Phylogenetic lineages in Pseudocercospora.

Pseudocercospora is a large cosmopolitan genus of plant pathogenic fungi that are commonly associated with leaf and fruit spots as well as blights on a wide range of plant hosts. They occur in arid as well as wet environments and in a wide range of climates including cool temperate, sub-tropical and tropical regions. Pseudocercospora is now treated as a genus in its own right, although formerly recognised as either an anamorphic state of Mycosphaerella or having mycosphaerella-like teleomorphs. The aim of this study was to sequence the partial 28S nuclear ribosomal RNA gene of a selected set of isolates to resolve phylogenetic generic limits within the Pseudocercospora complex. From these data, 14 clades are recognised, six of which cluster in Mycosphaerellaceae. Pseudocercospora s. str. represents a distinct clade, sister to Passalora eucalypti, and a clade representing the genera Scolecostigmina, Trochophora and Pallidocercospora gen. nov., taxa formerly accommodated in the Mycosphaerella heimii complex and characterised by smooth, pale brown conidia, as well as the formation of red crystals in agar media. Other clades in Mycosphaerellaceae include Sonderhenia, Microcyclosporella, and Paracercospora. Pseudocercosporella resides in a large clade along with Phloeospora, Miuraea, Cercospora and Septoria. Additional clades represent Dissoconiaceae, Teratosphaeriaceae, Cladosporiaceae, and the genera Xenostigmina, Strelitziana, Cyphellophora and Thedgonia. The genus Phaeomycocentrospora is introduced to accommodate Mycocentrospora cantuariensis, primarily distinguished from Pseudocercospora based on its hyaline hyphae, broad conidiogenous loci and hila. Host specificity was considered for 146 species of Pseudocercospora occurring on 115 host genera from 33 countries. Partial nucleotide sequence data for three gene loci, ITS, EF-1α, and ACT suggest that the majority of these species are host specific. Species identified on the basis of host, symptomatology and general morphology, within the same geographic region, frequently differed phylogenetically, indicating that the application of European and American names to Asian taxa, and vice versa, was often not warranted. TAXONOMIC NOVELTIES: New genera - Pallidocercospora Crous, Phaeomycocentrospora Crous, H.D. Shin & U. Braun; New species - Cercospora eucommiae Crous, U. Braun & H.D. Shin, Microcyclospora quercina Crous & Verkley, Pseudocercospora ampelopsis Crous, U. Braun & H.D. Shin, Pseudocercospora cercidicola Crous, U. Braun & C. Nakash., Pseudocercospora crispans G.C. Hunter & Crous, Pseudocercospora crocea Crous, U. Braun, G.C. Hunter & H.D. Shin, Pseudocercospora haiweiensis Crous & X. Zhou, Pseudocercospora humulicola Crous, U. Braun & H.D. Shin, Pseudocercospora marginalis G.C. Hunter, Crous, U. Braun & H.D. Shin, Pseudocercospora ocimi-basilici Crous, M.E. Palm & U. Braun, Pseudocercospora plectranthi G.C. Hunter, Crous, U. Braun & H.D. Shin, Pseudocercospora proteae Crous, Pseudocercospora pseudostigmina-platani Crous, U. Braun & H.D. Shin, Pseudocercospora pyracanthigena Crous, U. Braun & H.D. Shin, Pseudocercospora ravenalicola G.C. Hunter & Crous, Pseudocercospora rhamnellae G.C. Hunter, H.D. Shin, U. Braun & Crous, Pseudocercospora rhododendri-indici Crous, U. Braun & H.D. Shin, Pseudocercospora tibouchinigena Crous & U. Braun, Pseudocercospora xanthocercidis Crous, U. Braun & A. Wood, Pseudocercosporella koreana Crous, U. Braun & H.D. Shin; New combinations - Pallidocercospora acaciigena (Crous & M.J. Wingf.) Crous & M.J. Wingf., Pallidocercospora crystallina (Crous & M.J. Wingf.) Crous & M.J. Wingf., Pallidocercospora heimii (Crous) Crous, Pallidocercospora heimioides (Crous & M.J. Wingf.) Crous & M.J. Wingf., Pallidocercospora holualoana (Crous, Joanne E. Taylor & M.E. Palm) Crous, Pallidocercospora konae (Crous, Joanne E. Taylor & M.E. Palm) Crous, Pallidoocercospora irregulariramosa (Crous & M.J. Wingf.) Crous & M.J. Wingf., Phaeomycocentrospora cantuariensis (E.S. Salmon & Wormald) Crous, H.D. Shin & U. Braun, Pseudocercospora hakeae (U. Braun & Crous) U. Braun & Crous, Pseudocercospora leucadendri (Cooke) U. Braun & Crous, Pseudocercospora snelliana (Reichert) U. Braun, H.D. Shin, C. Nakash. & Crous, Pseudocercosporella chaenomelis (Y. Suto) C. Nakash., Crous, U. Braun & H.D. Shin; Typifications: Epitypifications - Pseudocercospora angolensis (T. Carvalho & O. Mendes) Crous & U. Braun, Pseudocercospora araliae (Henn.) Deighton, Pseudocercospora cercidis-chinensis H.D. Shin & U. Braun, Pseudocercospora corylopsidis (Togashi & Katsuki) C. Nakash. & Tak. Kobay., Pseudocercospora dovyalidis (Chupp & Doidge) Deighton, Pseudocercospora fukuokaensis (Chupp) X.J. Liu & Y.L. Guo, Pseudocercospora humuli (Hori) Y.L. Guo & X.J. Liu, Pseudocercospora kiggelariae (Syd.) Crous & U. Braun, Pseudocercospora lyoniae (Katsuki & Tak. Kobay.) Deighton, Pseudocercospora lythri H.D. Shin & U. Braun, Pseudocercospora sambucigena U. Braun, Crous & K. Schub., Pseudocercospora stephanandrae (Tak. Kobay. & H. Horie) C. Nakash. & Tak. Kobay., Pseudocercospora viburnigena U. Braun & Crous, Pseudocercosporella chaenomelis (Y. Suto) C. Nakash., Crous, U. Braun & H.D. Shin, Xenostigmina zilleri (A. Funk) Crous; Lectotypification - Pseudocercospora ocimicola (Petr. & Cif.) Deighton; Neotypifications - Pseudocercospora kiggelariae (Syd.) Crous & U. Braun, Pseudocercospora lonicericola (W. Yamam.) Deighton, Pseudocercospora zelkovae (Hori) X.J. Liu & Y.L. Guo.

