Morphochemical age-related changes in the nematode Caenorhabditis elegans: immunoperoxidase localization of cytokine- and growth factor-like molecules

Morphochemical age-related features in the hermaphrodite Caenorhabditis elegans are reported. The study of worms of different ages shows a gradual decline in response to the various histochemical reactions and a disorganization of the components of the gonad during ageing. Using an immunocytochemical procedure, we show for the first time the presence of immunoreactive IL-1alpha and PDGF-AB molecules in neurons from young adult C. elegans. Moreover, TNF-alpha- and PDGF-AB-like molecules are also present in the secretory cells of the pharyngeal terminal bulb. The number of positive cells to anti-cytokine and anti-growth factor antibodies decreases in older worms, suggesting that these molecules may play an important role in worm ageing. The present investigation therefore supports the findings in the literature obtained with different approaches on the crucial role of the nervous and reproductive systems in the life span of C. elegans.     

Synthesis, physicochemical properties, and preliminary biological characterizations of a novel amphoteric agmatine-based poly(amidoamine) with RGD-like repeating units

A linear, amphoteric poly(amidoamine) nicknamed AGMA1, based on 4-aminobutylguanidine, or agmatine, was successfully prepared by Michael-type polyaddition of monoprotonated agmatine and 2,2-bis(acrylamido)acetic acid (BAC). Copolymers between AGMA1 and the biocompatible poly(amidoamine) ISA23 (deriving from the polyaddition of 2-methylpiperazine with BAC) were also prepared. Acid-base titrations gave for AGMA1 three acid dissociation constants, with pKa values of 2.25, 7.45, and >or=12.1, corresponding to a strong acid, a medium-weak base, and a strong base, respectively. The charge distribution profiles show that this polymer is prevailingly cationic at all physiological pH values, the positive net average charge per unit varying from about 0.5 at pH 7.4 to about 1.0 at pH 5, with an isoelectric point at pH approximately 10. Zeta-potential measurements confirmed this. Despite that, AGMA1 is nontoxic and nonhemolytic in vitro within all pH ranges tested (4-7.5). This is in contrast with the previously observed behavior of amphoteric PAAs, for instance ISA23, that are weakly hemolytic at pH 7.4 but highly hemolytic at pH 5/5.5. The lack of hemolytic activity of AGMA1 even at acidic pH values seems typical of the agmatine-BAC sequences and may be ascribed to their RGD-like structure. In fact, AGMA1-ISA23 copolymers behave in a way increasingly similar to that of ISA23; that is, they become hemolytic at low pH values as their ISA23 content increases.     

Contrasting effects of low-dose IL-2 on vaccine-boosted simian immunodeficiency virus (SIV)-specific CD4+ and CD8+ T cells in macaques chronically infected with SIVmac251

IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1 infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we investigated how continuous low-dose IL-2 affected the CD4+ and CD8+ T cell response induced by two inoculations of a canarypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had normal levels of CD4+ T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly augmented Gag-specific CD8+ T cell responses, but actually reduced Gag-specific CD4+ T cell responses. Although IL-2 at low doses did not change the overall concentration of circulating CD4+ or CD8+ T cells, it expanded the frequency of CD4+CD25+ T cells. Depletion of the CD4+CD25+ T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4+ responses or augmentation of SIV-specific CD8+ T cell responses. Thus, we conclude that the decrease in virus-specific CD4+ T cell response may be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T regulatory population.     

Interleukin-12- and gamma interferon-dependent innate immunity are essential and sufficient for long-term survival of passively immunized mice infected with herpes simplex virus type 1

Interferon (IFN) type I (alpha/beta IFN [IFN-alpha/beta]) is very important in directly controlling herpes simplex virus type I (HSV-1) replication as well as in guiding and upregulating specific immunity against this virus. By contrast, the roles of IFN type II (IFN-gamma) and antibodies in the defense against HSV-1 are not clear. Mice without a functional IFN system and no mature B and T cells (AGR mice) did not survive HSV-1 infection in the presence or absence of neutralizing antibodies to the virus. Mice without a functional IFN type I system and with no mature B and T cells (AR129 mice) were unable to control infection with as little as 10 PFU of HSV-1 strain F. By contrast, in the presence of passively administered neutralizing murine antibodies to HSV-1, some AR129 mice survived infection with up to 10(4) PFU of HSV-1. This acute immune response was dependent on the presence of interleukin-12 (IL-12) p75. Interestingly, some virus-infected mice stayed healthy for several months, at which time antibody to HSV-1 was no longer detectable. Treatment of these virus-exposed mice with dexamethasone led to death in approximately 40% of the mice. HSV-1 was found in brains of mice that did not survive dexamethasone treatment, whereas HSV-1 was absent in those that survived the treatment. We conclude that in the presence of passively administered HSV-1-specific antibodies, the IL-12-induced IFN-gamma-dependent innate immune response is able to control low doses of virus infection. Surprisingly, in a significant proportion of these mice, HSV-1 appears to persist in the absence of antibodies and specific immunity.     

