Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.

B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity.

Increased Vascular Permeability in the Bone Marrow Microenvironment Contributes to Disease Progression and Drug Response in Acute Myeloid Leukemia.

The biological and clinical behaviors of hematological malignancies can be influenced by the active crosstalk with an altered bone marrow (BM) microenvironment. In the present study, we provide a detailed picture of the BM vasculature in acute myeloid leukemia using intravital two-photon microscopy. We found several abnormalities in the vascular architecture and function in patient-derived xenografts (PDX), such as vascular leakiness and increased hypoxia. Transcriptomic analysis in endothelial cells identified nitric oxide (NO) as major mediator of this phenotype in PDX and in patient-derived biopsies. Moreover, induction chemotherapy failing to restore normal vasculature was associated with a poor prognosis. Inhibition of NO production reduced vascular permeability, preserved normal hematopoietic stem cell function, and improved treatment response in PDX.

Host response. Inflammation-induced disruption of SCS macrophages impairs B cell responses to secondary infection.

The layer of macrophages at the subcapsular sinus (SCS) captures pathogens entering the lymph node, preventing their global dissemination and triggering an immune response. However, how infection affects SCS macrophages remains largely unexplored. Here we show that infection and inflammation disrupt the organization of SCS macrophages in a manner that involves the migration of mature dendritic cells to the lymph node. This disrupted organization reduces the capacity of SCS macrophages to retain and present antigen in a subsequent secondary infection, resulting in diminished B cell responses. Thus, the SCS macrophage layer may act as a sensor or valve during infection to temporarily shut down the lymph node to further antigenic challenge. This shutdown may increase an organism's susceptibility to secondary infections.

Reactive oxygen-mediated damage to a human DNA replication and repair protein.

Ultraviolet A (UVA) makes up more than 90% of incident terrestrial ultraviolet radiation. Unlike shorter wavelength UVB, which damages DNA directly, UVA is absorbed poorly by DNA and is therefore considered to be less hazardous. Organ transplant patients treated with the immunosuppressant azathioprine frequently develop skin cancer. Their DNA contains 6-thioguanine-a base analogue that generates DNA-damaging singlet oxygen ((1)O(2)) when exposed to UVA. Here, we show that this (1)O(2) damages proliferating cell nuclear antigen (PCNA), the homotrimeric DNA polymerase sliding clamp. It causes covalent oxidative crosslinking between the PCNA subunits through a histidine residue in the intersubunit domain. Crosslinking also occurs after treatment with higher-although still moderate-doses of UVA alone or with chemical oxidants. Chronic accumulation of oxidized proteins is linked to neurodegenerative disorders and ageing. Our findings identify oxidative damage to an important DNA replication and repair protein as a previously unrecognized hazard of acute oxidative stress.

Proteomic analysis of prodigiosin-induced apoptosis in a breast cancer mitoxantrone-resistant (MCF-7 MR) cell line.

Prodigiosin (PG) is a bacterial, red-pigmented antibiotic with immunosuppressive and apoptotic activities. To better understand its mechanisms of action, we tried to identify proteins associated with apoptosis induced by PG. For this purpose, the variation of protein expression on exposure to apoptotic concentrations of PG was examined, by high-resolution two-dimensional gel electrophoresis (2D-E), in the MCF-7 cancer cell line resistant to mitoxantrone (MCF-7-MR). Six PG apoptosis-associated protein spots were further characterized by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on a matrix-assisted laser desorption ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometer. The proteins identified were involved in various cellular functions, including cell defence, DNA repair and cellular organization. Our data provide novel information on cell response to PG, a new apoptotic drug with interesting anticancer activity.

Novel DNA lesions generated by the interaction between therapeutic thiopurines and UVA light.

The therapeutic effect of the thiopurines, 6-thioguanine (6-TG), 6-mercaptopurine, and its prodrug azathioprine, depends on the incorporation of 6-TG into cellular DNA. Unlike normal DNA bases, 6-TG absorbs UVA radiation, and UVA-mediated photochemical damage of DNA 6-TG has potentially harmful side effects. When free 6-TG is UVA irradiated in solution in the presence of molecular oxygen, reactive oxygen species are generated and 6-TG is oxidized to guanine-6-sulfonate (G(SO3)) and guanine-6-thioguanine in reactions involving singlet oxygen. This conversion is prevented by antioxidants, including the dietary vitamin ascorbate. DNA G(SO3) is also the major photoproduct of 6-TG in DNA and it can be selectively introduced into DNA or oligonucleotides in vitro by mild chemical oxidation. Thermal stability measurements indicate that G(SO3) does not form stable base pairs with any of the normal DNA bases in duplex oligonucleotides and is a powerful block for elongation by Klenow DNA polymerase in primer extension experiments. In cultured human cells, DNA damage produced by 6-TG and UVA treatment is associated with replication inhibition and provokes a p53-dependent DNA damage response.

