2016 Comprehensive Update of the Banff Working Group on Liver Allograft Pathology: Introduction of Antibody-Mediated Rejection.

The Banff Working Group on Liver Allograft Pathology reviewed and discussed literature evidence regarding antibody-mediated liver allograft rejection at the 11th (Paris, France, June 5-10, 2011), 12th (Comandatuba, Brazil, August 19-23, 2013), and 13th (Vancouver, British Columbia, Canada, October 5-10, 2015) meetings of the Banff Conference on Allograft Pathology. Discussion continued online. The primary goal was to introduce guidelines and consensus criteria for the diagnosis of liver allograft antibody-mediated rejection and provide a comprehensive update of all Banff Schema recommendations. Included are new recommendations for complement component 4d tissue staining and interpretation, staging liver allograft fibrosis, and findings related to immunosuppression minimization. In an effort to create a single reference document, previous unchanged criteria are also included.

Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes.

Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson's correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis.

Guidelines for the diagnosis of antibody-mediated rejection in pancreas allografts-updated Banff grading schema.

The first Banff proposal for the diagnosis of pancreas rejection (Am J Transplant 2008; 8: 237) dealt primarily with the diagnosis of acute T-cell-mediated rejection (ACMR), while only tentatively addressing issues pertaining to antibody-mediated rejection (AMR). This document presents comprehensive guidelines for the diagnosis of AMR, first proposed at the 10th Banff Conference on Allograft Pathology and refined by a broad-based multidisciplinary panel. Pancreatic AMR is best identified by a combination of serological and immunohistopathological findings consisting of (i) identification of circulating donor-specific antibodies, and histopathological data including (ii) morphological evidence of microvascular tissue injury and (iii) C4d staining in interacinar capillaries. Acute AMR is diagnosed conclusively if these three elements are present, whereas a diagnosis of suspicious for AMR is rendered if only two elements are identified. The identification of only one diagnostic element is not sufficient for the diagnosis of AMR but should prompt heightened clinical vigilance. AMR and ACMR may coexist, and should be recognized and graded independently. This proposal is based on our current knowledge of the pathogenesis of pancreas rejection and currently available tools for diagnosis. A systematized clinicopathological approach to AMR is essential for the development and assessment of much needed therapeutic interventions.

The CCR7 ligand elc (CCL19) is transcytosed in high endothelial venules and mediates T cell recruitment.

Lymphocyte homing to secondary lymphoid tissue is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial selectin-mediated tethering and rolling, firm adhesion of lymphocytes requires rapid upregulation of lymphocyte integrin adhesiveness. This step is mediated in part by the HEV-derived chemokine SLC (secondary lymphoid-tissue chemokine, or CCL21) that binds to the CC chemokine receptor (CCR)7 on lymphocytes. However, the CC chemokine ELC (Epstein-Barr virus-induced molecule 1 ligand chemokine, or CCL19) shares the same receptor, and ELC transcripts have been observed in the T cell areas of lymphoid organs. Here, we show that perivascular ELC is transcytosed to the luminal surfaces of HEVs and enables efficient T cell homing to lymph nodes. In situ hybridization on sections of human tonsil showed no ELC mRNA in HEVs, but immunostaining revealed ELC protein in cytoplasmic vesicles of HEV cells. Furthermore, ELC injected into the footpads of mice entered the draining lymph nodes and was presented by HEVs. Finally, intracutaneous injections of ELC in mice lacking functionally relevant ELC and SLC (plt/plt mice) restored T cell trafficking to draining lymph nodes as efficiently as SLC. We conclude that perivascular ELC is transcytosed to the luminal surfaces of HEVs and participates in CCR7-mediated triggering of lymphocyte arrest.

Gene expression and distribution of key bone turnover markers in the callus of estrogen-deficient, vitamin D-depleted rats.

An experimental rat model was used to test the hypothesis that in osteoporosis (OP) the molecular composition of the extracellular matrix in the fracture callus is disturbed. OP was induced at 10 weeks of age by ovariectomy and a vitamin D(3)-deficient diet, and sham-operated animals fed normal diet served as controls. Three months later a closed tibial fracture was made and stabilized with an intramedullary nail. After 3 and 6 weeks of healing, the animals were killed and the fracture calluses examined with global gene expression, in situ mRNA expression, and ultrastructural protein distribution of four bone turnover markers: osteopontin, bone sialoprotein, tartrate-resistant acid phosphatase, and cathepsin K. Global gene expression showed a relatively small number of differently regulated genes, mostly upregulated and at 3 weeks. The four chosen markers were not differently regulated, and only minor differences in the in situ mRNA expression and ultrastructural protein distribution were detected. Gene expression and composition of fracture calluses are not generally disturbed in experimental OP.

