First Report of Stagonosporiopsis cucurbitacearum Causing Fruit Rot of Luffa cylindrica in Brazil.

Luffa cylindrica (Cucurbitaceae) is an Asian vine widely known as the source of loofah (4). In Brazil (local name bucha), it is cultivated by small scale producers as a cash crop. In January 2012, samples of fruits were collected in three areas in the municipality of Cipotânea, state of Minas Gerais (Brazil) bearing rot symptoms. These had large necrotic areas with a grayish epidermis and slightly sunken tissue. Internally, the fibrous parts were necrosed, darkened, and unmarketable. Isolations by surface sterilization of necrotic tissue with 10% bleach and plating onto potato dextrose agar yielded colonies with consistent morphology. A representative culture was deposited in the culture collection of the Universidade Federal de Viçosa (UFV) as COAD1119. Inoculations of seven healthy-appearing L. cylindrica fruits were performed with culture disks obtained from 4-day-old cultures grown on PDA, which were placed onto two points on the epidermis of each of seven fruits. Each point was either intact or previously injured with a sterile needle. Controls consisted of two fruits treated equally but with tap water agar disks in place of fungal inoculum. Fruits were then placed on trays with water-soaked cotton and the trays were wrapped in plastic bags and left over a bench at room temperature for 2 days. The plastic bags were then removed. After 5 days, necrosis was evident and fungal fruit bodies appeared at points with injury. No symptoms appeared on controls. Isolation from diseased tissue yielded colonies identical to those of the inoculated fungus. A dried sample was deposited in the local herbarium at UFV (VIC 32053). Slides were mounted in lactophenol and observed. The fungus had subepidermal perithecia, globose to subglobose, from 75.5 to 134 µm diam.; asci bitunicate, cylindrical, 8-spored; pseudoparaphyses filiform; ascospores fusoid to ellipsoidal, from 26 to 45 µm long and 8 to 11.5 µm wide, one septate, and hyaline. This morphology is consistent with Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae) (3), a broad spectrum pathogen of cucurbits. Genomic DNA was extracted from the isolate growing in pure culture and ITS and LSU sequences were generated and deposited in GenBank under the accession numbers KC582022 and KC582021, respectively. Sequences were compared in BLASTn with other entries in GenBank, and the closest match for each region were S. cucurbitacearum strain CAP14C and D. bryoniae strain CBS 133.96 (JQ936326 and GU456335) with 100% of nucleotide homology for ITS and 100% of nucleotide homology for LSU. Cercospora citrullina and C. cucurbitae have been reported in Brazil on L. cylindrica and mistakenly indicated as synonyms of D. bryoniae (2). To our knowledge, this is the first valid report of S. cucurbitacearum causing fruit rot of loofah in Brazil and the first time pathogenicity to this host has been demonstrated. Losses due to the disease on the crop were reported to be high by growers and management to be difficult since there are no fungicides registered for this crop in Brazil. References: (1) M. M. Aveskamp et al. Stud. Mycol 65:1, 2010. (2) M. A. S. Mendes and A. F. Urben. Fungos em Plantas no Brasil. Brasília, Brazil: EMBRAPA-SPI. Retrieved from http://pragawall.cenargen.embrapa.br/aiqweb/michtml/micbanco01a.asp , 2012. (3) E. Puithalingam and P. Holliday. CMI Descriptions of Pathogenic Fungi and Bacteria 332:1, 1972. (4) J. W. Purseglove. Tropical Crops - Dicotyledons. Longman Group, London, 1968.

Comparisons between two monoclonal antibodies that bind to the same antigen but have differing affinities: uptake kinetics and 125I-antibody therapy efficacy in multicell spheroids.

It has been predicted that low affinity antibodies (Abs) should penetrate into tumors more readily than high affinity Abs. However, the absolute uptake and residence time of a high affinity Ab may be better. It is, therefore, not clear whether a high affinity Ab would have a therapeutic advantage. This is particularly relevant with 125I radioimmunotherapy, where targeting of every cell is important. This study compared the uptake kinetics and toxicity in multicell spheroids of two murine monoclonal Abs labeled with 125I. 17-1A was produced by immunization with a human colon cancer cell line and has an affinity of 5.15 x 10(7) M-1. 323/A3 was produced by immunization with a human breast cancer cell line and has an affinity of 1.87 x 10(9) M-1. Binding of both Abs to LS174T spheroids was similar at 4 degrees C, but binding of 17-1A was 8-10-fold less than that of 323/A3 at 37 degrees C. Despite this difference, the toxicity of 125I-17-1A in spheroids after 7 days of incubation was similar to that of 125I-323/A3. Autoradiography showed that 17-1A penetrated the spheroids much more deeply and evenly than did 323/A3. It appears that much of the radiation dose to spheroids treated with 125I-323/A3 was wasted because of the uneven Ab distribution. This study demonstrates the potential advantage of using Abs of lower affinity for 125I radioimmunotherapy, because of their more even distribution. It also suggests that a large number of binding sites per cell may be a disadvantage if more 125I is bound than is necessary to kill the cell, because this may slow Ab penetration.