Unravelling Mycosphaerella: do you believe in genera?

Many fungal genera have been defined based on single characters considered to be informative at the generic level. In addition, many unrelated taxa have been aggregated in genera because they shared apparently similar morphological characters arising from adaptation to similar niches and convergent evolution. This problem is aptly illustrated in Mycosphaerella. In its broadest definition, this genus of mainly leaf infecting fungi incorporates more than 30 form genera that share similar phenotypic characters mostly associated with structures produced on plant tissue or in culture. DNA sequence data derived from the LSU gene in the present study distinguish several clades and families in what has hitherto been considered to represent the Mycosphaerellaceae. In some cases, these clades represent recognisable monophyletic lineages linked to well circumscribed anamorphs. This association is complicated, however, by the fact that morphologically similar form genera are scattered throughout the order (Capnodiales), and for some species more than one morph is expressed depending on cultural conditions and media employed for cultivation. The present study shows that Mycosphaerella s.s. should best be limited to taxa with Ramularia anamorphs, with other well defined clades in the Mycosphaerellaceae representing Cercospora, Cercosporella, Dothistroma, Lecanosticta, Phaeophleospora, Polythrincium, Pseudocercospora, Ramulispora, Septoria and Sonderhenia. The genus Teratosphaeria accommodates taxa with Kirramyces anamorphs, while other clades supported in the Teratosphaeriaceae include Baudoinea, Capnobotryella, Devriesia, Penidiella, Phaeothecoidea, Readeriella, Staninwardia and Stenella. The genus Schizothyrium with Zygophiala anamorphs is supported as belonging to the Schizothyriaceae, while Dissoconium and Ramichloridium appear to represent a distinct family. Several clades remain unresolved due to limited sampling. Mycosphaerella, which has hitherto been used as a term of convenience to describe ascomycetes with solitary ascomata, bitunicate asci and 1-septate ascospores, represents numerous genera and several families yet to be defined in future studies.

Conversion from cyclosporine to tacrolimus results in improved creatinine but does not alter lipid profile.

