The present study sought to characterise the gut microbiota of subjects with kidney transplantation and healthy control to identify the distinct gut microbiota and analyse their potential function. We found that gut microbiota abundance had significant differences in subjects between the two groups. Line Discriminant Analysis (LDA) Effect Size (LEfSe) analysis showed that the bacterial taxa were differentially represented between the two groups, and the potential biomarkers at different taxonomic levels in kidney transplant recipients were Streptococcus, Enterococcaceae, and Ruminococcus. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) Functional Inference analyses suggested that the difference in gut microbiota between the two groups was correlated with bile acid metabolism. In conclusion, gut microbiota abundance is different between the two groups, which is related to bile acid metabolism, and may influence the metabolic homeostasis of allograft recipients.

Post-transplantation immune rejection remains an important factor for transplant patients. However, conventional immunosuppressants are associated with substantial adverse effects. Natural immunosuppressants present a promising alternative to conventional counterparts, boasting exceptional biological activity, minimal toxicity and reduced side effects. We identified carvacrol as a prospective immunosuppressive agent following T cell proliferation experiment and validated carvacrol's immunosuppressive efficacy in the murine allogeneic skin graft model. T cell proliferation assay was used to screen natural small molecule compounds and the immunosuppressive effect of compounds was evaluated in MHC-mismatched murine allogeneic skin graft model. H&E and immunohistochemical staining were applied to evaluate the pathological grade. Furthermore, flow cytometry was uitlized to analyse the immunophenotype changes of immune cells. Western blotting and q-PCR were used to detect the expression of key molecules in macrophages. In vitro, carvacrol demonstrates significant inhibition of the proliferation of CD4+ T and CD8+ T cells. It notably reduces inflammatory factor expression within the allografts, suppresses T cell differentiation toward Th1 phenotype and expansion. Furthermore, carvacrol prominently hinders M1-type macrophages polarization by activating Wnt signaling. Notably, the anti-rejection efficacy of carvacrol was significantly weakened upon the removal of macrophages in mice using chlorophosphate liposomes. Carvacrol could significantly inhibit T cell proliferation, alleviate graft rejection and has outstanding toxicological safety. The molecular mechanism of the anti-rejection effect of carvacrol is closely related to its mediating activation of macrophage Wnt pathway, inhibiting M1 polarization and inducing T cell differentiation.

In a series of studies, using an identical rat intestinal transplantation model, we evaluated the effects of several drugs. FK-506 caused a significant attenuation in the proliferation of allogeneic CD4+ T cells and IFN-γ secreting effector functions. FYT720 resulted in a marked reduction in the numbers of lymphocytes, associated with a reduction of T cell recruitment, in grafts. An anti-MAdCAM antibody was next reported to significantly down-regulate CD4+ T cell infiltration in intestinal grafts by blocking the adhesion molecule, and could be useful as an induction therapy. Concerning TAK-779, this CCR5 and CXCR3 antagonist diminished the number of graft-infiltrating cells by suppressing the expression of their receptors in the graft. As a result, it reduced the total number of recipient T cells involved in graft rejection. As the next step, we focused on the participation of monocytes/ macrophages in this field. PQA-18 has been the focus of a novel immunosuppressant that attenuates not only the production of various cytokines, such as IL-2 & TNF-α, on T cells, but the differentiation of macrophages by inhibiting PAK2 as well. In this report, we summarize our previous studies not only regarding the above drugs, but on an anti-complement drug and a JAK inhibitor as well.

Bisphosphonates are administered to post-transplantation patients with mineral and bone disorders; however, the association between bisphosphonate therapy and long-term renal graft survival remains unclear.

This nested case-control study investigated the effects of bisphosphonates on long-term graft outcomes after kidney transplantation. We enrolled 3836 kidney transplant recipients treated from April 1979 to June 2016 and matched patients with graft failure to those without (controls). Annual post-transplant bone mineral density assessments were performed and recipients with osteopenia or osteoporosis received bisphosphonate therapy. The associations between bisphosphonate use and long-term graft outcomes and graft survival were analyzed using conditional logistic regression and landmark analyses, respectively.

A landmark analysis demonstrated that death-censored graft survival was significantly higher in bisphosphonate users than in non-users in the entire cohort (log-rank test, P < 0.001). In the nested case-control matched cohort, bisphosphonate users had a significantly reduced risk of graft failure than did non-users (odds ratio = 0.38; 95% confidence interval 0.30-0.48). Bisphosphonate use, increased cumulative duration of bisphosphonate use >1 year and increased cumulative bisphosphonate dose above the first quartile were associated with a reduced risk of graft failure, after adjustments.

Bisphosphonates may improve long-term graft survival in kidney transplant recipients.

People who have chronic kidney disease (CKD) have important changes to bone structure, strength, and metabolism. Children experience bone deformity, pain, and delayed or impaired growth. Adults experience limb and vertebral fractures, avascular necrosis, and pain. The fracture risk after kidney transplantation is four times that of the general population and is related to Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD) occurring with end-stage kidney failure, steroid-induced bone loss, and persistent hyperparathyroidism after transplantation. Fractures may reduce quality of life and lead to being unable to work or contribute to community roles and responsibilities. Earlier versions of this review have found low certainty evidence for effects of treatment. This is an update of a review first published in 2005 and updated in 2007.

This review update evaluates the benefits and harms of interventions for preventing bone disease following kidney transplantation.