Local hyperthermia and SR 4233 enhance the antitumor effects of radioimmunotherapy in nude mice with human colonic adenocarcinoma xenografts.

Local hyperthermia and the hypoxic cytotoxin SR 4233 were administered to nude mice with 693 +/- 47 mm3 (mean +/- SE) s.c. HCT-8 human colonic adenocarcinoma xenografts in an attempt to enhance the antitumor effects of radioimmunotherapy. Biodistribution studies revealed preferential binding of NR-Lu-10, a murine monoclonal antibody, to the tumors compared with an isotype-matched control antibody, CCOO16-3.A single injection of 25 microCi 90Y-NR-Lu-10 significantly inhibited tumor growth (control versus 90Y-NR-Lu-10: P = 0.048). The administration of hyperthermia at 41.5 degrees C for 1 h immediately following the injection of 111In-labeled NR-Lu-10 up-regulated tumor-associated antigen expression and increased antibody uptake in the tumors by 73% (P = 0.001) without significantly affecting antibody uptake in normal tissues. However, the heat treatment did not produce a more homogeneous distribution of the antibodies in the tumors and did not significantly enhance the tumor growth delay produced by 90Y-NR-Lu-10 (P = 0.07). The administration of local hyperthermia at 43.0 degrees C for 1 h, on the other hand, had direct cytotoxic effects (P = 0.03) and enhanced the tumor growth delay produced by 90Y-NR-Lu-10 (P = 0.01). SR 4233 also enhanced the tumor growth delay produced by 90Y-NR-Lu-10 (P = 0.03). The greatest antitumor effects were observed when both hyperthermia at 43.0 degrees C and SR 4233 were administered in combination with 90Y-NR-Lu-10 (P = 0.002). No toxicity was produced by the local hyperthermia, and the only toxicities produced by 90Y-NR-Lu-10 and SR 4233 were neutropenia and weight loss.

Synergistic interaction between tirapazamine and cyclophosphamide in human breast cancer xenografts.

This study examined the efficacy of combining cyclophosphamide and the hypoxic cytotoxin, tirapazamine, in the treatment of human breast cancer xenografts grown in nude mice. A single dose of tirapazamine was followed 2 h later by a single dose of cyclophosphamide. As determined from tumor regrowth delay, the effectiveness of combined therapy was greater than the additive effects of each treatment given alone. Possible mechanisms of this synergistic interaction include enhancement of DNA damage, inhibition of repair of DNA damage, or induction of apoptosis. Apart from some loss in body weight, the only other toxicity of interest in mice treated with tirapazamine was necrosis of the skin on the distal tail, which appeared to be vascular in origin.

Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker.

We have investigated the hypoxia inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells using a monoclonal antibody to a fluorinated bioreductive drug, EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide], a chemical probe for hypoxia. We have shown that VEGF expression is predominantly localized in interior spheroid cells that are sufficiently hypoxic to bioreductively activate the 2-nitroimidazole and produce immunologically detectable adducts of the EF5 compound. Northern blotting analyses demonstrated that VEGF165 is the predominant form of VEGF produced by HT29 cells and that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate did not induce VEGF expression. This study demonstrates that VEGF expression is up-regulated in response to hypoxia and in the microenvironments found in human multicellular tumor spheroids. This investigation also illustrates the utility of the EF5 binding in multi-cellular tumor spheroids as a means of studying the expression and regulation of hypoxia-inducible genes.

Comparisons of the efficacy of 186Re- and 131I-labeled antibody in multicell spheroids.

Several radionuclides are being studied for use in radioimmunotherapy. Although 131I has been used most widely, there are several disadvantages to it including its large gamma-ray component, its rather long half-life, and its modest beta-particle energy. However, the beta-particle energy can be an advantage in very small tumors (less than 1-2 mm). 186Re has several potential advantages over 131I but it has never been directly compared with it experimentally. Dosimetry modeling predicted that 186Re would have a dose advantage over 131I at large tumor sizes but for tumors as small as 1 mm diameter, this advantage would be lost. In order to confirm these predictions experimentally, this study compared the relative efficacy of a pancarcinoma antibody, NR-LU-10 labeled with 186Re or 131I in 0.8-1.0 mm diameter LS174T human colon adenocarcinoma multicell spheroids. Spheroids were incubated for 90 hr at 37 degrees C and evaluated with clonogenic assay, autoradiography, and histology. When corrected for cumulative activity bound, both radionuclides were equally effective. Autoradiography demonstrated poor penetration of radionuclide into the depths of the spheroids. Because 186Re has a theoretical dose advantage in larger tumors and because it has been shown to be equivalent to 131I in tumors as small as 1 mm diameter, it may be superior to 131I in most clinical situations. However, in the treatment of micrometastases of less than 1 mm diameter, theoretical dosimetry modeling predicts that 131I or radionuclides with similar beta particle energies should be more effective.