Short-term perioperative parecoxib is not detrimental to shaft fracture healing in a rat model.

OBJECTIVES: Experimental studies indicate that non-steroidal anti-inflammatory drugs (NSAIDs) may have negative effects on fracture healing. This study aimed to assess the effect of immediate and delayed short-term administration of clinically relevant parecoxib doses and timing on fracture healing using an established animal fracture model. METHODS: A standardized closed tibia shaft fracture was induced and stabilized by reamed intramedullary nailing in 66 Wistar rats. A 'parecoxib immediate' (Pi) group received parecoxib (3.2 mg/kg bodyweight twice per day) on days 0, 1, and 2. A 'parecoxib delayed' (Pd) group received the same dose of parecoxib on days 3, 4, and 5. A control group received saline only. Fracture healing was evaluated by biomechanical tests, histomorphometry, and dual-energy x-ray absorptiometry (DXA) at four weeks. RESULTS: For ultimate bending moment, the median ratio between fractured and non-fractured tibia was 0.61 (interquartile range (IQR) 0.45 to 0.82) in the Pi group, 0.44 (IQR 0.42 to 0.52) in the Pd group, and 0.50 (IQR 0.41 to 0.75) in the control group (n = 44; p = 0.068). There were no differences between the groups for stiffness, energy, deflection, callus diameter, DXA measurements (n = 64), histomorphometrically osteoid/bone ratio, or callus area (n = 20). CONCLUSION: This study demonstrates no negative effect of immediate or delayed short-term administration of parecoxib on diaphyseal fracture healing in rats.Cite this article: G. A. Hjorthaug, E. Søreide, L. Nordsletten, J. E. Madsen, F. P. Reinholt, S. Niratisairak, S. Dimmen. Short-term perioperative parecoxib is not detrimental to shaft fracture healing in a rat model. Bone Joint Res 2019;8:472-480. DOI: 10.1302/2046-3758.810.BJR-2018-0341.R1.

Banff schema for grading pancreas allograft rejection: working proposal by a multi-disciplinary international consensus panel.

Accurate diagnosis and grading of rejection and other pathological processes are of paramount importance to guide therapeutic interventions in patients with pancreas allograft dysfunction. A multi-disciplinary panel of pathologists, surgeons and nephrologists was convened for the purpose of developing a consensus document delineating the histopathological features for diagnosis and grading of rejection in pancreas transplant biopsies. Based on the available published data and the collective experience, criteria for the diagnosis of acute cell-mediated allograft rejection (ACMR) were established. Three severity grades (I/mild, II/moderate and III/severe) were defined based on lesions known to be more or less responsive to treatment and associated with better- or worse-graft outcomes, respectively. The features of chronic rejection/graft sclerosis were reassessed, and three histological stages were established. Tentative criteria for the diagnosis of antibody-mediated rejection were also characterized, in anticipation of future studies that ought to provide more information on this process. Criteria for needle core biopsy adequacy and guidelines for pathology reporting were also defined. The availability of a simple, reproducible, clinically relevant and internationally accepted schema for grading rejection should improve the level of diagnostic accuracy and facilitate communication between all parties involved in the care of pancreas transplant recipients.

Growth inhibition of human osteosarcomas in nude mice by human interferon-alpha: significance of dose and tumor differentiation.

The sensitivity of 11 human osteosarcoma xenografts in nude mice to human interferon-alpha (IFN-alpha) was studied. Growth inhibition could be demonstrated in all tumors but the necessary IFN-alpha dose ranged from 1 X 10(5)-1 X 10(6) IU/day. IFN-alpha had to be given daily to attain growth arrest and growth resumed after reduction of the IFN-alpha dose. The xenografts could be divided in two groups based on their sensitivity to IFN-alpha: one group of five xenografts that were growth arrested by IFN-alpha, 2 X 10(5) IU/day, and another group of six xenografts in which this dose was insufficient to arrest growth. The proportions of S-phase cells, determined by DNA flow cytometry of untreated control xenografts, were lower in the former group compared to the latter less IFN-alpha sensitive group. Histological examination revealed that in four of the five more IFN-alpha sensitive xenografts, tumor tissue was replaced by normal bone and marrow tissue. This was not seen in the respective control xenografts and not in any of the six less sensitive IFN-alpha treated xenografts. It appears that less proliferative osteosarcoma xenografts are more sensitive to growth inhibition by IFN-alpha. Interestingly the antitumor effect by IFN-alpha on these xenografts was expressed not only by growth arrest but also by tumor differentiation.

