Clinical, Cytological, Histological and Immunohistochemical Features of Cutaneous Mast Cell Tumours in Ferrets (Mustela putorius furo).

Cutaneous mast cell tumours (cMCTs) are one of the most common cutaneous tumours in ferrets (Mustela putorius furo). However, limited information is available regarding cytological and histological features of these tumours and studies evaluating KIT expression are lacking in this species. The aims of this prospective study were to describe the most common clinical, cytological and histological features of cMCTs in ferrets and to compare the usefulness of different staining techniques in the diagnosis of these tumours in ferrets as well as evaluating KIT expression in neoplastic mast cells (MCs) by immunohistochemistry. Macroscopically, the tumours were small, round to plaque-like and frequently associated with surface crusting. The most common locations were the extremities and the trunk. MC granules were stained in all cases using toluidine blue (TB) and Wright-Giemsa stains in cytological specimens, but none stained with modified Wright's stain. Haematoxylin and eosin and TB on histological sections failed to stain MC granules in all the cases. Cytological and histological examination revealed low to moderate anisocytosis and anisokaryosis. An infiltrative rather than a delineated or encapsulated growth pattern was noted histologically in all cases. Eosinophilic infiltration was not uncommon and 'collagenolysis' was detected on cytological and histological examination. KIT expression was detected in all cases evaluated. In approximately one third of the cases the MCs exhibited KIT labelling pattern I and in the remaining ferrets, KIT pattern III. No correlation was found between KIT expression pattern and biological behaviour.

Gold-silver-alloy nanoprobes for one-pot multiplex DNA detection.

A specific colorimetric DNA detection method based on oligonucleotide functionalized gold-silver-alloy nanoparticles (AuAg-alloy-nanoprobes) is presented. The AuAg-alloy-nanoprobes were then used for the specific detection of a DNA sequence from TP53-a gene involved in cancer development. The AuAg-alloy-nanoprobes were then used in combination with Au-nanoprobes for a one-pot dual-colour detection strategy that allowed for the simultaneous differential detection of two distinct target sequences. This system poses an unprecedented opportunity to explore the combined use of metal nanoparticles with different composition towards the development of a multiplex one-pot colorimetric assay for DNA detection.

Diffusion-weighted imaging in patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the central nervous system due to JC polyoma virus infection of oligodendrocytes. PML develops in patients with impaired T-cell function as occurs in HIV, malignancy or immunosuppressive drugs users. Until now no imaging methods have been reported to correlate with clinical status. Diffusion-weighted imaging (DWI) is a robust MRI tool in investigating white matter architecture and diseases. The aim of our work was to assess diffusion abnormalities in focal white matter lesions in patients with PML and to correlate the lesion load measured with conventional MRI and DWI to clinical variables. We evaluated eight patients with a biopsy or laboratory-supported diagnosis of PML. All patients underwent MRI including conventional sequences (fluid attenuated inversion recovery-FLAIR) and DWI. Mean diffusivity (MD) maps were used to quantify diffusion on white matter lesions. Global lesion load was calculated by manually tracing lesions on FLAIR images, while total, central core and peripheral lesion loads were calculated by manually tracing lesions on DWI images. Lesion load obtained with the conventional or DWI-based methods were correlated with clinical variables such as disease duration, disease severity and survival. White matter focal lesions are characterized by a central core with low signal on DWI images and high MD (1.853 x 10(-3) mm2/s), surrounded by a rim of high signal intensity on DWI and lower MD (1.1 x 10(-3) mm2/s). The MD value of normal-appearing white matter is higher although not statistically significant (0.783 x 10(-3) mm2/s) with respect to control subjects (0.750 x 10(-3) mm2/s). Inter-rater correlations of global lesion load between FLAIR (3.96%) and DWI (3.43%) was excellent (ICC=0.87). Global lesion load on FLAIR and DWI correlates with disease duration and severity (respectively, p=0.037, p=0.0272 with Karnofsky scale and p=0.0338 with EDSS on FLAIR images; p=0.043, p=0.0296 with Karnofsky scale and p=0.0365 with EDSS on DW images). Central core lesion load on DWI correlates with disease duration and severity (respectively p=0.043, p=0.0103 with Karnofsky scale and p=0.0112 with EDSS), while peripheral lesion load does not correlate with any clinical variable. The global lesion load in PML correlates with disease duration and severity. DWI images, which can distinguish within lesions a central core from a peripheral rim, reveal that a larger central core component correlates to a worsened clinical status and longer disease duration. On the other hand the peripheral rim lesion load visualized on DWI images does not correlate with clinical variables and does not achieve obtaining further prognostic information with respect to conventional imaging.