Fused pyrazine mono-n-oxides as bioreductive drugs. II Cytotoxicity in human cells and oncogenicity in a rodent transformation assay.

PURPOSE: To determine what structural moieties of the fused pyrazine mono-N-oxides are determining factors in their in vitro cytotoxicity and oncogenicity. METHODS AND MATERIALS: A new series of experimental bioreductive drugs, fused pyrazine mono-N-oxides, was evaluated in vitro for aerobic and hypoxic cytotoxicity in the HT29 human colon adenocarcinoma cell line by using clonogenic assays. The relative oncogenicities of these compounds were also determined in aerobic cultures of C3H 10T1/2 mouse embryo fibroblasts by using a standard transformation assay. RESULTS: Removal of the 4-methyl piperazine side chain from the parent compound, RB 90740, reduced the potency of the hypoxic cytotoxin. Reduction of the N-oxide function increased the aerobic cytotoxicity and eliminated most of the hypoxic/aerobic cytotoxic differential. The reduced N-oxide also had significant oncogenicity, consistent with a mechanism of genotoxicity following bioreduction of RB 90740. CONCLUSION: This new series of bioreductive compounds may be effective in cancer therapy, particularly the lead compound RB 90740. The oncogenic potential of these compounds is similar to that for other cancer therapies. Further studies should include evaluation of these compounds in vivo and the development of analogs with reduced oncogenic potential and retention of the hypoxic/aerobic cytotoxicity differential.

The combined use of 131I-labeled antibody and the hypoxic cytotoxin SR 4233 in vitro and in vivo.

Radioimmunotherapy is hindered by the slow penetration of antibody molecules into tumors. Cells that are poorly targeted by antibody, because of their distance from feeding blood vessels, receive the lowest radiation dose, and this problem is compounded if there are radioresistant hypoxic cells present. It would be desirable to combine radioimmunotherapy with an agent that is preferentially toxic to these cells. SR 4233 is a potent hypoxic cytotoxin, and it was combined with 131I-NR-LU-10 to treat LS174T human colon adenocarcinoma multicell spheroids and nude mouse xenografts for these studies. Under conditions of severe hypoxia (< 0.01% O2), 2 h of pretreatment or 18 h of simultaneous treatment with SR 4233 did not significantly enhance the effectiveness of 131I-NR-LU-10 in spheroids. However, under aerobic conditions with a 10% fraction of hypoxic cells, there was more toxicity than would be predicted from simple additivity. Xenografts treated with 131I-NR-LU-10 + SR 4233 had a growth delay that was significantly longer than that achieved with 131I-NR-LU-10 alone. In both spheroids and xenografts, combined treatment produced about 10 times more cell killing than 131I-NR-LU-10 alone. The lack of enhancement in spheroids under complete hypoxia suggests that SR 4233 does not sensitize hypoxic cells to radiation damage. The results with aerobic spheroids and in vivo, where a portion of the cells were hypoxic, could be explained by the targeting of different cell populations (hypoxic and aerobic) by each therapeutic modality. This effect should also be enhanced by reoxygenation and reestablishment of the hypoxic fraction during treatment, thus allowing more than the initially hypoxic fraction of cells to be killed by the SR 4233.

Susceptibility of captive adult winter-run Chinook salmon Oncorhynchus tshawytscha to waterborne exposures with infectious hematopoietic necrosis virus (IHNV).

Sexually mature female Chinook salmon Oncorhynchus tshawytscha with no prior history of exposure to infectious hematopoietic necrosis virus (IHNV) were susceptible to experimental infection induced by additions of virus to the water. The resulting infections resembled those observed among naturally infected hatchery and wild populations of Chinook salmon. Virus was detected as early as 4 d post-exposure (p.e.) and subsequently in all virus-exposed fish that died or that were examined at 14 d p.e. when the study was terminated. The greatest concentrations of virus, up to 10(8) plaque-forming units (pfu) ml(-1), were found in the ovarian fluid at 13 to 14 d p.e., but the virus was also found in high concentrations in the gill, kidney/spleen and plasma. In contrast, the virus was not recovered from unexposed control adult salmon that died or were sampled at the end of the study. Despite detecting concentrations of IHNV in excess of 10(7) pfu g(-1) of tissue, no specific microscopic lesions were found in IHNV-exposed compared to unexposed control salmon. The results of this initial study suggest that virus in the spawning environment, either from adult salmon or other sources, may contribute to its rapid spread among adult Chinook salmon, thereby considerably increasing the prevalence of IHNV infection in both wild and hatchery populations of adult Chinook salmon.

