Evaluation of the efficacy and selectivity of Oberon (Spiromesifen) for the control of Tetranychus urticae on strawberry.

A study was performed in the period June to July 2009 by Bioagritest test facility according to EPPO guidelines and Principles of Good Experimental Practice (GEP), in the campaign of Tursi (MT), southern Italy, in order to evaluate the efficacy and selectivity of Spiromesifen on strawberry for the control of Tetranyichus urticae. Two different dosages of OBERON (a.i. Spiromesifen)--45 and 60 ml/hl--were compared with a unique dosage of two commercial formulates: VERTIMEC (a.i. Abamectine, Syngenta Crop Protection), 60 ml/hl, and MAGISTER (a.i. Fenazaquin, Dow AgroSciences), 60 ml/hl. The study has achieved the purpose of evaluating/measuring with a single application the activity of Spiromesifen (Oberon) to control T. urticae on strawberry and its selectivity on phytoseiids. All the treatments differed significantly from the checks and showed high ability to control mites, on all stages of the population. About selectivity on phytoseiids, only Spiromesifen (at both doses) has demonstrated a good selectivity, while Abamectine (in part) and Fenazaquin (totally), have limited the population of the predator. The study confirmed the usefulness, indeed the need for the new compounds, to get confirmation of their selectivity against the useful entomofauna. The experiment has allowed to demonstrate the suitability of Spiromesifen to be included in strategies for strawberry integrated pest management.

Effect of salinity on osmoregulatory patch epithelia in gills of the blue crab Callinectes sapidus.

In euryhaline crabs, ion-transporting cells are clustered into osmoregulatory patches on the lamellae of the posterior gills. To examine changes in the branchial osmoregulatory patch in the blue crab Callinectes sapidus in response to change in salinity and to correlate these changes with other osmoregulatory responses, crabs were acclimated to a range of salinities between 10 and 35 ppt. When crabs that had been acclimated to 35 ppt were subsequently transferred to 10 ppt, both the size of the osmoregulatory patch on individual gill lamellae and the specific activity of Na+, K+-ATPase in whole-gill homogenates increased only after the first 24 h of exposure to dilute seawater. Enzyme activity and size of patch area increased gradually and reached their maxima (increasing by 200% and 60%, respectively) 6 days following transfer to 10 ppt seawater and then remained at these levels. Patch size at acclimation varied inversely with the salinity for seawater dilutions below 26 ppt (the isosmotic point of the crab), although it did not vary in salinities at or above 26 ppt. Thus, the size of the patch clearly is modulated with acclimation salinity, but it increases only in those salinities in which the crab hyperosmoregulates. An increase in the total RNA/DNA ratio in gill homogenates, the lack of mitotic figures in the lamellae, and the lack of incorporation of bromodeoxyuridine into nuclei of lamellar epithelial cells during acclimation to dilute seawater were interpreted as evidence that no cell proliferation had occurred and that increases in the size of the osmoregulatory patch occurred through differentiation of existing gas exchange cells or of undifferentiated epithelial cells into ion-transporting cells.

Private or intimate relations between doctor and patient: is zero tolerance warranted?

This article reviews and comments on the five categories of arguments used to defend zero tolerance with regard to sexual contacts resulting from the physician-patient relationship as summarised by Cullen. In addition it puts forward a hypothesis-"fear of loss by third party"-as a psychological explanation for the collective insistence on a zero tolerance policy.

Utility of osteopontin as a biomarker in recurrent epithelial ovarian cancer.

