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Transplantation of donation after cardiac death (DCD) livers has higher rates of organ failure and complications, specifically ischemic biliary injuries. Reported large animal DCD models all employ active means to halt circulation, contrary to human DCD protocol. We report a DCD porcine model in which the animal passively progresses to cardiac death, thereby more closely mimicking human DCD scenario. Sixteen Yorkshire pigs (10 females, 6 males, 30-45 kg) had a mean time of 26:19 min ± 14:14 from withdrawal of ventilatory support (WVS) to circulatory arrest and 44:38 min ± 16:37 from WVS to electrical standstill. Cessation of hepatic flow (HF) occurred well before electrical standstill (22:15 min ± 10:09), previously not described in human or animal DCD. Histologically comparing livers from our DCD model demonstrated a dramatic increase in hepatocyte vacuolization, disorganization of endoplasmic reticulum, formation of mitochondrial inclusions and apoptosis compared with control specimens. Subtle changes were also evident in biliary epithelial cells (BEC). This results in severe cellular changes before reperfusion. Early histologic evidence suggests that there is severe hepatocyte and biliary cell disruption in our DCD model. Further research using this model may provide a deeper understanding of the pathophysiology of the DCD liver., (©2011 The Authors Journal compilation©2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Patients with Chagas' disease remain asymptomatic for many years, presumably by keeping the etiological agent Trypanosoma cruzi in check through protective immunity against. Recently, we found that T. cruzi uses TrkA, a receptor tyrosine kinase responsive to neurotrophin nerve growth factor in vertebrate nervous systems, to invade cells. We also found that TrkA, TrkB, and TrkC, but not T. cruzi, are targets of specific autoantibodies present in the sera of patients with chronic Chagas' disease. Here we show that TrkA-, TrkB-, and TrkC-specific autoantibodies isolated from the sera of four individuals with chronic indeterminate (asymptomatic) Chagas' disease potently blocked invasion of Trk-bearing neuronal PC12 cells, neuroglial astrocytes, enteroglial cells, and Schwann cells and Trk-expressing non-neural smooth muscle and dendritic cells. However, these autoantibodies did not inhibit T. cruzi invasion of mutant PC12 cells lacking TrkA or of normal cells lacking Trk receptors, suggesting that autoantibodies interfered with parasite/Trk cross talk to access the intracellular milieu. Passive immunization of susceptible and resistant mouse strains with very small doses of these autoantibodies reduced parasitemia and transferred resistance to an otherwise lethal trypanosome infection. Hence, this exquisitely sensitive and unique regulatory immunity against the host (instead of parasite) could benefit infected individuals by blocking cellular invasion of the obligatory intracellular pathogen, resulting in attenuation of tissue infection and clinical manifestations. Such action is contrary to the horror autotoxicus frequently associated with microbe-related autoimmune responses.

II3NeuAc-GgOse4Cer (GM1) gangliosidosis is an incurable lysosomal storage disease caused by a deficiency in acid beta-galactosidase (beta-gal), resulting in the accumulation of ganglioside GM1 and its asialo derivative GgOse4Cer (GA1) in the central nervous system, primarily in the brain. In this study, we investigated the effects of N-butyldeoxygalacto-nojirimycin (N B-DGJ), an imino sugar that inhibits ganglioside biosynthesis, in normal C57BL/6J mice and in beta-gal knockout (beta-gal-/-) mice from postnatal day 9 (p-9) to p-15. This is a period of active cerebellar development and central nervous system (CNS) myelinogenesis in the mouse and would be comparable to late-stage embryonic and early neonatal development in humans. N B-DGJ significantly reduced total ganglioside and GM1 content in cerebrum-brainstem (C-BS) and in cerebellum of normal and beta-gal-/- mice. N B-DGJ had no adverse effects on body weight or C-BS/cerebellar weight, water content, or thickness of the external cerebellar granule cell layer. Sphingomyelin was increased in C-BS and cerebellum, but no changes were found for cerebroside (a myelin-enriched glycosphingolipid), neutral phospholipids, or GA1 in the treated mice. Our findings indicate that the effects of N B-DGJ in the postnatal CNS are largely specific to gangliosides and suggest that N B-DGJ may be an effective early intervention therapy for GM1 gangliosidosis and other ganglioside storage disorders.

Twelve adrenocortical tumours with a variable spindle-cell component in ferrets (six spayed females, three intact females, two castrated males, and one intact male) were examined by light microscopy. One tumour with a moderate spindle-cell component was examined ultrastructurally, and three tumours were studied immunohistochemically. Light microscopy revealed a spindle-cell component in the tumours that varied from a few such cells occupying the stroma between packets of adrenocortical cells to cells in such large numbers that they formed almost the entire substance of the tumour. By light microscopy these spindle cells resembled smooth muscle cells, and the ultrastructural findings, particularly the presence of thin contractile filaments, suggested that the spindle cells were of smooth muscle origin. Immunohistochemical staining revealed that the spindle cells were negative for cytokeratins and S-100 protein but positive for smooth muscle actin. Desmin was readily demonstrated in two tumours but not in the other examined. Vimentin was variable, generally producing a small to moderate amount of reaction product.

