Region-specific sympatho-adrenergic regulation of specialized vasculature in bone homeostasis and regeneration.

Type H vessels couple angiogenesis with osteogenesis, while sympathetic cues regulate vascular and skeletal function. The crosstalk between sympathetic nerves and type H vessels in bone remains unclear. Here, we first identify close spatial connections between sympathetic nerves and type H vessels in bone, particularly in metaphysis. Sympathoexcitation, mimicked by isoproterenol (ISO) injection, reduces type H vessels and bone mass. Conversely, beta-2-adrenergic receptor (ADRB2) deficiency maintains type H vessels and bone mass in the physiological condition. In vitro experiments reveal indirect sympathetic modulation of angiogenesis via paracrine effects of mesenchymal stem cells (MSCs), which alter the transcription of multiple angiogenic genes in endothelial cells (ECs). Furthermore, Notch signaling in ECs underlies sympathoexcitation-regulated type H vessel formation, impacting osteogenesis and bone mass. Finally, propranolol (PRO) inhibits beta-adrenergic activity and protects type H vessels and bone mass against estrogen deficiency. These findings unravel the specialized neurovascular coupling in bone homeostasis and regeneration.

Optimized immunofluorescence staining protocol for identifying resident mesenchymal stem cells in bone using LacZ transgenic mice.

Glioma-associated oncogene homolog 1 (Gli1) marks a subpopulation of endogenous mesenchymal stem cells (MSCs) characterized by perivascular location. Here, we present an optimized immunofluorescence staining protocol to identify resident Gli1+ MSCs in fixed/frozen bone sections from LacZ transgenic mice. This protocol describes the preparation of fixed/frozen tissue sections and the use of LacZ immunofluorescent staining for the in vivo characterization of endogenous MSCs, regarding their specific identity and specialized niches, and is applicable to LacZ-expressing cells of diverse organs. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).

Trimethylamine N-oxide promotes hyperoxaluria-induced calcium oxalate deposition and kidney injury by activating autophagy.

Calcium oxalate (CaOx) is the most common component of kidney stones. Oxidative stress, inflammation and autophagy-induced cell death are the major causes of CaOx crystal deposition and CaOx crystal deposition can further lead to kidney injury. Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, plays an important role in the pathogenesis of many diseases, such as atherosclerosis, diabetes and chronic kidney disease, but the effect of TMAO on hyperoxaluria-induced CaOx crystal deposition and kidney injury remains unknown. We hypothesize that TMAO aggravates CaOx crystal deposition via promoting CaOx-mediated cell death. C57Bl/6 mice were given high-oxalate diet as a model of hyperoxaluria. TMAO was provided via drinking water. Serum TMAO levels increased 15 days after CaOx treatment (6.30 ± 0.17 µmol/L vs. 34.65 ± 8.95 µmol/L). High-oxalate diet induced inflammation, CaOx deposition and kidney injury, which TMAO aggravated. In accordance, TMAO intensified high-oxalate diet induced oxidative stress, autophagy and apoptosis. Moreover, TMAO enhanced CaOx crystal adhesion to HK-2 cells and reduced cell viability (from 88.9 ± 1.6% to 75.0 ± 2.7%). Protein kinase R-like endoplasmic reticulum kinase (PERK) may mediate these TMAO effects, as TMAO promoted PERK phosphorylation. Consistently, PERK knockdown alleviated TMAO-evoked CaOx-autophagy, apoptosis and oxidative stress in HK-2 cells. In conclusion, TMAO can aggravate hyperoxaluria-induced kidney injury by triggering the PERK/ROS pathway, which enhances autophagy, apoptosis and inflammation, and facilitates CaOx crystal deposition in renal tubular cells.

ADAMTS13 inhibits oxidative stress and ameliorates progressive chronic kidney disease following ischaemia/reperfusion injury.

