Structure determination of phi-Bi8Pb5O17 by electron and powder X-ray diffraction

The triclinic crystal structure of phi-Bi8Pb5O17, a ionic fast conductor material, has been determined by the synergy of both electron and powder X-ray crystallography. The heavy atom positions were found by direct methods on electron diffraction data and the structure was completed by iterative use of a priori information in direct methods and difference Fourier maps on both types of data. Structure refinement was performed by the Rietveld method on powder X-ray data. The results suggest that phi-Bi8Pb5O17 is an ordered phase, with Bi and Pb atoms occupying different sites of the lattice, at variance with the other structural phases known for similar composition in the Bi-Pb-O phase diagram, which are solid solutions characterised by a wide compositional range.

Clinical and molecular diagnosis of hereditary non-polyposis colorectal cancer: problems and pitfalls in an extended pedigree.

Hereditary non-polyposis colorectal cancer (or Lynch syndrome) is an autosomal dominant disease in which early onset colorectal carcinomas aggregate in families together with tumours of other organs. The genetic basis of the syndrome has been clarified with the identification of mutations in several DNA mismatch repair genes (MSH2, MLH1, PMS1, PMS2 and MSH6). We describe the clinical features and molecular characterization of a large hereditary non-polyposis colorectal cancer family which has been followed for almost 10 years. The kindred showed a striking aggregation of colorectal tumours in 3 successive generations; most of these neoplasms developed before the age of 50 years and were localized in the proximal colon. Molecular tests (carried out in ten individuals) showed specific alterations at the MLH1 gene, consisting in the insertion of a T nucleotide between bases 2,269 and 2,270; the mutation caused frameshift of the open reading frame and synthesis of a polypeptide longer than normal. The only tumour that could be analysed was positive for microsatellite instability. Physicians should become more confident with hereditary tumours and their implications, which are not limited to a single individual but concern all family members at risk of cancer. This family approach is different, and requires more expertise than the traditional individual approach. Common problems encountered in Hereditary Non-polyposis Colorectal Cancer families include: A) poor collaboration of subjects at risk (a situation which may cause some conflict between the doctor's duty to inform patients about their risk of disease and the rights of patients to choose and decide about their health); B) definition of the most appropriate surveillance programme for a given family (how many investigations to propose to the patients, and how often); C) possible interaction between genes and environmental factors (for instance, a gene carrier--in this family--developed an endometrial carcinoma after standard tamoxifen adjuvant therapy for breast cancer).

BRCA1 and BRCA2 genes: role in hereditary breast and ovarian cancer in Italy.

The heritable defects of BRCA1 and BRCA2 genes have been shown to predispose to breast and ovarian cancers. In a previous report, we analyzed 46 Italian families with breast and/or ovarian cancer for BRCA1 mutations. In the present study, those families and 11 others were screened for BRCA2 mutations; the newly enrolled families were also analyzed for the BRCA1 gene. The coding region and splice boundaries of BRCA2 and BRCA1 genes were assessed by the protein-truncation test and single-strand conformational polymorphism. A total of 20 different mutations were found in 21 families (37%). A total of 9 families (16%) showed mutations in the BRCA1 gene, including the one new mutation identified in this study (5382insC), and 12 families (21%) presented mutations in the BRCA2 gene. BRCA2-mutated families presented breast and ovarian cancers or breast cancers only, whereas most BRCA1-mutated families presented ovarian cancer alone or in association with breast cancer. All the BRCA2 mutations led to a truncated protein: 6 were frameshift mutations, 4 were non-sense mutations and 2 involved the intronic invariant region leading to splice variants. Therefore, in the Italian population, the cumulative proportion of BRCA1 and BRCA2 mutations was within the range observed in other studies (37%), with higher involvement of BRCA2 than of BRCA1. Many families in which no mutations were found presented a very high incidence of breast and/or ovarian cancer. Among the 36 BRCA1 and BRCA2 wild-type families, 24 presented at least 4 cancer cases, indicating the existence of other important predisposing genes.