Pneumocystis carinii, an extracellular parasite thriving in the lungs of immunosuppressed mammals, is a major cause of death in AIDS patients in the USA. As a prelude to growth, the parasite adheres mostly to type I pneumocytes lining the alveolar spaces. The mechanism of adherence remains unknown, largely because of difficulties in isolating type I pneumocytes and maintaining them in vitro. As a first step to understand P. carinii adherence to its natural substrate, we developed an in situ method to directly study parasite binding to lung alveolar cells. We used formaldehyde-fixed paraffin-embedded sections of normal rat lung as substrate for adhesion. As in its binding to the lungs in vivo, P. carinii adhered preferentially to type I pneumocytes. Adherence was saturable, time and dose dependent, and selectively blocked by glycoconjugates, in particular bovine submaxillary mucin, fetuin, and asialofetuin, suggesting that it may be mediated by a lectin type of interaction. Further, IgG of rats with P. carinii pneumonia inhibited adherence, suggesting that it may react with parasite ligands involved in the recognition of type I cell receptors. Our results demonstrate the usefulness of the in situ model for studying the mechanisms of P. carinii adherence to alveolar cells. In addition, this method may be valuable for identifying neutralizing antibodies and drugs potentially useful for controlling the infection in vivo.

The clinical, morphologic, histochemical, and biochemical features of GM1-gangliosidosis in two canine models, English Springer Spaniel (ESS) and Portuguese Water Dog (PWD), have been compared. The disease onset, its clinical course, and survival period of the affected dogs were similar in both models. Skeletal dysplasia was noted radiographically at 2 months of age, whereas at 4 1/2 months of age there was progressive neurologic impairment. However, dwarfism and coarse facial features were seen only in ESS. Both models had similar deficiency in activity of lysosomal beta-galactosidase, but possessed a normal protein activator for GM1-beta-galactosidase. Both models stored GM1-ganglioside, asialo-GM1, and oligosaccharides in brain. Furthermore, only the PWD stored glycoproteins containing polylactosaminoglycans in visceral organs, and neither model stored them in the brain. Morphologically, both models demonstrated similar storage material in multiple tissues and cell types. The ultrastructure of the storage material was cell-type specific and identical in both models. However, some differences in the lectin staining pattern were noted. Our clinical, biochemical, and histochemical findings indicate that PWD and ESS may represent two different mutations of the beta-galactosidase gene. Moreover, the authors conclude that it is difficult, and inappropriate, to apply the human classification of GM1-gangliosidosis (i.e. infantile, juvenile, and adult forms) to these canine models.

In lysosomal storage diseases that involve a defect in the catabolism of glycoconjugates, lectin histochemistry adds a new dimension to the characterization of stored carbohydrates as it identifies sugar residues in situ in the affected cells and, thus, determines which cell types are affected by storage. It may be combined with chemical and biochemical analysis by h.p.l.c. The present review summarizes recent results for a variety of storage diseases and presents new data for GM1-gangliosidosis.

Lysosomal alpha-mannosidase activity, oligosaccharide profiles, light and electron microscopy and lectin histochemistry studies were performed on full-term placentae obtained from five litters of cats. They resulted from breeding related cats who are obligate heterozygotes for lysosomal alpha-mannosidase deficiency. alpha-Mannosidase activity in placentae from affected kittens was less than 10 per cent of control, while in placentae from presumptive heterozygotes the activity was less than 50 per cent of control. High-pressure liquid chromatographic analysis of oligosaccharides revealed massive accumulation of undegraded oligosaccharides in placentae of affected kittens. A small elevation was found in placentae from presumptive heterozygous kittens, and none was detected in placentae of normal kittens. Light and electron microscopic examinations revealed vacuolization of fetal endothelial and mesenchymal cells only in placentae of affected kittens. Succinylated wheat germ agglutinin and concanavalin A stained the fetal fibroblasts only in placentae of affected kittens.

Four cases of spontaneous adenocarcinoma of the canine exocrine pancreas were studied by thin section and freeze-fracture electron microscopy. The neoplastic cells in all 4 cases were of acinar origin and showed many alterations of cytoarchitecture compared with normal acinar cells. In the neoplastic cells, zymogen granules varied in number and appearance and contained an abnormal secretory product characterized by 24-nm-thick microtubules. The nuclear volume and surface area and the nuclear cytoplasmic ratio were increased; although the nuclear pore density per nuclear surface area was significantly decreased, there was no change in the nuclear pore density per nuclear volume. There was a decrease in number and size of gap junctions; focal proliferation, fragmentation, and discontinuation of the tight junctions were also noted. The basal lamina (BL) of the neoplastic cells was discontinuous. The tumor microvasculature often appeared as sinusoids and had sparse discontinuous BL. Finally, the endothelium in both tumor and normal tissue contained "tubulo-reticular inclusions" (TRI) which simulated distemper virus and were located in the rough endoplasmic reticulum (RER) and the perinuclear cisternae.

