Peter E. Fisher

INDIRECT RECOGNITION OF DONOR HLA-DR PEPTIDES IN ORGAN ALLOGRAFT REJECTION

To determine whether indirect allorecognition is involved in heart allograft rejection T cells obtained from peripheral blood and graft biopsy tissues were expanded in the presence of IL-2 and tested in limiting dilution analysis (LDA) for reactivity to synthetic peptides corresponding to the hypervariable regions of the mismatched HLA-DR antigen(s) of the donor. Serial studies of 32 patients showed that T cell reactivity to donor allopeptides was strongly associated with episodes of acute rejection. The frequency of allopeptide reactive T cells was 10-50-fold higher in the graft than in the periphery indicating that T cells activated via the indirect allorecognition pathway participate actively in acute allograft rejection. In recipients carrying a graft differing by two HLA-DR alleles the response appeared to target only one of the mismatched antigens of the donor. Indirect allorecognition was restricted by a single HLA-DR antigen of the host and directed against one immunodominant peptide of donor HLA-DR protein. However, intermolecular spreading was demonstrated in patients with multiple rejection episodes by showing that they develop allopeptide reactivity against the second HLA-DR antigen. These data imply that early treatment to suppress T cell responses through the indirect pathway of allorecognition, such as tolerance induction to the dominant donor determinant, may be required to prevent amplification and perpetuation of the rejection process.

Defective insulin secretion in pancreatic β cells lacking type 1 IGF receptor

Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase of beta cell mass and impaired glucose-dependent insulin release. beta cell proliferation and secretion are thought to be regulated by signaling through receptor tyrosine kinases. In this regard, we sought to examine the potential proliferative and/or antiapoptotic role of IGFs in beta cells by tissue-specific conditional mutagenesis ablating type 1 IGF receptor (IGF1R) signaling. Unexpectedly, lack of functional IGF1R did not affect beta cell mass, but resulted in age-dependent impairment of glucose tolerance, associated with a decrease of glucose- and arginine-dependent insulin release. These observations reveal a requirement of IGF1R-mediated signaling for insulin secretion.

Does blood flow through holmium: YAG transmyocardial laser channels?

BACKGROUND Early reports indicate that transmyocardial laser revascularization improves symptoms in patients with refractory angina. However, there is little experimental evidence of whether blood flow through channels is the mechanism of action. METHODS Endocardial channels were made in the distribution of the left anterior descending coronary artery in canine hearts (n = 5) using a holmium:yttrium-aluminum garnet laser. Hearts were excised acutely while perfused in a retrograde fashion from a second dog so that the aortic valve always remained closed. The proximal left anterior descending coronary artery was ligated. To measure direct transmyocardial blood flow, colored microspheres were injected into the left ventricular chamber. RESULTS The number of spheres per gram of tissue in the channel region was significantly higher than in the control region (low load, 302.5 +/- 169.0 versus 41.8 +/- 59.4; high load, 208.4 +/- 138.3 versus 5.8 +/- 11.7; both, p < 0.05). However, the estimated regional blood flow through the channels was extremely low (<0.01 mL/g/min. In the chronic setting (n = 4) (2-week survival), no flow as detected through the channels, and the endocardial entry points were closed. CONCLUSIONS Transmyocardial blood flow does not appear to occur through channels made with the holmium:yttrium-aluminum garnet laser. It remains to be determined whether this is the case with other types of lasers.

Essential Role for ADAM19 in Cardiovascular Morphogenesis

ABSTRACT Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19−/− animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects.

Modulation of Notch Signaling Elicits Signature Tumors and Inhibits Hras1-Induced Oncogenesis in the Mouse Mammary Epithelium

Deregulation of Notch signaling, which normally affects a broad spectrum of cell fates, has been implicated in various neoplastic conditions. Here we describe a transgenic mouse model, which demonstrates that expression of a constitutively active form of the Notch1 receptor in the mammary epithelium induces the rapid development of pregnancy/lactation-dependent neoplasms that consistently exhibit a characteristic histopathological pattern. These signature tumors retain the ability to respond to apoptotic stimuli and regress on initiation of mammary gland involution, but eventually appear to progress in subsequent pregnancies to nonregressing malignant adenocarcinomas. Additionally, we present evidence indicating that cyclin D1 is an in vivo target of Notch signals in the mammary glands and demonstrate that we can effectively inhibit Hras1-driven, cyclin D1-dependent mammary oncogenesis by transgenic expression of the Notch antagonist Deltex.

Physiology, histology, and 2-week morphology of acute transmyocardial channels made with a CO2 laser

BACKGROUND Transmyocardial revascularization with a CO2 laser appears to improve symptoms in patients with refractory angina. However, it remains controversial as to whether blood flow through the channels is the mechanism of benefit, especially in the acute setting. METHODS AND RESULTS Three protocols were used to test whether blood flows through transmyocardial CO2 laser revascularization channels. First, channels were made in excised, cross-perfused dog hearts (n = 5) using a CO2 laser (The Heart Laser; PLC Systems Inc, Milford, MA; 40 J/pulse) followed by ligation of the proximal left anterior descending coronary artery. Colored microspheres injected into the left ventricular chamber failed to detect any significant transmyocardial blood flow. In the second protocol (n = 4), laser channels were created in the left anterior descending artery territory, the left anterior descending artery was ligated, and the hearts were excised after 24 hours. Triphenyltetrazolium chloride staining revealed that no viable myocardium was detected around the laser channels in the ischemic myocardium. Finally, channels examined 2 weeks after creation in normal (n = 6) or ischemic (n = 4) myocardium did not maintain their original caliber but were invaded by granulation tissue, which included a large amount of smaller vascular spaces and vessels of various sizes. CONCLUSIONS Transmyocardial laser revascularization channels made with this CO2 laser did not provide acute myocardial perfusion or preserve myocardial viability in the face of acute ischemia. Channel morphology changes dramatically within the first 2 weeks. To the degree that these findings pertain to human myocardium, the results suggest that transmyocardial blood flow may not be the mechanism of benefit of this procedure, particularly in the acute setting.

