Tacrolimus prevents von Willebrand factor secretion by allostimulated human glomerular endothelium.

Little is known about the endothelial injury caused directly by circulating donor-specific antibodies (DSAs) during antibody-mediated rejection. von Willebrand factor (vWF) is a highly thrombotic glycoprotein stored in Weibel-Palade bodies in endothelial cells. It has been shown that its secretion is triggered by allostimulation. Calcineurin-like phosphatases regulate pathways involved in vWF secretion. Therefore, we hypothesized that tacrolimus would prevent alloantibody-induced glomerular lesions, in part via inhibition of vWF secretion from endothelial cells. Here, we used a human in vitro model of glomerular endothelium expressing HLA class I and II antigens and demonstrated that anti-HLA class II antibodies elicit a higher endothelial release of vWF than do anti-HLA class I antibodies in cell supernatants. We observed that tacrolimus treatment decreased vWF secretion after stimulation with both classes of anti-HLA antibodies and decreased platelet adhesion on allostimulated endothelial cells in a microfluidic chamber. In kidney recipients, tacrolimus trough levels were negatively associated with vWF blood levels. These results indicate that direct disruption of hemostasis via vWF secretion is a potential mechanism of antibody-mediated injury in patients with DSAs. Our results further suggest that the targeting of microcirculation hemostasis may be beneficial to prevent the development of microangiopathic lesions in antibody-mediated rejection.

2013 Banff Criteria for Chronic Active Antibody-Mediated Rejection: Assessment in a Real-Life Setting.

Significant changes in the criteria for chronic active antibody-mediated rejection (CAABMR) were made in the Banff 2013 classification. These modifications expanded the number of patients diagnosed with CAABMR, with undetermined clinical significance. We compared the 2007 and 2013 criteria for the composite end point of death-censored graft failure or doubling of serum creatinine in 123 patients meeting the criterion related to the morphologic evidence of chronic tissue injury. In all, 18% and 36% of the patients met the 2007 and 2013 criteria, respectively. For the criterion related to antibody interaction with endothelium, only 25% were positive based on the 2007 definition compared with 82% using the 2013 definition. Cox modeling revealed that a 2013 but not a 2007 diagnosis was associated with the composite end point (adjusted hazard ratio 2.5 [95% confidence interval (CI) 1.2-5.2] vs. 1.6 [95% CI 0.7-3.8], respectively). The 2013 criterion based on both the C4d score and the glomerulitis plus peritubular capillaritis score (g+ptc) was more strongly associated with the end point than the 2007 criterion based only on C4d; however, when dissected by component, only the C4d component was significant. The association with clinical outcomes improved with the 2013 criteria. This is related to the new C4d threshold but not to the g+ptc ≥2 component.

A new diagnostic algorithm for antibody-mediated microcirculation inflammation in kidney transplants.

We studied the significance of microcirculation inflammation in kidney transplants, including 329 indication biopsies from 251 renal allograft recipients, who were mostly nonpresensitized (crossmatch negative). Glomerulitis (g) and peritubular capillaritis (ptc) were often associated with antibody-mediated rejection (65% and 75%, respectively), but were also found in other diseases in the absence of donor-specific antibody (DSA): T-cell-mediated rejection (ptc, g), glomerulonephritis (g) and acute tubular necrosis (ptc). To develop rules for reducing the nonspecificity of microcirculation inflammation and defining the best grading thresholds associated with DSA, we built and validated a decision tree to predict DSA. The decision tree revealed that g + ptc sum (addition of g-score plus ptc-score) was the best predictor of DSA, followed by time posttransplant, then C4d, which had a small role. Late biopsies with g + ptc > 0 showed higher frequency of DSA compared to early biopsies with g + ptc > 0 (79% vs. 27%). Microcirculation inflammation in early biopsies was often false positive (antibody-independent). The decision tree predicted DSA with higher sensitivity and accuracy than C4d staining. Microcirculation inflammation sum score predicted graft failure independently of time, C4d and transplant glomerulopathy. Thus any degree of microcirculation inflammation in late kidney transplant biopsies strongly indicates presence of DSA and predicts progression to graft failure.

Peritubular capillaritis in renal allografts: prevalence, scoring system, reproducibility and clinicopathological correlates.

While glomerulitis is graded according to the Banff classification, no criteria for scoring peritubular capillaritis (PTC) have been established. We retrospectively applied PTC-scoring criteria to 688 renal allograft (46 preimplantation, 461 protocol, 181 indication) biopsies. A total of 26.3% of all analyzed biopsies had peritubular capillaritis (implant 0%, protocol 17.6%, indication 45.5%; p < 0.0001). The most common capillaritis pattern was of moderate severity (5-10 luminal cells), focal in extent (10-50% of PTC), with a minority of neutrophils. A total of 24% of C4d- compared with 75% of C4d+ biopsies showed capillaritis (p < 0.0001). More than 80% of biopsies with glomerulitis had peritubular capillaritis. A total of 50.4% of biopsies with borderline or T-cell mediated rejection (TCMR) and 14.1% of biopsies without TCMR or antibody-mediated rejection (ABMR) showed capillaritis (p < 0.0001). The inter-observer reproducibility of the PTC-scoring features was fair to moderate. Diffuse capillaritis detected in early protocol biopsies had significant negative prognostic impact in terms of glomerular filtration rate 2 years posttransplantation. Indication biopsies show a significantly higher prevalence of capillaritis than protocol biopsies (45.5% vs. 17.6%; p < 0.0001). Capillaritis is more frequent and pronounced in ABMR, but can be observed in TCMR cases. Thus, scoring of peritubular capillaritis is feasible and can provide prognostic and diagnostic information in renal allograft biopsies.

