Anatomical and biochemical studies of Spartium junceum infected by Xylella fastidiosa subsp. multiplex ST 87.

Spartium junceum L. is a typical species of Mediterranean shrubland areas, also grown in gardens and parks as an ornamental. In recent years in Europe, S. junceum has been recurrently found to be infected by different subspecies and genotypes of the quarantine regulated bacterium Xylella fastidiosa (Xf). This work presents for the first time the anatomy of S. junceum plants that we found, by means of genetic and immunochemistry analysis, to be naturally infected by Xf subsp. multiplex ST87 (XfmST87) in Monte Argentario (Grosseto, Tuscany, Italy), a new outbreak area within the EU. Our anatomical observations showed that bacteria colonized exclusively the xylem conductive elements and moved horizontally to adjacent vessels through pits. Interestingly, a pink/violet matrix was observed with Toluidine blue staining in infected conduits indicating a high content of acidic polysaccharides. In particular, when this pink-staining matrix was observed, bacterial cells were either absent or degenerated, suggesting that the matrix was produced by the host plant as a defense response against bacterial spread. In addition, a blue-staining phenolic material was found in the vessels and, at high concentration, in the pits and inter-vessels. SEM micrographs confirmed that polysaccharide and phenolic components showed different structures, which appear to be related to two different morphologies: fibrillary and granular, respectively. Moreover, our LM observations revealed bacterial infection in xylem conductive elements of green shoots and leaves only, and not in those of other plant organs such as roots and flowers.

Influence of stomach mucosa tissue on the efficacy of intragastric antibacterial PDT.

In the field of photodynamic therapy (PDT), optimization of the in vivo therapeutic efficacy needs a comprehensive study of the photo-killing action spectrum that depends on both the photosensitizer (PS) absorption and the tissue optical properties. This is especially true in the case of gastric infections by Helicobacter pylori: PS absorption has been largely investigated in vitro, while the contribution of tissue optical properties and illumination geometry has been poorly studied, despite being parameters that reflect the specific in vivo conditions. To investigate their influence, we focussed on the case of a point-like light source positioned in the antrum. This models a therapeutic device developed by our team which consists of a LED-based ingestible pill. By a simple 3D illumination model, our approach mediates light-tissue interaction over the illuminated stomach wall surface, then calculates its average transmittance T by means of a 1D model representative of the mean gastric mucosa structure. Finally, by merging T(λ) with the photosensitizers' absorption we obtained the in vivo action spectrum. This shows two peaks at about 500 and 630 nm, indicating a noticeable influence of the tissue with respect to in vitro studies, where the action spectrum reflects PS absorption only. Our approach defines one average action spectrum for this specific therapeutic context, which reflects the need to choose one emission spectrum for the light source used. The proposed methodology could be applied to any other illumination geometry of cave organs, provided appropriate model modifications for the light source and tissue characteristics are made.

In vivo effects of rosiglitazone in a human neuroblastoma xenograft.

BACKGROUND: Neuroblastoma (NB) is the most common extra-cranial solid tumour in infants. Unfortunately, most children present with advanced disease and have a poor prognosis. There is in vitro evidence that the peroxisome proliferator-activated receptor gamma (PPARgamma) might be a target for pharmacological intervention in NB. We have previously demonstrated that the PPARgamma agonist rosiglitazone (RGZ) exerts strong anti-tumoural effects in the human NB cell line, SK-N-AS. The aim of this study was to evaluate whether RGZ maintains its anti-tumoural effects against SK-N-AS NB cells in vivo. METHODS AND RESULTS: For this purpose, tumour cells were subcutaneously implanted in nude mice, and RGZ (150 mg kg(-1)) was administered by gavage daily for 4 weeks. At the end of treatment, a significant tumour weight inhibition (70%) was observed in RGZ-treated mice compared with control mice. The inhibition of tumour growth was supported by a strong anti-angiogenic activity, as assessed by CD-31 immunostaining in tumour samples. The number of apoptotic cells, as determined by cleaved caspase-3 immunostaining, seemed lower in RGZ-treated animals at the end of the treatment period than in control mice, likely because of the large tumour size observed in the latter group. CONCLUSIONS: To our knowledge, this is the first demonstration that RGZ effectively inhibits tumour growth in a human NB xenograft and our results suggest that PPARgamma agonists may have a role in anti-tumoural strategies against NB.