Effects of essential oils on Aedes aegypti larvae: alternatives to environmentally safe insecticides.

The essential oils from leaves of Hyptis fruticosa (Lamiaceae) Salzm., H. pectinata (Lamiaceae) Poit., and Lippia gracilis (Verbenaceae) HBK were investigated for their larvicidal activity against Aedes aegypti and analyzed by GC/MS. Fifty-nine compounds, representing 91.28-98.39% of the essential oils, have been identified. A standard solution was used to make 20 mL solutions ranging from 30 to 2000 ppm. Twenty larvae between third and fourth stages were added to the essential oil solution. A mortality count was conducted 24 h after treatment. Essential oils LC50 and their confidence limits at 95% probability were calculated by the methods of Reed-Muench and Pizzi, respectively. The essential oil of Lippia gracilis showed potent insecticidal effect against Aedes aegypti larvae, the vector of dengue fever. Carvacrol and caryophyllene oxide were the main responsible for the activity of L. gracilis and H. pectinata. Minor compounds are probably acting synergistically to achieve H. fruticosa activity.

Nanodiagnostics: fast colorimetric method for single nucleotide polymorphism/mutation detection.

Advances in nanosciences are having a significant impact in many areas of research. The impact of new nanotechnologies has been particularly large in biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecules detection. To date, applications of nanoparticles have largely focused on DNA-functionalised gold nanoparticles used as the target-specific probes. These gold nanoparticle-based systems can be used for the detection of specific sequences of DNA (pathogen detection, characterisation of mutation and/or single nucleotide polymorphisms) or RNA (without prior retro-transcription and amplification). Here a rapid and inexpensive nanoparticle-based method for single-base mismatch detection (single nucleotide polymorphism/mutation) in DNA samples is reported. Gold nanoparticles derivatised with thiol modified oligonucleotides complementary to DNA targets -- Au-nanoprobes -- are used to distinguish fully complementary from mismatched sequences, with a single-base mismatch. The authors have successfully applied this strategy to detect common mutations within the beta-globin gene.

Increased levels of NF-kappaB inhibitors (IkappaBalpha and IkappaBgamma) in the intestinal mucosa of Crohn's disease patients during infliximab treatment.

The treatment with infliximab is employed successfully in Crohn's disease (CD) but predictors of efficacy are lacking. Activation of the transcription factor NF-kB has been demonstrated in CD and its inhibition is one of the mechanisms by which anti-inflammatory agents exert their effects. We evaluated the production of TNFalpha by peripheral blood mononuclear cells (PBMC) and the levels of NF-kappaB family molecules in the intestinal mucosa during infliximab therapy in 12 patients. TNFalpha was assayed on supernatants of PBMC culture stimulated with PHA or LPS. Immunohistochemistry was also done on intestinal biopsies. In six patients, Western blot analysis of the NF-kappaB subunit Rel-A, and its inhibitors IkappaBalpha and IkappaBgamma was performed on intestinal biopsies and PBMC. The TNFalpha production by LPS stimulated PBMC showed mild changes, while it was increased by PHA-stimulated PBMC after treatment. The number of inflammatory cells in the intestinal mucosa was reduced (p<0.002) by the treatment. In five out of six cases we detected an increase of the IkappaBalpha and IkappaBgamma)inhibitor levels in intestinal biopsies after treatment. An increase of IkappaB inhibitors levels could be one of the mechanisms by which infliximab decreases NF-kappaB activity and exerts its anti-inflammatory effects.

[Prevention and treatment of congenital toxoplasmosis: organization protocol]. / Prevenzione e trattamento della toxoplasmosi congenita. Modello organizzativo.