Cell cycle arrest and proapoptotic effects of the anticancer cyclodepsipeptide serratamolide (AT514) are independent of p53 status in breast cancer cells.

In a search for new anticancer agents, we have identified serratamolide (AT514), a cyclodepsipeptide from Serratia marcescens 2170 that induces cell cycle arrest and apoptosis in various cancer cell lines. A cell viability assay showed that the concentrations that cause 50% inhibition (IC50) in human cancer cell lines range from 5.6 to 11.5 microM depending on the cell line. Flow cytometry analysis revealed that AT514 caused cell cycle arrest in G0/G1 or cell death, depending on the cell type and the length of time for which the cells were exposed to the drug. Subsequent studies revealed that AT514-induced cell death is caused by apoptosis, as indicated by caspases activation (8, 9, 2 and 3) and cleavage of poly (ADP-ribose) polymerase (PARP), release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, and the appearance of apoptotic bodies and DNA laddering. Alterations in protein levels of Bcl-2 family members might be involved in the mitochondrial disruption observed. AT514 induced p53 accumulation in wild-type p53 cells but cell death was observed in both deficient and wild-type p53 cells. Our results indicate that AT514 induces cell cycle arrest and apoptosis in breast cancer cells irrespectively of p53 status, suggesting that it might represent a potential new chemotherapeutic agent.

Azathioprine and UVA light generate mutagenic oxidative DNA damage.

Oxidative stress and mutagenic DNA lesions formed by reactive oxygen species (ROS) are linked to human malignancy. Clinical treatments inducing chronic oxidative stress may therefore carry a risk of therapy-related cancer. We suggest that immunosuppression by azathioprine (Aza) may be one such treatment. Aza causes the accumulation of 6-thioguanine (6-TG) in patients' DNA. Here we demonstrate that biologically relevant doses of ultraviolet A (UVA) generate ROS in cultured cells with 6-TG-substituted DNA and that 6-TG and UVA are synergistically mutagenic. A replication-blocking DNA 6-TG photoproduct, guanine sulfonate, was bypassed by error-prone, Y-family DNA polymerases in vitro. A preliminary analysis revealed that in five of five cases, Aza treatment was associated with a selective UVA photosensitivity. These findings may partly explain the prevalence of skin cancer in long-term survivors of organ transplantation.

DNA interaction and dual topoisomerase I and II inhibition properties of the anti-tumor drug prodigiosin.

Prodigiosin is a red pigment produced by Serratia marcescens with apoptotic activity. We examined the mechanism of action of this tripyrrole alkaloid, focusing on its interaction with DNA and its ability to inhibit both topoisomerase I and topoisomerase II. We also evaluated the DNA damage induced in cancer cell lines. Prodigiosin-DNA intercalation was analyzed using a competition dialysis assay with different DNA base sequences. Topoisomerase I and II inhibition was studied in vitro by a cleavage assay, and in cultured cells, by analysis of its ability to form covalent complexes. Furthermore, we analyzed DNA damage by pulse-field gel electrophoresis and by immunocytochemistry. Apoptosis inducing factor (AIF)/phospho-H2AX (p-H2AX) double labeling by confocal microscopy was performed to determine the possible implication of AIF in the prodigiosin-DNA damage. Finally, we studied the ability of this drug to induce copper-mediated DNA damage at different pH by a DNA cleavage assay. Our results demonstrate prodigiosin-DNA interaction in vitro and in cultured cells. It involves prodigiosin-DNA intercalation, with some preference for the alternating base pairs but with no discrimination between AT or CG sequences, dual abolition of topoisomerase I and II activity and, as consequence, DNA cleavage. Prodigiosin-DNA damage is independent of AIF. Furthermore, we found that copper-mediated cleavage activity is associated with pH (occurring at pH 6.8 rather than pH 7.4) and with the Cu(2+) ion concentration. These results indicate DNA a therapeutic target for prodigiosin and could explain the apoptosis mechanism of action induced by this antineoplastic drug.

High cytotoxic sensitivity of the human small cell lung doxorubicin-resistant carcinoma (GLC4/ADR) cell line to prodigiosin through apoptosis activation.

