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Interspecific somatic hybrids produced by protoplast fusion between two wild Solanum species (S. acaule, acl; S. brevidens, brd) and cultivated potato Solanum tuberosum (tbr) were analyzed in terms of the starch nanometer-range structure and glycoalkaloid (GA) contents. The crystallinity of starch granules, the average size of starch crystallites, and the lamellar distances were obtained from tuber samples using wide-angle and small-angle X-ray scattering methods. These measurements showed that incorporation of wild genomes from either nontuberous (brd) or tuberous (acl) Solanum species caused no significant modifications of the nanostructure of potato starch. In contrast, the GA profiles of the hybrids, which were analyzed by LC-ESI-MS in both tuber and foliage samples, differed considerably from those of cultivated potato. Regardless of the low total tuber GA concentrations (approximately 9 mg/100 g of fresh weight), the somatic hybrids contained GAs not detected in the parental species. A high proportion of spirotype GAs consisting of 5,6-dihydrogenated aglycons, for example, alpha-tomatine and tomatidine bound with solatriose, and chacotriose were found in the hybrids. In conclusion, the foliage of interspecific hybrids contained a higher variation in the structures of GAs than did the tubers.

In a recent genome-wide linkage (GWL) analysis of Finnish families at high risk for prostate cancer, we found two novel putative susceptibility loci at 3p25-p26 and 11q14. Here, we report the fine-mapping of these two critical regions at high resolution with 39 microsatellite markers in 16 families, including multiplex families that were not used in the GWL scan. The maximum multipoint HLOD was 3.39 at 3p26 and 1.42 at 11q14. The highest LOD scores were seen around markers D3S1270 and D3S4559 (alpha=0.89), covering approximately two megabases. The two known genes in this region CHL1 (cell adhesion molecule with homology to L1CAM) and CNTN6 (contactin 6) were screened for exonic mutations in the families showing the strongest linkage, but no disease-segregating sequence variants were observed. The recombination map pointed to a region proximal to the area of best linkage, suggesting that more genes may need to be investigated as candidates. These results provide strong evidence for the existence of a prostate cancer susceptibility gene at 3p26 in Finnish prostate cancer families. This locus has not been strongly linked with hereditary prostate cancer in other populations. However, the mildly positive 3p LOD scores in a recent GWL analysis of patients from the United States suggest that the locus may also be important in other populations.

Purpose: Recently, Kruppel-like factor 6 gene (KLF6) has been shown to be inactivated in up to 77% of prostate carcinomas. KLF6 has an important role in regulating cell growth and differentiation. The function and high mutation frequency in sporadic prostate carcinomas make KLF6 an attractive candidate for prostate cancer predisposition and, therefore, DNA samples from 69 Finnish prostate cancer families were analyzed for KLF6 mutations., Materials and Methods: DNA samples from 69 Finnish prostate cancer families were screened for mutations in the KLF6 gene using single-strand conformation polymorphism analysis and confirmatory sequencing., Results: In 8 (11.6%) families single-strand conformation polymorphism shifts were present. Sequencing revealed 6 201G>A (R201R) polymorphisms, as well as a -4C>A and a 956T>C alteration in the 5'- and 3'-untranslated regions, respectively. Nonsense or missense mutations were not found., Conclusions: Our data suggest that KLF6 germ-line mutations are of marginal importance in prostate cancer predisposition in Finland.

Chlorosomes of green photosynthetic bacteria constitute the most efficient light harvesting complexes found in nature. In addition, the chlorosome is the only known photosynthetic system where the majority of pigments (BChl) is not organized in pigment-protein complexes but instead is assembled into aggregates. Because of the unusual organization, the chlorosome structure has not been resolved and only models, in which BChl pigments were organized into large rods, were proposed on the basis of freeze-fracture electron microscopy and spectroscopic constraints. We have obtained the first high-resolution images of chlorosomes from the green sulfur bacterium Chlorobium tepidum by cryoelectron microscopy. Cryoelectron microscopy images revealed dense striations approximately 20 A apart. X-ray scattering from chlorosomes exhibited a feature with the same approximately 20 A spacing. No evidence for the rod models was obtained. The observed spacing and tilt-series cryoelectron microscopy projections are compatible with a lamellar model, in which BChl molecules aggregate into semicrystalline lateral arrays. The diffraction data further indicate that arrays are built from BChl dimers. The arrays form undulating lamellae, which, in turn, are held together by interdigitated esterifying alcohol tails, carotenoids, and lipids. The lamellar model is consistent with earlier spectroscopic data and provides insight into chlorosome self-assembly.

Background: Obliterative bronchiolitis (OB) is the major cause of morbidity and mortality after lung transplantation. One initiating event in the development of obliteration of the airway lumen is epithelial injury. In our model of chronic rejection, initial ischemic injury and denudation of the epithelium occurs in the isografts, with eventual re-epithelialization and partial patency of the airway lumen. In contrast, allografts do not recover epithelium, and the airway lumen becomes obliterated. We hypothesized that because integrin alphaVbeta6 is expressed in healing epithelium, integrin alphaVbeta6 expression would be greatly increased in isografts, but not in allografts., Methods: Using a rat tracheal allograft rejection model as a source of 4- to 5-microm tissue sections, we compared integrin staining in allografts vs isografts from animals at post-transplant Days 7, 14, 28, and 60. We analyzed the sections using immunohistochemistry after incubation with a specific monoclonal antibody E7P6 against integrin alphaVbeta6. Negative control slides were processed identically, except that primary antibody was omitted., Results: The sections from healing, re-epithelializing isografts showed intense staining when using the antibody recognizing integrin alphaVbeta6, compared with the allografts studied. Days 7 and 14 isografts had increased epithelial expression of alphaVbeta6. As the isograft epithelium recovered, the intensity diminished at Days 28 and 60. In allografts, at Days 7 to 60, we detected only a comparatively low-level of expression in injured epithelium., Conclusions: Integrin alphaVbeta6 is readily detectable in healing isografts. Integrin alphaVbeta6 may be crucial in maintaining a viable epithelial cell layer, which is related to slowed progression of airway obliteration in OB.