We searched the Cochrane Kidney and Transplant Register of Studies up to 16 May 2019 through contact with the Information Specialist using search terms relevant to this review. Studies in the Register are identified through searches of CENTRAL, MEDLINE, and EMBASE, conference proceedings, the International Clinical Trials Register (ICTRP) Search Portal and ClinicalTrials.gov.

RCTs and quasi-RCTs evaluating treatments for bone disease among kidney transplant recipients of any age were eligible.

Two authors independently assessed trial risks of bias and extracted data. Statistical analyses were performed using random effects meta-analysis. The risk estimates were expressed as a risk ratio (RR) for dichotomous variables and mean difference (MD) for continuous outcomes together with the corresponding 95% confidence interval (CI). The primary efficacy outcome was bone fracture. The primary safety outcome was acute graft rejection. Secondary outcomes included death (all cause and cardiovascular), myocardial infarction, stroke, musculoskeletal disorders (e.g. skeletal deformity, bone pain), graft loss, nausea, hyper- or hypocalcaemia, kidney function, serum parathyroid hormone (PTH), and bone mineral density (BMD).

In this 2019 update, 65 studies (involving 3598 participants) were eligible; 45 studies contributed data to our meta-analyses (2698 participants). Treatments included bisphosphonates, vitamin D compounds, teriparatide, denosumab, cinacalcet, parathyroidectomy, and calcitonin. Median duration of follow-up was 12 months. Forty-three studies evaluated bone density or bone-related biomarkers, with more recent studies evaluating proteinuria and hyperparathyroidism. Bisphosphonate therapy was usually commenced in the perioperative transplantation period (within 3 weeks) and regardless of BMD. Risks of bias were generally high or unclear leading to lower certainty in the results. A single study reported outcomes among 60 children and adolescents. Studies were not designed to measure treatment effects on fracture, death or cardiovascular outcomes, or graft loss.Compared to placebo, bisphosphonate therapy administered over 12 months in transplant recipients may prevent fracture (RR 0.62, 95% CI 0.38 to 1.01; low certainty evidence) although the 95% CI included the possibility that bisphosphonate therapy might make little or no difference. Fracture events were principally vertebral fractures identified during routine radiographic surveillance. It was uncertain whether any other drug class decreased fracture (low or very low certainty evidence). It was uncertain whether interventions for bone disease in kidney transplantation reduce all-cause or cardiovascular death, myocardial infarction or stroke, or graft loss in very low certainty evidence. Bisphosphonate therapy may decrease acute graft rejection (RR 0.70, 95% CI 0.55 to 0.89; low certainty evidence), while it is uncertain whether any other treatment impacts graft rejection (very low certainty evidence). Bisphosphonate therapy may reduce bone pain (RR 0.20, 95% CI 0.04 to 0.93; very low certainty evidence), while it was very uncertain whether bisphosphonates prevent spinal deformity or avascular bone necrosis (very low certainty evidence). Bisphosphonates may increase to risk of hypocalcaemia (RR 5.59, 95% CI 1.00 to 31.06; low certainty evidence). It was uncertain whether vitamin D compounds had any effect on skeletal, cardiovascular, death, or transplant function outcomes (very low certainty or absence of evidence). Evidence for the benefits and harms of all other treatments was of very low certainty. Evidence for children and young adolescents was sparse.

Bisphosphonate therapy may reduce fracture and bone pain after kidney transplantation, however low certainty in the evidence indicates it is possible that treatment may make little or no difference. It is uncertain whether bisphosphonate therapy or other bone treatments prevent other skeletal complications after kidney transplantation, including spinal deformity or avascular bone necrosis. The effects of bone treatment for children and adolescents after kidney transplantation are very uncertain.

The aim of this study was to evaluate the effect of ibandronate administration on long-term graft function and graft survival after successful renal transplantation.

Seventy-two renal transplant recipients (36 patients each in the treatment and control group) were included and followed over a 15-year period. Data on graft function and death-censored transplant outcome were recorded at 1, 5, 10, and 15 years.

Death-censored Kaplan-Meier analysis showed significantly improved graft survival of the treatment group (p = 0.026), whereas Cox regression analysis showed that ibandronate was positively associated with improved transplant survival (p = 0.028, hazard ratio 0.24, 95% confidence interval 0.07-0.86). Although general linear modelling did not indicate that ibandronate had a significant effect on transplant function (calculated using the estimated glomerular filtration rate according to Chronic Kidney Disease Epidemiology Collaboration equation) over the entire 15-year period (p = 0.650), there was a tendency towards improved graft function 1-year post-transplantion (p = 0.056).

Ibandronate treatment within the first year of transplantation resulted in a trend towards better graft function within the first few year post-transplant, and was associated with increased transplant survival at long-term follow-up.

This review summarizes the phenotype and function of macrophages in the context of solid organ transplantation and will focus on fundamental insights into their paradoxical pro-inflammatory versus suppressive function. We will also discuss the therapeutic potential of regulatory macrophages in tolerance induction.

Macrophages are emerging as an essential element of solid organ transplantation. Macrophages are involved in the pathogenesis of ischemia reperfusion injury, as well as both acute and chronic rejection, exacerbating injury through secretion of inflammatory effectors and by amplifying adaptive immune responses. Notably, not all responses associated with macrophages are deleterious to the graft, and graft protection can in fact be conferred by macrophages. This has been attributed to the presence of macrophages with tissue-repair capabilities, as well as the effects of regulatory macrophages.