AIMS: Reduced A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif member 13 (ADAMTS13) levels are observed in kidney disease. We test whether recombinant human ADAMTS13 (rhADAMTS13) mitigates renal injury in chronic kidney disease (CKD) and the potential mechanisms. METHODS: CKD was established 3 months after ischaemia/reperfusion (IR). ADAMTS13 and von Willebrand factor (vWF) levels, renal function and morphological changes were analysed. Afferent arteriolar responses to angiotensin II (Ang II) and acetylcholine (ACh) were measured. Oxidative stress-related molecules were detected. RESULTS: Higher vWF and lower ADAMTS13 levels were observed in CKD mice, which were markedly attenuated by rhADAMTS13. rhADAMTS13 alleviated renal dysfunction, as documented by decreased blood urea nitrogen (BUN), serum creatinine, kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) levels in CKD mice. Moreover, rhADAMTS13 attenuated transforming growth factor (TGF)-ß1/Smad3 activation. Plasma vWF: ADAMTS13 ratio showed positive correlations with malondialdehyde (MDA), hydrogen peroxide (H2 O2 ) and proteinuria, and correlated inversely with superoxide dismutase (SOD) and catalase (CAT). Finally, rhADAMTS13 inhibited reactive oxygen species (ROS) levels and improved microvascular functional disorders, accompanied by the inhibition of glycogen synthase kinase (GSK) 3ß hyperactivity and upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) expression. CONCLUSIONS: Acute kidney injury (AKI) reduces the expression of ADAMTS13 that contributes to progressive CKD, microvascular dysfunction, oxidative stress, inhibition of Nrf2 activity and renal histopathological damage. All of which can be alleviated by administration of rhADAMTS13.

Osthole Ameliorates Renal Fibrosis in Mice by Suppressing Fibroblast Activation and Epithelial-Mesenchymal Transition.

Renal fibrosis is a common pathway of virtually all progressive kidney diseases. Osthole (OST, 7-Methoxy-8-(3-methylbut-2-enyl)-2-chromenone), a derivative of coumarin mainly found in plants of the Apiaceae family, has shown inhibitory effects on inflammation, oxidative stress, fibrosis and tumor progression. The present study investigated whether OST mediates its effect via suppressing fibroblast activation and epithelial-mesenchymal transition (EMT) in unilateral ureteral obstruction (UUO)-induced renal fibrosis in mice. Herein, we found that OST inhibited fibroblast activation in a dose-dependent manner by inhibiting the transforming growth factor-ß1 (TGFß1)-Smad pathway. OST also blocked fibroblast proliferation by reducing DNA synthesis and downregulating the expressions of proliferation- and cell cycle-related proteins including proliferating cell nuclear antigen (PCNA), CyclinD1 and p21 Waf1/Cip1. Meanwhile, in the murine model of renal interstitial fibrosis induced by UUO, myofibroblast activation with increased expression of α-smooth muscle actin (α-SMA) and proliferation were attenuated by OST treatment. Additionally, we provided in vivo evidence suggesting that OST repressed EMT with preserved E-cadherin and reduced Vimentin expression in obstructed kidney. UUO injury-induced upregulation of EMT-related transcription factors, Snail family transcriptional repressor-1(Snail 1) and Twist family basic helix-loop-helix (BHLH) transcription factor (Twist) as well as elevated G2/M arrest of tubular epithelial cell, were rescued by OST treatment. Further, OST treatment reversed aberrant expression of TGFß1-Smad signaling pathway, increased level of proinflammatory cytokines and NF-kappaB (NF-κB) activation in kidneys with obstructive nephropathy. Taken together, these findings suggest that OST hinder renal fibrosis in UUO mouse mainly through inhibition of fibroblast activation and EMT.

Mycophenolate Mofetil Combined With Prednisone Versus Full-Dose Prednisone in IgA Nephropathy With Active Proliferative Lesions: A Randomized Controlled Trial.