Influence of human alpha-interferon on four human osteosarcoma xenografts in nude mice.

Growth-inhibiting effects of human alpha-interferon (HuIFN-alpha) were investigated in four human osteosarcoma xenografts in nude mice. In addition to effects on growth, the HuIFN-alpha treatment was evaluated by histological examination and DNA flow cytometric analysis. Daily doses of 2 X 10(5) IU HuIFN-alpha completely arrested the growth of two osteosarcoma xenografts and partially inhibited one, whereas 1 X 10(6) IU/day were necessary to arrest the growth of the fourth. Growth inhibition was reversible and tumor size independent. The histological appearance, including mitotic indices, and S-phase proportions were unchanged in three xenografts. The mechanism of the HuIFN-alpha-induced growth inhibition of these three xenografts was therefore not considered to be a direct antiproliferative effect, but rather due to increased cell loss and/or increased cell cycle time. The modal DNA value of one xenograft was changed from aneuploid to diploid during HuIFN-alpha treatment. Histologically, these xenografts were partly replaced by normal appearing bone and bone marrow. The S-phase proportion was also reduced in these xenografts, implying that HuIFN-alpha can also have a direct antiproliferative effect.

Markers for pancreas-graft rejection in humans.

Rejection episodes were studied in 15 patients, in whom no kidney graft could serve as a marker for rejection, subjected to pancreas transplantation with pancreatoenterostomy and temporary exteriorization of the pancreatic juice (10 pancreas alone, 3 pancreas after kidney, and 2 combined pancreas and kidney in which the kidney was not functioning.) Twelve patients (80%) had a total of 18 rejection episodes. In the first 11 patients, 13 rejection episodes were diagnosed by a decline in amylase activity in the pancreatic juice, whereas in the next 4 patients, 5 rejection episodes were diagnosed by positive cytology in the pancreatic juice. Neopterin in pancreatic juice and immunoreactive anionic trypsin in serum showed promise as rejection markers, whereas serum neopterin, serum amylase, and serum immunoreactive cationic trypsin did not. Unspecific signs of rejections were an increase in white blood cell count, clinical symptoms such as fever, abdominal pain, and arthralgia. All acute rejection episodes were successfully reversed by antirejection treatment. However, late rejections diagnosed by impaired endocrine function were seen in 6 of the 15 (40%) patients, and the prognoses for these rejections were worse: 4 patients (27%) lost their grafts because of chronic rejections, and 2 patients still had impaired endocrine function.

Parathyroid cell number and size in hypercalcemic rats: a stereologic study employing modern unbiased estimators.

Recently developed stereologic methods for unbiased estimations of particle number and size were employed to study parathyroid growth in normal and hypercalcemic young rats. Thus, the parathyroid cell number and size of parathyroid secretory cells were estimated by both the disector method and the volume-weighted mean volume method. The glandular volume was calculated from serial sections, and the volume density of secretory cells was estimated by conventional stereologic techniques. Three groups of animals were studied: normal rats at 3 weeks of age, hypercalcemic rats at 7 weeks of age, and age-matched controls. Hypercalcemia was induced by feeding the animals a purified diet that was nutritionally adequate except for low amounts of phosphate (0.02%) from 3 weeks of age. During the period from 3 to 7 weeks of age, the number of parathyroid secretory cells increased by 100%, whereas the mean cell volume increased by 20%. However, when calculated per gram body weight the volume and number of cells were larger in the younger animals. The phosphate-depleted animals grew slowly and developed severe hypercalcemia. Their parathyroid secretory cells were smaller, and each gland contained fewer cells than in age-matched controls. The lower cell number and cell volume, however, were proportional to the reduced body weight. Data from the 3-week-old animals indicate that the reduced cell number and size in hypercalcemic rats reflected growth arrest rather than atrophy.

Ultrastructural localization of a tartrate-resistant acid ATPase in bone.

