Targeting tumor cells through chitosan-folate modified microcapsules loaded with camptothecin

Poly(vinyl alcohol) microcapsules have been tailored as carriers to deliver camptothecin, an anticancer drug poorly soluble in water. The capsules have been reacted with a chitosan--folate complex in order to selectively target cancer cells overexpressing the folic acid receptor. Microcapsules decorated with the chitosan--folate complex have been characterized in their uptake and release of camptothecin, following the absorption band at λ = 370 nm diagnostic of the drug molecule. The selectivity of chitosan-folate microcapsules in targeting cancer cells has been demonstrated by fluorescence microscopy using HeLa cells, overexpressing the folate receptor and NIH3t3 fibroblasts as a negative control. The chitosan--folate microcapsules loaded with camptothecin significantly reduce the proliferation of HeLa tumor cells, while they have a negligible effect on fibroblasts. This work demonstrates that the chitosan--folate microcapsules represent a promising system to selectively target hydrophobic drugs, such as camptothecin, to tumor cells.     

A Novel Alpha-Proteobacterium Resides in the Mitochondria of Ovarian Cells of the Tick Ixodes ricinus

An intracellular bacterium from Ixodes ricinus ticks collected in Italy was characterized by electron microscopy (EM), PCR sequencing of the 16S rRNA gene, molecular phylogenetic analysis, and in situ hybridization (ISH). This bacterium was shown by EM to be present in the cytoplasm, as well as in the mitochondria of ovarian cells. When universal 16S rRNA bacterial primers were used, PCR amplification of ovarian DNA followed by cloning and sequencing resulted in the same sequence being found in each sample. Phylogenetic analysis of this sequence showed that the bacterium from which it was derived, tentatively designated IricES1, is part of a novel clade in the alpha subdivision of the Proteobacteria. ISH and PCR assays of various tissues performed with oligonucleotides specific for the IricES1 16S rRNA showed that IricES1 is restricted to ovarian cells. Based on the results obtained, we inferred that the bacteria seen by EM in ovarian cells are a single type of bacteria, corresponding to IricES1. PCR screening of 166 ticks from various parts of Italy and one site in England showed that IricES1 was present in 96% of adult females and 44% of nymphs (unsexed). No adult males were found to be infected. Despite the apparent parasitism of host mitochondria by IricES1, the available information suggests that the bacterium has an obligate relationship with its host, although this must be confirmed.     

Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species

The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.     

IFN-alpha/beta signaling is required for polarization of cytokine responses toward a protective type 1 pattern during experimental cryptococcosis

The antiviral activities of type I IFNs have long been established. However, comparatively little is known of their role in defenses against nonviral pathogens. We examined here the effects of type I IFNs on host resistance against the model pathogenic yeast Cryptococcus neoformans. After intratracheal or i.v. challenge with this fungus, most mice lacking either the IFN-alpha/beta receptor (IFN-alpha/betaR) or IFN-beta died from unrestrained pneumonia and encephalitis, while all wild-type controls survived. The pulmonary immune response of IFN-alpha/betaR-/- mice was characterized by increased expression of IL-4, IL-13, and IL-10, decreased expression of TNF-alpha, IFN-gamma, inducible NO synthetase, and CXCL10, and similar levels of IL-12 mRNA, compared with wild-type controls. Histopathological analysis showed eosinophilic infiltrates in the lungs of IFN-alpha/betaR-/- mice, although this change was less extensive than that observed in similarly infected IFN-gammaR-deficient animals. Type I IFN responses could not be detected in the lung after intratracheal challenge. However, small, but statistically significant, elevations in IFN-beta levels were measured in the supernatants of bone marrow-derived macrophages or dendritic cells infected with C. neoformans. Our data demonstrate that type I IFN signaling is required for polarization of cytokine responses toward a protective type I pattern during cryptococcal infection.     