INTRODUCTION: While cyclosporine and tacrolimus use results in similar renal graft survival, the side effect profiles of the drugs are substantially different. We examined the electrolyte and lipid alterations that occurred in our patient population following conversion from cyclosporine to tacrolimus. METHODS: Data for electrolytes, lipid profile, and immunosuppression were analyzed from 98 patients with kidney or kidney-pancreas transplants who were converted from cyclosporine to tacrolimus between October 1994 and June 2001. Results, expressed as mean +/- SEM, were compared to baseline values using the Wilcoxon signed-rank test (P < .05 considered significant). RESULTS: Among these patients, there were 56 men, 42 women, 75 primary transplants, 15 repeat transplants, and 26 multiorgan transplants. The mean time to tacrolimus conversion was 769 +/- 122 days. Creatinine, BUN, and glucose improved after conversion to tacrolimus. Surprisingly, cholesterol, low-density lipoproteins, and high-density lipoproteins levels were not significantly altered, although triglyceride levels demonstrated a significant difference at 1 year. CONCLUSION: Significant improvements in creatinine and BUN were observed following conversion from cyclosporine to tacrolimus. While hypomagnesemia was also seen, there was surprisingly little alteration in lipid profile.

Effects of rapamycin on the arterial inflammatory response in atherosclerotic plaques in Apo-E knockout mice.

Because of its antiproliferative properties and its known effects on plasma lipids, we evaluated the mechanisms underlying the effect of rapamycin (RPM) on endothelial nitric oxide synthase (eNOS) and matrix metalloproteinases in Apo-E knockout mice. Apo-E-/- mice fed a high-cholesterol diet were given RPM (3 mg/kg per day intraperitoneally) or no treatment for 10 weeks (n = 8 each). Blood was drawn for serum lipid analysis. Protein was extracted from the abdominal aortas for Western immunoblotting and zymography. Cellular localization was assessed by histology and immunohistochemistry. The data, expressed as mean +/- SEM, were compared by Student's t test or analysis of variance (ANOVA). Lipid levels at 10 weeks were similar in both groups except for higher triglyceride levels in RPM-treated animals. RPM-treated mice expressed greater amounts of eNOS and p-eNOS compared with controls (P < .05). Akt, p-Akt, Caveolin-1, and p-Caveolin-1 were not significantly affected by RPM treatment. RPM treatment was associated with increased activation of pro-MMP-9, a significant decrease in MMP-2 tissue levels, and corresponding increases in TIMP-2 and TIMP-3 expression. The increased expression and phosphorylation of eNOS with RPM appears to be regulated by mechanisms other than Akt or Caveolin-1. Alterations in eNOS expression, in addition to changes in MMP/TIMP ratios and MMP-2 and MMP-9 activation, may partially explain the changes observed in the aorta of treated Apo-E-/- mice induced by RPM.

T-lymphocyte apoptosis is increased by non-interleukin-2-dependent induction in human mixed lymphocyte cultures.

INTRODUCTION: Transplant tolerance is dependent on the apoptotic deletion of allospecific T lymphocytes following interleukin-2 (IL-2)-dependent T-lymphocyte activation. Current immunosuppressive strategies block IL-2 and may prevent T-cell activation. We examined apoptotic alterations in mixed lymphocyte culture (MLC), a model of allospecific lymphocyte activation, by polyclonal rabbit antithymocyte antibody thymoglobulin (rATG) and monoclonal anti-IL-2 receptor antibody basiliximab. METHODS: Human lymphocytes were isolated using Ficoll-Paque gradient. Cesium-irradiated (2500 rad) stimulator cells (10(6) cells/mL) were cocultured with equal numbers of responder cells. Apoptosis was measured using annexin-V staining and propidium iodide exclusion using flow cytometry. Isolated protein was analyzed using Western blotting with densitometry. RESULTS: Apoptosis increased at days 3 and 7 in rATG MLC compared with control and basiliximab MLC. Fas was up-regulated in rATG MLC in a dose-dependent manner, whereas basiliximab did not alter fas. FasL was increased initially and at late time points in rATG MLC. CONCLUSIONS: Polyclonal rATG increased apoptosis and production of the proapoptotic proteins fas and fasL. In contrast, monoclonal basiliximab did not change lymphocyte apoptosis or apoptotic protein production. These results suggest that a specific IL-2 pathway blockade may prevent allospecific tolerance and that a non-IL-2 pathway blockade may encourage apoptosis of allospecifically activated T cells.

Vein adaptation to arterialization in an experimental model.

