small bile ducts in the liver. PBC patients express antibodies against mitochondrial PDC-E2 in serum (45). Interestingly, N. aromaticivorans, a member of the Sphingomonodaceae family found in human intestines, also expresses PDC-E2 (45). Serum from PBC patients reacts with N. aromaticivorans, but not with E. coli (45). Mice infected with N. aromaticivorans express antibodies against PDC-E2 and develop chronic inflammation in the small bile duct mediated by autoreactive T cells, iNKT cells being required in Selleck Antiinfection Compound Library this process (59). These

results indicate that iNKT cells play an important Selleck MLN8237 role in PBC pathogenesis. When iNKT cells are activated by αGalCer or its analogues, they stimulate many other cells, including APCs, NK cells, B cells and conventional T cells (1–4). Glycolipid mediated iNKT cell activation induces protective responses against various microbial pathogens including bacteria, fungi, parasites and viruses (1–4). For example, αGalCer treatment has a positive effect during certain microbial infections. In mouse pneumonia models with P. aeruginosa and S. pneumoniae,αGalCer treatment induces rapid clearance of bacteria from the lungs by activating alveolar macrophages and increasing neutrophil recruitment to the lungs, respectively (11, 60). In a urinary tract infection model with E. coli, P. aeruginosa, and methicillin resistant Staphylococcus aureus, αGalCer treatment enhances antibacterial effects (61). α−galactosylceramide treatment has also been shown to be protective in mice infected with intracellular fungi and bacteria. During C. neoformans infection, αGalCer treatment enhances clearance of fungi from the lungs and spleen through an enhanced Th1 response (62).

When mice infected with L. monocytogenes, an intracellular Gram-positive bacterium, are treated with αGalCer, bacterial numbers in the liver, before spleen and peritoneal cavity decrease compared to control mice (63). iNKT cells stimulated by αGalCer enhance the killing of L. monocytogenes in macrophages with an increased respiratory burst (63). Similarly, in M. tuberculosis infected mice, αGalCer treatment prolongs survival and decreases the bacterial burden and tissue injury in the lungs (64). Furthermore, a combination of αGalCer and isoniazid, a first line antibiotic for tuberculosis, reduces bacterial numbers in the spleen and lungs in mice significantly more than does isoniazid alone (65). Human iNKT cells have also been shown to have lytic activity involving granulysin (an antimicrobial peptide) against M. tuberculosis infected APCs, and this is greatly enhanced by αGalCer (22).

The resulting supernatant was resuspended in 10 μL of Solution A. The protein concentration of the nuclear and mitochondria/cytoplasm fractions was determined using the Biorad Protein Assay. These procedures were done as previously described 20. Quantitation of the Western blots was performed using Adobe Photoshop CS3 as described (http://lukemiller.org/journal/2007/08/quantifying-western-blots-without.html).

Briefly, the Adobe Photoshop lasso tool was used to outline each protein band and a background region on the membrane. The mean gray value and the pixel value were multiplied to determine the absolute intensity of the band. When no band was visible, the outlined region was made equal in pixel number to that of the JQ1 order background region. The background to be subtracted from a given band was determined by multiplying the mean gray value of the outlined background region by the pixel

measurement for the corresponding band. The authors thank Victor E. Marquez for his generous gift of HK434 and Yuefang Sun for taking care of the mouse colonies. BIBW2992 nmr This study was supported by a grant from the National Institute of Health (to A. W.) and the Research Supplement for underrepresented minorities from the National Cancer Institute (to J. T.). Conflict of interest: The authors Selleck Gefitinib declare no financial or commercial conflict of interest. “
“Monocytes are blood leukocytes that can differentiate into several phagocytic cell types, including DCs, which are instrumental to the inflammatory response and host defence against