The explosion of new information on the role of macrophages in solid organ transplantation has opened up new avenues of research and the possibility of therapeutic intervention. However, the role of myeloid cells in graft rejection, resolution of rejection and tissue repair remains poorly understood. A better understanding of plasticity and regulation of monocyte polarization is vital for the development of new therapies for the treatment of acute and chronic transplant rejection.

Sialoadhesin (CD169) facilitates T-cell priming when overexpressed on inflammatory monocytes. Monocyte-derived macrophages prime acute cellular rejection after intestine transplantation (ITx).The purpose of this study was to evaluate whether CD169-expressing activated monocytes associate with or predict ITx rejection.

After informed consent (ClinicalTrials.gov NCT No. 01163578), activated CD169+CD14+monocytes were measured by flow cytometry in five normal healthy adult volunteers (group A), and 56 children with ITx sampled cross-sectionally (group B, 26), longitudinally (group C, 18), or during infection/inflammation without rejection (group D: acute enteritis, 9; Helicobacter pylori, 1; Streptococcal pharyngitis 1; and posttransplant lymphoma, 1). Activated monocytes were tested for correlations with donor-specific alloreactivity in simultaneous mixed lymphocyte co-cultures.

Median age was 3 years (range 0.5-21 yr), and distribution of ITx-alone:combined liver-ITx was 25:31. Higher frequencies (%) of activated monocytes were seen during rejection in group B and infection/inflammation without rejection in group D (58 ± 28 and 73 ± 26), compared with nonrejectors or normal controls (10.6 ± 7.9 or 10.7 ± 6.5, P=0.001). In longitudinal monitoring, rejectors also showed higher activated monocyte frequencies (%) before ITx (64 ± 26 vs. 13.4 ± 8.6, P=0.0007) and during acute cellular rejection (55 ± 28 vs. 22.4 ± 15, P=0.006) when compared with nonrejectors. Activated monocytes correlated significantly with allospecific CD154+T-cytotoxic memory cells (Spearman r=0.688, P=7.1E-05) and CD154+B cells (r=0.518, P=0.005) in ITx recipients without inflammation/infection but not in group D.

Monocytes overexpress sialoadhesin nonspecifically during ITx rejection and systemic or enteritic inflammatory states. When combined with allospecific T and B cells, this information may differentiate between rejection and other enteritides.

Recurrent rejection shortens graft survival after intestinal transplantation (ITx) in children, most of whom also experience early acute cellular rejection (rejectors). To elucidate mechanisms common to early and recurrent rejection, we used a test cohort of 20 recipients to test the hypothesis that candidate peripheral blood leukocyte genes that trigger rejection episodes would be evident late after ITx during quiescent periods in genome-wide gene expression analysis and would achieve quantitative real-time PCR replication pre-ITx (another quiescent period) and in the early post-ITx period during first rejection episodes. Eight genes were significantly up-regulated among rejectors in the late post-ITx and pre-ITx periods, compared with nonrejectors: TBX21, CCL5, GNLY, SLAMF7, TGFBR3, NKG7, SYNE1, and GK5. Only CCL5 was also up-regulated in the early post-ITx period. Among resting peripheral blood leukocyte subsets in randomly sampled nonrejectors, CD14(+) monocytes expressed the CCL5 protein maximally. Compared with nonrejectors, rejectors demonstrated higher counts of both circulating CCL5(+)CD14(+) monocytes and intragraft CD14(+) monocyte-derived macrophages in immunohistochemistry of postperfusion and early post-ITx biopsies from the test and an independent replication cohort. Donor-specific alloreactivity measured with CD154(+) T-cytotoxic memory cells correlated with the CCL5 gene and intragraft CD14(+) monocyte-derived macrophages at graft reperfusion and early post-ITx. CCL5 gene up-regulation and CD14(+) macrophages likely prime cellular ITx rejection. Infiltration of reperfused intestine allografts with CD14(+) macrophages may predict rejection events.

The nucleotide-binding oligomerization protein 2 (NOD2) gene single nucleotide polymorphisms (SNPs) associated with Crohn's disease were recently associated with severe rejection after small-bowel transplantation (SBTx). The purpose of this study was to re-test this association and explore whether deficient innate immunity suggested by the NOD2 SNPs predisposes to intestine failure requiring isolated SBTx or combined liver-intestine failure requiring combined liver-SBTx (LSBTx).

Archived DNA from 85 children with primary isolated SBTx or LSBTx was genotyped with Taqman biallelic discrimination assays. To minimize confounding effects of racial differences in minor allele frequencies (MAFs), allelic associations were tested in 60 Caucasian recipients (discovery cohort). Replication was sought in an independent cohort of 39 Caucasian pediatric and adult SBTx patients.