BACKGROUND: Observational studies suggest that patients with immunoglobulin A nephropathy (IgAN) with active proliferative lesions show a good response to immunosuppressive treatment. STUDY DESIGN: Multicenter, prospective, randomized, controlled trial. SETTING & PARTICIPANTS: 176 patients with IgAN with active proliferative lesions (cellular and fibrocellular crescents, endocapillary hypercellularity, or necrosis), proteinuria with protein excretion ≥ 1.0g/24h, and estimated glomerular filtration rate > 30mL/min/1.73m2. INTERVENTION: Mycophenolate mofetil (MMF) group: MMF, 1.5g/d, for 6 months and prednisone, 0.4 to 0.6mg/kg/d, for 2 months and then tapered by 20% per month for the next 4 months; prednisone group: prednisone, 0.8 to 1.0mg/kg/d, for 2 months and then tapered by 20% per month for the next 4 months. All patients were followed up for another 6 months. OUTCOMES: The primary end point was complete remission rate at 6 and 12 months. RESULTS: At baseline, median estimated glomerular filtration rates were 90.2 and 94.3mL/min/1.73m2 and mean proteinuria was protein excretion of 2.37 and 2.47g/24h in the MMF and prednisone groups, respectively. At 6 months, complete remission rates were 37% (32 of 86 patients) and 38% (33 of 88 patients); the between-group difference was not statistically significant (P=0.9). At 12 months, complete remission rates were 48% (35 of 73 patients) and 53% (38 of 72 patients) in the MMF and prednisone groups, respectively; the between-group difference was not statistically significant (P=0.6). Incidences of Cushing syndrome and newly diagnosed diabetes mellitus were lower in the MMF group than in the prednisone group. LIMITATIONS: Not all participants were treated with renin-angiotensin system blockers, relatively short follow-up. CONCLUSIONS: MMF plus prednisone versus full-dose prednisone did not differ in reducing proteinuria, but patients treated with the former had fewer adverse events in patients with IgAN with active proliferative lesions.

Modified Si-Miao-San () regulates adipokine expression and ameliorates insulin resistance by targeting IKKß/Insulin receptor substrate-1 in mice.

OBJECTIVE: To evaluate modified Si-Miao-San (mSMS, ) regulation of insulin sensitivity and explore the molecular mechanism by which mSMS inhibits inflammation and improves insulin action in mice. METHODS: Insulin resistant model in mice was prepared by stimulation with macrophage-derived condition medium (Mac-CM) and the effects of mSMS on oral glucose tolerance, insulin sensitivity and liver glycogen content in mice was observed. The mice adipose tissue was isolated and the regulation of inflammation-related adipokine expression and insulin phosphatidylinositol 3-kinase (PI3K) signaling transduction by mSMS was investigated. Effect of mSMS on insulin-mediated glucose uptake was also investigated in adipocytes. RESULTS: Oral administration of mSMS improved glucose tolerance in mice. Treatment of mice with Mac-CM resulted in glucose intolerance in mice and this change was effectively reversed by mSMS. Meanwhile, mSMS enhanced insulin sensitivity and increased glucose load-stimulated liver glycogen when mice were exposed to Mac-CM. Mac-CM stimulation induced dysregulation of adipokine expression in adipose tissue of mice. mSMS downregulated tumor necrosis factor α and interleukin 6 (IL-6) overexpression and upregulated adiponectin and peroxisomal proliferator activated receptor γ with inhibition of inhibitory kappa B kinase-ß (IKKß) and p65 phophsphorylation. Meanwhile, mSMS inhibited IL-6 production and increased adiponectin secretion in adipocytes against Mac-CM insult. Mac-CM challenge impaired insulin phosphatidylinositol 3 kinase (PI3K) signaling in adipose tissue. Oral administration mSMS inhibited inflammation-induced serine phosphorylation of insulin receptor substrate-1 (IRS-1) and restored insulin-mediated tyrosine phosphorylation, and thereby facilitated insulin PI3K signaling manifested by restoration of Akt phosphorylation. The resultant improvement of insulin sensitivity promoted insulin-stimulated glucose uptake when adipocytes were exposed to Mac-CM. CONCLUSIONS: mSMS improves glucose tolerance in mice by enhancing insulin sensitivity in mice. mSMS inhibits IKKß/NF κ B (p65)-dependent inflammatory response with beneficial regulation of adipokine expression in adipose tissue. mSMS inhibits inflammation and improves insulin sensitivity by blocking inflammatory interaction between IKKß/IRS-1.