AIM: Prevention and treatment of congenital toxoplasmosis are still a matter of debate among obstetricians, pediatricians and epidemiologists. There is no consensus about antenatal screening and diagnostic tests, nor there is about treatment for presumed infection in pregnancy. As an example of this type of organisation for health care delivery, a regional model has been promoted as a multidisciplinary approach for prenatal diagnosis of congenital toxoplasmosis. The model had been designed on the national guidelines of the National Health Institute (Istituto Superiore di Sanità, ISS). METHODS: Suspected maternal infections are referred and seen as outpatients at our centre on a specific day of the week; maternal investigation (specific IgG, IgM, IgA and IgG avidity titres) are performed at the Institute of Virology of the University of Bari, and patients are started on spiramycin. All cases of true or presumed seroconversion are counselled for amniotic fluid sampling and the sample is sent to ISS. In cases of late seroconversion and positive amniotic fluid results, patients are prescribed pyrimethamine+sulphonamide+folinic acid and alternate spiramycin until the end of pregnancy. A fetal-neonatal follow-up is performed in all cases. RESULTS: During the period 1999-2001, 180 cases of presumed toxoplasmosis infection have been referred (average 60 cases per year). We have been able to reclute, since the adoption of the national network protocol, 1/3 of presumed regional cases with a positive increasing trend. CONCLUSION: The service for prenatal diagnosis of toxoplasma gondii infection has definitely benefitted from the adoption of this protocol, which combines adherence to a national network and pays respect to regional requirements.

[Second trimester termination of pregnancy with gemeprost: a review of 306 cases]. / Interruzione di gravidanza nel secondo trimestre con il gemeprost. Analisi di 306 casi.

AIM: Prostaglandin analogues provide an effective method for induction of abortion in the second trimester of pregnancy. The clinical outcome and the risk of complications were evaluated in a group of women having a medical termination of pregnancy with gemeprost. METHODS: Three-hundred and six women undergoing second trimester termination of pregnancy, between January 1998 and July 2002 in our center, were studied. All women were given 1 mg vaginal gemeprost every 3 h up to a maximum of 3 doses in the first 24 hours. If the abortion did not occur within the first 24 hours after initiation of treatment, they were given a 2(nd) course of gemeprost. Outcome measures included failure of the 1st course of gemeprost, length of hospital stay after expulsion of conceptus, heavy blood loss with or without necessity of uterine packing or blood transfusion, and failure of induction. RESULTS: There was a significant difference, with better results in women with previous deliveries (vaginal or abdominal), as to the failure to abort after the 1(st) course of PG (P<0.01). Length of hospital stay, complications and failure of induction were independent from parity. Twelve (3.9%) women failed to abort with gemeprost and required other methods for abortion. CONCLUSIONS: The study confirms the efficacy of gemeprost for mid-trimester termination of pregnancy, although it is a risky and costly procedure, requiring hospitalisation and is associated with higher complication rate than the first trimester surgical abortion.

Activity of a nitroxylated analog of daunorubicin, ruboxyl, in B-lymphoproliferative disorders.

A nitroxylated analog of daunorubicin, ruboxyl (RBX), showed low toxicity but significant lympholytic effect in preclinical evaluations. A series of studies in vitro and in animals demonstrate that RBX is a putative agent in the treatment of many neoplasms. We report the results of a study in mice in which RBX showed selective B-lymphocyte immunosuppression. On the basis of this experience, RBX was administered to 3 patients with multiple myeloma and two patients with Waldenström's disease. The results of this pilot clinical study show that this compound has good activity and low myelotoxicity and cardiotoxicity, but seems to be characterized by a threatening immunosuppressive effect.

Role of DNA-dependent protein kinase in recognition of radiation-induced DNA damage in human peripheral blood mononuclear cells.

The DNA-dependent protein kinase (DNA-PK) complex plays a crucial role in radiation-induced DNA damage recognition. The complex includes the ku heterodimer, which comprises ku 70 and ku 80 subunits, that binds DNA termini of breaks without sequence specificity, and the catalytic subunit DNA-PKCS: The activation of the DNA-PK complex was studied in X-irradiated peripheral blood mononuclear cells (PBMC) from subjects of different ages. Radiation-induced changes in the DNA-binding activity of the ku heterodimer, and in the concentrations of ku 70, ku 80, DNA-PKcs and phosphorylated ku 80 were determined in nuclear and cytoplasmic extracts. DNA-binding activity was increased by irradiation only in the nuclear extract of PBMC from young, but not from elderly subjects, whereas it was found unchanged in cytoplasmic extracts regardless of age. The radiation-induced activation of the DNA-PK complex may result from the increased concentrations of ku 80 and DNA-PKcs in the cytoplasm of PBMC from young, but not from elderly subjects, leading to a higher concentration of phosphorylated ku 80 which readily migrates to the nucleus where, after dimerization with ku 70, binds to DNA breaks. These findings suggest major steps involved in DNA-PK activation, and the intracellular and molecular changes that may account for the age-dependent impairment of DNA repair capacity in irradiated mammalian cells.