Protective immunization against group B meningococci using anti-idiotypic mimics of the capsular polysaccharide

Use of the serogroup B meningococcal capsular polysaccharide (MenB CP) as a vaccine is hampered by the presence of epitopes that cross-react with human polysialic acid. As non-cross-reactive, protective capsular epitopes have also been described, we set out to develop protein mimics of one of such epitopes using as a template a highly protective mAb (mAb Seam 3) raised against a chemically modified form of the MenB CP (N-Pr MenB CP). Using phage display, anti-idiotypic single-chain Ab fragments (scFvs) were obtained from spleen cells of mice immunized with the Seam 3 mAb. Two Seam 3-specific scFvs competed with N-Pr MenB CP for binding to either mAb Seam 3 or rabbit Abs present in typing sera. Moreover, in mice and rabbits the scFvs elicited the production of Abs reacting with both N-Pr MenB CP and whole meningococci, but not with human polysialic acid. These scFv-induced Ab responses were boostable and of the Th1 type, as shown by a predominance of IgG2a. In addition, passive immunization with sera from scFv-immunized animals partially protected neonatal mice from experimental infection with group B meningococci. In conclusion, we have produced anti-idiotypic scFvs that mimic a protective MenB CP epitope and may be useful in the development of an alternative group B meningococcal vaccine.     

The transglutaminase family: an overview: Minireview article

Summary. The knowledge that very different processes such as normal and neoplastic cell growth, reproduction and death are dependent on the presence of adequate levels of transglutaminases (TGase: EC 2.3.2.13) and that they are capable of affecting the differentiation and proliferative capability of several cell types, has prompted a multitude of researchers to study these fascinating molecules. In the following overview we intend to summarize the currently known information on the biological significance of these enzymes.     

Somatostatin analogues, a series of tissue transglutaminase inducers, as a new tool for therapy of mesenchimal tumors of the gastrointestinal tract

Summary. Imatinib, a tyrosine kinase inhibitor directed against the enzymatic domain of KIT protein, was found to produce dramatic clinical responses in metastatic gastrointestinal stromal tumors (GISTs). However, resistance usually develops thus determining treatment failure. The present study was performed to analyse the expression of somatostatin receptor (SSTR) subtypes, modulators of tissue transglutaminase, in a series of GISTs and leiomyosarcomas by immunohistochemistry to identify a new potential therapeutic target. Sixteen cases (8 males and 8 females, age range: 38–73; 11 GISTs, 4 leiomyosarcomas, 1 leiomyoma) were studied. Immunohistochemical detection of the relevant SSTRs was performed on paraffin-embedded tissue sections, stained with polyclonal antibodies directed against the five somatostatin receptor subtypes. We found 7 out of 16 (44%) tumors expressing all SSTRs and 14 out of 16 (87%) tumors positive for at least 3 subtypes. SSTR2_A was the most represented subtype in the tumors studied, being expressed in approximately 70% of cases exhibiting an intense labeling in most of these cases. The significant expression of SSTRs shown in this series of GISTs and gastrointestinal leiomyosarcomas suggests a potential therapeutic target to be explored alone and/or in combination with other therapeutic agents in the setting of refractory GI stromal tumors.     

Enhancement of transglutaminase activity and polyamine depletion in B16-F10 melanoma cells by flavonoids naringenin and hesperitin correlate to reduction of the <Emphasis Type="Italic">in vivo</Emphasis> metastatic potential

Summary. The in vitro and in vivo effects of two flavonons, naringenin (NG) and hesperitin (HP) on the proliferation rate of highly metastatic murine B16-F10 melanoma cell were investigated. NG or HP treatment of melanoma cells produced a remarkable reduction of cell proliferation, paralleled with both the lowering of the intracellular levels of polyamine, spermidine and spermine and the enhancement of transglutaminase (TGase, EC 2.3.2.13) activity. Orally administered NG or HP in C57BL6/N mice inoculated with B16-F10 cells affected the pulmonary invasion of melanoma cells in an in vivo metastatic assay. The number of lung metastases detected by a computerized image analyzer was reduced, compared to untreated animals, by about 69% in NG-treated mice and by about 36% in HP-treated mice. Survival studies showed that 50% of the NG-treated animals died 38 ± 3.1 days after tumor cell injection (control group: 18 ± 1.5 days) and HP-treated mice died 27 ± 2.3 days after cell inoculation. Taken together, these findings provide further evidences for the potential anticancer properties of dietary flavonoids as chemopreventive agents against malignant melanoma.