[Superhigh spectral resolution measurement of spectrometer].

The spectral resolution is one of the most important indexes of spectrometer. A new method is put forward for measuring the superhigh spectral resolution based on the Rayleigh criterion and the optical heterodyne, and the uncertainty of this method is analyzed. The spectral resolution of some spectrometer was measured using this method, and the experimental results show that the spectral resolution is higher than 18.9 pm, and the standard uncertainty is 2.3 pm. When showed using wave number, the spectral resolution is higher than 0.078 8 cm(-1), and the standard uncertainty is 0.009 6 cm(-1).

A multicenter application and evaluation of the oxford classification of IgA nephropathy in adult chinese patients.

BACKGROUND: The Oxford classification of immunoglobulin A (IgA) nephropathy (IgAN) provides a histopathologic grading system that is associated with kidney disease outcomes independent of clinical features. We evaluated the Oxford IgAN classification in a large cohort of patients from China. STUDY DESIGN: Retrospective study. SETTING & PARTICIPANTS: 1,026 adults with IgAN from 18 referral centers in China. Inclusion criteria and statistical analysis were similar to the Oxford study. PREDICTORS: Histologic findings of mesangial hypercellularity score, endocapillary proliferation, segmental sclerosis or adhesion, crescents, necrosis, and tubular atrophy/interstitial fibrosis. Clinical features, blood pressure, estimated glomerular filtration rate (eGFR), proteinuria, and treatment modalities. OUTCOMES: Time to a 50% reduction in eGFR or end-stage renal disease (the combined event); the rate of eGFR decline (slope of eGFR); proteinuria during follow-up. RESULTS: Compared with the Oxford cohort, the Chinese cohort had a lower proportion of patients with mesangial hypercellularity (43%) and endocapillary proliferation (11%), higher proportion with segmental sclerosis or adhesion (83%) and necrosis (15%), and similar proportion with crescents (48%) and tubular atrophy/interstitial fibrosis (moderate, 24%; severe, 3.3%). During a median follow-up of 53 (25th-75th percentile, 36-67) months, 159 (15.5%) patients reached the combined event. Our study showed that patients with a mesangial hypercellularity score higher than 0.5 were associated with a 2.0-fold (95% CI, 1.5-2.8; P<0.001) higher risk of the combined event than patients with a score of 0.5 or lower. Patients with tubular atrophy/interstitial fibrosis of 25%-50% and >50% versus <25% were associated with a 3.7-fold (95% CI, 2.6-5.1; P<0.001) and 15.1-fold (95% CI, 9.5-24.2; P<0.001) higher risk of the combined event, respectively. Endocapillary proliferation, glomerular crescents, and necrosis were not significant. LIMITATIONS: Retrospective study; the therapeutic interventions were miscellaneous. CONCLUSIONS: We confirmed the associations of mesangial hypercellularity and tubular atrophy/interstitial fibrosis with kidney disease outcomes.

Kupffer cells contribute to concanavalin A-induced hepatic injury through a Th1 but not Th17 type response-dependent pathway in mice.