Inhibition of IgG1 and IgE production by stimulation of the B cell CTLA-4 receptor.

Although a large amount of information is available on the activity of CTLA-4 in T cells, the role of this receptor in B cells has not been previously characterized. Our results show that CD40 or LPS stimulation in the presence of IL-4 induces CTLA-4 expression in purified B cells; the maximum level is reached in both membrane and intracellular compartments after 48-72 h. Engagement of the B cell CTLA-4 by immobilized mAb inhibits IgG1 and IgE production and reduces the frequency of IgG1- and IgE-expressing B cells. Cepsilon and Cgamma(1) germline mRNA expression as well as NF-kappaB and STAT6 activation, events required for isotype switching, are also inhibited by CTLA-4 engagement. Together these findings show the critical role of CTLA-4 in the control of IL-4-driven isotype switching and suggest new approaches for modulating immediate-type hypersensitivity responses.

Linkage disequilibrium and haplotype mapping of a skin cancer susceptibility locus in outbred mice.

Car-R (carcinogenesis-resistant) and Car-S (carcinogenesis-susceptible) outbred mice, obtained by phenotypic selection from an initial intercross of eight inbred strains, show a >100-fold difference in their susceptibility to two-stage skin tumorigenesis. We found that the lines carry a high degree of genetic polymorphism. with an average heterozygosity of 0.39. This polymorphism allowed the use of linkage disequilibrium (LD) and haplotype analysis for the mapping of a skin cancer modifier locus on Chr 7, in a short region of 6 cM, around the Tyr gene. Car-S mice inherited the susceptibility allele at this locus from the A/J, BALB/c, SJL/J, and SWR/J strains. Our results demonstrate the feasibility and usefulness of mapping disease genes by LD in phenotypically selected, genetically heterogeneous animals.

Genetics of chemical carcinogenesis: analysis of bidirectional selective breeding inducing maximal resistance or maximal susceptibility to 2-stage skin tumorigenesis in the mouse.

We report on bidirectional selective breeding, initiated from a genetically defined foundation population and carried out to selection limit, for producing lines of mice endowed with maximal resistance (Car-R) or maximal susceptibility (Car-S) to 2-stage skin tumorigenesis. The initial population resulted from a balanced intercrossing of 8 inbred strains of mice. The tumors, induced by a single application of DMBA (initiation) and twice weekly applications of TPA (promotion), were benign papillomas; their number at the end of the promotion period was the phenotype chosen for assortative mating. Afterward, the majority of them regressed while others progressed to malignant carcinomas. The Car-R line was selected through a strong challenge, while the Car-S line selection was based on responses to decreasing concentrations of DMBA and TPA. The selection limit was reached after 14 or 15 generations showing progressive interline divergence, which strongly suggests the interaction of several quantitative trait loci (QTL). The phenotypic difference was extremely large: the tumor response was 73 times higher in Car-S than in Car-R mice, though the applied concentrations of DMBA and TPA were 100 and 40 times lower, respectively. The mean heritability realized during the selective breeding was 0.20 in Car-R and 0.49 in Car-S. Our results are compatible with a minimal QTL estimate of 8 in the Car-R line and of 9 or 10 in the Car-S line. The Car-S line is also much more susceptible to carcinoma induction. An association of coat color with tumorigenesis was observed in interline F2 segregants. The Car-R and Car-S lines, obtained through a long-lasting breeding program, are a unique model for identifying the QTL involved in chemical tumorigenesis and will be provided to interested investigators.

Hematopoietic reconstitution after lethal irradiation and bone marrow transplantation: effects of different hematopoietic cytokines on the recovery of thymus, spleen and blood cells.

Lethally irradiated mice were grafted with syngeneic bone marrow cells or left ungrafted. Mice of each group were injected with different hematopoietic cytokines for 5 consecutive days starting immediately after irradiation or left uninjected. The recovery of lymphoid tissues induced by hematopoietic cytokines 7 days after irradiation and bone marrow cell transplantation was comparable to that observed at days 21-28 in irradiated, bone marrow-grafted, but cytokine-uninjected mice. IL-11 or IL-6, in combination with IL-3, was able to hasten thymus, spleen and blood cell numbers and functions. SCF also displayed a detectable effect when used with IL-3. Conversely, the IL-6 superagonist K-7/D-6 was able, when injected alone, to induce significant recovery of thymus, spleen and blood cells. Thus, K-7/D-6 appears to be a most efficient cytokine for fast reconstitution of lymphoid tissues after irradiation and bone marrow transplantation.