Low incidence of BRCA1 mutations among Italian families with breast and ovarian cancer.

Most familial breast or ovarian cancers are thought to be due to highly penetrant mutations in the predisposing genes BRCA1 and BRCA2. The cloning of these genes has opened a new era for the genetic counseling of women with a family history of breast or ovarian cancer. To estimate the incidence of detectable BRCA1 mutations and to define the eligibility criteria for genetic testing in the Italian population, a total of 53 patients belonging to 46 families clustering multiple cases of breast and/or ovarian cancer were investigated. Seven families presented with ovarian cancer only, 16 had both ovarian and breast cancers, and 23 were characterized by breast cancer only. Using a combination of protein truncation test (PTT) and single strand conformational polymorphism (SSCP) analysis followed, when necessary, by direct sequencing, we found 8 distinct mutations, 2 of these not reported before. Five frameshift and 2 nonsense mutations led to a truncated protein. One mutation was a missense substitution involving a cysteine in the zinc finger domain. One variant creating an ETS binding site in intron I was found but its role was not defined. The percentage of families carrying mutations was 17%. Among the families characterized by ovarian cancer only and by breast and ovarian cancer, the percentage of BRCA1 mutations was 57% and 12.5%, respectively. In contrast, the percentage of altered BRCA1 in families with only breast cancers was 9%. In the 46 Italian families studied, BRCA1 mutations were detected in fewer kindreds than those previously hypothesized based on linkage analysis, especially when these were characterized by breast cancers only. Our results indicate that families with a low number of cancer patients should be referred for BRCA1 genetic testing mainly when ovarian cancer is present.

Lack of PMS2 gene-truncating mutations in patients with hereditary colorectal cancer.

Hereditary non-polyposis colorectal cancer (HNPCC) is a genetically heterogeneous disease for which PMS2 gene, a member of the human PMS gene family, is believed to have a marginal role. To better define the contribution of PMS2 to hereditary colorectal cancer, we investigated this gene in 22 unrelated Italian patients that, despite a positive family history and/or early onset and development of tumors with microsatellite instability (MSI), did not carry constitutional mutations of MLH1 and MSH2 genes. No mutations with clear-cut pathogenetic significance were detected in the coding regions of PMS2 gene, but only 8 polymorphisms (7 common and 1 rare, 3 silent and 5 missense) and 3 unique molecular variants (2 missense substitutions and one 3-nucleotide deletion) were seen. Lack of PMS2 truncating mutations in our study does not disagree with its supposed marginal involvement in hereditary colorectal cancer, but at the same time points out the need to investigate the phenotypic molecular and clinical characteristics more specifically associated with PMS2 mutations.

Expression of heat shock protein 72 in renal cell carcinoma: possible role and prognostic implications in cancer patients.

Renal cancer cells from 43 patients and normal renal cells from 10 of them were studied for the expression of highly stress-inducible heat shock protein 72 (HSP72) by means of immunoperoxidase analysis. It was found that HSP72 was expressed in a significantly higher percentage of renal cancer cells than normal renal cells (P = 0.0001), the mean percentage of positive cells being 33.1 +/- 18% and 8 +/- 5%, respectively. Moreover, a percentage of HSP72-positive cells that was less than the cut-off point (18%, mean value of normal cells +2 S.D.) significantly correlated with shorter disease-free survival (P = 0.002). The renal cancer cell populations taken from the 21 patients who relapsed after a median time of 13 months (range 3-73 months) had a significantly lower percentage of HSP72-positive cells (mean value 25.1 +/- 17%) than the cells taken from the patients who remained tumour-free (mean value 40.8 +/- 15%) after a median period of 72 months (range 19-96 months, P = 0.003). It was also demonstrated that HSP72 expression can be significantly increased by 48-h in vitro incubation with rIFN-gamma (P = 0.007). These data suggest that HSP72 may represent a favourable prognostic factor regardless of stage and histological grade and its expression may be increased by treatment with rIFN-gamma. Further studies are needed in order to investigate the relationship between HSP72 and the immunoeffector cells.