BACKGROUND: Increasing evidence suggests that a close interaction of Kupffer cells with T cells plays a central role in concanavalin A-induced hepatic injury in mice, but the underlying mechanisms remain obscure. The present study aimed to determine the relative roles of Th1 and Th17 type responses in concanavalin A-induced hepatic injury in mice, and to investigate whether or not Kupffer cells contribute to hepatic injury via a Th1 or Th17 type response-dependent pathway. METHODS: Immune-mediated hepatic injury was induced in C57BL/6 mice by intravenous injection of concanavalin A. Kupffer cells were inactivated by pretreatment with gadolinium chloride 24 hours before the concanavalin A injection. The interferon-gamma (IFN-gamma) and interleukin-17 (IL-17) pathways were blocked by specific neutralizing antibodies. Hepatic injury was assessed using serum transferase activity and pathological analysis. Expression of inflammatory cytokines within the liver was detected by real-time polymerase chain reaction and immunohistochemistry. RESULTS: Neutralization of IFN-gamma significantly attenuated concanavalin A-induced hepatic injury. However, neutralization of IL-17 failed to suppress the injury. Inactivation of Kupffer cells by gadolinium chloride pretreatment protected against concanavalin A-induced injury and significantly reduced hepatic cytokine levels including TNF-alpha, IL-6 and IFN-gamma but not IL-17. CONCLUSION: Our findings suggest that Kupffer cells contribute to concanavalin A-induced hepatic injury via a Th1 type response-dependent pathway and production of inflammatory cytokines including TNF-alpha, IL-6 and IFN-gamma.

ADAP deficiency combined with costimulation blockade synergistically protects intestinal allografts.

Adhesion and degranulation promoting adapter protein (ADAP) plays an important role in T cell activation. ADAP deficiency was recently found to prolong heart graft survival in mice. We investigated the role of ADAP in intestinal transplantation and the synergistic effect of ADAP deficiency and Costimulation blockade (CB). T cell proliferation and cytotoxic T lymphocyte (CTL) activity were determined. MHC mismatched intestinal allografts was transplanted heterotopically. Anti-CD40L antibody was applied to the recipient. Upon stimulation with allogenic dendritic cells (DC), ADAP-deficient (ADAP-/-) T cells displayed impaired proliferative responses compared with that of wild-type (WT) T cells. In contrast, the CTL activity in ADAP-/- mice was comparable with that of WT mice. Rejection of intestinal allografts was ameliorated, but not prevented in ADAP-/- mice. Although CB alone was not sufficient to mitigate the rejection, the combination of CB and ADAP deficiency profoundly inhibited rejection. This was accompanied by less infiltration and activation of host lymphocytes in the gut-associated lymphoid tissue of intestinal allografts. ADAP deficiency combined with CB protected the intestinal allografts synergistically. ADAP could be a novel target in the induction phase of the immune responses in organ transplantation.

A novel and knotless technique for heterotopic cardiac transplantation in mice.

BACKGROUND: The development of microsurgical techniques has facilitated the establishment of fully vascularized cardiac transplantation models in small mammals. Anastomotic stenosis and bleeding continue to hamper procedures and limit long-term graft survival. In this study we assess a novel technique to improve outcome after cardiac transplantation in mice. METHODS: Our novel technique of murine heterotopic cardiac transplantation consists of three critical steps: (i) a novel procedure for graft harvest; (ii) a modified method for recipient vessel preparation; and (iii) a novel suturing procedure for graft implantation. Importantly, a new knotless suturing technique for end-to-side vascular anastomosis was applied, which allows for adjustment of the anastomosis after transplantation, thus reducing the risk of anastomotic bleeding or stenosis. RESULTS: The recipient survival rate based on this novel technique was between 90% and 98%, depending on physician expertise. Graft implantation time varied between 20 and 25 minutes after the initial 200 training cases. In comparing the standard knot microvascular suturing technique to the new knotless technique carried out by an experienced surgeon, the latter was found to be more efficient by significantly reducing the rate of anastomotic stenosis (0% vs 8% with knot, p < 0.001, n = 200) and anastomotic bleeding (2% vs 7% with knot, p < 0.05, n = 200). CONCLUSIONS: This novel technique offers a rapid, easy and effective method for murine heterotopic cardiac transplantation.