Purpose: Steroid hormones, particularly androgens, are suspected to have a major role in prostate carcinogenesis. Since androgen receptor mediates androgenic effects on cells and recent studies suggest that the androgen receptor gene is a putative prostate cancer susceptibility locus, we screened the coding region of the androgen receptor gene for germline mutations using the genomic DNA of patients with prostate cancer., Materials and Methods: DNA samples from 38 patients with early onset prostate cancer and 36 from Finnish prostate cancer families showing no male-to-male transmission of prostate cancer were analyzed for mutations in the androgen receptor gene using single strand conformation polymorphism analysis and subsequent sequencing., Results: R726L substitution in the hormone binding region of androgen receptor was found in 1 prostate cancer family but no previously uncharacterized germline mutations were detected., Conclusions: Our results indicate that constitutional androgen receptor mutations explain only a small fraction of familial and early onset prostate cancer cases in Finland.

Purpose: The MSR1 gene maps to 8p22-23, a novel susceptibility locus for hereditary prostate cancer (HPC). Mutations in MSR1 have been reported to associate with prostate cancer (PRCA) risk. Here we report a follow-up study from Finland to evaluate the association between PRCA and MSR1 gene., Experimental Design: The youngest affected patient from each of 120 HPC families was initially used for the screening of MSR1 mutations by single-strand conformational polymorphism analysis. Selected variants of MSR1 gene were then screened in 537 unselected PRCA cases and in 480 controls., Results: Among 120 HPC families, five MSR1 sequence variants were identified. The carrier frequencies of the R293X, P275A, and -14743A>G variants were compared between the probands with HPC, unselected PRCA cases, and healthy male blood donors. No significant differences were observed. The odds ratios for R293X, P275A, and -14743A>G mutations were also calculated to estimate the PRCA risk. No significantly elevated or lowered risks for PRCA among these three variants were detected. However, the mean age at diagnosis of the R293X mutation carriers among HPC probands was significantly lower compared with noncarriers (55.4 versus 65.4 years; t test, P = 0.04). The same trend was observed among unselected PRCA cases (65.7 versus 68.7 years; t test, P = 0.37)., Conclusions: Our results do not support a major role for the MSR1 gene in the causation of hereditary or unselected PRCAs but suggest a possible modifying role in cancer predisposition.

Effects of systematic environmental factors and milk production and quality traits on milk coagulation properties (MCP), and on repeatability of those traits were estimated from 979 milk samples collected once a month over a period of 2 years from 83 Finnish Ayrshire cows. Estimation was based on a multitrait animal model and REML methodology. In addition, persistence of non-coagulation of milk in individual cows, and factors associated with it were established from a sub sample of 24 cows producing non-coagulating (NC) milk at least once. MCP were at their best during the first lactation, at the beginning and at the end of lactation, and during grazing seasons. Variation in MCP with systematic environmental factors was partly due to variation in composition and quality of milk, especially in pH and ln (somatic cell count, SCC). Coefficients of repeatability for milk coagulation time and curd firmness were 0.65 and 0.68. These estimates were of the same magnitude as those for protein content, but were higher than those for daily milk yield, fat content, pH, and SCC. Based on the repeatability estimates for the milk coagulation traits and effects of the environmental factors, cows should be sampled at least three times during a lactation to estimate reliably breeding values for the milk coagulation traits. A total of 10% of the milk samples did not coagulate in 30 min after addition of rennet. Cows that produced NC milk at least once (30% of the cows) could be classified into those that produced NC milk only a few times during a lactation and those that produced NC milk at almost every sampling. Based on logistic regression analyses, peak and mid-lactation, high milk yield, low protein and fat content and high pH increased the risk of non-coagulation of milk.

A new principle in constructing molecular complexes from the known high-resolution domain structures joining data from NMR and small-angle x-ray scattering (SAXS) measurements is described. Structure of calmodulin in complex with trifluoperazine was built from N- and C-terminal domains oriented based on residual dipolar couplings measured by NMR in a dilute liquid crystal, and the overall shape of the complex was derived from SAXS data. The residual dipolar coupling data serves to reduce angular degrees of freedom, and the small-angle scattering data serves to confine the translational degrees of freedom. The complex built by this method was found to be consistent with the known crystal structure. The study demonstrates how approximate tertiary structures of modular proteins or quaternary structures composed of subunits can be assembled from high-resolution structures of domains or subunits using mutually complementary NMR and SAXS data.

Recent studies have suggested that polymorphisms of the androgen receptor gene ( AR) may influence the risk of prostate cancer (PC) development and progression. Here, we analyzed the length of the CAG repeat of the AR gene in 1363 individuals, including patients with PC, benign prostate hyperplasia (BPH), and population controls. There was a tendency for short CAG repeats to be associated with PC. The Odds Ratio (OR) for PC was 1.47 ( P=0.05) when individuals with short CAG repeats (18). CAG repeat length was not significantly associated with family history, disease stage, grade, age at diagnosis, prostate-specific antigen (PSA) level at diagnosis, or prognosis of the patients. Unexpectedly, short CAG repeats were significantly less common in patients with BPH compared with controls (OR=0.47, P=0.03). Our results suggest that the CAG polymorphism of the AR gene is unlikely to have a major role in the development or progression of PC in the Finnish population. The association of CAG repeats with the risk of BPH warrants further study.

The RNASEL gene (2',5'-oligoisoadenylate-synthetase dependent) encodes a ribonuclease that mediates the antiviral and apoptotic activities of interferons. The RNASEL gene maps to the hereditary-prostate-cancer (HPC)-predisposition locus at 1q24-q25 (HPC1) and was recently shown to harbor truncating mutations in two families with linkage to HPC1. Here, we screened for RNASEL germline mutations in 66 Finnish patients with HPC, and we determined the frequency of the changes in the index patients from 116 families with HPC, in 492 patients with unselected prostate cancer (PRCA), in 223 patients with benign prostatic hyperplasia (BPH), and in 566 controls. A truncating mutation, E265X, was found in 5 (4.3%) of the 116 patients from families with HPC. This was significantly higher (odds ratio [OR] =4.56; P=.04) than the frequency of E265X in controls (1.8%). The highest mutation frequency (9.5%) was found in patients from families with four or more affected members. Possible segregation was detected only in a single family. However, the median age at disease onset for E265X carriers was 11 years less than that for noncarriers in the same families. In addition, of the four missense variants found, R462Q showed an association with HPC (OR=1.96; P=.07). None of the variants showed any differences between controls and either patients with BPH or patients with PRCA. We conclude that, although RNASEL mutations do not explain disease segregation in Finnish families with HPC, the variants are enriched in families with HPC that include more than two affected members and may also be associated with the age at disease onset. This suggests a possible modifying role in cancer predisposition. The impact that the RNASEL sequence variants have on PRCA burden at the population level seems small but deserves further study.