The glycoconjugates of regenerative acinar cells, acinic cell carcinomas, islet cell tumors, and normal canine and feline pancreas were studied. The authors used biotinylated lectins as probes and avidin-biotin-peroxidase complex as visualant to identify and to compare the distribution of carbohydrate residues on paraffin sections from 74 cases. The findings demonstrate a difference in the staining pattern between normal acinar, islet, and ductal cells in each species and small differences in the staining pattern between the species. It is shown that in nodules of regenerative acinar cells and acinic cell carcinomas there is an increased staining intensity with Concanavalia ensiformis agglutinin, Ricinus communis agglutinin-I, and wheat germ agglutinin. The pattern of lectin staining in regenerative cells and malignant acinar cells reflects the degree of cellular differentiation. Intensive apical staining characterizes a higher degree of differentiation, while dispersed staining is a major feature of poor differentiation. These findings suggest that malignant transformation of pancreatic acinar cells is associated with enhanced expression of glycoconjugates, which resembles that seen in a normal immature acinar cells.

In the preceding paper the authors demonstrated that the lectin staining patterns of normal colonic epithelium obtained from colitis and carcinoma-prone cotton top tamarins (CTTs), Saguinus oedipus, a New World primate, differs from colitis- and carcinoma-resistant primate species. In this study they determined the usefulness of cytochemical features in inflamed epithelium as indicators for malignant change. They compared the lectin staining pattern in inflamed mucosa and adjacent mucosa with colonic carcinoma from 8 CTTs with that of 9 clinically healthy CTTs with no histologic evidence of colitis. Deparaffinized sections were labeled with ten biotinylated lectins and stained by the avidin-biotin peroxidase complex method. Numerous significant differences were demonstrated in the lectin staining pattern between normal epithelium and colonic carcinoma; fewer between normal and chronic inflamed epithelium. However, between chronic inflamed epithelium and colonic carcinoma significant staining differences were observed with only two lectins, peanut agglutinin (PNA) and Ulex europaeus agglutinin-I (UEA-I). These findings suggest that there is a progression in alteration of lectin staining pattern from normal epithelium, via chronic colitis, to colonic carcinoma. Furthermore, the differences between chronic colitis and colonic carcinoma are expressed only with those lectins that are associated with malignant transformation of human colonic epithelium.

Differences in colonic secretory glycoconjugates (ie, mucin) between normal and ulcerative colitis-prone patients have been noted. Similar differences may occur in a corresponding primate model, the cotton-top tamarin (CTT), Saguinus oedipus, a New World monkey which suffers from spontaneous chronic colitis and colon cancer. Lectin reagents were used to characterize and compare colonic cell surface, cytoplasmic, and secretory glycoconjugates of 9 clinically healthy cotton-top tamarins, 7 colitis-susceptible, cancer-resistant tamarins (Callithrix jacchus, Saguinus fuscicollis), and 8 colitis and cancer-resistant primates (Aotus trivirgatus, Saimiri sciureus, Macaca fascicularis, and Macaca mulatta). Paraffin-embedded colonic sections were labeled with ten different biotinylated lectins and visualized by the avidin-biotin peroxidase (ABC) method. Significant differences were demonstrated in the pattern of lectin staining between the colitis-resistant and colitis-prone groups of primates. The differences were noted with Griffonia simplicifolia-I (GS-I), Dolichos biflorus agglutinin (DBA), peanut agglutinin (PNA) before and after neuraminidase, Ricinus communis agglutinin-I (RCA-I), soybean agglutinin (SBA), Ulex europaeus agglutinin-I (UEA-I), wheat germ agglutinin (WGA), and succinylated WGA (S-WGA). Significant differences between the CTT and phylogenetically related colitis-prone but cancer-resistant tamarins were demonstrated with SBA, UEA-I, and PNA after desialylation with neuraminidase. These results suggest that differences in colonic cellular glycoconjugates between colitis- and cancer-susceptible species versus colitis-susceptible, cancer-resistant species may be associated with risk of cancer.

Oligosaccharides were extracted from tissues and body fluids of five kittens with alpha-mannosidosis, three being from the same litter. The kittens were all of different ages at death and were compared to normal and heterozygote cats. The oligosaccharides were analyzed by high-pressure liquid chromatography after perbenzoylation and were identified by comparison with compounds of known structure. This provided a detailed picture of the distribution of oligosaccharides in each tissue, and a method for quantitation of the total oligosaccharides. With the exception of the youngest animal (death at day 2), the oligosaccharide elution profiles were broadly similar for all tissues and fluids, and were typical of feline alpha-mannosidosis. In contrast, concentrations of total oligosaccharides diverged widely from one source to another, from a high of 17.3 mumol/g to a low of 0.04 mumol/g. The results are interpreted in the context of glycoprotein catabolism.