MAF for rs2066845 and rs2066847 was similar to that seen in 538 healthy North American Caucasians. In the discovery cohort, MAF for rs2066844 was significantly higher in LSBTx (13.5 vs. 3.6%, P=0.0007, Fisher's exact test), but not in isolated SBTx recipients (2.2 vs. 3.6%, P=NS), when compared with 538 healthy Caucasians. In addition, among LSBTx recipients who received identical immunosuppression, the minor allele of rs2066844 associated with early rejection in linear regression analysis (P=0.028) (all but one of the risk alleles were found in rejectors), decreased survival (P=0.015, log-rank, Kaplan-Meier analysis), and a 20-fold greater hazard of septic death in proportional hazard analysis (P=0.030). Steroid-resistant (severe) rejection and graft loss were associated with isolated SBTx (P=0.036 and 0.082, respectively), but not with NOD2 SNPs. The association between rs2066844 and combined liver-intestine failure requiring LSBTx was significant in the replication cohort (P=0.014), and achieved greater significance in the combined cohort (P=0.00006).

The NOD2 SNP rs2066844 associates with combined liver and intestinal failure in subjects with short-gut syndrome, who require combined liver-intestine transplantation, and secondarily with early rejection and septic deaths.

This review summarizes the outcomes and known adverse effects of current immunosuppression strategies in use in pediatric intestinal transplantation. Intestinal transplantation has evolved from an experimental therapy to a highly successful treatment for children with intestinal failure who have complications with total parenteral nutrition. Because of continued success with intestinal transplantation over the past decade, the focus of clinicians and researchers is shifting from short-term patient survival to optimizing long-term outcomes. Current 5-year patient and graft survival rates after intestinal transplantation are 58% and 40%, respectively, in the US; single centers have reported nearly 80% patient and 60% graft survival rates at 5 years. The immunosuppression strategy in intestinal transplantation includes a tacrolimus-based regimen, usually in conjunction with an antibody induction therapy such as rabbit-antithymocyte globulin, interleukin-2 receptor antagonists, or alemtuzumab. The use of these immunosuppressive regimens, along with improved medical and surgical care, has contributed significantly toward improved outcomes. Optimization of post-transplant immunosuppression strategies to reduce adverse effects while minimizing acute and chronic graft rejection is a strong clinical and research focus.

The multivisceral liver-intestine-pancreas-stomach allograft was first described by Starzl nearly 50 years ago. Since then, over 1000 children have received small bowel transplantation (SBTx), alone or with the liver and other organs, for refractory short gut syndrome (SGS) because of a variety of congenital conditions. In 2001, SBTx was approved as definitive therapy for SGS by Medicare. Currently, 1- and 5-year graft survival routinely exceeds 90% and 80%, respectively. The expected outcomes also include freedom from parenteral nutrition, normalization of growth parameters, and quality of life. However, recurrent rejection, complications of high-dose immunosuppression, or chronic rejection, which is more likely to occur after SBTx without a liver graft, account for differences between early and late survival. Future efforts aimed at overcoming such challenges include preventing SBTx through early referral to comprehensive SGS management programs and understanding why the liver protects the small bowel allograft from rejection. Finally, inflammatory mechanisms, which predispose the highly immunogenic small bowel allograft to a protracted risk of resistant rejection must be elucidated, in order to ensure durable success.

Ischemia/reperfusion evokes a functionally relevant inflammatory response within the muscularis propria of small bowel grafts by activation of resident macrophages and leukocyte recruitment. We hypothesized that immunomodulatory perioperative treatment with glycine attenuates the proinflammatory cascade and improves smooth muscle dysfunction of small bowel grafts.

Orthotopic SBTx was performed in Lewis rats. Glycine (1 mg/g body weight) was infused (0.1 mL/g/hr) for 2 hr before harvest as preconditioning in the donor, and for 2 hr from the onset of reperfusion in the recipient. Transplanted vehicle (isotonic saline)-treated animals and naive animals served as controls. Rats were sacrificed after 3 hr and 24 hr. Leukocyte infiltration was investigated in muscularis whole mounts by immunohistochemistry. Mediator mRNA expression was determined by real-time-PCR. Jejunal circular smooth muscle contractility was assessed in a standard organ bath.

Compared with vehicle controls, glycine-treated graft muscularis expressed a significant alleviation in mRNA peak expression for IL-6, IL-1beta, ICAM-1, MCP-1, TNFalpha, COX-2, and iNOS. Also glycine-treated grafts exhibited significantly less infiltration with ED-1-positive macrophages and MPO-positive neutrophils as well as reduced apoptosis. Concurrent to these results, vehicle controls showed an 80% decrease in smooth muscle contractility, whereas glycine-treated animals exhibited only a 40% decrease in contractile activity compared with controls.

The data indicate that perioperative glycine treatment reduces the molecular and cellular inflammatory response within the grafts and improves smooth muscle dysfunction after transplantation. Therefore, the glycine-activated chloride channel on resident and infiltrating leukocytes could be a promising pharmacologic target to attenuate ischemia/reperfusion injury after ITx.

Patients with chronic kidney disease have significant abnormalities of bone remodeling and mineral homeostasis and are at increased risk of fracture. The fracture risk for a kidney transplant recipient is four times that of the general population and higher than for a patient on dialysis. Randomised controlled trials (RCTs) report the use of bisphosphonates, vitamin D sterols, calcitonin, and hormone replacement therapy to treat bone disease following transplantation.

To evaluate the use of interventions for treating bone disease following kidney transplantation.

We searched the Cochrane Central Register of Controlled Trials (CENTRAL in The Cochrane Library), Cochrane Renal Group's specialised register, MEDLINE, EMBASE, reference lists, and conference proceedings abstracts without language restriction. Date of last search: May 2006

RCTs and quasi-RCTs comparing different treatments for kidney transplant recipients of any age were selected. We excluded all other transplant recipients, including kidney-pancreas transplant recipients.