In our recent cancer registry-based study, the incidence of gastric carcinoma was increased up to 5-fold in male relatives of early-onset prostate cancer (PCA) patients. This association may reflect the influence of genetic factors predisposing individuals to both tumor types. Germ-line mutations of the CDH1 gene at 16q have recently been associated with familial gastric cancer. Furthermore, two genome-wide linkage studies of PCA recently reported positivity at 16q. We therefore identified families and individual patients with both gastric and PCA and investigated whether the CDH1 gene mutations were involved in cancer predisposition in these cases. Fifteen of the 180 Finnish hereditary PCA families (8.3%) had one or more gastric cancer cases. No truncating or splice site CDH1 mutations were identified by PCR single-strand conformational polymorphism in these families or in eight individual patients who had both prostate and gastric cancer. However, a novel S270A missense mutation in exon 6 of the CDH1 gene was seen in a single family with four prostate and two gastric cancers. A large-scale population-based survey indicated a higher prevalence of S270A among both familial PCA cases (3.3%; n = 120; P = 0.01) and unselected PCA patients (1.5%; n = 472; P = 0.12) as compared with blood donors serving as population controls (0.5%; n = 923). We conclude that individual rare mutations and polymorphisms in the CDH1 gene, such as S270A, may contribute to the onset of PCA and warrant further investigations in other populations. However, the CDH1 gene does not appear to explain the link between prostate and gastric cancer.

The main extracellular matrix components of the lung, type I and III collagens, were studied in chronic allograft rejection developing in a porcine heterotopic bronchial transplantation model. Specific porcine complementary DNA probes were constructed for detection of the expression of type I and III procollagen messenger RNAs in the bronchial wall structures and in the obliterative plug by in situ hybridization. In autografts, and in allografts immunosuppressed with 40-O-(2-hydroxyethyl)-rapamycin, cyclosporine A, and methylprednisolone, no histological changes of obliterative bronchiolitis (OB) developed, and the number of fibroblast-like cells expressing type I and III procollagen mRNA remained low. In nontreated allografts obliterating within 21 d, a preponderance of fibroblast-like cells showing positivity for type III procollagen mRNA existed in the obliterative plug and bronchial wall. This study shows for the first time the temporal and spatial activation of type I and III procollagen genes during the course of obliterative bronchiolitis. The number of cells expressing procollagen III mRNA increased parallel to developing obliteration and fibrosis in nontreated allografts, whereas autografts and immunosuppressed allografts exhibited no such trend. This finding suggests a positive association between type III collagen mRNA expression in fibroblast-like cells and development of obliterative bronchiolitis.

The ELAC2/HPC2 gene at 17p11 is the first candidate gene identified for human prostate cancer (PRCA) based on linkage analysis and positional cloning (S. V. Tavtigian et al. Nat. Genet., 27:172-180, 2001). A truncating mutation was found in one hereditary prostate cancer (HPC) family, whereas two missense variants, Ser217Leu and Ala541Thr, were reported to be associated with increased PRCA risk in the general population. Here, we screened for mutations of the ELAC2/HPC2 gene in 66 Finnish HPC families. Several sequence variants, including a new exonic variant (Glu622Val) were found, but none of the mutations were truncating. We then analyzed the frequency of the three found missense variants in 1365 individuals, including hereditary (n = 107) and unselected (n = 467) PRCA, benign prostatic hyperplasia (n = 223), and population controls (568 healthy male blood donors). Ser217Leu and Ala541Thr variants carried no significantly elevated risk for HPC or PRCA, although the latter variant was associated with benign prostatic hyperplasia. The previously undescribed Glu622Val variant had a 1.0% population prevalence, but a significantly higher frequency in PRCA cases (3.0% odds ratio, 2.94; 95% confidence interval, 1.05-8.23). We conclude that ELAC2/HPC2 truncating mutations are rare in HPC, but that rare variants of the ELAC2/HPC2 require additional study as risk factors for PRCA in the general population.

Background and Aims: Results of endovascular procedures to the superficial femoral artery and popliteal artery are not as good as at the iliac level. The purpose of this study is to evaluate the long-term results of a new endoluminal stent-graft for the management of lesions of the femoropopliteal segment., Material and Methods: Fifteen patients underwent endoluminal treatment of femoropopliteal lesions with a PTFE covered stent-graft. Nine patients had critical leg ischaemia, two patients had acute leg ischaemia, two patients were claudicants, one patient had a ruptured popliteal artery aneurysm, and one had a large adjuvant therapeutic arteriovenous fistula., Results: The procedure was successfully carried out in all patients. Thrombosis occurred in two stent-grafts 4.5 months and 12.5 months after the procedure. One of them was successfully thrombolysed. During the two-year follow-up, a haemodynamically significant decrease in ankle/brachial index was observed in two patients. Ankle/brachial index was the same or higher than the immediately postoperative value in six patients. Primary patency rates at 1 month, 1 year and 2 year follow-up were 100%, 93% and 84%, respectively. Assisted primary patency rates at the same intervals were 100%, 93% and 93%, respectively. No patient had limb loss during the follow-up period., Conclusions: Endoluminal femoropopliteal bypass using PTFE covered stent-graft can be successfully performed with good two-year results.