OBJECTIVE: Osteopontin (OPN) is overexpressed in tumors and serum of ovarian cancer patients and may serve as a biomarker. To evaluate the utility of serum osteopontin in monitoring disease status, we evaluated 234 serum samples from post-oophorectomy patients with ovarian cancer and 38 samples from healthy controls. METHODS: Serum samples were collected from 203 women with recurrent ovarian cancer and 31 newly diagnosed women participating in an experimental chemotherapeutic clinical trial. Controls included 11 young healthy women and 27 peri- or postmenopausal women without ovarian cancer. Samples were assayed for osteopontin using an enzyme-linked immunosorbent assay (ELISA) kit. Statistical analyses for group comparisons of biomarker distribution used the nonparametric Wilcoxon's rank sum test for two-group comparisons and the Kruskal-Wallis test for three-group comparisons. RESULTS: Osteopontin values ranged from 25 to 1463 ng/ml for patients and 25 to 617 ng/ml for controls. Mean patient levels were lower than mean control levels (74 ng/ml vs. 147 ng/ml, respectively, P = 0.0006). Serum osteopontin levels correlated with recurrent disease versus remission (68 ng/ml vs. 34 ng/ml, P = 0.0034), presence of ascites versus absence (71 ng/ml vs. 53 ng/ml, P = 0.0002), and bulky disease vs. nonbulky disease (75 ng/ml vs. 38 ng/ml, P = 0.0005). CA-125 values yielded the same trends with greater statistical difference. CONCLUSIONS: These results demonstrate that serum osteopontin concentrations in post-oophorectomy patients with recurrent ovarian cancer are not greater than in healthy controls. Nevertheless, within this heterogeneous patient population, the values do correlate with bulk of disease. The potential utility of this assay in monitoring women with CA-125 negative disease is worthy of exploration.

A phase II trial of three sequential doublets for the treatment of advanced müllerian malignancies.

OBJECTIVES: In an effort to improve the results of primary chemotherapy for müllerian malignancies a novel chemotherapy program was piloted that delivered three sequential chemotherapy doublets. The primary endpoints were surgically defined response rates and evaluation of toxicity. METHODS: After primary cytoreductive surgery patients were treated with three sequential doublets including three initial cycles of carboplatin and paclitaxel (doublet 1) and then two cycles of cisplatin (day 1) and gemcitabine (days 1 and 8; doublet 2), and finally two cycles of doxorubicin (day 1) and topotecan (days 3,4, and 5; doublet 3). Cycles 4 through 7 were given with G-CSF (Neupogen) support at a dose of 5 mcg/kg/day. After therapy, all women were clinically staged and evaluated by second-look laparoscopy/laparotomy (SLO) if clinical staging was negative for residual disease. RESULTS: A total of 49 eligible patients were enrolled with a median age of 52 (SD 9). Forty-four women had either ovarian cancer or primary peritoneal carcinoma with 3 women diagnosed with fallopian tube carcinoma and 2 with papillary serous carcinoma of the uterus. Eighty-four percent of patients had stage IIIc/IV tumors, with 29% having >1 cm residual disease after primary cytoreductive surgery. Thirty-nine of 49 (80%) patients completed therapy. A total of 283 cycles of chemotherapy were delivered with acceptable toxicities. There were no toxic deaths. Five women were withdrawn from trial (3 for Taxol hypersensitivity, 1 for gemcitabine pulmonary hypersensitivity, and 1 for serious line infection). Neutropenia, typically without fever, was relatively frequent in the first doublet. Nausea and thrombocytopenia were the predominant toxicities in doublet 2. Thirty-nine women completed all cycles of treatment. Thirty-six women had restaging results consistent with a clinical complete response (CR) and underwent SLO. The pathologic CR rate of the patients undergoing SLO was 38%. CONCLUSIONS: Treatment with this sequential doublet regimen is feasible with a 38% pathologic CR rate.

Manipulation of avidity to improve effectiveness of adoptively transferred CD8(+) T cells for melanoma immunotherapy in human MHC class I-transgenic mice.