PURPOSE: The events preceding myointimal thickening in vein grafts after vascular reconstructions are not well characterized. Indeed, the injury response associated with vein graft arterialization may be different than that observed in the balloon angioplasty model. Therefore, we used a rat model to study the early cellular response after arterialization of vein grafts. METHODS: Epigastric veins were placed as femoral artery interposition grafts in 37 male Lewis rats (weight range, 350-400 g). Vein grafts and contralateral epigastric veins were harvested at different time points (6 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, 30 days, and 70 days). Tissue specimens were processed for histology and immunohistochemistry with antibodies for the proliferating cell nuclear antigen (PCNA) and for different cell types. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used as a means of determining the presence of apoptosis. Electron microscopy was used as means of assessing the integrity of the endothelial cell surface (SEM) and confirming the presence of apoptosis (TEM). Specimens were also snap frozen in liquid nitrogen for RNA isolation and molecular analysis. RESULTS: At 1 day, endothelial denudation with platelet deposition on the surface was shown by means of SEM. Both apoptosis and necrosis of smooth muscle cells (SMCs) were present in the media, along with monocyte infiltration. Cellular proliferation and apoptosis were most intense within the first week of implantation. PCNA staining was first seen in the adventitial fibroblasts and microvessels, then in the medial SMCs at 3 days. With reverse transcriptase polymerase chain reaction, upregulation of vascular endothelial growth factor (VEGF) messenger RNA (mRNA) was noted at 1 day. Myointimal thickening progressively developed, with no apparent diminution of the luminal area as long as 70 days after implantation. By means of the analysis of the transforming growth factor beta1, mRNA showed expression during intimal thickening and accumulation of extracellular matrix. Reendothelialization was complete at 30 days. CONCLUSIONS: These observations indicate that the cellular composition in our vein graft model is similar to human stenotic explants. Endothelial denudation is observed in rat vein grafts with complete regeneration by 30 days. VEGF mRNA is upregulated at 1 day, followed by proliferation of microvessel endothelial cells in the adventitia. Cellular proliferation and apoptosis are minimal after 21 days, with progressive intimal thickening likely to be the result of matrix accumulation.

Intimal growth and neovascularization in human stenotic vein grafts.

BACKGROUND: Myointimal thickening and microvessel ingrowth are commonly observed in vein graft stenosis, which complicates a third of infrainguinal bypass procedures. But a direct correlation between these two features has not been established. Our purpose was to analyze the relationship between neovascularity and intimal thickness in human vein grafts. STUDY DESIGN: Twenty-two explant stenotic vein grafts (STVG), 8 nonstenotic arterialized vein grafts (AVG), and 20 age-matched control greater saphenous veins (CGSV) were analyzed histologically and compared morphologically by light microscopy. Digitized computer image analysis was used to measure intimal thickness and quantitate microvessel ingrowth. Immunolocalization of endothelial cells around the lumen and in microvessels was determined using antibodies to factor VIII and to endothelial nitric oxide synthase (eNOS), respectively. RESULTS: Focal areas of endothelial disruption and thrombus deposition were present in 23% (5 of 22) of stenotic vein grafts. The neointima of STVG grafts was two- and fourfold thicker than that of AVG and CGSV, respectively (p < 0.0001). Microvessels were most frequently observed in the adventitia and media of STVG and increased in number with increasing intimal thickness (p < 0.001 by ANOVA). CONCLUSIONS: A fourfold increased neointimal thickness in critically stenotic vein grafts is associated with increased medial and adventitial neovascularization. Remodeling alone with doubling of the intimal thickness in nonstenotic arterialized vein grafts does not appear to be associated with enhancement of the graft microvasculature. More specific observations using an experimental model may allow us to further define the role of angiogenesis in vein graft stenosis and to determine the therapeutic implications of such observations.

The contribution of inducible nitric oxide and cytomegalovirus to the stability of complex carotid plaque.

BACKGROUND: Although the association between inflammation and atherosclerosis is well established, the biologic events that trigger the local inflammatory response within plaque are not fully understood. Cytotoxic free radicals and infectious agents, both of which are associated with an inflammatory response, have previously been implicated in the initiation and progression of atherosclerosis. In this study, we analyzed carotid plaque for evidence of oxidative vascular injury by determining the presence and distribution of inducible nitric oxide synthase (iNOS) expression and nitrotyrosine formation and for evidence of infection with cytomegalovirus. METHODS: Carotid plaque from 51 patients who underwent endarterectomy for either primary (n = 37) or recurrent (n = 14) stenosis were examined histologically (hematoxylin-eosin staining and Masson's trichrome staining) and with immunohistochemistry with specific antibodies to alpha-smooth muscle actin, macrophages (CD68), T-lymphocytes (CD3), and T-cell activation (human leukocyte antigen-DR). Twenty-eight specimens from patients with primary (n = 15) and recurrent (n = 13) stenosis were examined for the presence of iNOS and nitrotyrosine with immunohistochemistry and in situ hybridization (iNOS). Twenty-three additional specimens (22 primary, and 1 recurrent) were analyzed with antibodies to p53, cytomegalovirus, and the polymerase chain reaction (cytomegalovirus, n = 8). RESULTS: Primary atherosclerotic lesions were either complex heterogenous cellular plaques (n = 29) or relatively acellular fibrous plaques (n = 8). Ten of 14 recurrent plaques were either complex or fibrous lesions, and the remaining four were typical of myointimal thickening. CD68-positive staining cells were detected in all specimens regardless of their structural morphology. CD3-positive cells were interspersed between macrophages in all heterogeneous cellular plaques and only infrequently noted in fibrous plaques. iNOS and nitrotyrosine immunoreactivity were detected in macrophages and smooth muscle cells in all complex and fibrous plaques and in two of four myointimal plaques. The presence of iNOS and nitrotyrosine in plaque correlated with the existence of symptoms in 80% of primary and 62% of recurrent lesions. Cytomegalovirus was detected in only two of 23 carotid specimens (9%). CONCLUSION: The association between ischemic cerebrovascular symptoms and iNOS and nitrotyrosine immunoreactivity in complex primary and recurrent carotid plaque and the infrequent occurrence of cytomegalovirus in primary carotid lesions suggests that ongoing free radical oxidative damage rather than viral infection may contribute to plaque instability in patients with complex and fibrous carotid plaques.

