Abscisic acid, cold and salt stimulate conserved metabolic regulation in the moss Physcomitrella patens.

Salt and cold are major abiotic stresses that have adverse effects on plant growth and development. To cope with these stresses and their detrimental effects plants have evolved several metabolic, biochemical and physiological processes that are mainly triggered and mediated by the plant hormone abscisic acid (ABA). To elucidate the metabolic responses of the moss Physcomitrella patens, which serves as a model plant for abiotic stress adaptation, we performed GC-MS-based metabolic profiling of plants challenged for 5 and 28 h with either salt, cold or ABA. Our results indicate significant changes in the accumulation of several sugars including maltose, isomaltose and trehalose, amino acids including arginine, histidine, ornithine, tryptophan and tyrosine, and organic acids mainly citric acid and malonic acid. The metabolic responses provoked by ABA, cold and salt show considerable similarities. The accumulation of certain metabolites positively correlates with gene expression data whereas some metabolites do not show correlation with cognate transcript abundance. To place our results into an evolutionary context we compared the ABA- and stress-induced metabolic changes in moss to available metabolic profiles of the seed plant Arabidopsis thaliana. We detected considerable conservation between the species, indicating early evolution of stress-associated metabolic adaptations that probably occurred at the plant water-to-land transition.

Bcl-xL anti-apoptotic network is dispensable for development and maintenance of CML but is required for disease progression where it represents a new therapeutic target.

The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the development of drug resistance. Altered expression of the anti-apoptoticBcl-xL has been correlated with BCR-ABL leukemogenesis; however, its involvement in the pathogenesis and evolution of CML has not been formally demonstrated yet. Thus, we generated an inducible mouse model in which simultaneous expression of p210-BCR-ABL1 and deletion of bcl-x occurs within hematopoietic stem and progenitor cells. Absence of Bcl-xL did not affect development of the chronic phase-like myeloproliferative disease, but none of the deficient mice progressed to an advanced phenotype, suggesting the importance of Bcl-xL in survival of progressing early progenitor cells. Indeed, pharmacological antagonism of Bcl-xL, with ABT-263, combined with PP242-induced activation of BAD markedly augmented apoptosis of CML-BC cell lines and primary CD34(+) progenitors but not those from healthy donors, regardless of drug resistance induced by bone marrow stromal cell-generated signals. Moreover, studies in which BAD or Bcl-xL expression was molecularly altered strongly support their involvement in ABT-263/PP242-induced apoptosis of CML-BC progenitors. Thus, suppression of the antiapoptotic potential of Bcl-xL together with BAD activation represents an effective pharmacological approach for patients undergoing blastic transformation.

The natural history of clinical operational tolerance after kidney transplantation through twenty-seven cases.

We report here on a European cohort of 27 kidney transplant recipients displaying operational tolerance, compared to two cohorts of matched kidney transplant recipients under immunosuppression and patients who stopped immunosuppressive drugs and presented with rejection. We report that a lower proportion of operationally tolerant patients received induction therapy (52% without induction therapy vs. 78.3%[p = 0.0455] and 96.7%[p = 0.0001], respectively), a difference likely due to the higher proportion (18.5%) of HLA matched recipients in the tolerant cohort. These patients were also significantly older at the time of transplantation (p = 0.0211) and immunosuppression withdrawal (p = 0.0002) than recipients who rejected their graft after weaning. Finally, these patients were at lower risk of infectious disease. Among the 27 patients defined as operationally tolerant at the time of inclusion, 19 still display stable graft function (mean 9 ± 4 years after transplantation) whereas 30% presented slow deterioration of graft function. Six of these patients tested positive for pre-graft anti-HLA antibodies. Biopsy histology studies revealed an active immunologically driven mechanism for half of them, associated with DSA in the absence of C4d. This study suggests that operational tolerance can persist as a robust phenomenon, although eventual graft loss does occur in some patients, particularly in the setting of donor-specific alloantibody.

Biomarkers for the diagnosis of the stable kidney transplant and chronic transplant injury using the ProtoArray® technology.

Transplant glomerulopathy (TG), a form of chronic renal transplant rejection, carries a poor prognosis. It must be differentiated from the entity defined by the Banff '05 classification, interstitial fibrosis/tubular atrophy (IF/TA). Sequential transplant biopsies have shown that these lesions are subclinical long before clinical manifestations. The availability of biomarkers may provide an earlier diagnosis and subsequent treatment. The aim of our study was to identify serum biomarkers in kidney recipients showing TG compared with IF/TA or stable patients, using protein microarray technology. This technology detects auto- or alloantibodies in patient sera. With a high degree of statistical significance, we identified 18 antibody reactivities specific for TG; 11 for IF/TA; and 10 among stable patients. Target proteins were involved in signal transduction, transcription regulation, DNA replication and repair, cell cycle, endocytosis, cell redox, as well as glycolysis. Some markers, such as podocan and collagen XXIII among TG and tubular cell ion channels among IF/TA, possibly provide insights into the pathogenesis of the lesions.