Genetic factors in immunity and aging.

Maximum life span is controlled by genes that regulate molecular mechanisms accounting for the synchrony of structural and functional changes in different cells and tissues of each member of a given species. The role of immune response genes was investigated in aging mice genetically selected for high (H) or low (L) antibody response (Biozzi mice). Results from genetic selection of over 1000 mice showed that genes expressed in the immune system affect life span and diseases. In most cases, the life span is longer in H than in L mice whereas the lymphoma incidence is remarkably higher in L than in H mice. Since DNA repair capacity is a property positively correlated with the maximum life span in several mammalian species, DNA repair was studied by use of hydroxyurea, a cell-synchronizing agent, and found to take place in irradiated human PBMC from young and, to a lesser extent, from adult subjects. Conversely, no repair was detected in irradiated PBMC from elderly subjects. DNA damage recognition and repair pathways involve several nuclear proteins, as double strand breaks are firstly recognized by proteins displaying helicase activity, such as ku 70/80, and then repair is carried out under the control of other proteins. Radiation-induced expression of activated ku(70/80) proteins, in terms of DNA-binding, was found in PBMC from young-adults but not from elderly subjects. Maintenance of DNA integrity is fundamental for normal immune functions, as suggested by the lack of V(D)J recombination in lymphocytes of knock-out mice deficient in ku 70 or ku 80 protein. However, whether the link between genetic factors and life span is mediated by the performance of the immune system remains to be demonstrated.

Modulation of X-ray-induced damage recognition and repair in ageing human peripheral blood mononuclear cells by an interleukin-6-type cytokine.

We have investigated the effects of an interleukin (IL)-6-type cytokine on the DNA-binding activity of ku and on unscheduled DNA repair in X-ray-treated peripheral blood mononuclear cells (PBMC) from human subjects of different ages. The cytokine used, called K-7/D-6, is an IL-6 variant with increased in vivo and in vitro biological activity compared to the wild type molecule. Ku is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK). It binds the ends of various types of DNA discontinuity and is involved in the repair of DNA breaks caused by V(D)J recombination, isotype switching, physiological oxidation reactions, ionizing radiation and some chemotherapeutic drugs. The ku-dependent repair process, called non-homologous end joining, is the main DNA double strand break repair mechanism in irradiated mammalian cells. Results show that K-7/D-6 significantly increases DNA-binding activity of ku in irradiated PBMC from young but not from elderly subjects. However, K-7/D-6 is able to induce unscheduled DNA repair in irradiated PBMC from both young and elderly subjects. These effects of K-7/D-6 are relevant to the mechanisms of the cellular response to DNA damage.

Neuropeptide Y plasma levels and immunological changes during academic stress.

Academic stress is a good model of psychological stress in humans for studying psychoneuroimmune correlations. We looked for correlations between psychological scores, immune tests and plasma levels of cortisol and neuropeptide Y (NPY). A group of medical students were evaluated at the beginning of the academic year (Baseline) and the day before an examination (Stress). They underwent evaluation by The Profile of Mood States (POMS), The Malaise Inventory, The Self Efficacy Scale and A Global Assessment of Recent Stress (GARS). The lymphocyte subsets, the lymphocyte proliferative response and the cytokine production were also evaluated. We detected modifications of some psychological test scores between the Baseline and Stress evaluation, a significant reduction of lymphocyte proliferation, IL-2 production and percentage of the lymphocyte CD19, and an increase in plasma cortisol levels during stress. The lymphocyte proliferation negatively correlated with the POMS score as well as the percentage of CD16+ cells with NPY plasma levels. NPY levels were not different from Baseline. The emotional and mood states seem to influence immunity. Copyrightz1999S.KargerAG,Basel

Effect of age on DNA binding of the ku protein in irradiated human peripheral blood mononuclear cells (PBMC).

DNA binding of the ku protein was investigated in peripheral blood mononuclear cells (PBMC) from 24 subjects of different ages (20-89 years old) displaying age-related changes in DNA repair, mitotic responsiveness, and cytokine production. Ku is an heterodimeric protein composed of two subunits of 70 and 80 kDa, which is involved in the earliest steps of DNA damage recognition. DNA binding of ku 70/80 was found unchanged in normal PBMC from aging subjects but progressively declined in x-ray-irradiated PBMC from young to adult, and elderly subjects. This finding was concomitant with the age-related fall of DNA repair in the whole population.