Antioxidant enzyme activity in human abdominal aortic aneurysmal and occlusive disease.

The present study further investigates evidence for lipid peroxidation in atherosclerotic aortic tissue by determining the activity of antioxidant enzymes and concentrations of lipid peroxide fluorochromes in abdominal aortas from 15 patients with abdominal aortic aneurysms (AAA), an additional 7 patients with ruptured abdominal aneurysms, and 12 patients with atherosclerotic occlusive disease (AOD). Aortas from nonatherosclerotic organ donors served as nondiseased controls. Cu, Zn-superoxide dismutase (Cu,Zn-SOD) activities in AAA and AOD tissues were 16% and 25% of control activity, respectively. Mn-SOD activity in diseased aortae were about 65% of controls. CuZn-SOD protein in AAA and AOD was 56% and 100% of controls, respectively, resulting in significantly lower CuZn-SOD specific activity in these tissues. Ruptured AAA tissue also had low SOD activity and protein. Glutathione peroxidase (GPx) activity in AAA and AOD aortas was 70% and 65% of controls, respectively, and glutathione reductase (GR) activity in AAA and AOD aortas was 80% and 65% of control activities, respectively. These results were associated with significantly higher lipid peroxide fluorochromes, expressed as U/g aorta, in both groups of atherosclerotic aortas than in controls. AOD aortas had 33% higher fluorescence than AAA aortas, but the highest levels were seen in ruptured AAA. These data further support the involvement of free radicals and lipid peroxidation in atherosclerotic aortic disease, but do not indicate that these mechanisms are specifically involved in aneurysm formation versus development of occlusive disease.

Topography of cell replication in human vein graft stenoses.

BACKGROUND: Analysis of the cellular composition of human autogenous vein graft lesions at the time of revision provides an opportunity to identify the cellular processes leading to the development of stenosis in humans after vascular reconstruction. METHODS AND RESULTS: Human vein graft-threatening stenotic lesions were identified by duplex scanning within 3 to 18 months after infrainguinal bypass and surgically removed. They were serially studied by immunocytochemistry for expression of the proliferating cell nuclear antigen (PCNA) in different cell types: alpha-actin-positive smooth muscle cells (SMCs), endothelial cells (ECs), monocytes, and macrophages. Proliferation indexes were separately obtained for each layer of the vessel wall by determining the mean percentage of PCNA-positive nuclei among the total number of nuclei present within the intima, the media, and the adventitia, respectively. The percentage distribution of the replicating cell types was also determined. We report that in autogenous vein graft (n = 14) the intima of the lesion displayed fewer PCNA + nuclei (1.03 +/- 0.88) than the underlying media (3.14 +/- 0.74) or the adventitia (3.01 +/- 0.74). Replicating SMCs were predominantly in the medial layer (68% of PCNA + cells) of stenotic vein grafts. In the adventitia, the proliferation was most intense in the endothelium of microvessels (65% of PCNA + nuclei). CONCLUSIONS: Our findings reveal a 3-fold greater proliferative activity in the media and the adventitia as compared with the intima of autogenous vein graft lesions, in contrast to cellular proliferation identified in recurrent coronary stenotic plaques. Moreover, there are distinctive patterns of distribution of the different cell populations among the 3 layers. The results indicate a proliferative response of the media and the adventitia of autogenous vein grafts transplanted into the arterial circulation, in addition to the cellular proliferation observed in the intima of the lesion.

Current trends in the detection and management of carotid body tumors.