Background: Current immunosuppressive protocols fail to prevent chronic rejection often manifested as graft vascular disease (GVD) in solid organ transplant recipients. Several new immunosuppressants including sirolimus, a dual function growth factor antagonist, have been discovered, but studies of drug efficacy have been hampered by the lack of a model of GVD in primates, as a prelude to clinical trials. As described earlier, we have developed a novel non-human primate model of GVD where progression of GVD is quantified by intravascular ultrasound (IVUS)., Methods: Twelve cynomolgus monkeys underwent aortic transplantation from blood group compatible but mixed lymphocyte reaction-mismatched donors. To allow the development of GVD in the allograft, no treatment was administered for the first 6 weeks. Six monkeys were treated orally with sirolimus from day 45 after transplantation to day 105., Results: Progression of GVD measured as change in intimal area from day 42 to 105 was halted in sirolimus-treated monkeys compared to untreated monkeys (P<0.001, general linear model). On day 105, the intimal area +/- SEM was 3.7+/-1.0 and 6.4+/-0.5 mm2, respectively (P<0.05, t test). The magnitude of allograft intimal area on day 105 correlated inversely with sirolimus trough levels (R2=0.67, P<0.05). Regression of the intimal area was seen in four of six sirolimus-treated monkeys, which was significantly different from the untreated monkeys (P<0.05)., Conclusions: Our results in the first non-human primate model of GVD showed that treatment with sirolimus not only halted the progression of preexisting GVD but also was associated with partial regression. Sirolimus trough blood levels were correlated with efficacy. Therefore, sirolimus has the potential to control clinical chronic allograft rejection.

Background: Because epithelial cells are targets of alloimmune injury leading ultimately to airway obliteration, we tested whether epithelial re-growth could prevent obliterative airway disease (OAD) in orthotopic tracheal allografts., Methods: Brown Norway tracheal segments were orthotopically transplanted into nonimmunosuppressed Lewis rats. Allografts were removed on days 2-10 (n=13), 30 (n=4), and 60 (n=5) for histology, computerized morphometry (obliteration), and immunohistochemical detection of mononuclear cells, smooth muscle alpha-actin, and tissue phenotype. Normal tracheas, host tracheas, and heterotopically transplanted allografts served as controls., Results: Orthotopic allografts removed on days 2-10 exhibited epithelial damage and re-growth and mononuclear cell infiltration. On days 30 and 60, partially ciliated cuboidal or attenuated epithelium completely covered the lumen. Although mononuclear cells declined, numerous T cells with a high CD4/CD8 ratio were found in the epithelium till day 60. Orthotopic allograft epithelium expressed donor phenotype on day 7, but recipient phenotype on days 30 and 60. Despite subepithelial alpha-actin positive myofibroblast proliferation, obliteration did not progress from day 7 to 30 and 60 (35, 30, and 33%, respectively). Although more than in normal or host tracheas, the obliteration in orthotopic allografts on days 30 and 60 was significantly less (P<0.001) than in heterotopic allografts., Conclusions: We describe, for the first time, longterm patency of fully histoincompatible orthotopic tracheal allografts in nonimmunosuppressed rats. Despite acute alloimmune injury and induction of myofibroblast proliferation, epithelial re-growth from the host limited the progression of OAD, thus emphasizing the role of epithelium in the control of airway obliteration.

Background: Graft vascular disease (GVD) is an incompletely understood process and the primary cause of late allograft failure. A nonhuman primate model was established to study the progression of GVD by using serial intravascular ultrasound (IVUS)., Methods: Aortic allografts were transplanted below the inferior mesenteric arteries (IMA) into 6 rhesus monkeys. Removed and re-implanted aortic segments between renal arteries, and the inferior mesenteric arteries served as autografts. IVUS was performed at days 0, 24, 52, 80, and 98 after transplantation. Vessel area (VA) and lumen area (LA) were measured from each cross-section at 0.5 mm intervals. Intimal index (II=100x (VA-LA/VA)) and corresponding vessel volumes were calculated for the whole grafts. Histologic features were assessed from autopsy samples using computerized morphometric method and a score from 0 to 3 for GVD (0=none, 3=severe)., Results: In allografts, vessel volume and luminal volume decreased significantly (P<0.05 for both) and the intimal index increased from 12% to 59% by day 98. These parameters remained unchanged in autografts. Histologic analysis of allografts showed concentric intimal hyperplasia and scattered mononuclear cell accumulations, whereas the autografts had only occasional eccentric intimal changes. The GVD-scores were significantly higher in allografts than in autografts (median 3 vs. 1, P=0.042)., Conclusions: We introduce a nonhuman primate model of GVD that enables serial IVUS assessments of multiple parameters of GVD. Concentric intimal proliferation and decrease of vessel dimensions was observed in allografts as a consequence of alloimmunity. This is a potential new model for studying new therapies to prevent GVD or halt its progression.

The main cause of mortality following lung transplantation is chronic rejection, manifesting morphologically as obliterative bronchiolitis (OB). It has been suggested that damage to the respiratory epithelium initiates proliferation of mesenchymal cells, leading to dense collagenous scarring in small airways. Inducible nitric oxide synthase (iNOS) is strongly expressed in the damaged epithelium in human OB, along with high levels of peroxynitrite, suggesting that endogenous NO mediates the epithelial destruction. To examine further the role of iNOS in this process, heterotopic airway implants were studied in rats, an acknowledged disease model. Specimens of iso- or allografted trachea, collected 3-60 days after implantation, were processed for histology and immunocytochemistry for iNOS and, as a marker of peroxynitrite formation, nitrotyrosine. In both iso- and allografts at the earliest stage (day 3), ischaemia was associated with severe epithelial damage or loss. These changes progressed until day 7 and were accompanied by strong expression of iNOS and nitrotyrosine in epithelial cells. In isografts, epithelial recovery was seen, with abundant iNOS immunoreactivity but little nitrotyrosine. In contrast, the epithelium in allografts did not regenerate and progressive inflammation and fibroproliferation occurred until complete obliteration of the tracheal lumen at day 60. The fibroproliferation was associated with changes in morphology of fibroblasts that were accompanied by alterations in their iNOS expression. iNOS immunoreactivity was dense in the plump fibroblasts of early lesions, in some cases as early as post-operative day 5, but very weak in elongated fibroblasts in totally occluded grafts. The intensity of immunoreactivity for nitrotyrosine corresponded to that of iNOS. These results indicate a dual role for NO in the airway obliteration that follows transplantation, through destruction of epithelium and stimulation of fibroblast activity., (Copyright 2000 John Wiley & Sons, Ltd.)