Lymph node metastases in low-grade endometrial stromal sarcoma.

OBJECTIVES: The objective of this study was to review our experience on lymphatic dissemination in patients with low-grade endometrial stromal sarcoma. METHODS: All cases diagnosed as low-grade endometrial stromal sarcoma or endolymphatic stromal myosis before October 2003 and who had lymph node sampling at some point in their evolution were retrieved from the files of the pathology and gynecologic oncology departments of l'Hotel-Dieu de Quebec University Hospital (HDQ). RESULTS: Fifteen patients with either limited lymph node biopsies or a complete lymph node dissection at some point in the course of their disease were found. Five of these patients (33%) presented lymph node metastases either at the initial hysterectomy, during a subsequent staging procedure, or at the time of a recurrence. CONCLUSION: These findings suggest that the incidence of lymph node involvement in low-grade endometrial stromal sarcoma is higher than expected. More extensive sampling of lymph nodes in a larger number of patients may allow a better understanding of the frequency and prognostic significance of these metastases.

Granulocyte-macrophage colony-stimulating factor-deficient mice have impaired resistance to blood-stage malaria.

The contribution of granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic and immunoregulatory cytokine, to resistance to blood-stage malaria was investigated by infecting GM-CSF-deficient (knockout [KO]) mice with Plasmodium chabaudi AS. KO mice were more susceptible to infection than wild-type (WT) mice, as evidenced by higher peak parasitemia, recurrent recrudescent parasitemia, and high mortality. P. chabaudi AS-infected KO mice had impaired splenomegaly and lower leukocytosis but equivalent levels of anemia compared to infected WT mice. Both bone marrow and splenic erythropoiesis were normal in infected KO mice. However, granulocyte-macrophage colony formation was significantly decreased in these tissues of uninfected and infected KO mice, and the numbers of macrophages in the spleen and peritoneal cavity were significantly lower than in infected WT mice. Serum levels of gamma interferon (IFN-gamma) were found to be significantly higher in uninfected KO mice, and the level of this cytokine was not increased during infection. In contrast, IFN-gamma levels were significantly above normal levels in infected WT mice. During infection, tumor necrosis factor alpha (TNF-alpha) levels were significantly increased in KO mice and were significantly higher than TNF-alpha levels in infected WT mice. Our results indicate that GM-CSF contributes to resistance to P. chabaudi AS infection and that it is involved in the development of splenomegaly, leukocytosis, and granulocyte-macrophage hematopoiesis. GM-CSF may also regulate IFN-gamma and TNF-alpha production and activity in response to infection. The abnormal responses seen in infected KO mice may be due to the lack of GM-CSF during development, to the lack of GM-CSF in the infected mature mice, or to both.

NRSA-1: a resistance gene homolog expressed in roots of non-host plants following parasitism by Striga asiatica (witchweed).

Studies of the initial interactions of Striga asiatica with the non-host plant species Tagetes erecta (marigold) established that parasite penetration through the root is arrested most frequently in the cortex. The arrest of parasite ingress is associated with browning and necrosis of root cortical cells flanking the invading endophyte and with increased intracellular wall appositions on the root cell walls directly adjacent to the plant-parasite interface. Using a polymerase chain reaction-based differential cDNA amplification strategy followed by 5'-RACE, we have identified several gene products whose expression is induced in marigold roots during attempted parasitism by Striga. Among these was a 917 bp cDNA encoding a 221 amino acid protein with significant homology to proteins encoded by disease resistance genes from other plant species, including N, RPP5, L6 and M. This cDNA was subsequently used to isolate a nuclear gene, designated NRSA-1, for non-host resistance to Striga asiatica. NRSA-1 is a member of a small gene family in marigold consisting of two to four members. RNA gel blot analysis showed that NRSA-1 transcripts accumulate to high levels in roots near the site of Striga invasion within 120 h after parasite attachment, and appear at lower levels throughout the rest of the plant under Striga parasitism. NRSA-1 expression is rapidly induced by treatment with jasmonic acid (JA), but not by mechanical wounding, treatment with salicylic acid, paraquat or ABA. A possible role for NRSA-1 in the non-host resistance mechanism is discussed.

Primary Haustorial Development of Striga asiatica on Host and Nonhost Species.

ABSTRACT Initial interactions of Striga asiatica with a susceptible host and non-host plants were examined by histological methods. Haustorial development was initiated when radicles of S. asiatica were placed in contact with host or nonhost roots. Reorganization of the S. asiatica root apical meristem was rapid and involved the formation of a distal group of cells that penetrated the host or nonhost root. Penetration of the epidermis of the host (sorghum) roots and advance into the cortex occurred within 24 to 48 h of inoculation. Penetration of the endodermis by the developing endophyte was delayed for 72 to 96 h after initial contact. However, upon penetration vascular continuity was established between parasite and host. In contrast, interactions with nonhosts provided evidence of active resistance mechanisms. Penetration of lettuce, marigold, and cowpea roots by S. asiatica was most frequently arrested in the cortex, and endophytic cells were necrotic 72 h after inoculation. Some species-specific differences were observed in the reactions of nonhosts to penetration, although in their general nature the interactions with S. asiatica were similar.