Two authors independently assessed trial quality and extracted data. Statistical analyses were performed using the random effects model and the results expressed as relative risk (RR) with 95% confidence intervals (CI) for dichotomous variables and mean difference (MD) for continuous outcomes.

Twenty-four trials (1,299 patients) were included. No individual intervention (bisphosphonates, vitamin D sterol or calcitonin) was associated with a reduction in fracture risk compared with placebo. Combining results for all active interventions against placebo demonstrated any treatment of bone disease was associated with a reduction in the RR of fracture (RR 0.51, 95% CI 0.27 to 0.99). Bisphosphonates (any route), vitamin D sterol, and calcitonin all had a beneficial effect on the bone mineral density at the lumbar spine. Bisphosphonates and vitamin D sterol also had a beneficial effect on the bone mineral density at the femoral neck. Bisphosphonates had greater efficacy for preventing bone mineral density loss when compared head-to-head with vitamin D sterols. Few or no data were available for combined hormone replacement, testosterone, selective oestrogen receptor modulators, fluoride or anabolic steroids. Other outcomes including all-cause mortality and drug-related toxicity were reported infrequently.

Treatment with a bisphosphonate, vitamin D sterol or calcitonin after kidney transplantation may protect against immunosuppression-induced reductions in bone mineral density and prevent fracture. Adequately powered trials are required to determine whether bisphosphonates are better than vitamin D sterols for fracture prevention in this population. The optimal route, timing, and duration of administration of these interventions remains unknown.

Gut manipulation and ischemia/reperfusion evoke an inflammatory response within the intestinal muscularis that contributes to dysmotility. We hypothesize that resident macrophages play a key role in initiating the inflammatory cascade. Isogenic small bowel transplantation was performed in Lewis rats. The impact of recovery of organs on muscularis inflammation was investigated by comparing cold whole-body perfusion after versus prior to recovery. The role of macrophages was investigated by transplantation of macrophage-depleted gut. Leukocytes were counted using muscularis whole mounts. Mediator expression was determined by real-time RT-PCR. Contractility was assessed in a standard organ bath. Both organ recovery and ischemia/reperfusion induced leukocyte recruitment and a significant upregulation in IL-6, MCP-1, ICAM-1 and iNOS mRNAs. Although organ recovery in cold ischemia prevented early gene expression, peak expression was not changed by modification of the recovery technique. Compared to controls, transplanted animals showed a 65% decrease in smooth muscle contractility. In contrast, transplanted macrophage-depleted isografts exhibited significant less leukocyte infiltration and only a 19% decrease in contractile activity. In summary, intestinal manipulation during recovery of organs initiates a functionally relevant inflammatory response within the intestinal muscularis that is massively intensified by the ischemia reperfusion injury. Resident muscularis macrophages participate in initiating this inflammatory response.

Because no biomarker that reflects small bowel allograft rejection is available, we applied surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to develop noninvasive markers required for routine diagnosis.

Heterotopic small bowel transplantation (SBT) was performed in rats, and they were divided into four experimental groups: sham-operated rats (sham), syngeneic transplants (syngeneic), allogeneic transplants (allogeneic), allogeneic transplants received FK506 (allo+FK). Plasma samples were analyzed with SELDI ProteinChip arrays to detect peaks that predominated in the allogeneic model. Possible biomarkers were identified in combination with SELDI retentate chromatography mass spectrometry (RCMS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The identified protein was further analyzed by immunohistochemistry.

An increase in the level of a 14.8-kDa protein, identified as lysozyme, was observed specifically in the plasma of the allogeneic group; the levels of this protein remained unchanged in the plasma of the other groups. On the other hand, the levels of a 10.1-kDa and a 13.0-kDa protein, identified as migration inhibitory factor-related proteins (MRP), MRP-8 and MRP-14, respectively, began to increase from an early stage of acute rejection. We also observed that lysozyme-positive macrophages had strongly infiltrated the lamina propria during acute rejection.

We identified three plasma proteins-MRP-8, MRP-14, and lysozyme-that increased during small bowel allograft rejection. The identified proteins appeared to be markers for inflammation associated with allograft rejection. This proteomic approach will be useful for the identification of candidate biomarkers.

Inflammatory events within the gut muscularis contribute to dysmotility. We hypothesized that manipulation during organ harvesting initiated an inflammatory response via muscularis macrophages and that this cascade was amplified during reperfusion.

Small bowel transplantation was performed in Lewis rats. To investigate the impact of organ harvesting on muscularis inflammation, cold whole-body perfusion was performed after versus prior to organ harvesting. The role of macrophages was investigated by transplantation of the macrophage-depleted gut. Leukocyte infiltration was investigated in muscularis whole mounts. Mediator mRNA expression was determined by real-time reverse transcriptase polymerase chain reaction. Contractility was assessed in a standard organ bath.

Organ harvesting and ischemia-reperfusion induced leukocyte recruitment and mRNA upregulation in the muscularis: interleukin-6 12217-fold, monocyte chemoattractant protein-1 62-fold, ICAM-1 12-fold, cyclooxygenase-2: 8-fold, iNOS: 150-fold. Although organ harvesting with cold ischemia prevented early gene expression, peak expression at 3-hour reperfusion was not changed by modification of the harvesting technique. Compared to controls, transplanted animals showed a 63% decrease in smooth muscle contractility. In contrast, transplanted macrophage-depleted gut exhibited significantly fewer leukocytes and only a 16% decrease in contractility.