Osteoclasts are effector cells in bone breakdown, and the active bone resorption is confined to the ruffled border zone of these cells. An acid milieu is maintained in this zone which is probably a prerequisite for bone resorption. Tartrate-resistant acid phosphatase (TRAP) activity has been recognized as a characteristic property of osteoclasts and in several studies proposed as a cytochemical marker of osteoclasts. We have previously isolated and characterized a tartrate-resistant and iron-activated acid ATPase (TrATPase) from rat bone, the enzyme being a member of the TRAP family. In the present study the ultrastructural localization of this enzyme was delineated by employing immunogold technique on low temperature-embedded maxillar rat bone. Intensive immunolabeling was seen on the bone surfaces facing the ruffled border zone while lower amounts of marker were seen in adjacent bone areas, that is, on the bone surfaces facing the clear zone and deeper-into the bone. Within the osteoclasts gold markers were observed mainly in vesicular structures interpreted as lysosomes. Immunolabeling was also observed in the recently described endocytic cells located near osteoblasts and osteoclasts. Also in these cells, the marker was confined to lysosomelike structures. The amount of label in bone facing osteoblasts was low, as was the amount within osteoblasts. Our observation of extracellular localization, in particular accumulation of TrATPase in bone matrix facing the ruffled border area of the osteoclasts, favors the view that the enzyme is exported to areas of active bone resorption, thereby indicating a potential role for the enzyme in this process.

Distribution of integrin subunits on rat metaphyseal osteoclasts and osteoblasts.

A key event in bone resorption is the attachment of osteoclasts to the bone matrix. The process apparently involves integrin plasma membrane receptors for extracellular matrix proteins. In the present study the distribution of some integrin subunits among different osteoclastic and osteoblastic domains was determined using immunocytochemistry on rat metaphyseal bone. Ultrathin cryosections were incubated with polyclonal antibodies against the alpha v beta 3 integrin and against the individual integrin subunits, alpha v, beta 1, beta 3, and beta 5. Bound antibodies were detected with 10-nm colloidal gold coated with protein A. The results show that alpha v- and beta 3-subunits are enriched at the osteoclast plasma membrane facing the bone matrix, i.e., the clear zone. Furthermore, beta 1- and beta 5-subunits were somewhat enriched at the osteoblast plasma membrane facing the bone surface. The present observations corroborate and extend our previous data indicating that the alpha v beta 3 integrin mediates tight attachment of the osteoclast to the bone matrix. Moreover, a role for integrins in the attachment and interactions of the osteoblast is suggested.

Structural and functional integrity of chondrocytes immediately following isolation.

Chondrocytes were isolated from costal cartilage in young rats after digestion with collagenase and hyaluronidase. The immediate survival of the cells was investigated with the use of different criteria for viability, namely structural integrity and metabolic activity. Structural integrity was studied by transmission and scanning electron microscopy, trypan blue exclusion and NADH oxidation. Metabolic activity was measured both as O2 consumption and as proline and sulphate incorporation, as indicators of collagen and proteoglycan synthesis. The cellular content of glutathione was also measured. The chondrocytes isolated were found to be structurally intact and metabolically active. Early after isolation the chondrocytes varied considerably in size similarly to the native tissue. A selective loss of the larger sized cells was observed during further incubation for 24 h.

Distribution and synthesis of bone sialoprotein in metaphyseal bone of young rats show a distinctly different pattern from that of osteopontin.

Bone sialoprotein (BSP) and osteopontin (OPN) are two phosphorylated and highly glycosylated cell-binding proteins in bone. Both proteins bind to hydroxylapatite. The cell binding is mediated via an Arg-Gly-Asp (RGD) sequence and previous work indicates that both proteins can bind to the vitronectin receptor (alpha v beta 3). The present work shows that a prevailing localization of BSP in metaphyseal bone of the young rat is at the interface between calcified cartilage and bone. Thus BSP shows a conspicuous enrichment in the osteoid laid down by the invading osteoblasts immediately next to the calcified cartilage. Furthermore, the most prominent amount of BSP mRNA was detected in cells at the epiphyseal/metaphyseal border. As opposed to OPN, no prominent accumulation of BSP immunoreactivity was observed at bone surfaces that face cells. Also the synthesis OPN was most pronounced at sites very different from those of BSP. Thus, the most prominent amount of OPN mRNA was observed in cells close to the metaphyseal/diaphyseal border, where osteoclastic bone resorption is particularly active. Indeed, message was often found in cells surrounding osteoclasts without any detectable message. The distinctly different patterns of synthesis and expression of the two proteins indicate different roles in bone turnover at this stage of development. Thus, it appears that BSP has a specific role during the initial phases of bone formation at the cartilage/bone interface. On the other hand, the pattern of OPN synthesis and expression support and extend our previous data showing OPN particularly enriched at attachment sites of osteoclasts resorbing bone.

Fibromodulin distribution and association with collagen.