PURPOSE: Because the natural history of carotid body tumors is believed to be unpredictable, immediate surgical removal has been recommended. The present study reviews our experience in the diagnosis and treatment of these uncommon lesions. METHODS: The medical records of patients who appeared for treatment with carotid body tumors between 1981 and 1997 were reviewed. Patients demographics, mode of presentation, imaging and treatment modalities, Shamblin classification, and neurologic complications (stroke, cranial nerve injuries) were analyzed. RESULTS: Over the past 16 years, 31 patients with 32 carotid body tumors have been evaluated, with an average follow-up of 3.2 years. The patients were arbitrarily classified into two groups on the basis of the mode of detection. Seventy percent (23 of 32) of the tumors discovered on clinical or self-examination were classified as Group 1; 28% (9 of 32) of the tumors detected during duplex scanning for carotid artery disease (8) or MRI (1) were classified as Group 2. The mean size of chemodectomas found on palpation (4.3 +/- 1.7 cm) was larger than that of those detected by duplex ultrasound (2.7 +/- 1.0 cm; p < 0.05, by paired t test). Preoperative embolization was successfully performed in 5 of 6 instances of large tumors; the remaining patient suffered a procedure-related stroke. Thirty-one carotid body tumors were resected. In one case, the tumor was felt by the primary surgeon to be too small (0.9 x 0.7 cm on duplex scan) to warrant immediate excision; this patient is being followed by periodic duplex scanning. Five neurologic complications were noted in Group 1, one after preoperative embolization and four after surgery. One cranial nerve injury occurred in Group 2. One patient had a large recurrent chemodectoma with clinical evidence of metastatic disease. CONCLUSION: The increasing use of sophisticated imaging modalities may allow earlier discovery of carotid body tumors before they can be clinically detected. Resection of carotid body tumors of all sizes in appropriate surgical candidates remains the standard of care. Unfortunately, resection of even small tumors is associated with a low but constant incidence of neurologic complications.

The histologic characteristics of primary and restenotic carotid plaque.

BACKGROUND: Although smooth muscle cell proliferation is a prominent feature of restenosis in experimental models, the role of cellular proliferation in the initiation and progression of carotid restenosis is not well documented. METHODS: Between 1985 and 1995, 35 carotid endarterectomies (CEA) in 34 patients were performed for restenosis. Patient risk factors, cerebrovascular symptoms, and operative findings were recorded. Tissue specimens from 29 of these cases and 14 original specimens from the same patient were examined by light microscopy (H&E, trichrome, elastochrome, and Alcian blue) and immunohistochemistry (alpha actin, CD 68, vWF, and proliferating nuclear cell antigen (PCNA)) in order to determine the morphologic characteristics and cellular proliferative activity of the plaque. RESULTS: Hemodynamically significant recurrent stenosis occurred in the 29 patients (69% symptomatic) between 2 months and 30 years after their initial CEAs. Eleven of 29 (38%) lesions were removed early (< 3 years). Recurrent lesions were characterized based on their components as neointimal thickening, 24% (7/29), neointimal thickening and atherosclerosis, 55% (16/29), or atherosclerotic, 21% (6/29). Nineteen of 29 (66%) plaques were complicated by mural thrombus or intraplaque hemorrhage. An inflammatory cell infiltrate consisting of macrophages and T lymphocytes was observed adjacent to areas of recurrent atherosclerosis and macrophages in regions of intimal thickening. Although infrequently present (generally 1-3% of cells) PCNA-positive cells were detected in 41% (12 of 29) of recurrent and 14% (2 of 14) of primary plaques. No PCNA-positive cells were detected in the remaining 67% (29 of 43) of specimens. There was no statistical difference in the number of PCNA-positive cells in early recurrent lesions compared to those recurring after 3 years (36% vs 44%). PCNA immunoreactivity when present was most commonly noted in macrophages associated with thrombus or atheroma rather than smooth muscle cells. CONCLUSIONS: Although evidence of cellular proliferation was observed in 40% of recurrent carotid endarterectomy lesions, the proliferation rate was low (1-3%) and unrelated to the time interval of recurrence. Proliferative activity was most pronounced in macrophages associated with intraplaque hemorrhage or atheroma. The contribution of inflammatory cells to the biologic behavior of restenotic lesions requires further investigation.

Edgar J. Poth Memorial/W.L. Gore and Associates, Inc. Lectureship. The clinical and pathological spectrum of recurrent carotid stenosis.