Outbreak of cyclosporiasis in British Columbia associated with imported Thai basil.

Sporadic outbreaks of cyclosporiasis, a common cause of protracted diarrhoea in underdeveloped countries, are often undetected and undiagnosed in industrial countries. In May 2001, an outbreak of Cyclospora cayetanensis gastroenteritis was identified in British Columbia, Canada, with 17 reported cases. We conducted a case-control study involving 12 out of the 17 reported and confirmed case patients. Eleven (92%) of the patients had consumed Thai basil, an essential ingredient in Vietnamese cuisine, compared to 3 out of 16 (19%) of the control patients (P = 0.003). Trace-back investigations implicated Thai basil imported via the United States as the vehicle for this outbreak. This is the first documented sporadic outbreak of cyclosporiasis linked to Thai basil in Canada, and the first outbreak of cyclosporiasis identified in an ethnic immigrant population. This outbreak provides the opportunity to increase our understanding of this emerging pathogen and improve on our prevention and control for future outbreaks.

Identification of BARD1 as mediator between proapoptotic stress and p53-dependent apoptosis.

The BRCA1-associated protein BARD1 is a putative tumor suppressor. We suggest that BARD1 is a mediator of apoptosis since (1) cell death in vivo (ischemic stroke) and in vitro is accompanied by increased levels of BARD1 protein and mRNA; (2) overexpression of BARD1 induces cell death with all features of apoptosis; and (3) BARD1-repressed cells are defective for the apoptotic response to genotoxic stress. The proapoptotic activity of BARD1 involves binding to and elevations of p53. BRCA1 is not required for but partially counteracts apoptosis induction by BARD1. A tumor-associated mutation Q564H of BARD1 is defective in apoptosis induction, thus suggesting a role of BARD1 in tumor suppression by mediating the signaling from proapoptotic stress toward induction of apoptosis.

Management of shotgun injuries to the pelvis and lower genitourinary system.

OBJECTIVES: Shotgun injuries are rare, with the extent of injury best determined at time of surgical exploration. There are no defined workup or management guidelines for patients with shotgun injuries to the genitourinary system. Injuries are usually treated on an individual basis. This study was conducted to determine the management and extent of genitourinary tract injuries in 10 patients with shotgun injuries to the pelvis during a 6-year interval. METHODS: Between September 1990 and December 1996, 140 patients were treated for firearm injuries to the lower genitourinary tract, of which 10 were secondary to shotgun blasts. We performed a retrospective hospital and clinic chart review and telephone interview to assess organs injured, initial treatment, follow-up surgeries, mortality, and erectile function. RESULTS: Mean patient age was 20 years at the time of the injury. The mean follow-up was 4 years (range 1 to 7). Two patients died, both with major vascular injuries, one in the operating room and the other 1 week later from sepsis. Eight patients underwent radiographic examinations (1 intravenous urogram and 7 urethrocystograms). The bladder was injured in 5 patients, 2 with concomitant complete posterior urethral transection. Of the 5 patients without bladder injury, one had an incomplete penile urethral injury and one had a complete bulbar urethral transection. The initial management consisted of repairing nongenitourinary injuries in 8 cases (80%), most commonly involving injuries to the rectum and small bowel. All patients were treated operatively, including 8 who required laparotomy and 4 who required suprapubic cystotomy. A total of four urethral injuries were noted. Subsequent reconstructive surgeries included two urethroplasties and one permanent supravesical diversion for 3 patients with extensive urethral loss. Erectile dysfunction was present in 3 of 6 patients available for telephone interview. CONCLUSIONS: Shotgun injuries involving the lower genitourinary tract are associated with significant soft tissue injury and morbidity. Death usually results from major associated vascular injuries. All hemodynamically stable patients should undergo retrograde urethrograms and cystograms to evaluate possible urethral and bladder injuries. Open primary repair should be attempted for distal urethral, testicular, and corporal injuries. Delayed repair with staged urethral reconstruction should be reserved for patients with extensive loss of urethral tissue. Impotence is common in patients with extensive perineal injuries.