Fibromodulin, an acidic 59-kDa proteoglycan, binds to collagen and inhibits collagen fibril formation in vitro. To determine whether fibromodulin is also bound to collagen in vivo, we used immunocytochemical methods to study the spatial relation of the proteoglycan to collagen fibrils in cartilage and tendon. We also studied the quantitative distribution of fibromodulin among compartments in articular cartilage at the ultrastructural level. Fibromodulin was identified with polyclonal antibodies raised in rabbits, and immunoreactivity was detected with protein-A gold. As the major proportion of fibromodulin immunoreactivity was localized along the periphery of the collagen fibrils, the relationship to the banding pattern of the collagen fibrils was mapped. The proteoglycan showed a non-random distribution, with preference to the gap region, axially within the D-period. Reactivity differed among the tissue compartments, with the lowest degree of labelling pericellularly, increasing with distance from the cell, the highest levels being observed in the interterritorial matrix. Labelling density was highest at the articular surface, gradually decreasing towards the cartilage-bone junction. The correlation between collagen fibril diameter and fibromodulin concentration also varied among compartments. Thus, the ratio of fibromodulin to collagen surface density was highest at the surface of the joint cartilage, exhibiting a gradient with higher values in the territorial matrix, decreasing towards the cell in all layers. These findings indicate that fibromodulin represents a factor used by chondrocytes to regulate assembly and function of collagen fibrils.

Vascularisation and osteochondrosis of the epiphyseal growth cartilage of the distal femur in pigs--development with age, growth rate, weight and joint shape.

Until recently, the cartilage canals of the epiphyseal growth cartilage have not been associated with any specific disease. However, data support the hypothesis that osteochondrosis could be related to inadequate blood supply from vessels in these channels. We have done a study to investigate the relationship between the regression of cartilage canals and the formation of osteochondrosis latens in the epiphyseal growth cartilage of the distal femur in pigs, and the relationship between these events and age, growth rate, weight and femoral shape of the individual animals. This involved, in part, a comprehensive study of the distribution and pattern of regression of the cartilage canals. We found that the regression is a highly predictable process that follows an age-dependent pattern. However, we failed to prove any association between overall vascular regression and osteochondrosis, between vascular regression and weight, growth rate or femoral shape or between osteochondrosis and weight, growth rate or femoral shape. This may indicate that osteochondrosis latens is not caused by a general failure of vascular supply or general factors such as growth rate, but rather a consequence of local conditions affecting a limited number of vessels. A factor fitting this description is local compression.

Comparison of fine-needle aspiration biopsy and histology in human liver transplants.

The findings in 50 fine-needle aspiration biopsies obtained from 27 liver transplants in 24 patients were compared with concomitant histologic findings. When histology showed rejection, rejection was diagnosed in 20 out of 29 FNAB specimens; when rejection was absent histologically, FNAB was negative in 20 out of 21 specimens (sensitivity 69%, specificity 95%). There was a statistically significant correlation between the degree of immunoactivation as reflected by total corrected increment (TCI) in FNAB specimens and the portal triad cell density during rejection, while no correlation existed between the 2 parameters in situations other than rejection. When cholestasis was demonstrated histologically, FNAB showed cholestasis in 30 out of 31 specimens. Furthermore, FNAB showed fatty change in the hepatocytes in 29 out of 43 specimens from livers with histologically evident steatosis. Histology showed necrotic areas in 40 specimens; however, only in 12 concomitant FNAB specimens were necrotic clumps observed. In conclusion, FNAB is a good method for diagnosing acute liver transplant rejection as well as for evaluating intracellular cholestasis or fatty change. Furthermore, TCI seems to reflect the severity of cellular infiltration in the portal triads during rejection.

The correlation between cytological patterns in bile and histological findings in liver transplantation.

The cytological patterns in bile were compared with the histological findings in concomitant specimens from liver transplants. In cases where cytology showed a moderate (n = 10) or high cell density (n = 8), histology demonstrated rejection in 14 of 18 specimens and cholangitis in 4. When the cell density was low (n = 22), histology was nearly normal in 3 specimens and showed cholangitis in 9, while rejection was observed in 10 specimens. Cell density in bile did not correlate quantitatively with the severity of cellular infiltration in the portal triads or with the percentage of bile ducts attacked by inflammatory cells. The results of the present study support our hypothesis that an increased concentration of cells in bile is indicative of liver transplant rejection (sensitivity 58%), while normal cytology does not rule out the possibility of rejection (specificity 75%).