Carborane derivatives loaded into liposomes as efficient delivery systems for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is an anticancer therapy based on the incorporation of (10)B in tumors, followed by neutron irradiation. Recently, the synthesis and delivery of new boronated compounds have been recognized as some of the main challenges in BNCT application. Here, we report on the use of liposomes as carriers for BNCT active compounds. Two carborane derivatives, i.e., o-closocarboranyl beta-lactoside (LCOB) and 1-methyl-o-closocarboranyl-2-hexylthioporphyrazine (H(2)PzCOB), were loaded into liposomes bearing different surface charges. The efficacy of these formulations was tested on model cell cultures, that is, DHD/K12/TRb rat colon carcinoma and B16-F10 murine melanoma. These induce liver and lung metastases, respectively, and are used to study the uptake of standard BNCT drugs, including borophenylalanine (BPA). Boron concentration in treated cells was measured by alpha spectrometry at the TRIGA mark II reactor (University of Pavia). Results showed high performance of the proposed formulations. In particular, the use of cationic liposomes increased the cellular concentration of (10)B by at least 30 times more than that achieved by BPA.

Expression of Id helix-loop-helix proteins in colorectal adenocarcinoma correlates with p53 expression and mitotic index.

Id helix-loop-helix (HLH) proteins function as regulators of cell growth and differentiation and when overexpressed can induce malignant transformation. In a series of 34 cases of primary human colorectal adenocarcinoma, immunoreactivity for Id1, Id2, and Id3 was found to be significantly elevated in tumor compared with normal mucosa (P = 0.001 for Id1 and Id2; P = 0.002 for Id3). No elevation of Id expression was observed in 17 cases of adenoma. Expression of Id1 and to a lesser extent of Id2 was correlated with mitotic index (P = 0.005 for Id1; P = 0.042 for Id2) in human adenocarcinomas, and expression of all three Id proteins was correlated with p53 immunoreactivity (a marker of mutational 'inactivation' of p53 function; P = 0.002 for Id1; P = 0.006 for Id2; P = 0.016 for Id3). In normal intestinal mucosa of p53-null mice and in spontaneous tumors arising in Min+/- mice, expression of all three Id proteins was also found to be up-regulated. Antisense oligonucleotide blockade of Id protein expression inhibited the proliferation of human adenocarcinoma cells. Enforced, ectopic expression of the E47 basic HLH (bHLH) protein in human adenocarcinoma cell lines efficiently sequestered endogenous Id proteins as Id-bHLH heterodimers, as shown by coimmunoprecipitation and subcellular colocalization studies. This led to growth arrest of the cells. Enforced overexpression of a mutant E47 protein, deficient in transactivation and DNA binding function, also partially inhibited cell growth. Taken together, these data imply that deregulated expression of Id proteins in colorectal adenocarcinoma arises at least in part as a consequence of loss of p53 function and contributes to the uncontrolled proliferation of tumor cells in colorectal cancer.

Polyamines as biochemical indicators of radiation injury.

The search for parameters of different nature to quantify radiation damage is carrying on from many years in humans and lab animals. The polyamines (spermidine and spermine) are ubiquitous polycations with many metabolic functions and can be easily assayed by HPLC method. Their involvement in cell proliferation has been evidenced in healthy and tumour tissues. Statistically significant reductions have been demonstrated in tissues and in red blood cells (RBC), in animals and in patients treated by total body irradiation (TBI) before bone marrow transplantation (BMT). In rats submitted to TBI with 3 Gy of gamma radiations, tissue polyamines significantly decrease during the early phase of injury in tissues with high proliferative activity (small intestine, spleen) whereas do not show any modification in kidney. When recovery takes place, the polyamines significantly increase and return to control levels when a normal morphology is restored. In patients submitted to radiation therapy, polyamines have been determined in urine and in RBC of patients with carcinoma of uterine cervix, head and neck and prostate, treated by external radiotherapy, and with thyroid cancer treated with iodine-131 therapy. The most interesting results has been obtained with RBC: in patients treated on the pelvis for prostate cancer a significant reduction during radiotherapy occurs, followed by the maintenance of low levels in patients with favourable outcome. It should be noted that polyamine levels before treatment appeared significantly higher than in healthy controls. After TBI the RBC polyamines show a dramatic fall to extremely low levels during the phase of marrow aplasia. The values show an increase corresponding to the engraftment of transplanted cells and to the following marrow repopulation. These evidences make the RBC polyamines very interesting parameters to monitor the radiation effects on humans.