Paclitaxel as a radiosensitiser: a proposed schedule of administration based on in vitro data and pharmacokinetic calculations.

Paclitaxel is efficacious against many human cancers. Because it blocks cells at the radiosensitive G2-M interface, paclitaxel has been investigated as a radiosensitiser. The results have been equivocal and somewhat contradictory. It is impossible to obtain proper pharmacokinetic calculations, aimed at obtaining maximum cytotoxicity and/or radiosensitisation, without knowing (i) how long the drug must be in contact with the cells, (ii) how long the effect lasts after the drug is removed from the cellular environment, (iii) whether the drug acts as a radiosensitiser even when, like cis-platinum, it is added after the radiation and (iv) what the minimum quantity of drug in the cellular environment is required for both chemotoxicity and radiosensitisation. The present work addresses the above questions. Two radioresistant cell lines of human origin were used, A375 melanoma and S549 lung carcinoma, in a clonogenic assay where only colonies with 50 or more cells were counted. For the irradiation, 6 MV X-rays were used. Any G2-M block was quantified by cell cycle kinetics analysis. From the results, a simulation of pharmacokinetics was conducted to calculate the schedule of administration of paclitaxel most likely to achieve and maintain significant chemotoxocity and radiosensitisation. The minimum concentration of paclitaxel for measurable cytotoxicity was 3 nM for both cell lines, but the drug was more toxic to the A549 cells. The minimum concentration for measurable radiosensitisation was 3 nM for A375 and approximately 0.1 nM for A549, but whereas above 3 nM the radiosensitivity increased in A375, it decreased above 1 nM for A549. A minimum of 18 h incubation with the drug was necessary for measurable effects and the radiosensitising effects were lost soon after its removal. There was no radiosensitisation if paclitaxel was added after the radiation, and, at the minimum effective concentrations, it caused only a minor and transient G2-M block. The pharmacokinetic calculations predict that 15 mg/m2 paclitaxel given as a 1 h infusion 5 days/week for 3 weeks during the radiotherapy should achieve both cytotoxicity and radiosensitisation. The mechanism of radiosensitisation by paclitaxel at the concentrations suggested by our results does not appear to be via a G2-M block and is probably concentration dependent. The results imply that low-dose, daily infusions of paclitaxel for as long as possible during a course of radiotherapy are more likely to result in radiosensitisation and prolonged cytotoxicity than high-dose infusions given once a week.

Interleukin-6 and soluble intercellular adhesion molecule-1 in renal cancer patients and cultured renal cancer cells.

Serum interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1) levels were measured by enzyme-linked immunosorbent assay in 62 renal cancer patients: 30 were tumor-free after radical nephrectomy and 32 presented with metastatic disease. Serum IL-6 was undetectable in all but one of the tumor-free patients, whereas 41% (13 of 32) of the metastatic patients presented serum IL-6 levels. Furthermore, there was a significant correlation between serum IL-6 levels and a shorter overall survival (p = 0.009). Moreover, serum sICAM-1 levels were significantly higher (p = 0.05) in the metastatic patients with detectable serum IL-6 than in those without IL-6, suggesting a possible link between IL-6 and sICAM-1. The probability of a shorter overall survival was greater in the metastatic patients with both serum IL-6 and elevated sICAM-1 levels (>635 ng/ml), than in those with elevated sICAM-1 but without IL-6 (p = 0.01). The production of IL-6 by 16 freshly dissociated renal cancer cells cultured in vitro was also observed. It appeared that IL-6 levels did not correlate with the expression and release of ICAM-1 by cultured cells, although the highest values of ICAM-1 release were found in cultures synthesizing the highest values of IL-6. In conclusion, in vivo presence of serum IL-6 and elevated sICAM-1 levels is related to an unfavorable prognosis; it can be speculated that the cells capable of releasing high levels of sICAM-1 and IL-6 may negatively influence the antitumor response.