The adoptive transfer of tumor-reactive CD8(+) T cells into tumor-bearing hosts provides an attractive alternative to vaccination-based active immunotherapy of melanoma. The development of techniques that result in the preferential expansion of tumor-reactive T cells is therefore of great importance. In this study, we report the generation of HLA-A*0201-restricted CD8(+) T cell populations that recognize either tyrosinase(369-376) or gp100(209-217) from tolerant human class I MHC-transgenic mice by using single amino acid-substituted variant peptides. Low peptide concentration or restimulation with the parent peptide was used to enhance the functional avidity, defined by stimulation of IFN-gamma accumulation, and cross-reactivity of the resulting T cell populations. We found a direct correlation between the ability of a T cell population to respond in vitro to low concentrations of the precise peptide expressed on the tumor and its ability to delay the outgrowth of B16 melanoma after adoptive transfer. Surprisingly, we found that some T cells that exhibited high functional avidity and were effective in controlling tumor outgrowth exhibited low structural avidity, as judged by MHC-tetramer staining. Our results establish strategies for the development and selection of CD8(+) T cell populations that persist despite peripheral tolerance, and that can control melanoma outgrowth. Furthermore, they support the use of human MHC class I-transgenic mice as a preclinical model for developing effective immunotherapies that can be rapidly extended into therapeutic settings.

Immune responses to the HLA-A*0201-restricted epitopes of tyrosinase and glycoprotein 100 enable control of melanoma outgrowth in HLA-A*0201-transgenic mice.

Many of the Ags recognized by human melanoma-reactive CTL are derived from proteins that are also expressed in melanocytes. The possibility of self-tolerance to these epitopes has led to questions about their utility for antitumor immunotherapy. To investigate the issue, we established a preclinical model based on transgenic mice expressing a recombinant HLA-A*0201 molecule and B16 melanoma transfected to express this molecule. HLA-A*0201-restricted epitopes from the melanocyte differentiation proteins (MDP) tyrosinase and gp100 are expressed in both tumor cells and melanocytes, and the former is associated with self-tolerance. However, adoptive transfer of tyrosinase or gp100-reactive CTL developed from tolerant mice delayed tumor outgrowth, as did immunization with MDP peptide-pulsed dendritic cells. Protection was enhanced by the use of peptide ligands containing conservative substitutions that were cross-reactive with the original Ags. These data establish that CTL populations reactive against MDP-derived self-Ags can be activated to mount effective antitumor immunity and strongly support their continued development for tumor immunotherapy in humans.

Direct identification of human tumor-associated peptide antigens and a preclinical model to evaluate their use.

Although the arsenal of a healthy immune system includes both circulating antibodies and cellular components such as T cells, the latter seem to be particularly important in tumor immunology. Under normal conditions, the immune system does not react to the body's cells, which may be described as expressing "self" antigens on the cell surface. When a cell becomes cancerous, however, novel antigens are expressed on the cell surface. These novel "tumor" antigens are recognized as foreign by the body's immune system, and the cells that express them are destroyed or incapacitated. Whereas antibodies may react directly with protein antigens, T cells instead recognize peptide antigens presented by class I and class II molecules of the major histocompatibility complex (MHC). All cells normally break down proteins that they have made. The class I antigen-processing pathway has evolved to display peptides produced by this breakdown process as a way to provide information to cytotoxic T cells about what the cell is making. The display of new peptides as a result of infection or transformation can stimulate cytotoxic T cells to kill the cell. In addition, antigen-processing cells such as dendritic cells engulf dead or dying cells and degradeproteins into peptide fragments. These peptides are then displayed by the MHC class II molecules and presented to T helper cells, which augment the activity of the cytotoxic T cells. Cytotoxic T lymphocytes have recently been isolated from human tumors (especially melanoma) and are critical to the development of promising immunotherapeutic agents. As we shall discuss, these cells can recognize antigens that are common to tumors from different patients. We shall also explore how advances in instrumentation and the use of transgenic mice have increased our understanding of tumor-associated peptides to the point where we can begin to strive for a peptide-based therapeutic vaccine. The caveats for such therapy will also be addressed.

Self-tolerance to the murine homologue of a tyrosinase-derived melanoma antigen: implications for tumor immunotherapy.

