Immune complex formation after xenotransplantation : evidence of type III as well as type II immune reactions provide clues to pathophysiology.

Rejection of renal and cardiac xenografts is initiated when natural antibodies of the recipient bind to donor endothelium, activating complement on the surface of endothelial cells. Pulmonary xenotransplants, however, reveal less evidence of antibody binding and complement activation and, in contrast to other xenografts, fare worse when the complement of the graft recipient is depleted. Accordingly, we asked whether distinct immunochemical reactions might occur after xenotransplantation of the lung and what implications such reactions might have for pulmonary pathophysiology. Analysis of serum from baboons after transplantation with porcine lungs revealed complexes containing baboon IgM and porcine von Willebrand factor. The baboon IgM in these complexes was specific for Galalpha1-3Gal. Immune complexes were also seen, albeit to a lesser extent, in the serum of kidney and heart xenotransplant recipients. Deposits of porcine von Willebrand factor and baboon C3 were detected in livers and spleens of transplanted baboons. These results indicate pulmonary xenotransplantation eventuates in formation of immune complexes and in the deposition of those complexes at distant sites. Immune complex formation could explain the peculiar fate of xenoreactive antibodies after pulmonary xenotransplantation and might contribute to the pathophysiology of the lung and systemic changes not previously considered a complication of xenotransplantation.

Heparanase expression in invasive trophoblasts and acute vascular damage.

Heparan sulfate proteoglycans play a pivotal role in tissue function, development, inflammation, and immunity. We have identified a novel cDNA encoding human heparanase, an enzyme thought to cleave heparan sulfate in physiology and disease, and have located the HEP gene on human chromosome 4q21. Monoclonal antibodies against human heparanase located the enzyme along invasive extravillous trophoblasts of human placenta and along endothelial cells in organ xenografts targeted by hyperacute rejection, both sites of heparan sulfate digestion. Heparanase deposition was evident in arterial walls in normal tissues; however, vascular heparan sulfate cleavage was coincident with heparanase enzyme during inflammatory episodes. These findings suggest that heparanase elaboration and control of catalytic activity may contribute to the development and pathogenesis of vascular disease and suggest that heparanase intervention might be a useful therapeutic target.

Expression of tissue factor mRNA in cardiac xenografts: clues to the pathogenesis of acute vascular rejection.

BACKGROUND: Acute vascular rejection destroys vascularized xenografts over a period of hours to days and is now considered the major hurdle to the clinical application of xenotransplantation. The hallmark of acute vascular rejection is diffuse intravascular coagulation; however, the pathogenesis of coagulation is a matter of controversy. One line of evidence points to activated endothelial cells and another to activated inflammatory cells as a source of tissue factor and thus as a primary cause of this lesion. The distinction between the two mechanisms inducing coagulation in the xenograft provides an opportunity for specific intervention. METHODS: To explore these mechanisms, we studied the expression of tissue factor mRNA by in situ reverse transcriptase-polymerase chain reaction in relation to the histopathologic manifestations of acute vascular rejection in guinea pig hearts transplanted into rats treated by cobra venom factor to avoid the hyperacute rejection. RESULTS: Three hours after transplantation and before the deposition of fibrin, tissue factor mRNA was expressed in the endothelial cells lining small and medium blood vessels and in smooth muscle cells of guinea pig cardiac xenografts. Sixteen hours after transplantation, while rat tissue factor mRNA was expressed only in occasional infiltrating cells, cardiac xenografts showed prominent deposits of fibrin in small vessels. Maximum expression of tissue factor on rat infiltrating cells was observed 48 hr after transplantation. CONCLUSIONS: These results suggest that in acute vascular rejection, coagulation is initiated on the donor vascular system, while the procoagulant characteristics of infiltrating cells may reflect a response to tissue injury rather than a cause.

In situ hybridization detection of low copy nucleic acid sequences using catalyzed reporter deposition and its usefulness in clinical human papillomavirus typing.