Adjuvant immunotherapy treatment of renal carcinoma patients with autologous tumor cells and bacillus Calmette-Guèrin: five-year results of a prospective randomized study.

BACKGROUND: Active specific immunotherapy (ASI) is a strategy that attempts to boost the host's immune response specifically against its own tumor. The purpose of this study was to investigate the effect of adjuvant ASI in patients with renal carcinoma. METHODS: Of 120 consecutive patients, 60 were randomized to a control group and 60 to receive ASI comprised of 3 intradermal injections of 10(7) autologous irradiated tumor cells mixed with 10(7) Bacillus Calmette-Guèrin (in the first 2 vaccinations) or alone. At randomization and 1, 6, and 12 months after completing immunotherapy, the treated patients were evaluated for the development of delayed type cutaneous hypersensitivity (DTCH) response to autologous tumor and autologous normal renal cells. RESULTS: The baseline DTCH responses were negative in all patients. One month after completing ASI, 38 of 54 immunized patients showed a significant (P<0.01) DTCH response to autologous tumor but not to autologous normal renal cells. The response was persistent at 6 months in 25 of 44 patients and at 12 months in 16 of 28 patients. DTCH response remained negative in the nonimmunized control patients. There was no systemic toxicity but local ulcerations that healed in 2 months were observed. After a median follow-up of 61 months, the probability of 5-year disease free survival (DFS) was 63% for treated patients and 72% for control patients. The corresponding probability of 5-year overall survival (OS) was 69% and 78%, respectively. These differences were not statistically significant. CONCLUSIONS: This is the first prospective randomized study of ASI in a large population of patients with renal carcinoma after radical nephrectomy. Our data clearly indicate that ASI can increase the reactivity to autologous tumor, as measured by the DTCH test, but it appears unable to affect DFS and OS of patients.

Expression and release of intercellular adhesion molecule-1 in renal-cancer patients.

We examined ICAM-1 expression in 37 freshly dissociated renal-cancer cell populations. Immunoperoxidase analysis revealed that 31 of the 37 renal tumors expressed ICAM-1 to various degrees; ICAM-1 expression was significantly lower in tumor cells obtained from patients remaining tumor-free after a median follow-up of 60 months (mean value 24.4% +/- 21) than in tumor cells obtained from the relapsed patients (mean value 40.8% +/- 22), and the low expression of this molecule on the cell surface seemed to correlate with favorable clinical behavior. In 41 patients, the mean level of sICAM-I was 551 +/- 260 ng/ml, significantly higher than normal. However, sICAM-1 levels were significantly lower in the 20 tumor-free (mean 467 +/- 158 ng/ml) than in the 21 metastatic patients (mean 631 +/- 318 ng/ml). Eleven renal-cancer cell populations were cultured in order to examine the expression and release of ICAM-1. All of these cells were positive for ICAM-1 expression, which was elevated in 6 cases (> 50%) and low in the remaining 5 cases (18-35%). However, only the 5 cell populations expressing low levels of ICAM-1 released this molecule, showing an inverse correlation with cellular expression. Five of the cell populations were treated for 48 hr with rIFN-gamma, in these cells, both ICAM-1 expression and sICAM-1 levels increased, although sICAM-1 levels in the supernatants of the cell populations with constitutive high ICAM-1 expression remained very low.

Soluble intercellular adhesion molecule-1 and serum cytokines in melanoma patients treated with liposomes containing muramyl tripeptide.