Development of mammary adenocarcinomas by tissue-specific knockout of Brca2 in mice.

Cre-mediated deletion of exons 3 and 4 of the mouse Brca2 gene occurring specifically in mammary epithelial cells of conditional female mutants carrying a combination of loxP-modified and null Brca2 alleles resulted in a high incidence (77%) of breast tumors that were often palpable and developed in one or more glands after long latency (time for median tumor-free survival of ∼1.4 years; total of 40 tumors in 20 animals). These invasive adenocarcinomas were histologically quite uniform, exhibiting predominantly a solid, nodular tumor pattern with very few variants, in striking contrast to the morphological heterogeneity of analogous Brca1-associated tumors. The karyotypes of tumor cells lacking Brca2 had various chromosomal aberrations and ranged from diploid to hypertetraploid, but this wide variability was incongruous with the histological appearance of carcinomas that was comparable between specimens. The implications of these observations in the context of models positing that Brca2 is involved in the maintenance of genomic stability are discussed.

The regulatory protein PhoP controls susceptibility to the host inflammatory response in Shigella flexneri

The PhoP/PhoQ two‐component regulatory system controls transcription of several key virulence genes essential for Salmonella survival in the host cell phagosome. Here, we determine that the PhoP/PhoQ system also regulates virulence in the aetiological agent of bacillary dysentery, Shigella flexneri, even though this pathogen escapes from the phagosome into the cytoplasm of the host cell. A phoP mutant of Shigella established infections and induced an acute inflammatory response in two different animal models. However, infections with phoP mutant bacteria were resolved more rapidly than infections with wild‐type Shigella. Moreover, the Shigella phoP mutant was more sensitive than the wild‐type strain to killing by polymorphonuclear leucocytes (PMNs), cationic polypeptides extracted from PMNs and other animal‐derived antimicrobial peptides. The phoP mutant, however, invaded epithelial cells, spread intercellularly, induced apoptosis in macrophages and tolerated extreme acid pH as efficiently as the wild‐type strain. PhoP appears to regulate Shigella susceptibility to PMNs and antimicrobial molecules that are important for the late stages of infection with this enteric bacterium.

New strategies for early diagnosis of heart allograft rejection

BACKGROUND Allograft rejection is mediated by T cells that recognize allogeneic major histocompatibility complex (MHC) molecules via the direct and indirect pathway. The direct pathway involves T cells that react against MHC/peptide complexes expressed on the surface of donor antigen-presenting cells (APCs). In contrast, T cells involved in the indirect pathway recognize peptides derived from processing and presentation of allogeneic MHC molecules by self (recipient) APCs. To explore the relative contribution of these two pathways to rejection, we have evaluated the response of peripheral blood T cells from 50 heart transplant recipients against donor APCs (direct recognition) and against self APCs pulsed with synthetic peptides corresponding to the hypervariable region of the mismatched HLA-DR antigens of the donor (indirect recognition). METHODS T cell reactivity against donor APCs was quantitated by measuring the expression of CD69 on allostimulated CD3+ LDA1+ cells. Reactivity to synthetic allopeptides was determined in limited dilution assays. RESULTS Serial studies of the kinetics of direct and indirect recognition showed that both pathways contribute to early acute rejection episodes. Primary rejection was accompanied invariably by indirect recognition of a dominant allopeptide. Intermolecular spreading of T cell epitopes was observed during recurrent rejections. Enhanced recognition of donor alloantigens via the direct pathway was found predominantly during early rejection episodes. A single form of allorecognition was shown to occur in some rejection episodes. CONCLUSIONS Monitoring of the direct and indirect pathway of allorecognition provides a reliable method for prediction and differential diagnosis of acute rejection of heart allografts.

Assessment of myocardial recovery in a patient with acute myocarditis supported with a left ventricular assist device: a case report

Acute myocarditis may present with profound hemodynamic compromise; however, spontaneous resolution of the inflammatory process may occur in up to half of such patients. In patients with fulminant myocarditis, mechanical circulatory support may serve as a bridge to myocardial recovery. In this report we describe a 35-year-old man with acute myocarditis who required left ventricular assist device support as a bridge to recovery, and suggest a method for determining the suitability and timing of device explantation. A combination of echocardiography, right heart catheterization, exercise testing and serial endomyocardial biopsies was used to determine the resolution of myocarditis, recovery of myocardial function and timing for device explantation. Successful device explantation was performed after 37 days of device support. Further study is required to assess the role of ventricular assist devices in combination with immunosuppressive therapy in the management of fulminant myocarditis.