Proposal for biochemical dosimeter for prolonged space flights.

Radiation dosimetry has been developed by means of physical, chemical and biological methods. A different approach to calculate the absorbed dose is related to the assay in body fluids of some molecules that modify their concentration after irradiation. The salivary glands in humans appear particularly radiosensitive and the effects of ionizing radiation can be evaluated by means of the determination of serum amylase (produced by acinar cells) and Tissue Polypeptide Antigen (TPA, synthesized by ductal cells). Patients submitted to external radiotherapy for tumours localized in the head and neck region show early and late effects on salivary glands. The modification of amylase activity and TPA appear as a progressive statistically significant increase within two days. Levels of 200-300% of baseline value are reached, followed by a rapid return to preirradiation levels. The use of different doses per fraction and fractionation schedules (conventional or multiple daily fractionations) confirm the direct correlation between the absorbed dose and serum amylase and TPA levels. It is worth noting that the irradiation of pancreas region did not produce any effect on amylase activity. The correlation may be assumed as linear for a short dose range (2-6 Gy) whereas in the range from 0.5 to 10 Gy a sigmoid curve represents the experimental data. Both molecules confirm their capability to quantify the absorbed dose in patients with thyroid carcinoma submitted to metabolic treatment with iodine-131. The effects of radiation are species-specific and are absent in laboratory mammals. The easiness of the determination of serum amylase and TPA lead us to propose the test as biochemical dosimeter for cosmic rays exposure during prolonged staying in the space.

Bcl-w expression in colorectal adenocarcinoma.

We have found that the anti-apoptotic Bcl-2 family protein, Bcl-w, was frequently expressed in colorectal adenocarcinomas, with 69/75 showing positive staining with anti-Bcl-w IgG. Adenomas demonstrated a much lower frequency of Bcl-w expression (only 1 of 17), as did adenocarcinomas from other epithelial tissues such as breast (0/8), stomach (1112) and cervix (0/12). Bcl-w status could be related to the histopathological classification of the tumours, with TNM stage III tumours showing significantly higher levels of expression than tumours of better prognostic grade (at P = 0.009). Those patients with node involvement also had tumours with significantly elevated levels of Bcl-w (at P = 0.02), compared to those which were node-negative. The results suggest that Bcl-w could play a general role in the progression from adenoma to adenocarcinoma in the colorectal epithelium. Currently, more data are being collected to allow us to assess the importance of Bcl-w for disease progression and patient survival.

Correlation between DNA content and p53 deletion in colorectal cancer.

OBJECTIVE: To find out whether tumour DNA content correlates with allelic loss of p53 and other pathological features in primary colorectal carcinomas. DESIGN: Ongoing prospective study. SETTING: University hospital, Italy. SUBJECTS: 128 patients who had undergone radical resections for colorectal carcinoma. INTERVENTIONS: Flow cytometric measurement of tumour DNA content and detection of allelic loss on the short arm of chromosome 17 by Southern blot (restriction fragment length polymorphism) analysis in fresh tumour specimens. MAIN OUTCOME MEASURES: Correlation between DNA ploidy and deletion of p53, as well as between these two genetic events and clinicopathological variables. RESULTS: Interpretable DNA histograms were obtained for 122 tumour specimens. Forty-three tumours (35%) were diploid and 79 (65%) aneuploid. The diploid tumours were significantly more common in the proximal colon (from the caecum to the splenic flexure) than in the distal colon (from the descending colon to the rectum) (p = 0.002). The allelic state on the short arm of chromosome 17 was evaluated in 80 heterozygous patients. Forty-four tumour specimens (55%) showed deletion of 17p. Allelic loss of p53 was significantly more common in the distal and rectal tumours than in the proximal ones (p < 0.0001). Aneuploidy was more common among those tumours which had shown deletion of p53 than in those that had not (p = 0.0008). CONCLUSIONS: DNA aneuploidy was significantly associated with the deletion of the p53 gene. This suggests that the functional loss of p53 may favour the growth and establishment of an aneuploid cell population within tumours. Tumours of the proximal and distal colon differ in their genetic nature.