Objective: to evaluate the frequency of potential oral foci of infection in patients scheduled for elective abdominal aortic surgery., Design: prospective clinical study., Materials: oral health and dentures of 50 patients (33 males and 17 females, mean age 65 years) were examined before aortic surgery., Chief Outcome Measures: radiographic and clinical examination with special emphasis on identifying acute and chronic oral and ontogenic conditions which may contribute to aortic prosthesis infection., Results: eighty-two per cent of the patients had some oral infection foci. The mean number of remaining teeth in the cohort was 9.3, and 21% of these were potential infectious foci (62% in the patients). Twenty-six per cent of the patients suffered from oral Candida infection. Seventy-four per cent of the patients had total or partial dentures, 45% of which were ill-fitting and needed repair., Conclusions: oral infectious foci occur frequently in patients needing aortic surgery. Untreated foci may contribute to aortic prosthesis infection. Preoperative oral evaluation and elimination of intraoral infection is recommended for patients scheduled for abdominal aortic repair., (Copyright 2000 Harcourt Publishers Ltd.)

Representative animal models are needed for the study of posttransplant obliterative bronchiolitis (OB). Because human OB originates in terminal bronchi, the validity of tracheal models can be questioned. Using our hetrotopic model, we implanted pieces of a lobar bronchus subcutaneously into domestic pigs. Five groups were included: autograft implants, allograft implants, allograft implants with 2 regimens of cyclosporine (CsA)azathioprine (AZA)-methylprednisolone (MP), and allograft implants with CsA-SDZ RAD-MP. Samples were harvested at 2 weeks and at 1, 2, and 3 months. The histological findings were graded from 0 to 3. Following initial ischemic epithelial damage, autograft implants recovered, but untreated allografts and those treated with CsA-AZA-MP were totally and permanently damaged within one month. In the group treated with CsA-SDZ RAD-MP, a maximal grade 1.5 +/- 0.5 epithelial injury was seen at one month. Epithelial damage preceded and correlated with luminal obliteration. The obliterative lesions histologically resembled human OB. Differences from our previous findings with terminal bronchioles were minor. This study supports the use of larger-size airways in the study of OB.

Background: Vascular endothelial growth factor (VEGF) is a hypoxia-induced endothelial cell-specific mitogen, which is angiogenic in vivo and up-regulated in several malignancies. VEGF can be used as a prognostic marker, but the effect of surgical trauma on serum VEGF (S-VEGF) concentrations is unknown and might reduce the value of VEGF as a serum marker., Methods: We monitored S-VEGF levels by enzyme-linked immunosorbent assay in patients undergoing surgery., Results: Eighteen patients with major surgery had slightly elevated S-VEGF compared with the preoperative level (median 9.5 pg/mL) on the first (median 35 pg/mL; P = 0.0002) and third (median 19 pg/mL; P = 0.004) postoperative day, but not in later samples. The levels measured in 8 patients after minor surgery did not differ from the preoperative levels (P = 0.14)., Conclusions: Even major surgery is associated only with a slight and transient increase in S-VEGF levels, and, therefore, is unlikely to interfere markedly with the use of VEGF as a prognostic marker.

Background: Unprocessed ultrasound radiofrequency (RF) signal analysis has been shown to distinguish different tissue structures more reliably than gray-scale interpretation of conventional ultrasound images., Methods and Results: The objective of this study was to test the feasibility of in vivo intravascular ultrasound (IVUS) RF signal analysis in an animal model of allograft rejection. Six cynomolgus monkeys underwent transplantation of 3-cm aortic allograft segments distal to the renal arteries from immunologically mismatched donors. IVUS imaging with a 30-MHz system was performed 84 to 105 days after the operation. RF signals were acquired from cross sections of the recipient and the allograft aortas in real time with a digitizer at 500 MHz with 8-bit resolution. Sixty-five cross sections and 68 regions of interest (31 in host aorta and 37 in allograft) were analyzed in the adventitial layer with a total number of 8568 vectors processed. For each region of interest, a weighted-average attenuation was calculated on the basis of the attenuation and length for each individual vector. Histological examination was performed at every cross section imaged by IVUS. When the gray-scale images of conventional IVUS scored by an independent observer were compared, no distinction between adventitia of the native aorta and allograft was possible. Analysis of the average RF backscatter power also showed no significant difference (70.32+/-3.55 versus 70.72+/-3.38 dB). However, the average attenuation of allografts was significantly lower than that of the host aortas (2.64+/-1.38 versus 4.02+/-1.16 dB/mm, P<0.001). Histology demonstrated a marked adventitial inflammatory response in all allografts, with no inflammation observed in the host aortas., Conclusions: In vivo IVUS tissue characterization can be performed during routine imaging. In this model of transplant vasculopathy, RF attenuation measurements were more sensitive than visual or quantitative gray-scale analysis.

Background: Obliterative bronchiolitis (OB), the major long-term complication of lung transplantation, has thus far lacked a good large-animal model. Our goal was to develop such a model on the basis of previous rodent models with tracheal implants., Methods: Fragments of pulmonary tissue with structures of terminal bronchi were subcutaneously transplanted to four random-bred domestic piglets. Each animal received 10 autograft and 10 allograft implants. The histologic findings were graded from 0 to 3 for implants harvested repeatedly over 2 months., Results: In autografts, partial destruction of the respiratory epithelium and gradual luminal obliteration as well as mild damage to the cartilage and the bronchial wall underwent rapid reversal after initial ischemic injury. In the allografts, epithelial destruction and gradual obliteration were total within 14 days, the difference being statistically significant (P<0.05) in both. The histologic features of the obliterative plug were similar to those of human OB. In the allografts, cartilaginous destruction and pericartilaginous inflammation increased gradually to severe levels, significantly worse than in the autografts (P<0.05). Necrosis and inflammation of the bronchial wall were also more severe in the allografts (P<0.05)., Conclusions: At the end of follow-up, all autografts were vital, whereas the allografts were almost totally rejected and were without native structures. All bronchi in the allografts exhibited accelerated obliteration with histologic features characteristic of human OB, thus providing a model for research into OB and its prevention.