Gut manipulation during organ harvesting initiates an inflammatory response within the muscularis that is massively intensified during reperfusion. This change contributes to muscular dysfunction. Furthermore, the results suggested that resident macrophages play a key role in initiating this process.

Patients with chronic kidney disease have significant abnormalities of bone remodelling and calcium homeostasis and are at increased risk of fracture. The fracture risk for a kidney transplant recipient is four times that of the general population and higher than that for a patient on dialysis. Trials reporting the use of bisphosphonates, vitamin D analogues, calcitonin, and hormone replacement therapy to treat bone disease following engraftment exist.

To evaluate the use of interventions for the treatment of bone disease following kidney transplantation.

The Cochrane CENTRAL Register of Controlled Trials (The Cochrane Library - Issue 3 2004), MEDLINE (1966 to August 2004), EMBASE (1980- August 2004) and reference lists were searched without language restriction.

Randomised trials of treatment of bone disease following kidney transplantation were included. Trials of recipients of any transplant other than a kidney transplant including trials of kidney-pancreas transplants were excluded.

Two authors assessed independently trial quality and extracted data. Statistical analyses were performed using the random effects model and the results expressed as relative risk (RR) with 95% confidence intervals (CI) for dichotomous variables. For continuous variables the weighted mean difference (WMD) and its 95% CI was used.

Twenty-three eligible trials (1,209 patients) were identified. Seven trials compared more than two interventions. Nineteen trials compared active treatment with placebo, five vitamin D analogue and calcium, six vitamin D analogue alone, twelve bisphosphonates, and four nasal calcitonin. Eight trials compared two active treatments, one 17-beta oestradiol and medroxyprogesterone versus vitamin D analogue, five bisphosphonate versus vitamin D analogue, two vitamin D analogue and calcium versus calcium and one bisphosphonate versus calcitonin. Methodological quality was suboptimal. There were no significant differences between any treatment group for risk of fracture. Bisphosphonate, administered by any route, vitamin D analogue and calcitonin all had a beneficial effect on the bone mineral density at the lumbar spine. Bisphosphonates and vitamin D analogue had a beneficial effect on the bone mineral density at the femoral neck. Few or no data were available for combined hormone replacement, testosterone, selective oestrogen receptor modulators, fluoride or anabolic steroids. All-cause mortality and drug-related toxicity were reported infrequently and there was no statistical difference between treatment groups.

No benefit from any intervention known to reduce risk of fracture from bone disease could be demonstrated to reduce fracture incidence in kidney transplant recipients.

Before renal transplantation complex abnormalities of bone metabolism exist and lead to increased risk for fracture after transplantation. This study was conducted to assess the evidence available to guide targeted treatment to reduce bone disease in transplant recipients.

The Cochrane CENTRAL Registry, MEDLINE, and EMBASE were searched for randomized trials of interventions for bone disease after renal transplantation. Data were extracted on fracture, bone mineral density (BMD) by means of dual-energy X-ray absorptiometry, acute graft rejection, and adverse events. Analysis was performed with a random-effects model, and all results are expressed as relative risk with 95% confidence intervals (CIs).

Twenty-three eligible trials (1,209 patients) were identified. No trial found a reduction in risk for fracture. Bisphosphonates (7 trials; 268 patients; weighted mean difference [WMD], 7.66; 95% CI, 4.82 to 10.50), vitamin D analogues (2 trials; 51 patients; WMD, 6.13; 95% CI, 4.97 to 7.29), and calcitonin (1 trial; 31 patients; WMD, 5.00; 95% CI, 0.88 to 9.12) favorably affected the percentage of change in BMD at the lumbar spine compared with no treatment. Bisphosphonates (4 trials; 149 patients; WMD, 7.18; 95% CI, 6.22 to 8.13) and vitamin D analogues (2 trials; 51 patients; WMD, 3.73; 95% CI, 2.71 to 4.75), but not calcitonin (1 trial; 31 patients; WMD, -0.30; 95% CI, -5.00 to 4.40), had a favorable effect on BMD measured at the femoral neck compared with no treatment. The incidence of reported toxicity was low.

The trials were inadequately powered to show a reduction in risk for fracture. Bisphosphonates and vitamin D have a beneficial effect on BMD at the lumbar spine and femoral neck. With increasing survival after renal transplantation, this study stresses the importance of randomized controlled trial evidence of interventions of bone disease after renal transplantation.