Colonic cell proliferation in normal mucosa of patients with colon cancer.

Cell kinetics parameters have been analysed in colonic mucosa at different distances from a tumour in patients with colon carcinoma. Total cell number (TCN), 3H thymidine labelling index (TLI), mitotic index (MI), Goblet cell index (GCI) and the distribution of labelled cells along the crypt column (cell position frequency plot) were determined in well-aligned crypts. Total cell number, GCI and the labelled cell position frequency plots were similar in different samples from the same individual. A negative linear correlation between TCN and TLI was observed. The analysis of the cell position plots showed two patterns 1) with a high concentration in the bottom fifth of the crypt and 2) with frequent labelled cells at high positions. Whereas a negative correlation between overall TLI and the percent contribution to the TLI of the lowermost fifth was seen, the correlation was positive for the next 3 fifths and labelling was absent in the last part of the crypt.

3H-thymidine labelling index (TLI) as a marker of tumour growth heterogeneity: evaluation in human solid carcinomas.

Many studies deal with the analysis of cell kinetic, cytogenetic, biochemical and molecular cell biology parameters to identify prognostic factors relating to tumour growth but all methods use only a small part of the total tumour mass. This study is devoted to the analysis of the heterogeneity of the growth of human solid tumours assaying proliferative activity by means of 3H-thymidine labelling index (TLI) in a fixed number of samples collected in different areas of the lesion (larynx and colon cancers), or in different lesions of the same subject (breast and bladder cancers). Each sample (at the macroscopic level) was divided into small fragments (at the microscopic level) and proliferative activity was determined. The analysis of variance for hierarchical designs demonstrated that in all cases a high component of the variance is attributable to the subjects and to the fragments whereas the variance attributable to the different areas is very low. The heterogeneity of proliferative activity displays a higher focal variability among the fragments (microscopic level) compared with that among areas (macroscopic level) within subjects, provided an adequate number of fragments and cells are counted. In multiple synchronous carcinoma of the bladder the wide variability of proliferation among the single lesions demonstrated that it is necessary to analyse all the tumours in a subject because each one is characterized by a different cell growth potential.

Potentiated bradykinin-induced increase of 1,2-diacylglycerol generation and phospholipase D activity in human senescent fibroblasts.

1. The comparative study of the effect of bradykinin (BK) in young and old IMR-90 human fibroblasts shows that old cells are characterized by a reduced increase in 1,2-diacylglycerol (1,2-DAG) generation upon stimulation after short-term treatment and a significant higher increase after long-term agonist treatment. BK-induced activation of phospholipase D (PLD), the major enzyme involved in sustained 1,2-DAG generation, was 2.5-fold higher in old cells, strongly suggesting that it is involved in the potentiated increase of 1,2-DAG formation. The increased activation of PLD by BK in old cells was specific, since in parallel experiments the effect of thrombin was not significantly different in young and old cells. PLD activity in old cells was only reduced by down-regulation of protein kinase C (PKC) activity, in contrast to what was observed in young cells where it was completely abolished. This indicates that the enzyme activity in old cells was partially PKC-independent. BK was also able to increase the release of [14C]ethanolamine, a water-soluble product of hydrolysis of phosphatidylethanolamine (PtdEtn), through PLD activation in young and old cells. The BK effect was significantly higher in old cells and, very likely, PKC-independent, since phorbol 12-myristate 13-acetate failed to induce PtdEtn hydrolysis. 2. The present results indicate that the PLD/1,2-DAG pathway is specifically potentiated by BK in old fibroblasts, demonstrating that the formation of positive effectors of PKC activation is not necessarily decreased in cellular senescence. It remains to be established whether the increased generation of DAG upon BK stimulation plays any role in the altered PKC signalling pathway which characterizes old fibroblasts.