A comparison of a standard-dose prednisone regimen and mycophenolate mofetil combined with a lower prednisone dose in Chinese adults with idiopathic nephrotic syndrome who were carriers of hepatitis B surface antigen: a prospective cohort study.

BACKGROUND: When receiving immunosuppressive therapy, patients with idiopathic nephrotic syndrome who are also carriers of hepatitis B virus (HBV) surface antigen (HBsAg) are at risk for reactivation of HBV. OBJECTIVE: This study compared the effectiveness and tolerability of a standard-dose prednisone regimen with those of the combination of mycophenolate mofetil (MMF) and a lower prednisone dose for the treatment of idiopathic nephrotic syndrome characterized by minimal-change nephropathy or slight mesangial proliferative glomerulonephritis in Chinese adults who were also carriers of HBsAg, a combination here termed MSNS-HBV. METHODS: This was a prospective, open-label cohort study in Chinese adults with MSNS-HBV. Patients were self-assigned to 1 of 2 treatment groups: the standard prednisone regimen of 1 mg/kg daily or oral MMF 0.5 to 1.0 g BID combined with the lower prednisone dose of 0.5 mg/kg daily. The planned duration of treatment was 36 weeks, with an additional 60 weeks of follow-up. The primary outcome measures were rates of complete remission of idiopathic nephrotic syndrome (a decrease in daily proteinuria to within the normal range [<0.3 g]) and rates of HBV reactivation (detectable serum HBV DNA). Secondary outcome measures included relapse rates (>1+ albuminuria on dipstick urinalysis on 3 consecutive days), alanine aminotransferase (ALT) elevations (>50 U/L), use of lamivudine 100 mg/d (added if HBV DNA titers reached >or=10(5) copies/mL), and adverse effects. RESULTS: The intent-to-treat population included 41 patients (22 prednisone, 19 MMF). In patients who completed the study, rates of complete remission after 24 weeks of treatment were 78.9% (15/19) in the prednisone group and 76.5% (13/17) in the MMF group; 2 and 3 patients in the respective groups had a partial remission, and 2 and 1 patient had no response. HBV reactivation occurred in 63.6% (14/22) and 36.8% (7/19) of patients (P = 0.047). The only significant difference in the study was in the probability of HBV reactivation between groups (P = 0.043, log-rank test). During follow-up, at least 1 relapse occurred in 46.7% (7/15) and 30.8% (4/13) of patients. Elevations in ALT were observed in 36.4% (8/22) and 26.3% (5/19) of patients, and the addition of lamivudine was required in 40.9% (9/22) and 21.1% (4/19) of patients. The most frequent adverse effects in both groups were infections (27.3% and 26.3%), followed by gastrointestinal symptoms (13.6% and 21.1%). Two MMF patients developed leukopenia. One patient in the prednisone group discontinued treatment because of severe hepatitis, and 1 patient in the MMF group discontinued because of severe pulmonary infection. CONCLUSIONS: Among the adult Chinese patients with MSNS-HBV who completed this study, there were no significant differences in remission rates of idiopathic nephrotic syndrome between the standard prednisone regimen and the combination of MMF and a reduced prednisone dose. Rates of HBV reactivation, however, were significantly lower in the combination-therapy group.

Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans.