Objective: To investigate the emptying of solids after total gastrectomy with pouch reconstruction., Design: Randomised, prospective study., Setting: University hospital, Finland., Subjects: 49 patients with gastric carcinoma operated on for cure between 1988 and 1992., Interventions: After total gastrectomy 26 patients were randomised to have a pouch reconstruction and 23 a Roux-en-Y reconstruction. 15 months after the operation 24 patients with a pouch (pouch group) and 21 patients with a simple Roux-en-Y reconstruction (Roux-en-Y group) were alive and available to have an emptying test by the solid isotope method. Emptying of the pouch and Roux-en-Y limb was assessed by measuring the activity at 3 hours., Main Outcome Measures: Postoperative symptoms, size of meal, and postoperative nutrition., Results: Emptying was significantly slower after pouch reconstruction than after Roux-en-Y (mean activity at 3 hours 82% and 44%, respectively, p < 0.01). Early satiety occurred in 10 (48%) of the patients after Roux-en-Y reconstruction and 6 (25%) in the pouch group. Dumping was more common in the Roux-en-Y group (n = 12, 57% compared with n = 6, 25%, p = 0.04). 19 (79%) of the patients in the pouch group were able to eat normal sized meals compared with 3 (14%) in the Roux-en-Y group (p < 0.0001). Weight gain and haemoglobin and albumin concentrations were similar in the two groups., Conclusion: Jejunal pouch reconstruction after total gastrectomy delays passage of solid food in the upper intestine compared with Roux-en-Y reconstruction. It is associated with better eating capacity and fewer postoperative symptoms.

In recent years, many new immunosuppressive drugs have been discovered and developed for clinical use in transplantation. This review focuses on those drugs (leflunomide, mycophenolate mofetil, sirolimus, tacrolimus) that have been shown to have immunosuppressive activity in patients. Different anti-interleukin-2 receptor antibodies are also reviewed as an example of a resurgence of development in the area of monoclonal antibodies. The price for reducing the incidence of allograft rejection by improved immunosuppression was thought to be a proportional increase in the incidence of infection and malignancy. Data from Phase III clinical trials of new immunosuppressants, however, show a statistically significant reduction in the incidence of acute rejection produced by these new drugs, which has not been accompanied by increases in infection and malignancy rates. The wide array of new drugs offers the opportunity to use combinations that block different pathways of immune activation while at the same time selecting drug combinations with nonoverlapping toxicity profiles so that doses of each single drug can be reduced below toxicity levels. The immunosuppressive therapy for patients can be tailored according to their individual needs.

Genotypic effects of beta-casein (CN), kappa-CN, and beta-lactoglobulin (LG) on milk, fat, and protein production and fat and protein percentages were estimated for 18,686 Finnish Ayrshire cows in first lactation using an animal model. Casein genotype effects were estimated including individual beta-CN and kappa-CN simultaneously in a model and then as composite beta-kappa-CN. The A2 allele of beta-CN and the A allele of kappa-CN, as well as the A1 allele of beta-CN and the B or E allele of kappa-CN, appeared together more frequently than was expected. Because of linkage disequilibrium in the casein loci and, consequently, unbalanced data, some contradictory effects of casein genotypes were obtained with the two models. A well-founded way to estimate the effects of casein genotypes was to use beta-kappa-CN genotypes. Composite casein genotypes including the A2 allele of beta-CN were associated with the highest milk and protein production and the lowest fat content, those including the B allele of kappa-CN with the highest protein content, and those including the E allele of kappa-CN with the lowest protein content. The effect of the beta-kappa-CN genotypes on protein content was moderately strong, and the effect was somewhat smaller for other traits. The AA genotype of beta-LG had a favorable effect on milk and protein production, and the BB genotype had a favorable effect on fat content.

Background: We studied a heterotopic large-animal model with obliterative airway lesions caused by allograft rejection., Methods: Lung fragments (1 cm3) with airways (LB), and 1 to 2 mm diameter bronchi alone (B) were implanted subcutaneously in 11 domestic piglets weighing 20 kg. Six animals each received 40 implants from nonrelated donors without immunosuppression (group A). Another 5 animals had autograft implants (group U). The implants were harvested consecutively for histologic analysis over 3 months in group A and 6 months in group U., Results: In group U, the initial ischemia caused mild to moderate epithelial damage with moderate metaplasia but with a return to normal ciliary epithelium within 1 month. Transient mild luminal obliteration with granulation tissue and mononuclear cells was observed during the first weeks, but after 4 weeks the lumen was patent and filled with mucus. In the bronchial wall, moderate fibrosis developed in LB implants, whereas mild fibrosis was seen in B implants. In group A, the epithelium was totally absent by 2 weeks, and mild inflammation, fibrosis, and destruction of the cartilage with pericartilaginous mononuclear accumulation were observed in the bronchial wall. Small airways were gradually obliterated between days 7 and 21, initially by granulation tissue and mononuclear cells and later by progressive fibrosis., Conclusions: In this model, autografted airway implants stayed patent for at least 6 months, whereas total luminal obliteration histologically resembling obliterative bronchiolitis developed in allografts within 21 days. Because small airways, including bronchioli, can be transplanted with the use of this model, it may be useful for research into the causes of airway obliteration, which may be relevant to the pathogenesis of obliterative bronchiolitis in lung recipients.

Uridine diphosphate (UDP)-glucuronosyltransferases (UGTs), presently called UDP-glycosyltransferases, catalyse the detoxification of many toxic and carcinogenic compounds. Glucuronidation is also a major metabolic pathway for numerous drugs. The UGT1A6 gene (formerly known as UGT1*06 and UGT1A1) has been suggested to belong to the aryl hydrocarbon (Ah) gene battery, which consists of several genes encoding for drug-metabolising enzymes regulated by dioxin and other ligands of the Ah receptor. In this study, we analysed the localisation of UGT1A6 expression in rat liver by in situ hybridisation to mRNA. Two different RNA probes were used, one which was specific to UGT1A6 and the other against the C terminal sequence shared by all UGT1 genes. In this study, no UGT1A6 mRNA was detected in the control animals. However, other gene(s) of the UGT1 family were expressed in the perivenous region surrounding the central veins as detected by hybridisation with the probe against the common region of the UGT1 genes. Treatment with the lower dose (5 mg/kg) of 3-methylcholanthrene (3MC) induced expression of UGT1A6 perivenously. Treatment with the higher dose (25 mg/kg) of 3-Methylcholanthrene resulted in a more panacinar expression pattern. In contrast to the perivenous induction observed with 3-methylcholanthrene, treatment with 15 mg/kg of beta-naphthoflavone (BNF) resulted in strong induction in the periportal region. The results reveal an inducer-specific pattern of UGT1A6 expression similar to that demonstrated earlier for other Ah battery genes, namely CYP1A1, CYP1A2, GSTYalpha and ALDH3. The finding further supports the notion that common factors regulate the regional hepatic expression of Ah battery genes.