In situ hybridization (ISH) detection of low copy DNA and RNA sequences using nonisotopic probes has been difficult in the past because of a lack of sensitivity. Several techniques, such as ISH with radioisotopic-labeled probes, in situ polymerase chain reaction, in situ reverse transcription polymerase chain reaction, self-sustained sequence replication, and chemiluminescence, have allowed increased sensitivity but have required specialized and often expensive equipment, lengthy protocols, and in the case of radioactive probes, there has been an associated increased health risk. Catalyzed reporter deposition (CARD) combined with ISH (CARD-ISH) increases the signal-generating potential of labeled hybridized probes and allows the detection of low copy sequences of nucleic acids in formalin-fixed, paraffin-embedded tissue sections. To determine the sensitivity of CARD-ISH to detect nucleic acids in routinely processed specimens, we analyzed the detection of HPV 16 and 18 infection in formalin-fixed, paraffin-embedded sections of cultured cell lines, including CaSki cells with 400-600 copies of HPV 16, HeLa 229 cells with 10-50 copies of HPV 18, and SiHa cells with 1-2 copies of HPV 16 using a conventional ISH method and by CARD-ISH. In addition, 20 cases of clinical specimens previously analyzed for HPV 6, 11, 16, 18, 31, 33, and 51 with the Enzo PathoGene kit (Enzo Diagnostics, Inc., Farmingdale, NY, U.S.A.) were reexamined with the CARD-ISH method. The CARD-ISH system detected one to two copies of HPV 16 in the SiHa cells whereas the conventional ISH method did not. Both methods detected HPV 16 and 18 in CaSki and HeLa 229 cells, respectively. Three clinical cases that were previously negative and two weakly positive cases of HPV infection were all strongly positive with the CARD-ISH system, a 25% increase in the detection of positive cases by CARD-ISH. We also showed for the first time that a cocktail of six biotinylated oligonucleotide probes was capable of detecting one to two copies of HPV 16 in SiHa cells. These results show that the CARD-ISH method increases the sensitivity of nonisotopic ISH to the level of detecting one to two copies of HPV DNA in formalin-fixed, paraffin-embedded tissue sections using biotinylated cDNA or oligonucleotide probes.

Placental site trophoblastic tumor: human placental lactogen and pregnancy-associated major basic protein as immunohistologic markers.

Placental site trophoblastic tumor (PSTT) consists of a neoplastic proliferation of intermediate or extravillous trophoblast (also known as X cells). Pregnancy-associated major basic protein (pMBP) is a marker for placental intermediate trophoblast. We compared the distribution of pMBP and human placental lactogen (hPL) in 24 PSTT and 3 exaggerated placental site (EPS) specimens using two distinct immunohistologic methods. Statistical analyses were used to compare staining intensities in metastatic and nonmetastatic lesions. By immunofluorescence, 77% of the PSTT specimens and 100% of the EPS specimens stained with antibodies to pMBP, and the pMBP was localized in intermediate trophoblast and surrounding extracellular areas. By immunohistochemistry, 78% of the PSTT specimens and 100% of the EPS specimens stained for pMBP with a pattern comparable with that of immunofluorescence. Likewise, by immunohistochemistry, hPL stained 96% of the PSTT specimens and 100% of the EPS specimens. Immunohistochemical staining intensities for pMBP and hPL correlated (r2 = +.24; P = .013), but hPL staining was mainly confined to intermediate trophoblast and was more intense. Anti-pMBP tended to stain metastatic PSTT weakly. Thus, pMBP is a useful marker for intermediate trophoblast tumors and could help distinguish these from other forms of trophoblastic disease.

An immunohistochemical survey for neuroendocrine cells in regional pancreatic lymph nodes: a plausible explanation for primary nodal gastrinomas? Mayo Clinic Pancreatic Surgery Group.

BACKGROUND: The existence of primary gastrinomas in lymph nodes continues to be controversial, because distinction from metastases from occult or regressed primary tumors may be difficult to exclude with certainty. If primary nodal gastrinomas do indeed occur, precursor neuroendocrine cells should be identifiable within lymph nodes. In a effort to detect these cells we undertook an immunohistochemical survey of regional pancreatic lymph nodes by using antibody against chromogranin, a highly specific marker for mature neuroendocrine cells. METHODS: We retrospectively identified consecutive cases from five surgeons in which Whipple resections had been performed for nonendocrine pathologic conditions. All formalin-fixed, paraffin-embedded lymph nodes were identified from these 106 cases, excluding lymph nodes with metastases. Immunoperoxidase staining with monoclonal antibody against chromogranin A was performed on single tissue sections. All slides were reviewed by a single pathologist; chromogranin-positive cells were identified and, when possible, further evaluated immunohistochemically with additional neuroendocrine markers, including sequential synaptophysin staining. RESULTS: A total of 1026 lymph nodes were available for review. Although well-formed intranodal chromogranin-positive nests were not identified, six nodes from three patients contained 13 strongly chromogranin-positive cells representing putative neuroendocrine cells. Mast cells showed weak nonspecific staining with chromogranin but were morphologically distinct. Perinodal adipose tissue contained a single paraganglion and several small neuroendocrine clusters. CONCLUSIONS: Putative neuroendocrine cells are rarely (less than 1%) found in regional pancreatic lymph nodes. Although these may be embryonic rests and might represent precursors of primary nodal gastrinomas, specific hormones have yet to be identified in these cells. Although these data provide support for the concept of primary nodal gastrinomas, this remains a diagnosis of exclusion.