A functionally distinct subset of CD103(+) dendritic cells (DCs) has recently been identified in murine mesenteric lymph nodes (MLN) that induces enhanced FoxP3(+) T cell differentiation, retinoic acid receptor signaling, and gut-homing receptor (CCR9 and alpha4beta7) expression in responding T cells. We show that this function is specific to small intestinal lamina propria (SI-LP) and MLN CD103(+) DCs. CD103(+) SI-LP DCs appeared to derive from circulating DC precursors that continually seed the SI-LP. BrdU pulse-chase experiments suggested that most CD103(+) DCs do not derive from a CD103(-) SI-LP DC intermediate. The majority of CD103(+) MLN DCs appear to represent a tissue-derived migratory population that plays a central role in presenting orally derived soluble antigen to CD8(+) and CD4(+) T cells. In contrast, most CD103(-) MLN DCs appear to derive from blood precursors, and these cells could proliferate within the MLN and present systemic soluble antigen. Critically, CD103(+) DCs with similar phenotype and functional properties were present in human MLN, and their selective ability to induce CCR9 was maintained by CD103(+) MLN DCs isolated from SB Crohn's patients. Thus, small intestinal CD103(+) DCs represent a potential novel target for regulating human intestinal inflammatory responses.

PD-1/PD-L1, PD-1/PD-L2, and other co-inhibitory signaling pathways in transplantation.

Transplantation of cells, tissues and vascularized solid organs is a successful therapeutic intervention for many end-stage chronic diseases. The combination of co-stimulatory blockade with the delivery of negative signals to T cells through co-inhibitory receptors would provide a robust approach to modulating T-cell receptor signaling and improving alloantigen-specific control of transplant rejection. This approach based on fundamental knowledge of APC/T-cell interactions may complement conventional therapies in the near future to reinforce long-term allograft survival, and permit minimal immunosuppression. The focus of this review was primarily on two major co-inhibitory signaling pathways, namely PD-1/PD-L1/PD-L2 and BTLA/CD160/HVEM/LIGHT that have been thoroughly characterized in murine models of transplantation using genetically modified mice, specific monoclonal antibodies and fusion proteins.

Inactivation of T-cell receptor-mediated integrin activation prolongs allograft survival in ADAP-deficient mice.

BACKGROUND: Signaling through the T cell receptor (TCR) leads to profound changes in the function and properties of T cells, including integrin activation. Adhesion and degranulation promoting adapter protein (ADAP) is an adapter protein linking T cell receptor stimulation to integrin activation. We aim to clarify how disruption of TCR-mediated integrin activation affects alloreactive immune responses. METHODS: In vitro T cell proliferation and the cytokine production was determined. In vivo cytotoxic T lymphocyte (CTL) activity was measured as well. Allogenic skin and heart transplantation was used to test the in vivo role of ADAP in alloimmune responses. Histology and flow cytometry was applied to analyze the graft infiltrating lymphocytes. RESULTS: Upon stimulation with allogenic dendritic cells ADAP-deficient T cells displayed impaired proliferative responses compared to wild type (WT) T cells. This was accompanied by significantly decreased production of the cytokine interleukin-2. In contrast, the in vivo CTL activity in ADAP-deficient mice was comparable to that of WT mice. Consistently, we observed a prolongation of fully major histocompatibility complex (MHC)-mismatched heart transplants in ADAP deficient mice. Protection of allogenic heart grafts in ADAP-deficient mice was accompanied by a decrease in the infiltration, proliferation and activation of T cells in the allograft. However, no effect was observed after fully MHC-mismatched skin transplantation. CONCLUSIONS: We have shown that although ADAP is dispensable for the rejection of allografts, ADAP function plays an important role for the efficacy of graft rejection. ADAP's main function appears to affect the induction phase of the immune response.

Blockade of the PD-1/PD-1L pathway reverses the protective effect of anti-CD40L therapy in a rat to mouse concordant islet xenotransplantation model.