Identification of an apoptotic cleavage product of BARD1 as an autoantigen: a potential factor in the antitumoral response mediated by apoptotic bodies.

We have shown previously that rats can be cured from induced peritoneal colon carcinomatosis by injections of apoptotic bodies derived from tumor cells and interleukin 2. This curative treatment generated a tumor-specific cytotoxic T-cell response associated with a humoral response. Autoantibodies from sera of cured rats strongly recognized a Mr 67,000 protein from apoptotic bodies and weakly reacted with a protein of Mr approximately 97,000 in PROb parental cells. We now show that these autoantibodies are directed against BARD1, originally identified as a protein interacting with the product of the breast cancer gene 1, BRCA1. We demonstrate that the Mr 67,000 antigen is a cleaved form of BARD1 present in apoptotic bodies derived from rat and human colon and mammary carcinoma cell lines. Moreover, we show that the cleavage site of BARD1 is located NH2 terminally but downstream of the RING domain essential for BARD1 and BRCA1 protein interaction. In vitro studies using [35S]methionine-labeled human BARD1 and apoptotic cellular extracts derived from SW48 carcinoma cells indicate that BARD1 proteolysis occurs at an early stage of apoptosis and in a cell cycle-dependent manner. This hydrolysis is inhibited by EGTA, and the calpain inhibitor I, N-acetyl-leu-leu-norleucinal, but not by several caspases inhibitors, suggesting that BARD1 is hydrolyzed by the calcium-dependent cysteine proteases, calpains. Thus, the highly immunogenic form of cleaved BARD1 could contribute to the antitumoral response mediated by apoptotic bodies.

Production and characterisation of a monoclonal antibody specific for apoptotic bodies derived from several tumour cell lines.

We have recently shown that apoptotic bodies (apobodies) derived from rat colon carcinoma cell lines (PROb) after sodium butyrate (NaB) treatment were able to cure rats with induced peritoneal carcinomatosis ( [BOISTEAU] ). A specific immune response was assumed to be involved since the serum of cured rats contained antibodies specific for apobodies. In the present study, a mAb (clone 6E8) produced by immunisation of rats with apobodies strongly recognized apobodies but had little reactivity with parental tumour cell lines, as demonstrated by enzyme-linked immunosorbent assay (ELISA), immunostaining and flow cytometry. Immunoelectron microscopy showed that 6E8 mAb mainly stained the hyaloplasm or cytosol of apobodies. A protein was detected at 67 kDa by immunoprecipitation of apobodies with mAb, followed by immunoblotting, using serum of rats immunised with apobodies. The 6E8 mAb recognized apobodies derived from several rat or human colon cancer cell lines and a rat glioma cell line, regardless of the apoptosis stimulus used (NaB, staurosporine or UV). Our results clearly show that 6E8 mAb defines an epitope specifically generated during apoptosis, which suggests that the protein recognized may be involved in the molecular cascade of apoptotic cell death.

Severe combined immunodeficient-hu model of human prostate cancer metastasis to human bone.

Commonly used in vivo models of prostate cancer metastasis include syngeneic rodent cancers and xenografts of human cancer in immunodeficient mice. However, the occurrence of osseous metastases in these models is rare, and in xenograft models, species-specific factors may limit the ability of human cells to metastasize to rodent bones. We have modified the severe combined immunodeficient (SCID)-human model to test the ability of circulating human prostate cancer cells to home to macroscopic fragments of human bone and other organs previously implanted into SCID mice. We have also compared the growth of human prostate cancer cells in various human and mouse tissue microenvironments in vivo. Macroscopic fragments of human fetal bone, lung, or intestine (16-22 weeks gestation) or mouse bone were implanted s.c. into male CB.17 SCID mice. Four weeks later, human prostate cancer cells were injected either i.v. via the tail vein (circulating cell colonization assay) or directly into the implanted tissue fragments transdermally (end organ growth assay). Tumor growth was followed for 6 weeks by palpation and magnetic resonance imaging. After 6 weeks, tumors were enumerated in implanted human and mouse organ fragments and native mouse tissue. Tumors were characterized by histology, immunohistochemistry, and chromosomal analysis. After i.v. injection, circulating PC3 cells successfully colonized implanted human bone fragments in 5 of 19 mice. Tumors were easily followed by palpation and imaging and had an average volume of 258 mm3 at autopsy. Histological examination revealed osteolysis and a strong desmoplastic stromal response, which indicated intense stromal-epithelial interaction. Bone tumors were subcultured, and chromosomal analysis demonstrated that the tumors were derived from the parental prostate cancer cell line. Microscopic tumor colonies were also found in a few mouse lungs after i.v. injection of PC3, DU145, and LNCaP cells, however the volume of the lung nodules was less than 1 mm3 in all of the cases. No colonization of human lung or intestine implants, the mouse skeleton, or other mouse organs was detected, demonstrating a species- and tissue-specific colonization of human bone by PC3 cells. Direct injection of 10(4) prostate cancer cells into human bone implants resulted in large tumors in 75-100% of mice. PC3 and DU145 bone tumors were primarily osteolytic, whereas LNCaP bone tumors were both osteoblastic and osteolytic. PC3 and LNCaP bone tumors showed a desmoplastic stromal response, which indicated intense stromal-epithelial interaction. All three of the cell lines formed tumors in implanted human lung tissue; however, the tumors were all < or = 10 mm3 in volume and showed minimal stromal involvement. No tumors formed after either s.c. injection or injection of cells into implanted mouse bone demonstrating both species- and tissue-specific enhancement of growth of human prostate cancer cells by human bone. The severe combined immunodeficient-human model provides a useful system to study species-specific mechanisms involved in the homing of human prostate cancer cells to human bone and the growth of human prostate cancer cells in human bone.