Objective: Our aim was to evaluate the development of changes on high-resolution CT (HRCT) associated with chronic pulmonary rejection., Materials and Methods: Repeated HRCT examinations were performed 140 times on 13 consecutive lung transplant recipients during a mean observation period of 26 months. The postoperative time interval to the first detection of each chronic change on CT was calculated and compared with the onset of chronic rejection. Bronchiolitis obliterans syndrome (BOS) or the histologic diagnosis of obliterative bronchiolitis was assessed by the published criteria of the International Society for Heart and Lung Transplantation., Results: BOS developed in eight patients, on an average, within 11.6 (+/-5.0) months. Histologic diagnosis was available from five patients. On HRCT, among the first identifiable chronic changes were volume contraction, decreased peripheral vascular and bronchial markings, and thickening of septal lines, all of which appeared between 7 (+/-5.0) and 11 (+/-6.8) months postoperatively. The mean interval for appearance of bronchodilatation was 12.5 (+/-8.7) months. Hyperlucency and mosaic phenomenon were identified, on an average, 16 (+/-6.3) and 21 (+/-7.3) months after transplantation., Conclusion: On radiologic monitoring of lung recipients with HRCT, in addition to bronchodilatation. a special attention should be paid to the early chronic changes, including diminution of peripheral bronchovascular markings, thickening of septal lines, and volume reduction, which usually precede the establishment of the diagnosis of chronic rejection, whereas hyperlucency and mosaic phenomenon usually appear during more advanced BOS.

Aim of this experimental study was to evaluate acute rejection in a porcine single-lung transplantation model by using 133Xe radiospirometry. Left lung allotransplantation was performed on 11 and autotransplantation on 5 piglets. Postoperative monitoring consisted of 77 radiospirometric examinations with assessments of regional perfusion (Qtx), ventilation (V) and ventilation/perfusion ratio (V/Qtx). Results were compared with transbronchial biopsy. A significant decrease in Qtx (p < 0.01) and an increase in V/Qtx (p < 0.01) were observed during acute rejection, whereas V remained unaltered. These changes were significant already at minimal rejection (grade A1), and further increased at moderate rejection (grade A3). Sensitivity and specificity of Qtx were 82 and 74%, and those of V/Qtx, 87 and 65%. 133Xe radiospirometry detects even minimal acute rejection after single lung transplantation.

In order to characterize the two new kappa-casein variants F and G (CSN3F and CSN3G) recently detected in Ayrshire and Pinzgauer cattle, exon IV of CSN3 from heterozygous animals was amplified by polymerase chain reaction (PCR), cloned and sequenced. The sequencing data revealed single point mutations at nucleotide positions 10530 (G-->A) for CSN3F and 10790 (C-->T) for CSN3G, corresponding to amino acid exchanges in positions 10 (Arg-->His) and 97 (Arg-->Cys) respectively. These mutations alter recognition sites for the restriction enzymes HhaI and MaeII, which were subsequently used to confirm these polymorphisms in cattle carrying CSN3F or CSN3G. A PCR-restriction fragment length polymorphism (RFLP) genotyping procedure for all currently known CSN3 alleles (CSN3A, CSN3B, CSN3C, CSN3E, CSN3F, CSN3G) was developed.

Decreased exercise capacity is the main factor restricting the daily life of patients with chronic congestive heart failure (CHF). We performed a controlled, randomized study to evaluate the effect of dynamic exercise training of moderate intensity on exercise capacity and gas exchange in patients with CHF. Twenty-seven patients with stable CHF, New York Heart Association (NYHA) functional class II and III, were randomized to training (n = 12) and control (n = 15) groups. During a 3-month period, the training group underwent a supervised physical training program using a bicycle ergometer for 30 min 3 times a week at a load corresponding to 50 to 60% of their peak oxygen consumption. Thereafter, they were advised to continue training at home for the next 3 months. The control group did not change their previous physical activity. A graded maximal exercise test with respiratory gas analysis and an endurance test with constant submaximal workload were performed at baseline and after 3 and 6 months. The exercise endurance increased from 14.7 +/- 2.0 to 27.8 +/- 2.7 min (p < 0.01) and the peak oxygen consumption tended to improve from 19.3 +/- 1.6 to 21.7 +/- 2.3 mL/kg/min (p = 0.09) during the supervised training period. At submaximal workloads, minute ventilation was reduced by 16% per se (p < 0.01) and by 7% in proportion to carbon dioxide production (p < 0.05). Oxygen consumption at the anaerobic threshold increased from 10.5 +/- 0.8 to 12.7 +/- 1.0 mL/kg/min (p < 0.05). The positive training effects were associated with an improvement in the NYHA functional class. The effects of supervised training were preserved during the home-based training period. The results indicate that physical training of moderate intensity significantly improves the exercise capacity and reduces the exaggerated ventilatory response to exercise, particularly at submaximal working levels in patients with CHF. This is associated with alleviation of symptoms.

Cross-modulation between androgen receptor (AR) and NF-kappaB/Rel proteins was studied using various androgen- and NF-kappaB-regulated reporter genes under transient transfection conditions. In COS-1 cells, elevated expression of RelA (p65) repressed AR-mediated transactivation in a dose-dependent manner, whereas NFkappaB1 (p50), another major member of the NF-kappaB family, did not influence transactivation. The repression of AR appeared to involve the N-terminal region of the protein between residue 297 and the DNA-binding domain. RelA-mediated transrepression could not be overcome by increasing the amount of AR. Transcriptional interference between RelA and AR was mutual in that cotransfected AR was able to attenuate transactivation by RelA in a dose- and steroid-dependent fashion. An excess of RelA was able to rescue the repression to some extent. Immunological analyses of RelA and AR protein levels indicated that transrepression was not due to reciprocal decrease in their amounts. Neither did AR increase the concentration of IkappaBalpha, which can sequester and inactivate RelA. Electrophoretic mobility shift assays using extracts from cotransfected cells and purified recombinant proteins showed that AR and RelA did not significantly influence each other's DNA binding activity. Nevertheless, protein-protein interaction experiments demonstrated a weak association between AR and RelA. Collectively, these data suggest that the mutual repression in intact cells is due to formation of AR-RelA complexes that are held together by another partner or to competition for a coactivator required for transcription.