BACKGROUND: We have previously demonstrated that costimulatory blockade with anti-CD40L monoclonal antibody (mAb) prolongs the survival of non-vascularized concordant rat to mouse islet xenografts. Here, we examine whether signaling through the PD-1/PD-1L pathway is required for the anti-CD40L therapy to prolong concordant islet graft survival using a novel anti-murine PD-1 mAb (clone 4F10). METHODS: C57BL/6 mice received a cellular concordant islet xenograft under the left kidney capsule and four experimental groups were prepared. Group I: untreated control; group II: recipient mice were treated with three doses of 0.5 mg of anti-CD40L mAb (clone MR1) on days 0, 2 and 4; group III: mice were treated with 0.5 mg of anti-PD-1 (CD279) mAb (clone 4F10) every other day for 8 days; and finally group IV: mice received the combined treatment that consisted of anti-CD40L plus anti-PD-1 mAb. RESULTS: Concordant islet xenografts transplanted in control untreated mice showed a median survival time (MST) of 17 +/- 7.43 days, whereas anti-CD40L treatment led to a significant prolongation of graft survival (MST: 154 +/- 65.56, P < 0.0001). The administration of anti-PD-1 alone significantly accelerated graft rejection compared to non-treated controls (MST: 10 +/- 2.24 vs. MST: 17 +/- 7.43, P < 0.0004). Remarkably, the combined administration of anti-CD40L and anti-PD-1 reversed the protective effect obtained with anti-CD40L alone (anti-CD40L, MST: 154 +/- 65.56 vs. anti-CD40L plus anti-PD-1, MST: 10 +/- 7.72, P < 0.0002). CONCLUSION: Overall, our data indicate that the PD-1/PD-1L pathway is required for the achievement of prolonged graft survival in anti-CD40L-treated mice in a setting of rat to mouse concordant islet xenotransplantation.

The circadian rhythm of glucocorticoids is regulated by a gating mechanism residing in the adrenal cortical clock.

In mammals, the master clock of the suprachiasmatic nuclei (SCN) and subordinate clocks found throughout the body coordinate circadian rhythms of behavior and physiology. We characterize the clock of the adrenal, an important endocrine gland that synchronizes physiological and metabolic rhythms. Clock gene expression was detected in the outer adrenal cortex prefiguring a role of the clock in regulating gluco- and mineral corticoid biogenesis. In Per2/Cry1 double mutant mice, which lack a circadian clock, hypothalamus/pituitary/adrenal axis regulation was defective. Organ culture and tissue transplantation suggest that the adrenal pacemaker gates glucocorticoid production in response to adrenocorticotropin (ACTH). In vivo the adrenal circadian clock can be entrained by light. Transcriptome profiling identified rhythmically expressed genes located at diverse nodes of steroid biogenesis that may mediate gating of the ACTH response by the adrenal clock.

Lipid peroxidation in IgA nephropathy and the effect of lipo-prostaglandin E1.

BACKGROUND: Lipid peroxidation (LPO) plays a role in glomerulonephritis pathogenesis. The role of LPO in IgA nephropathy (IgAN) and the effect of prostaglandin E1 on it, is not determined. METHODS: Levels of malondialdehyde (MDA), superoxide dismutase (SOD) and vitamin E (vitE) were measured in 42 patients with IgAN and in 31 healthy controls. Clinical nephropathy parameters such as daily proteinuria (DP), serum creatinine (Cr) and Cr clearance were obtained. Forty-two IgAN patients were divided into two subgroups according to renal pathology severity. Twenty-one patients with IgAN received lipo-prostaglandin E1 (PGE1) treatment and the other 21 patients received saline as a control. RESULTS: The serum activity of SOD and vitE in IgAN patients was significantly lower than in healthy controls, while the MDA level was higher in the patient groups. DP-related positively to MDA, while it related negatively to SOD and vitE. Cr clearance related negatively to MDA, while it related positively to SOD and vitE. In the moderate pathological group, the MDA level was significantly higher and the SOD activity was lower than in the mild pathological group. The MDA level in the PGE1 treated group was lower than in the control group, but the activity of SOD and vitE did not differ significantly from the control group. DP declined and Cr clearance increased in the PGE1 treated group, while no significant change in serum Cr was noted. CONCLUSIONS: LPO in IgAN was evident and was at least attributed to the decline in antioxidant ability. LPO could play a role in IgAN pathogenesis, and PGE1 is able to ameliorate the LPO in IgAN patients.