BACKGROUND: Hemodynamically significant (> or =50%) carotid restenosis occurs in approximately 10% to 12% of individuals undergoing carotid endarterectomy. The underlying pathology is usually neointimal thickening within 3 years and recurrent atherosclerosis thereafter. Although a number of etiologic factors have been implicated in the development of restenosis, the etiology remains unclear and preventative measures relatively ineffective. METHODS: A review of the English literature was undertaken to determine the incidence, clinical presentation, and pathologic features of carotid restenosis. CONCLUSIONS: Carotid restenosis is the major factor limiting long-term patency after carotid endarterectomy. Although drug therapy has been shown to be effective in preventing restenosis in animal models, the results of clinical human trials have been disappointing. Delineation of the biochemical and molecular mechanisms contributing to the development of restenosis is essential if effective therapeutic interventions are to be developed.

Free tissue transfer to extend the limits of limb salvage for lower extremity tissue loss.

BACKGROUND: The extent of tissue loss amenable to primary healing after revascularization is unknown. Salvage of limbs with large soft-tissue defects with exposed tendon, joint, or bone lies beyond the limits of conventional techniques. We report our results using free tissue transfer as an adjunct to lower extremity vascular reconstruction in patients with complex ischemic or infected wounds. METHODS: Retrospective chart review of patient and wound characteristics. RESULTS: From January 1992 to June 1996, 585 procedures were performed in 544 patients, including 27 free flaps in 26 patients: 17 free flaps combined with distal bypass (7 staged, 10 simultaneous) and 10 isolated free flaps. Flap donor sites included radial forearm (8), latissimus dorsi (7), rectus abdominus (9), and scapula (3). Surgical indications included extensive ischemic/neurotrophic ulcers, and nonhealing vein graft harvest incision or transmetatarsal amputation site. Mean area of tissue loss was 70 cm2, mean ulcer duration was 5 months, and 92% of patients had exposed tendon, joint, or bone. During a mean follow-up of 14 months, 2 patients died of cardiopulmonary disease and 3 flaps failed, resulting in below-knee amputation. Six flaps were revised for graft stenosis (1), venous thrombosis (1), or flap edge necrosis (4). Limb salvage rate was 70% at 24 months by life-table analysis. Functional ambulation was achieved in 21 of 24 (88%) patients, including 7 of 8 with diabetes, end-stage renal disease, and heel ulcers. CONCLUSION: In select ambulatory patients with large soft-tissue defects and exposed deep structures, functional limb salvage is obtainable in more than 80% of patients. For lesions not amenable to vascular reconstruction with conventional methods of wound coverage, free tissue transfer extends the limits of limb salvage and is a viable alternative to amputation.

Distal revascularization-interval ligation for limb salvage and maintenance of dialysis access in ischemic steal syndrome.

PURPOSE: Traditional options for treating ischemic steal syndrome related to a functioning dialysis access graft or fistula include banding or ligation. Unfortunately, these techniques usually result in inconsistent limb salvage, loss of a functional access, or both. We report our experience with an alternative method of limb revascularization that eliminates steal while maintaining continuous dialysis access. METHODS: Patients who had critical limb ischemia and functioning arteriovenous fistulae (AVF) underwent color-flow duplex scanning, digital photoplethysmography, and arteriography. Arterial ligation distal to the AVF origin eliminated the steal physiologic mechanism while arterial bypass grafting from above to below the AVF revascularized the extremity (distal revascularization-interval ligation [DRIL] procedure). RESULTS: From March 1994 through December 1996, 21 patients with functioning extremity AVFs presented with critical ischemia and steal syndrome. Eleven patients had chronic ischemia with rest pain, paresthesias, or ulcerations related to nine native fistulae (six brachiocephalic, two basilic vein transpositions, one radiocephalic) and two prosthetic bridge grafts (one upper arm, one lower extremity). Acute ischemia developed in 10 patients related to three native fistulae (two brachiocephalic, one radiocephalic) and seven prosthetic bridge grafts (three forearm, three lower extremity, one upper arm). All 21 patients were treated with the DRIL technique. Three of these patients required treatment for ischemia at the time of AVF construction. Nineteen of 21 bypass procedures were performed with autogenous vein, including nine brachial-brachial, three brachial-radial, two radial-radial, two brachial-ulnar, one popliteal-popliteal, one femoral-popliteal, and one femoral-peroneal. Polytetrafluoroethylene grafts were used for one external iliac-popliteal bypass graft and one axillary-brachial bypass graft. Limb salvage and maintenance of a functional fistula were achieved in 100% and 94%, respectively, at 18 months by life-table analysis. CONCLUSION: The DRIL technique reliably restores antegrade flow to the ischemic limb, eliminates the potential pathway for the steal physiologic mechanism, and maintains continuous dialysis access in these difficult patients.