The human tyrosinase-derived peptide YMDGTMSQV is presented on the surface of human histocompatibility leukocyte antigen (HLA)-A*0201(+) melanomas and has been suggested to be a tumor antigen despite the fact that tyrosinase is also expressed in melanocytes. To gain information about immunoreactivity and self-tolerance to this antigen, we established a model using the murine tyrosinase-derived homologue of this peptide FMDGTMSQV, together with transgenic mice expressing the HLA-A*0201 recombinant molecule AAD. The murine peptide was processed and presented by AAD similarly to its human counterpart. After immunization with recombinant vaccinia virus encoding murine tyrosinase, we detected a robust AAD-restricted cytotoxic T lymphocyte (CTL) response to FMDGTMSQV in AAD transgenic mice in which the entire tyrosinase gene had been deleted by a radiation-induced mutation. A residual response was observed in the AAD(+)tyrosinase(+) mice after activation under certain conditions. At least some of these residual CTLs in AAD(+)tyrosinase(+) mice were of high avidity and induced vitiligo upon adoptive transfer into AAD(+)tyrosinase(+) hosts. Collectively, these data suggest that FMDGTMSQV is naturally processed and presented in vivo, and that this presentation leads to substantial but incomplete self-tolerance. The relevance of this model to an understanding of the human immune response to tyrosinase is discussed.

The density of peptides displayed by dendritic cells affects immune responses to human tyrosinase and gp100 in HLA-A2 transgenic mice.

Several HLA-A*0201-restricted peptide epitopes that can be used as targets for active immunotherapy have been identified within melanocyte differentiation proteins. However, uncertainty exists as to the most effective way to elicit CD8+ T cells with these epitopes in vivo. We report the use of transgenic mice expressing a derivative of HLA-A*0201, and dendritic cells, to enhance the activation of CD8+ T cells that recognize peptide epitopes derived from human tyrosinase and glycoprotein 100. We find that by altering the cell surface density of the immunizing peptide on the dendritic cells, either by pulsing with higher concentrations of peptide, or by changing the MHC-peptide-binding affinity by generating variants of the parent peptides, the size of the activated CD8+ T cell populations can be modulated in vivo. Significantly, the density of peptide that produced the largest response was less than the maximum density achievable through short-term peptide pulsing. We have also found, however, that while some variant peptides are effective at eliciting both primary and recall CD8+ T cell responses that can recognize the parental epitope, other variant epitopes lead to the outgrowth of CD8+ T cells that only recognize the variant. HLA-A*0201 transgenic mice provide an important model to define which peptide variants are most likely to stimulate CD8+ T cell populations that recognize the parental, melanoma-specific peptide.

Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens.

Melanoma-reactive HLA-A x 0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pme117/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A x 0201+ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pme117/gp100, gp75/ trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLAA x 0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A x 0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A x 0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP.

Proteasomes can either generate or destroy MHC class I epitopes: evidence for nonproteasomal epitope generation in the cytosol.

Proteasomes have been implicated in the production of the majority of peptides that associate with MHC class I molecules. We used two different proteasome inhibitors, the peptide aldehyde N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL) and the highly specific inhibitor lactacystin, to examine the role of proteasomes in generating peptide epitopes associated with HLA-A*0201. Neither LLnL nor lactacystin was able to completely block the expression of the HLA-A*0201. Furthermore, the effects of LLnL and lactacystin on the expression of different categories of specific epitopes, TAP independent vs TAP dependent and derived from either cytosolic or membrane proteins, were assessed. As predicted, presentation of two TAP-dependent epitopes was blocked by LLnL and lactacystin, while a TAP-independent epitope that is processed in the endoplasmic reticulum was unaffected by either inhibitor. Surprisingly, both LLnL and lactacystin increased rather than inhibited the expression of a cytosolically transcribed and TAP-dependent peptide from the influenza A virus M1 protein. Mass spectrometric analyses of in vitro proteasome digests of a synthetic 24 mer containing this epitope revealed no digestion products of any length that included the intact epitope. Instead, the major species resulted from cleavage sites within the epitope. Although cleavage at these sites was inhibitable by LLnL and lactacystin, epitope-containing species were still not produced. We conclude that proteasomes may in some cases actually destroy epitopes that would otherwise be destined for presentation by class I molecules. These results suggest that some epitopes are generated by nonproteasomal proteases in the cytosol.

Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues: implications for tumor vaccine development.

To identify shared epitopes for melanoma-reactive CTL restricted by MHC molecules other than HLA-A*0201, six human melanoma patient CTL lines expressing HLA-A1 were screened for reactivity against the melanocyte differentiation proteins Pmel-17/gp100, MART-1/Melan-A, and tyrosinase, expressed via recombinant vaccinia virus vectors. CTL from five of the six patients recognized epitopes from tyrosinase, and recognition of HLA-A1+ target cells was strongly correlated with tyrosinase expression. Restriction by HLA-A1 was further demonstrated for two of those tyrosinase-reactive CTL lines. Screening of 119 synthetic tyrosinase peptides with the HLA-A1 binding motif demonstrated that nonamer, decamer, and dodecamer peptides containing the sequence KCDICTDEY (residues 243-251) all reconstituted the CTL epitope in vitro. Epitope reconstitution in vitro required high concentrations of these peptides, which was hypothesized to be a result of spontaneous modification of cysteine residues, interfering with MHC binding. Substitution of serine or alanine for the more N-terminal cysteine prevented modification at that residue and permitted target cell sensitization at peptide concentrations 2 to 3 orders of magnitude lower than that required for the wild-type peptide. Because spontaneous modification of sulfhydryl groups may also occur in vivo, tumor vaccines using this or other cysteine-containing peptides may be improved by amino acid substitutions at cysteine residues.

Differences in potentially preventable risk factors for stroke amongst ethnic groups

AIM: To assess the prevalence of risk factors for stroke potentially amenable to health service intervention (hypertension, diabetes, smoking, increased alcohol consumption, physical inactivity, atrial fibrillation, cardiac disease, previous TIA) in black Caribbean, black African and white population of an inner-city health authority. METHODS: A cross-sectional survey was conducted, involving 16 GP Practices serving an area with a high proportion of black residents as identified by the 1991 Census. The FHSA list for these practices was used as the sampling frame. A random sample of 8000 residents (45-74 years old) was selected for a postal survey, which collected data on age, sex, occupation and ethnic group. Responders were stratified by ethnic group. A random sample of 450 subjects in white and black Caribbean group was selected, and together with 193 black African responders invited for screening. RESULTS: There were 725 responders: 303 whites (41.8 percent), 316 black Caribbean (43.6 percent) and 106 black Africans (14.6 percent). Black Caribbeans and Africans were less likely to have a normal blood pressure than whites (OR=0.42, p=0.0001, and OR=0.39, p=0.001 respectively.) Black Caribbeans and were also less likely to have a normal ECG (OR=0.55, p=0.0011, and OR=0.41, p=0.007 respectively.) Left ventricular strain was more common in black Caribbeans (OR=15.81, p=0.008) and Africans (OR=19.97, p=0.007), ischaemic changes were more common in black Caribbeans (OR=2.8, p=0.0001) and myocardial infarction in Africans were found in the prevalence of reported risk factors, such as diabetes (5 percent, 15.2 percent, 10.4 percent respectively, p>0.00001), smoking (31.4 percent, 21 percent, 11.3 percent, p<0.00001, alcohol drinking (80.9 percent, 77.8 percent, 61.3 percent, p<0.00001) and physical activity (75.3 percent, 83.2 percent, 79.3 percent, p=0.048). No difference was found in prevalence of atrial fibrillation and previous TIA. CONCLUSION: Black Caribbeans and black Africans have significantly higher prevalence rates of the important risk factors for stroke. This may partially explain higher mortality rates for stroke in these ethnic groups. Strategies for stroke prevention will be considered in the context of the on-going study on cultural attitudes to risk factor reduction. (AU)