microbes. A study published in this issue of the European Journal of Immunology by Balboa et al. [Eur. J. Immunol. 2013. 43: 335-347] suggests that a shift of the CD16− monocyte population toward a CD16+ subpopulation may represent an immune evasion strategy that ultimately favors persistence of Mycobacterium tuberculosis. Together with other recent reports, the article by Balboa et al. sheds new light on the function of CD16+ monocytes in health and disease; in this commentary, we discuss the implications stemming from these findings. Immunity to pathogens and inflammatory reactions relies on the coordinated action of several immune cell populations including lymphoid cells and monocyte-derived phagocytes, such as macrophages and DCs. Monocytes are generated in the marrow and circulate in the blood where they can patrol the whole body for signs of infection or inflammation, and migrate to injured tissues upon attraction by several chemokines and microbial ligands. Monocytes exhibit high plasticity and can differentiate into a variety of cell subsets depending on their microenvironment in infected or inflamed tissues [1, 2].

78 Most studies on the location of effector/memory T cells in non-lymphoid tissues have focused on entry (homing) or proliferation and survival as determining factors of lymphocyte content in a given tissue. Recent findings have shown that exit from the tissue is an find more active process controlled by chemotactic mechanisms.

The chemokine receptor CCR7 was shown to be required for T-cell exit from inflamed peripheral tissue.79,80 Another chemotactic agent, sphingosin-1-phosphate (S1P), and its receptors are required for the exit from lymph nodes, a finding emerging from studies with the drug FTY 720, which displays immunosuppressive effects. Both CCR7 and S1P receptors are modulated in the course of T-cell activation, and thereby might cause the transient retention of recently activated T cells in the lymph node.81 When CCR7 is knocked out, the number of T cells retained in an inflamed tissue doubles, confirming its importance for continuous circulation.82 For technical reasons, quantification of exit rates for specific subsets of cells and specific tissues is

more difficult. However, a variety of data are available from early studies in which cannulation of the thoracic duct or even single lymph nodes was applied, Ku-0059436 molecular weight which provided clear evidence not only that naïve cells entering a lymph node via the high endothelium pass through the tissue within half a day and exit it, but also that large numbers of effector/memory cells attracted to an inflamed tissue, or generated by local proliferation, exit the tissue via the efferent lymph.83 It is conceivable that

the process of emigration also underlies a variety of regulatory influences; T-cell activation upon antigen encounter within the tissue may be one factor, but an influence of inflammation-generated mediators such as prostaglandins has also been described.84 The directional movement towards a chemical Casein kinase 1 compound plays a major role in the recruitment as well as the egress of T cells from the site of an immune response. Leucocytes are able to integrate signals from multiple chemoattractants in their migration.85 In fact, cells migrating away from a local chemoattractant source actually chemotax towards distant attractants. The ability to navigate through chemoattractant arrays may be sufficient to explain entry and egress of T cells during an immune response. However, recent evidence supports the existence of both chemoattractants and chemorepellents that guide the directed movement of leucocytes into and out of tissues. Chemorepulsion is defined as the migration away from peak concentrations of a chemokine and was initially studied in the context of axonal guidance, where the same molecule may act as a chemoattractive or chemorepulsive cue depending on the receptor expressed on the cell surface.

We next examined the mannan structure of CMWS and compared it to that of CAWS, because we have previously found that the mannan moiety might be responsible for these activities (9–15), and many reports have indicated that Candida cell wall mannan contributes to its antigenicity and pathogenicity (30). In addition, the structure of

mannan from Candida differs between species (21, 31–35) and can also be altered by environmental conditions such as growth temperature (18), pH (19), and osmotic pressure (20). As revealed by the reactivity of Candida serum factors (Table 3), CMWS reacted to antisera against α-mannan but not β-mannan. Moreover, NMR analysis of CMWS confirmed that CMWS contains only α-mannosyl, Atezolizumab datasheet and not β-mannosyl, residues. These serum reactivity and NMR data are similar to those of CAWS. These results strongly indicate that α-mannan, but not β-mannan, contributes to these pathogenic