Identification of predictors for lower extremity vein graft stenosis.

BACKGROUND: The cause of intrinsic vein graft stenosis, which develops in at least 20% of infrainguinal autogenous bypass grafts during the intermediate follow-up interval, is unknown. We performed standard duplex surveillance of all lower extremity bypass grafts and evaluated the potential of comorbid patient risk factors that might predict development of vein graft flow disturbance or high-grade graft stenosis. METHODS: Patients with at least 6 months of postoperative duplex surveillance were identified through our vascular registry. The association of clinical and hemodynamic profiles of graft performance were compared with specific patient risk factors, including demographics, cigarette smoking, antihypertensive medical therapy, type and quality of conduit, degree of ischemia, bypass run-off, and presence of infection, using stepwise logistic regression analysis. RESULTS: Ninety-three patients (55 male, 38 female; mean age 69) underwent 100 infrainguinal bypasses. Twenty-six high-grade graft stenoses (>70%) were identified in 26 patients during follow-up (mean 21 months) by graft-flow peak systolic velocity (PSV) >300 cm/sec on more than one duplex examination, and were electively revised. Graft flow disturbances (180 cm/sec >PSV <300 cm/sec) were identified in an additional 13 grafts (6 regressed, 7 observed). The need for graft revision was associated with an early graft flow disturbance (P = 0.02), or drop in ankle-brachial index >0.15 (P = 0.03), and the use of an alternative conduit in 13 of 100 grafts (P = 0.04). Only smoking was associated with the development of a duplex detected graft flow disturbance during follow up (P = 0.03). CONCLUSION: Grafts with early flow disturbances warrant close duplex surveillance to identify graft-threatening stenosis. Risk factors that may predict future lower extremity bypass graft stenosis are smoking and the use of alternative bypass conduits.

MRI and NMR spectroscopy of the lipids of atherosclerotic plaque in rabbits and humans.

The early stages of atherosclerosis are characterized by the deposition of cholesteryl esters and triglycerides into the arterial wall. In the excised human atherosclerotic plaque these lipids are in a liquid-like state at body temperature and observable via MRI and NMR spectroscopy. To assess the ability of MRI to quantitatively image the lipids of atherosclerotic plaque in vivo, we have investigated eight New Zealand White rabbits fed atherogenic diets (2 weight (wt)% cholesterol, 1 wt% cholesterol + 6 wt% peanut oil, and 1 wt% cholesterol + 6 wt% com oil). Postmortem examination indicated that all rabbits developed atherosclerosis in the aorta. Except for one animal, magnetic resonance angiography showed no noticeable obstruction in the aorta. MRI was carried out in an attempt to image atherosclerotic plaque lipids directly, but no signal was detected in vivo. However, a plaque lipid signal was observed from excised tissue using a small diameter RF coil. 1H NMR spectroscopy of the atherosclerotic plaque from excised aortas indicated that the major fraction of plaque lipids in rabbits is not in a liquid state at physiological temperature and are only marginally MRI-visible compared to human plaque lipid. The differences in the MRI characteristics of rabbit and human plaque are due to differences in the fatty acid profile of the cholesteryl esters, chiefly a decrease of linoleic acid in rabbit lesions.

Lower extremity iatrogenic nerve injury due to compression during intraabdominal surgery.

BACKGROUND: latrogenic nerve injury due to poor positioning and external compression is a common surgical complication. However, sciatic neuropathy from external compression and femoral nerve injury after self-retaining retraction are less-published complications. METHODS: Surgical Morbidity and Mortality Reports from 1986 through 1995 were reviewed to identify femoral and sciatic neuropathies following intraabdominal vascular and general surgeries. RESULTS: Two sciatic and 5 femoral neuropathies were reported, an incidence of approximately 0.17% of abdominal cases. Sciatic injuries were attributed to external compression, whereas femoral neuropathies were due to compression by self-retaining retraction. The 3 female and 4 male patients had a mean age of 53.4 years, and no patient had a prior history of peripheral neuropathy. Mean operating time for sciatic injuries was 8.2 hours, versus 4.3 hours for femoral neuropathies. Both patients with sciatic neuropathy had complete resolution of symptoms, compared with 1 femoral neuropathy patient. Two femoral neuropathies were permanent, 1 had partial resolution and 1 had improvement at 4 months but was lost to follow-up. CONCLUSIONS: Sciatic and femoral compression neuropathies are rare but serious complications of abdominal surgery. When retracting in the deep pelvis, consideration should be given to using small, well-padded retractor blades and repositioning these regularly. Prevention of sciatic nerve compression requires careful padding of the table surface, especially for longer cases.