Jejunal diverticulosis with massive hemorrhage.

A case of a relatively uncommon disease, jejunal, diverticulosis, is reported. The case presented an unusual complication, massive melena. The patient was successfully treated and cured by resection of the involved portion of jejunum. In the management of gastrointestinal hemorrhage in the elderly one must always have in mind this infrequent pathological entity, especially when the small bowel is suspected as the site of bleeding.

Perforation of the colon in unsuspected amebic colitis: report of two cases.

Two cases of amebic colitis that resulted in perforation of the colon, an ominous complication, are presented. The first was diagnosed preoperatively as acute ulcerative colitis with toxic megacolon, and the second as peritonitis complicating acute cholecystitis. In both instances the correct diagnosis was made after operation. The first patient recovered after colectomy and antiamebic therapy, but the second patient died in the early postoperative period, in septic shock. Amebic colitis occurs infrequently in the United States, and the diagnosis is rarely considered. In most cases an initial diagnosis of ulcerative or granulomatous colitis is made and the true diagnosis is recognized only after operation for colonic perforation or hepatic abscess. It is suggested that amebic colitis should be considered more frequently in cases of patients who have diarrhea. Stool examination for ova and parasites is often negative in amebic colitis. The IHA is usually positive in emebiasis, and should be performed early in casesof patients who have bloody diarrhea or other clinical symptons when amebiasis is suspected. Rectal biopsy is also a useful diagnostic approach, but failed to reveal amebae in one of our cases. Finally, it is suggested that operation be performed urgently when fulminating amebic colitis is not reversed by antiamebic therapy, when peritonitis occurs even with antiamebic treatment in progess, and for colonic perforation or toxic megacolon even when antiamebic therapy has not been indicated.

Mitogen-activated protein kinase phosphatase-1 (MKP-1) expression is induced by low oxygen conditions found in solid tumor microenvironments. A candidate MKP for the inactivation of hypoxia-inducible stress-activated protein kinase/c-Jun N-terminal protein kinase activity.

Pathophysiological hypoxia is an important modulator of gene expression in solid tumors and other pathologic conditions. We observed that transcriptional activation of the c-jun proto-oncogene in hypoxic tumor cells correlates with phosphorylation of the ATF2 transcription factor. This finding suggested that hypoxic signals transmitted to c-jun involve protein kinases that target AP-1 complexes (c-Jun and ATF2) that bind to its promoter region. Stress-inducible protein kinases capable of activating c-jun expression include stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 members of the mitogen-activated protein kinase (MAPK) superfamily of signaling molecules. To investigate the potential role of MAPKs in the regulation of c-jun by tumor hypoxia, we focused on the activation SAPK/JNKs in SiHa human squamous carcinoma cells. Here, we describe the transient activation of SAPK/JNKs by tumor-like hypoxia, and the concurrent transcriptional activation of MKP-1, a stress-inducible member of the MAPK phosphatase (MKP) family of dual specificity protein-tyrosine phosphatases. MKP-1 antagonizes SAPK/JNK activation in response to diverse environmental stresses. Together, these findings identify MKP-1 as a hypoxia-responsive gene and suggest a critical role in the regulation of SAPK/JNK activity in the tumor microenvironment.

c-jun cooperates with SV40 T-antigen to sustain MMP-2 expression in immortalized cells.

The c-jun gene is a major regulator of proliferative and stress responses of both normal and transformed cells. In general, during immortalization/transformation c-jun cooperates with oncogenic signals rather than acting as an oncogene itself. Here we report a novel example of this cooperation, the requirement for c-jun to sustain expression of the matrix metalloproteinase-2 (MMP-2) gene in cells immortalized by SV40 large T-antigen (TAg). MMP-2 encodes a type IV collagenase that is secreted by cells within normal and tumor microenvironments. We used wild-type and c-jun null primary and TAg-immortalized mouse embryonic fibroblasts (mEFs) to investigate the importance of c-jun for the regulation of this activity, and observed that c-jun is essential for MMP-2 expression in immortalized but not primary mEFs. This finding directly demonstrates a cooperative interaction of c-jun with an oncogene, and suggests that TAg dependent immortalization/transformation may require other c-Jun/AP-1-dependent genes.