Repair of molluscan tissue injury: role of PDGF and TGF-beta1

Various cellular and humoral activities of the wound repair process and the effects of PDGF-AB and TGF-beta1 on tissue repair mechanisms in the mollusc Limax maximus are studied by histological and immunocytochemical procedures. Histological examination at different times after the wound production demonstrates that tissue repair is the result of successive and related activities distinguishable by different morphological pictures. In the first hours, an infiltration phase is activated 24 h after the incision, hemocytes stratify at wound margins and actively phagocitize cell debris and damaged tissue in the surrounding area. Moreover, the cells are immunoreactive to anti-IL-1alpha, IL-8 and TNF-alpha antibodies. After 24-72 h, granulation tissue rich in small blood lacunae is formed and the provisional matrix is synthesized and deposited on the base of the new tissue. In histological sections 72 h after injury, the incision is filled with granulation tissue, and at the wound base, a layer of fibrous connective tissue is formed. Hemocytes present in the newly formed blood lacunae and fibroblasts are involved in the synthesis and deposit of extracellular matrix components, i.e. fibronectin, reticular and collagen fibres. Ninety-six h after wound production, the repair process continues and the granulation tissue is more developed. At 192 h, re-epithelialization begins, and this is more evident in the histological sections after 14 days. Hemocytes are immunoreactive to the cytokines at all the times examined. Exogenous administration of PDGF-AB and TGF-beta1 stimulates the tissue healing process through a general acceleration of the activities involved. A larger closing area of clumped hemocytes and a smaller damaged tissue area are observed 24 h after treatment of the wound. At 72 h, the granulation tissue is more developed and more extracellular matrix components are deposited than in the control incision. A larger number of cells express cytokine-like molecules, and re-epithelialization of the wound is accelerated, as 14 days after growth factor treatments almost all the wound area is covered by a layer of cubic epithelial cells, and the alcianophilic cell coat is restored. No differences in the responses of the two growth factors are observed.     

Targeting the vaginal mucosa with human papillomavirus pseudovirion vaccines delivering simian immunodeficiency virus DNA

The majority of HIV infections occur via mucosal transmission. Vaccines that induce memory T and B cells in the female genital tract may prevent the establishment and systemic dissemination of HIV. We tested the immunogenicity of a vaccine that uses human papillomavirus (HPV)-based gene transfer vectors, also called pseudovirions (PsVs), to deliver SIV genes to the vaginal epithelium. Our findings demonstrate that this vaccine platform induces gene expression in the genital tract in both cynomolgus and rhesus macaques. Intravaginal vaccination with HPV16, HPV45, and HPV58 PsVs delivering SIV Gag DNA induced Gag-specific Abs in serum and the vaginal tract, and T cell responses in blood, vaginal mucosa, and draining lymph nodes that rapidly expanded following intravaginal exposure to SIV(mac251.) HPV PsV-based vehicles are immunogenic, which warrant further testing as vaccine candidates for HIV and may provide a useful model to evaluate the benefits and risks of inducing high levels of SIV-specific immune responses at mucosal sites prior to SIV infection.     

Effect of pollution on some freshwater species. II. Bioaccumulation and toxic effects of experimental lead pollution on the ganglia in Viviparus ater (Mollusca, Gastropoda)

The effect of lead on ganglia of Viviparus ater were studied by histochemical and histomorphological procedures. The pollution experiment should be considered a "short-time static bioassay" because of its experimental characteristics. There was considerable accumulation of lead in the ganglia as determined by atomic absorbance (A.A.S.). The cytological damage principally affected the neuronal cell bodies which undergo degenerative processes. The most serious cytopathological changes occurred in the following sequence: nuclear damage leading to pyknosis; nucleolar damage until disappearance; changes in Nissl bodies, at times forming a uniform mass. These cytological disorders led to markedly altered protein synthesis. Nerve fibers and neuroglia did not appear affected by lead exposure, even at higher doses. Membrane enzymes, phosphorylase, NADHDH, NADPHDH and SDH activities were decreased, whereas D-LDH, G-6-PDH, G-6-Pase and MAO activities increased. GDH was unchanged. Changes in polar lipid composition were also observed with an increase of phospholipids and a decrease of sulpholipids and cerebrosides.     

A convenient synthesis of lubeluzole and its enantiomer: Evaluation as chemosensitizing agents on human ovarian adenocarcinoma and lung carcinoma cells

Lubeluzole, a neuroprotective anti-ischemic drug, and its enantiomer were prepared following a convenient procedure based on hydrolytic kinetic resolution. The ee values were >99% and 96%, respectively, as assessed by HPLC analysis. The chemosensitizing effects of both enantiomers were evaluated in combination with either doxorubicin (human ovarian adenocarcinoma A2780 cells) or paclitaxel (human lung carcinoma A549 cells) by the MTT assay. At the lowest concentrations used, lubeluzole showed an overall and remarkable tendency to synergize with both anticancer drugs. In ovarian cancer cells a clear prevalence of antagonistic effect was observed for the R-enantiomer. The synergistic effects of lubeluzole for both drugs were observed over a wide concentration window (0.005–5 μM), the lowest limit being at least 40 times lower than human plasma concentrations previously reported as causing serious side effects.     

Exhaustive CoMFA and CoMSIA analyses around different chemical entities: a ligand-based study exploring the affinity and selectivity profiles of 5-HT1A ligands

The 5-hydroxytryptamine (5-HT_1A) receptors represent an attractive target in drug discovery. In particular, 5-HT_1A agonists and partial agonists are deeply investigated for their potential role in the treatment of anxiety, depression, ischaemic brain disorder and more recently, of pain. On the other hand, 5-HT_1A antagonists have been revealed promising compounds in cognition disorders and, lately, in cancer. Thus, the discovery of 5HT_1A ligands is nowadays an appealing research activity in medicinal chemistry. In this work, Comparative Molecular Fields Analysis (CoMFA) and Comparative Molecular Similarity Index Analysis (CoMSIA) were applied on an in-house library of 5-HT_1A ligands bearing different chemical scaffolds in order to elucidate their affinity and selectivity for the target_. Following this procedure, a number of structural modifications have been drawn for the development of much more effective 5-HT_1AR ligands.