Background: Chronic rejection is a major long-term complication after lung transplantation. The purpose of our study was to evaluate the role of repeated high-resolution computed tomographic examinations in monitoring the development of bronchiolitis obliterans syndrome after lung transplantation., Methods: A total of 126 high-resolution computed tomographic examination in 13 lung transplant recipients was analyzed. During a mean follow-up period of 23 months, bronchiolitis obliterans syndrome developed in eight of the patients. A scoring system from 0 to 10 based on the number of chronic changes on high-resolution computed tomography was developed, and the score of each patient was compared with decline in the forced expiratory volume in 1 second and maximal forced expiratory flow rate of 50% of the forced vital capacity., Results: The score of chronic changes, measured at 1 year after transplantation, correlated inversely with the values of forced expiratory volume in 1 second and maximal forced expiratory flow rate at 50% of the forced vital capacity (p < 0.05). Stage I bronchiolitis obliterans syndrome was associated with scores of 4 to 6 (mean 5.0), stage 2 with scores of 6 to 9 (mean 7.0), and stage 3 with scores of 6 to 9 (mean 7.7). The sensitivity of high-resolution computed tomography was 93% and its specificity was 92% when five chronic changes were used as a cutoff level., Conclusions: The progress of chronic changes on high-resolution computed tomography occurs concurrently with the development of bronchiolitis obliterans syndrome. High-resolution computed tomography may provide additional morphologic information for noninvasive evaluation of chronic lung rejection.

In single-lung transplant recipients, the usefulness of spirometric indexes in detecting acute events involving the lung graft is limited due to the bias caused by the native lung. Selective functional monitoring is needed for the proper evaluation of complications after transplantation, but thus far, to our knowledge, no clinically feasible methods for selective graft-function assessment have been presented. In ten single-lung recipients, of whom six had a parenchymal lung disease and four had pulmonary hypertension, the relative ventilation (Vtx), perfusion (Qtx), and ventilation/perfusion ratio of the transplanted lung (V/Qtx) were determined with multidetector 133Xe radiospirometry. Additionally, the fractions of FEV1, FVC, and diffusing capacity for carbon monoxide (Dco) of the transplant (FEV1tx, FVCtx, Dcotx, respectively) were determined by using corresponding radiospirometric parameters for the calculation of their distribution between the lungs. The analysis included seven episodes of acute rejection and nine episodes of infection. The Qtx decreased during acute rejection but did not change during infection (p=0.001). Compared with the figures during infection, the V/Qtx increased during acute rejection significantly (p<0.05) in patients with underlying fibrosis or emphysema, but not in those with pulmonary hypertension. In detection of acute events, the sensitivity of the selective parameters, ie, FEV1tx (86%) and FVCtx (73%), was higher than that of the sum-function parameters, FEV1 (66%) and FVC (40%). Moreover, the sensitivity of Dcotx (80%) was higher than that of Dco (60%) in detecting acute rejection. The findings indicate that, in single-lung recipients with a parenchymal lung disease, the assessment of Qtx, V/Qtx, and Dcotx with a radioactive tracer can help to distinguish acute rejection from infection. The graft-selective parameters, ie, FEV1tx, FVCtx, and Dcotx, tended to be more sensitive than the corresponding sum-function parameters in detecting acute events, thus providing a more accurate functional profile of the single-lung graft.

Background: Staging of bronchiolitis obliterans syndrome is based on the decline of forced expiratory volume in 1 second, a measure of overall ventilatory capacity. A single staging system is applied to all lung recipients, regardless of the bias which can be caused by the native lung after single lung transplantation., Methods: We determined the decline of graft function in single lung recipients by a combination of two methods: 133-Xe radiospirometry and dynamic spirometry. The forced expiratory volume in 1 second fraction of the transplant (FEV1tx) was calculated from the proportion of ventilation of the transplant (Vtx) and forced expiratory volume in 1 second. Eight single lung recipients were followed up for a median observation period of 17 months; bronchiolitis obliterans syndrome developed in four of them., Results: The fractional decline of forced expiratory volume in 1 second of the transplant was significantly greater than the decline of forced expiratory volume in 1 second (p = 0.016) in all patients during the follow-up. In the patients with bronchiolitis obliterans syndrome, the mean decline in forced expiratory volume in 1 second was 1.1 L (39.5%), and in forced expiratory volume in 1 second of the transplant it was 0.9 L (55.8%). The measurement of forced expiratory volume in 1 second of the transplant suggested stage 2a and 3a dysfunction in two grafts in which the assessment of forced expiratory volume in 1 second indicated stage 1a bronchiolitis obliterans syndrome. In one patient, decrease of forced expiratory volume in 1 second of the transplant was suggestive of chronic dysfunction 4 months before it was diagnosed by biopsy and declined lung function., Conclusions: The assessment of forced expiratory volume in 1 second seems to underestimate the severity of chronic dysfunction in single lung grafts. Instead, the determination of forced expiratory volume in 1 second of the transplant with radioactive tracers provides selective information of the graft function, which could be used for clinical evaluation of bronchiolitis obliterans syndrome in single lung recipients.

Thoracoscopic fundoplication and hiatoplasty were performed by a videoassisted technique in a patient with longstanding pathologic gastro-oesophageal reflux, reflux oesophagitis and hiatal hernia. Ten months later the patient was asymptomatic and had normal findings by oesophageal endoscopy and 24-hour pH-monitoring. Thoracoscopic fundoplication may be useful in patients with intra-abdominal adhesions due to previous abdominal surgery. Also obese patients with large hiatal hernias and perioesophageal adhesions may benefit from the thoracoscopic approach.