Chronic ethanol intake has been shown to be associated with immune suppression and impairment of epithelial barrier function. We investigated the effects of ethanol on intestinal immunity and its relation to bacterial translocation (BT). Male Wistar rats were assigned to one of three groups and received respective diets for 28 days. The ethanol-fed group [(EG); n=11] received a liquid diet containing 5% [volume/volume (vol./vol.)] ethanol; a pair-fed group [(PFG); n=11] received an isocaloric diet without ethanol; and a third (control) group [(CG); n=11] received water and chow ad libitum. On experimental day 29, animals in the EG and the PFG underwent distal ileum ligature and small intestine inoculation of a tetracycline-resistant Escherichia coli strain (TcR E. coli R6), by means of gastric intubation, followed by duodenal ligature. One hour after inoculation, mesenteric lymph nodes, right lobe of liver, spleen, and left kidney were excised for bacterial studies. Sections of jejunum and colon were immunostained, with the use of antibodies against immunoglobulin (Ig) A, T cells, macrophages, and proliferating cell nuclear antigen (PCNA). Apoptosis was determined by the terminal deoxynucleotidyltransferase TdT-mediated dUDP-biotin nick end labeling (TUNEL) method. Bacterial translocation rates were greater in the PFG compared with findings for the EG. Lamina propria of the jejunum of the EG showed a reduction in the densities of IgA+ cells and T cells compared with findings for the PFG and the CG. Colonic lamina propria of the EG showed reduced densities of IgA+ cells and macrophages compared with findings for the PFG and the CG. Apoptotic index was increased in the EG compared with findings for the PFG and the CG, in both jejunum and colon. Proliferation index was not significantly different among groups. Results of the current study show that chronic ethanol ingestion led to a reduction of cellular and humoral components of the intestinal mucosa, possibly by cell loss as a result of ethanol-induced apoptosis. The reduced rates of BT observed after chronic ethanol intake seem to indicate that factors irrespective of immune function might be involved in BT inhibition.

To establish a successful model of heterotopic total small intestinal transplantation (SIT) in rats in order to reduce the complications and increase the survival rate.

A total of 196 Wistar rats underwent heterotopic SIT with microsurgical technique. Technical modifications included shortening fasting time and supplying energy before surgery, administering optimal volume of crystalloid fluid to the donor and recipient during surgical procedures, reducing mechanical and ischemic injuries to donor intestine, revascularizing small intestinal graft with a combination of conventional aorta to aorta anastomosis and a cuffed portal vein to left renal vein anastomosis which resulted in an acceptably short warm ischemic time, and also an adequate blood supply and drainage of the graft.

The average time for the donor surgery was 86 min +/- 20 min, the mean operative time for the recipient was 115 min +/-20 min and warm ischemia time was shortened to 40 min +/- 5 min. There was a shorter revascularizing time of the graft, the abdominal aorta (AA) to AA anastomosis being 21 min +/- 10 min, and the cuffed portal vein (PV) to the renal vein anastomosis being 5 min +/- 5 min. The one-week survival rate of 98 rats with SIT was 88.78% (87/98), without thrombosis and stenosis of anastomosis. The longest survival time of recipient rats was more than 389 days after SIT, the rats were maintaining normal weight, with perfect intestinal function and intact intestinal histology.

These modified techniques for SIT would remarkably reduce the complications and improve survival rate in rats, which provided a potentially more consistent and practical model for experimental and clinical studies.

The allograft inflammatory factor (AIF-1/daintain) is a hormone-like peptide produced by activated monocytic cells in a variety of traumatic, inflammatory and degenerative lesions. Gut-derived AIF-1 has been shown to modulate insulin production and to attenuate autoimmune diabetes. As the localization of this gastrointestinal peptide in the porcine duodenum is not known and the pig is a convenient model for the study of nutritional modulation of the mucosal immune compartment, we have localized expression of AIF-1 by immunohistology in the duodenum of either malnourished (energy and protein supply 50% of demands, n = 5) or optimally fed pigs (n = 5). AIF-1 macrophages were predominantly located at the villus tip. The number of positively stained cells per high-power field was significantly (P < or = 0.001) higher in the malnourished pigs (74.6 +/- 2.44; least square means +/- SEM) compared to optimally fed pigs (32.56 +/- 1.99). It is likely that the effect in malnourished pigs can be explained by a more pronounced antigen contact of macrophages due to loss of epithelial integrity. Thus, AIF-1 is a novel marker for the study of the nutritional regulation of the mucosal immune system of the pig. AIF-1 expression in the duodenum was further validated by polymerase chain reaction and sequencing. Surprisingly, we detected a slight deviation from the original sequence (probably representing an allelic variation) and an AIF-1 splice variant, previously not known to occur in pigs.

Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival after lung transplantation. Acute rejection, its main risk factor, is characterized by perivascular/bronchiolar leukocyte infiltration. BOS is characterized by persistent peribronchiolar leukocyte recruitment leading to airway fibrosis and obliteration. The specific mechanism(s) by which these leukocytes are recruited are unknown. Because MCP-1, acting through its receptor CCR2, is a potent mononuclear cell chemoattractant, we hypothesized that expression of this chemokine during an allogeneic-response promotes persistent recruitment of leukocytes and, ultimately, rejection. We found that elevated levels of biologically active MCP-1 in human bronchial lavage fluid (BALF) were associated with the continuum from acute to chronic allograft rejection. Translational studies in a murine model of BOS demonstrated increased MCP-1 expression paralleling mononuclear cell recruitment and CCR2 expression. Loss of MCP-1/CCR2 signaling, as seen in CCR2(-/-) mice or in WT mice treated with neutralizing antibodies to MCP-1, significantly reduced recruitment of mononuclear phagocytes following tracheal transplantation and led to attenuation of BOS. Lymphocyte infiltration was not reduced under these conditions. We suggest that MCP-1/CCR2 signaling plays an important role in recruitment of mononuclear phagocytes, a pivotal event in the pathogenesis of BOS.