AIMS AND BACKGROUND: A soluble form of intercellular adhesion molecule-1 (sICAM-1) has been recently identified in patients with malignant melanoma. It has been demonstrated that inflammatory cytokines can modulate the cellular expression of ICAM-1 and the shedding of this molecule by cells. To our knowledge, few data exist on serum sICAM-1 levels in cancer patients treated with immunomodulators. Liposomes containing muramyl tripeptide (MLV MTP-PE) can activate monocytes from cancer patients in vitro and in vivo, making them cytotoxic such as tumor necrosis factor-alpha (TNF-alpha) and Interleukin-6 (IL-6). The purpose of the present study was to evaluate the levels of sICAM-1 and their possible correlation with serum inflammatory cytokine levels in melanoma patients treated with MLV MTP-PE. METHODS: The sera from 9 patients with metastatic melanoma treated with MLV MTP-PE, 4 mg i.v. twice a week for 12 weeks, were tested in ELISA system to detect sICAM-1, TNF-alpha, IL-6, Interleukin-1 beta (IL-1 beta) and Interferon-gamma (IFN-gamma) before, and 2 and 24 h after the 1st, 12th and 24th infusion of MLV MTP-PE. RESULTS: Baseline levels of sICAM-1 were elevated in all patients (median 540 ng/ml: range 400-1030 ng/ml). Twenty-four h after the 1st infusion of MLV MTP-PE, we observed 6 increases in sICAM-1 levels, 1 decrease and 2 stable values (median 720 ng/ml: range 410-1820; P = 0.060). Twenty-four h after the 12th infusion, sICAM-1 increased in 3 patients and did not change in 4 (median 790 ng/ml: range 495-1650 ng/ml; P = 0.069). At the 24th infusion, sICAM-1 increased in 4 of 6 evaluable patients and remained stable in 2 (median 802 ng/ml: range 510-1450 ng/ml; P = 0.045). To better analyze the variations in sICAM-1, the patients were arbitrarily divided into two groups according to their clinical behavior: 4 presented stabilization (all lesions, n = 2; some lesions, n = 2) (Group A); 5 presented progressive disease (Group B). In Group A, sICAM-1 levels remained stable or showed a modest increase during treatment (except in 1 patient, who exhibited a substantial variation after the 12th infusion). In contrast, in Group B very high levels of sICAM-1 were observed at the beginning of the study therapy in 1 patient and after the 1st infusion in 3 patients; these values remained high until the 24th infusion. In most of the patients, TNF-alpha and IL-6 increased after the 1st infusion, but not thereafter. IFN-gamma was never detected; IL-1 beta was detectable in a few cases, but only before the infusions. CONCLUSIONS: baseline levels of sICAM-1 were elevated in all patients and further increased during treatment only in patients with more aggressive disease. No correlation was found between sICAM-1 and inflammatory cytokines. It would therefore seem that in patients with advanced disease, higher levels and a progressive increase in sICAM-1 may be unfavorable prognostic factors.

Induction and maintenance of monocyte cytotoxicity during treatment with liposomes containing muramyl tripeptide despite tachyphylaxis to the cytokine response.

Monocyte-mediated cytotoxicity (determined in a 72-h111In release assay) and the circulating levels of tumor necrosis factor alpha (TNF-alpha), interleukin (IL) 1beta, IL-6, IFN-gamma, C-reactive protein, and beta2-microglobulin were determined in 14 melanoma patients treated with multilamellar vesicle liposomes containing muramyl tripeptide phosphatidylethanolamine, 4 mg twice a week for 12 weeks. Monocyte-mediated cytotoxicity increased 24 h after the first infusion in 9 of 14 patients and had reached maximum levels (mean, 44% +/- 8) in all patients by the sixth week; similar values were observed at the 12th week. Once increased in vivo, peripheral blood monocyte cytotoxicity was not susceptible to any further increase after a subsequent in vitro incubation of the monocytes with liposomes. However, the peripheral blood monocytes which were not cytotoxic in vivo were activated by in vitro incubation with liposomes and not by medium. TNF-alpha and IL-6 peaked 2 h after the first infusion and returned to baseline values at 24 h; they were not significantly increased by subsequent treatments. The induction of fever in patients, observed 2 h after the first infusion, correlated with TNF-alpha and IL-6 levels. Similarly, C-reactive protein levels also increased at 24 h, but only after the first dose. No increase in beta2-microglobulin and IL-1beta levels was observed, and IFN-gamma was never detected in serum. Two patients experienced stable disease lasting 7 and 12 months, and 12 patients progressed. These results show that multilamellar vesicle muramyl tripeptide phosphatidylethanolamine administration activates monocyte cytotoxicity and cytokine production (TNF-alpha, IL-6). Chronic treatment with multilamellar vesicle muramyl tripeptide phosphatidylethanolamine results in tachyphylaxis in terms of cytokine secretion but not cytotoxicity. There was no difference between the maximum cytotoxicity levels obtained in vivo and those obtained in vitro using the same agent. A better understanding of immunoregulation is required for a rational application of this and related immunotherapies.