Ureterocele arising from a lower-pole moiety.

We report on an uncommon case of a ureterocele arising from the lower-pole ureter in a duplex system. To our knowledge, this represents the 3rd such case reported in the English literature. Ultrasonography and retrograde pyelogram established the diagnosis. The patient underwent left upper-pole nephroureterectomy with excision of the ureterocele and cross-trigonal ureteral reimplantation.

Apoptosis induced by sodium butyrate treatment increases immunogenicity of a rat colon tumor cell line.

We have recently demonstrated that a treatment combining the cell differentiating agent sodium butyrate (NaBut) and interleukin-2 (IL2) resulted in a remission of established peritoneal colorectal carcinomatosis in rats. NaBut or IL2 treatment alone, never cured these tumour-bearing rats. In the present investigation, we report that NaBut-treatments induce apoptosis in the colonic cancer cells both in vitro and in vivo. We postulated that the significant therapeutic effect of NaBut/IL2 treatment can be mainly attributed to a NaBut-induced apoptosis of the tumoural cells increasing their immunogenicity. Indeed, treatment which combined apoptotic bodies (apobodies) as cell vaccine, plus IL2 immunotherapy significantly increased tumour remission and survival rate of the vaccinated rats, whereas IL2 treatment alone did not. We observed that the cured rats presented long-term protection against subsequent challenge with the parental tumour cells. This latter result suggests that these treatments generate an immune protection. This was confirmed by the presence, in the sera of the cured rats, of anti-tumoural antibodies directed against both the apobodies and the tumour cells, but not against normal colonocytes. In addition, we show that injections of apobodies before administration of the parental tumour cells results in a partial protection. We provide the first evidence that apobodies, derived from cancer cells after NaBut-treatment, induce a specific immune response against parental tumours cells. These data suggest that the distinctive immunologic properties of apobodies could provide a valuable tool in colorectal cancer immunotherapy.

Asphyxial death caused by a laryngeal schwannoma: a case report.

Neurogenic tumours of the larynx are unusual, with approximately 115 cases reported in the literature to date. Most of these lesions are benign, solitary submucosal nodules which present with hoarseness and are amenable to surgical resection. We present a case of a large pedunculated schwannoma arising in the aryepiglottic fold associated with sudden asphyxial death in an otherwise healthy young female.

Induction of fibroblast gelatinase B expression by direct contact with cell lines derived from primary tumor but not from metastases.

During cancer progression, tumor cells interact with stromal cells. As a consequence, matrix metalloproteinases are produced that contribute to the degradation of the extracellular matrix. This study used coculture systems to investigate fibroblast interaction with three colon cancer cell lines isolated from a single patient. Cells from primary colorectal carcinoma, but not from corresponding liver or lymph node metastases, induced gelatinase B expression by fibroblasts of different tissue origin. Remarkably, direct cell-cell contact was required for this induction, which occurred at the pretranslational level (as revealed by Northern blot analysis) and was completely blocked by anti-beta1 integrin monoclonal antibody, but only partially blocked by anti-alpha5 or anti-alpha(v). Induction was also inhibited by cytochalasin D, staurosporine, or dexamethasone, suggesting the need, respectively, for an organized actin cytoskeleton, protein kinase C, and AP-1-driven gene transcription. Our data suggest that direct tumor-stromal cell contact is one inductive event involved in matrix metalloproteinase expression by stromal cells.