Severe osteoporosis frequently is observed after organ transplantation. In kidney transplantation, it adds to pre-existing renal bone disease and strategies to prevent osteoporosis are not established. Eighty kidney recipients were included in a randomized controlled prospective intervention trial. Treated patients (n = 40) received an injection of ibandronate, a bisphosphonate, immediately before and at 3, 6, and 9 mo after transplantation. The primary outcome measured was the change in bone mineral density. Secondary measures included graft outcome, spinal deformities, fracture rate, body height, and hormonal and metabolic data. Loss of spongy and cortical bone after transplantation was prevented by ibandronate. Changes of bone mineral density (ibandronate versus controls) were as follows: lumbar spine, -0.9 +/- 6.1% versus -6.5 +/- 5.4% (P < 0.0001); femoral neck, +0.5 +/- 5.2% versus -7.7 +/- 6.5% (P < 0.0001); and midfemoral shaft, +2.7 +/- 12.2% versus -4.0 +/- 10.9% (P = 0.024). Fewer spinal deformities developed with ibandronate (7 patients with 7 deformities versus 12 patients with 23 deformities; P = 0.047). Loss of body height was 0.5 +/- 1.0 cm versus 1.1 +/- 1.0 cm in control subjects (P = 0.040). Two bone fractures occurred in each group. There were fewer acute rejection episodes with ibandronate (11 versus 22; P = 0.009). Graft function after 1 yr was comparable. Bone loss, spinal deformation, and loss of body height during the first year after kidney transplantation are prevented by injection of ibandronate at intervals of 3 mo. The smaller number of rejection episodes of the ibandronate-treated group should be confirmed and its mechanism should be explored in additional studies.

Studies indicate that lymphoid tissue (e.g., thymus, bone marrow, and Peyer's patches) shows evidence of increase apoptosis (Ao, a form of nonnecrotic cell death) during sepsis. However, it is not known if mucosal lymphoid tissue, such as lamina propria (LP), also shows evidence of increased Ao and if so, is this associated with functional changes, i.e., cytokine gene expression in the LP. To examine this, male C3H/HeN mice were subjected to cecal ligation and puncture (CLP) and lamina propria mononuclear cells (LPMC) were harvested at 4 h (early sepsis) or 24 h (late sepsis). Alterations in the cell phenotype as well as Ao (Tunel assay) were determined by three-color flow cytometry. Cytokine gene expression was assessed by multiprobe RNase protection assay. Sham LPMC preparations were found to be 34.4 +/- 2.4% B220(+) (B-cells), while 12.4 +/- 2.1% were CD8(+) (cytotoxic T-cells), 22.0 +/- 0.8% were CD4(+) (helper T-cells), and 6.4 +/- 0.7% were F4/80(+) (macrophages). The frequency of B220(+) (9%* upward arrow) and CD8 (6%* upward arrow) populations increased markedly at 4 h after CLP; however, this increase was not seen at 24 h. The percentage of Ao+ in CD8(+), B220(+), and F4/80(+) cells increased markedly at both 4 and 24 h. CD4(+) cells showed a marked increase in Ao only at 24 h after CLP. When LPMC mRNA expression was examined, a significant increase in IL-2, -10, and -15 gene expression was observed only at 24 h but not 4 h after CLP. Thus, the early phenotypic changes associated with increased Ao may be a reflection of localized immune cell activation in early sepsis contributing to the increased cytokine gene expression seen in late sepsis. This localized activation may contribute to gastrointestinal inflammation and/or immune dysfunction in sepsis.

Endothelial cells are known to be an early target of preservation/reperfusion injury and acute rejection, whereas the extracellular matrix (ECM) may also play an equally important role in the sequelae of both events.

Syngeneic and allogeneic rat small bowel transplantations (SBTX) were performed after 6 hr of preservation. Animals were subsequently killed at defined time points for determination of ECM parameters within the graft and in plasma.

Laminin levels were significantly increased 20 min after reperfusion (syngeneic SBTX: 357+/-65.9 ng/ml; allogeneic SBTX: 361+/-79.6 ng/ml; P< or =0.01). After syngeneic transplantation, laminin levels normalized by postoperative day (POD) 7, whereas there was a rejection-induced increase after allogeneic SBTX (POD 7: 179+/-60.1 ng/ml; POD 9: 333+/-13.6 ng/ml; P< or =0.01 vs. syngeneic SBTX). This increase was accompanied by an increase in tumor necrosis factor-alpha levels at POD 9. Hyaluronic acid levels were significantly elevated after 24 hr (syngeneic SBTX: 1086+/-176 microg/L; allogeneic SBTX: 918+/-108 microg/L; P< or =0.01). After syngeneic SBTX, hyaluronic acid levels normalized by POD 7, whereas persistently higher levels were observed after allogeneic SBTX. Immunohistochemistry confirmed early changes (20 min after reperfusion) at the ECM. Anti-laminin and anti-CD44 staining normalized at POD 5 after syngeneic SBTX. After allogeneic SBTX, rejection-specific changes were evident with anti-laminin staining commencing on POD 5 and progressing until POD 9. At similar time points, increased expression of fibronectin- and interferon-gamma-positive material was evident.

The ECM can be considered to be an early target of preservation/reperfusion injury and acute rejection. Plasma parameters reliably reflected the changes observed within the graft. Laminin and hyaluronic acid levels may be used as indicators of initial graft function. Furthermore, the increase in laminin levels was an early indicator of acute rejection. Determination of these parameters may significantly improve monitoring after SBTX.