effects of Maraviroc order CMWS. Numerous studies on the antigenicity and pathogenicity of fungal cell wall mannans, especially those from C. albicans and Saccharomyces cerevisiae, have been reported. Kind et al. reported that the lethal toxicity and increased vascular permeability of some yeast mannans, including that of C. albicans, seem to depend on the 1,2-α-, 1,6-α-linkage in their main chain (30). Garner et al. reported that tumor necrosis factor-α is produced in vivo in response to mannan derived from C. albicans (36). These effects can be regulated by mannan ligands such as anti-mannan antibodies and corticosteroids. On the other hand, numerous studies have shown that 1,2-β-linked mannans, which are only expressed by pathogenic yeasts such as C. albicans, are vital for cell adhesion to host cells (27) and cytokine Fludarabine production from various cells (37). This specific glycan does not bind

to typical mannan receptors such as the macrophage mannose receptor or mannose-binding lectin. However, some studies have recently reported that galectin-3 is the receptor for 1,2-β-linked mannan (38), and may contribute to some biological effects of mannan (39). In our studies, CAWS, an extracellular polysaccharide fraction obtained from the culture supernatant of C. albicans, has been found to induce coronary arteritis and acute anaphylactoid shock (10–17). These biological effects depend on the pH of the culture process (15). CAWS synthesized in neutral pH conditions that result in the expression of 1,2-β-mannosyl residues produces significantly reduced acute anaphylactoid shock, coronary arteritis, and complement activation. This pattern was most definitely matched by the results of investigations of the activities of mannan from C. albicans cell wall (9). Our previous studies have clearly suggested that the β-mannosyl residue attached to nonreducing terminal α-mannosyl branched chains within an acid-stable region is very different in biologically active versus inactive mannan (9, 15).

Blood glucose concentrations were determined with test reagent strips (Medisense™; Medisense Sweden, Stockholm, Sweden). Serum insulin concentrations were measured with ELISA (Rat Insulin ELISA; Mercodia AB, Uppsala, Sweden). Statistical calculations.  All values are given as means ± SEM. Probabilities (P) of chance differences were calculated with Students paired

or unpaired t-test or anova with Bonferroni’s correction for multiple comparisons (Sigmastat; SSPD, Erfart, Germany). A value of P < 0.05 was considered to be statistically significant. On day 2 after transplantation, GSK-3 signaling pathway both HA (Fig. 1) and water contents (Fig. 2) were increased in the transplanted pancreas when compared to the endogenous gland. These differences had, however, disappeared on days 4 and 7 post-transplantation (Figs. 1 and 2). There was no statistically significant correlation between HA and water contents on day 2, 4 and 7, respectively (data not shown). However, when all data from the three observation days were pooled, there was such

a correlation (r = 0.48; P < 0.05). Hyaluronidase treatment decreased the content of HA in the transplanted click here pancreas 2 days after implantation, but did not affect that of the endogenous gland in the transplanted rats (Fig. 3). In rats not treated with hyaluronidase, the HA contents of the pancreas were similar to that of the endogenous pancreas in transplanted rats (Fig. 3). Hyaluronidase treatment induced a decrease in HA content of the pancreas of non-transplanted control rats (Fig. 3). Hyaluronidase treatment did not, however, influence the water content of the pancreases irrespective of whether endogenous or transplanted glands were investigated (Fig. 4). It is worthy of note, however, that the pancreas Oxymatrine of the non-transplanted rats contained less than both the pancreas grafts and the endogenous

pancreas of the grafted animals. Macroscopically, the grafted pancreases were swollen, and occasional haemorrhages as well as calcified infiltrates were seen on day 2 post-transplantation. Small (2–3 mm) sterile abscesses in association with the sutures in the anastomosis between the intestines occurred in some of the animals. The endogenous glands were slightly swollen in some of the animals, but there were no haemorrhages or calcifications. There were no macroscopic differences between PBS- and hyaluronidase-treated rats. Microscopically, there were interstitial oedema and occasional haemorrhages. Vacuoles were found in some of the exocrine cells of the transplanted pancreases (Fig. 5). The endogenous pancreases of transplanted rats had sometimes a mild oedema, but vacuoles or haemorrhages were rarely seen. Hyaluronidase treatment affected none of the morphological changes referred to above. A total of 17 of 20 of the transplanted animals allocated for blood flow measurements tolerated the surgical procedures well and showed no signs of infirmity.