In vitro synergic effect of interferon gamma combined with liposomes containing muramyl tripeptide on human monocyte cytotoxicity against fresh allogeneic and autologous tumor cells.

AIMS: The purpose of the present study was to investigate whether human recombinant interferon-gamma (hrIFN-gamma) can act synergically with various activators in increasing the cytotoxicity of cancer patient monocytes against fresh autologous and allogeneic tumor cells. METHODS: Fresh target cells were obtained by means on the mechanical and enzymatic dissociation of human renal carcinomas. A 375 and SW 626 cell lines were used as positive controls. Monocytes from renal cancer patients and normal volunteers were activated in vitro with lipopolysaccharide, muramyl tripeptide (MTP-PE) or liposomes containing MTP-PE (MTP-PE liposomes), with or without a pre-incubation with hrIFN-gamma and were tested for cytotoxicity by means of a 72-hr 111indium-release assay. All of the patients were tumor free at the time of the study. RESULTS: Cancer patient peripheral blood monocytes were activated in vitro by different immunomodulators and became cytotoxic to freshly dissociated autologous or allogeneic tumor cells. A synergic effect producing maximal cytotoxicity was obtained with an appropriately scheduled combination of hrIFN-gamma (10 U/ml) and MTP-PE liposomes (50 nm/ml), free lipopolysaccharide (10 micrograms/ml) or MTP-PE (100 micrograms/ml). The synergic cytotoxicity was observed against fresh allogeneic and autologous tumor cells, as well as against cultured cells. CONCLUSIONS: All of these data support the possibility of a combined treatment using hrIFN-gamma and MTP-PE liposomes in human studies, particularly when it is borne in mind that liposomes can prevent the direct toxicity of many immunomodulators and that the low levels of hrIFN-gamma required for the synergic activation are not toxic in vivo.

Activation of cytolytic activity in peripheral blood monocytes of renal cancer patients against non-cultured autologous tumor cells.

Our purpose was to evaluate the ability of blood monocytes of renal cancer patients to become cytotoxic against fresh, autologous tumor cells. Fresh target cells were obtained by mechanical enzymatic dissociation of tumor and normal renal tissue. The A375 cell line, derived from a human melanoma, and the SW626 cell line, derived from a human ovarian carcinoma, were used as positive target cell controls. Monocytes from renal cancer patients and normal volunteers were activated in vitro with lipopolysaccharide (LPS), or muramyl tripeptide (MTP-PE), or multilamellar vesicle liposomes containing MTP-PE (MLV-MTP-PE), with or without a pre-incubation with r-IFN-gamma, and tested for cytotoxicity in a 72-hr 111Indium-release assay. All patients were tumor-free at the time of the monocyte study. No difference in cytotoxic activity was observed between monocytes from healthy volunteers and those from cancer patients. Freshly dissociated tumor cells were as susceptible to tumoricidal monocytes as the 2 cell lines. Moreover, no cell population appeared to be resistant to activated monocytes, which were cytotoxic to both allogeneic and autologous fresh tumor cells. Activated monocytes maintained their ability to discriminate between normal and neoplastic cells and were not cytotoxic against autologous or allogeneic normal non-neoplastic cells. Our data indicate that MLV MTP-PE liposomes activate peripheral blood monocytes from cancer patients to a tumoricidal status against fresh, dissociated non-cultured autologous tumor cells.