In the present study, we describe the cytotoxicity of the new drug prodigiosin (PG) in two small cell lung carcinoma (SCLC) cell lines, GLC4 and its derived doxorubicin-resistant GLC4/ADR cell line, which overexpresses multidrug-related protein 1 (MRP-1). We observed through Western blot that PG mediated cytochrome c release, caspase cascade activation and PARP cleavage, thereby leading to apoptosis in a dose-response manner. MRP-1 expression increased after PG treatment, although that does not lead to protein accumulation. The MTT assay showed no difference in sensitivity to PG between the two cell lines. Our results support PG as a potential drug for the treatment of lung cancer as it overcomes the multidrug resistance phenotype produced by MRP-1 overexpression.

Mitochondria-mediated apoptosis operating irrespective of multidrug resistance in breast cancer cells by the anticancer agent prodigiosin.

Prodigiosin (PG) is a red pigment produced by Serratia marcescens with pro-apoptotic activity in haematopoietic and gastrointestinal cancer cell lines, but no marked toxicity in non-malignant cells. Breast cancer is the most frequent malignancy among women in the European Union and better therapies are needed, especially for metastatic tumors. Moreover, multidrug resistance is a common phenomenon that appears during chemotherapy, necessitating more aggressive treatment as prognosis worsens. In this work, we extend our experiments on PG-induced apoptosis to breast cancer cells. PG was potently cytotoxic in both estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cell lines. Cytochrome c release, activation of caspases-9, -8 and -7 and cleavage of poly (ADP-ribose) polymerase protein typified the apoptotic event and caspase inhibition revealed that PG acts via the mitochondrial pathway. In a multidrug-resistant subline of MCF-7 cells that over-expresses the breast cancer resistance protein, the cytotoxic activity of PG was slightly reduced. However, flow-cytometry analysis of PG accumulation and efflux in MCF-7 sublines showed that PG is not a substrate for this resistance protein. These results suggest that PG is an interesting and potent new pro-apoptotic agent for the treatment of breast cancer even when multidrug resistance transporter molecules are present.

The prodigiosins, proapoptotic drugs with anticancer properties.

The family of natural red pigments, called prodigiosins (PGs), characterised by a common pyrrolylpyrromethene skeleton, are produced by various bacteria. Some members have immunosuppressive properties and apoptotic effects in vitro and they have also displayed antitumour activity in vivo. Understanding the mechanism of action of PGs is essential for drug development and will require the identification and characterisation of their still unidentified cell target. Four possible mechanisms of action have been suggested for these molecules: (i) PGs as pH modulators; (ii) PGs as cell cycle inhibitors; (iii) PGs as DNA cleavage agents; (iv) PGs as mitogen-activated protein kinase regulators. Here, we review the pharmacological activity of PG and related compounds, including novel synthetic PG derivatives with lower toxicity and discuss the mechanisms of action and the molecular targets of those molecules. The results reported in this review suggest that PGs are a new class of anticancer drugs, which hold out considerable promise for the Pharmacological Industry.

The prodigiosins: a new family of anticancer drugs.

Apoptosis is involved in the action of several (and perhaps all) cancer-chemotherapeutic agents. Prodigiosins, a family of natural red pigments characterized by a common pyrrolylpyrromethene skeleton, are produced by various bacteria. Three members of the prodigiosin family, viz. prodigiosin (PG), undecylprodigiosin (UP) and cycloprodigiosin hydrochloride (cPrG.HCl), have immunosuppressive properties and apoptotic effects on cancer cells in vitro and in vivo. Their cytotoxic effect is attributed to the presence of the C-6 methoxy substituent. The A-pyrrole ring plays a key role in both the copper nuclease activity and the cytotoxicity of prodigiosins. Here, we have reviewed the pharmacological activity of PG and related compounds, including novel synthetic PG-derivatives with lower toxicity. The mechanism of action for these molecules is a current topic in biomedicine. The molecular targets of prodigiosins are also discussed.

Prodigiosin induces apoptosis by acting on mitochondria in human lung cancer cells.

Prodigiosin (PG) is a secondary metabolite, isolated from a culture of Serratia marcescens, which has shown potent cytotoxicity against various human cancer cell lines as well as immunosuppressive activity. The purpose of this study was to evaluate the role of mitochondria in PG-induced apoptosis. Therefore, we evaluated the apoptotic action of PG in GLC4 small cell lung cancer cell line by Hoechst 33342 staining. In these cells, we examined mitochondrial apoptosis-inducing factor (AIF) and cytochrome c (cyt c) release to the cytosol in PG time-response studies. These findings suggest that PG induces apoptosis in both caspase-dependent and caspase-independent pathways.

Peptide dendrimers based on polyproline helices.

We present a new family of peptide dendrimers based on polyproline helices and cis-4-amino-L-proline as a branching unit. Dendrimers were synthesized by a convergent solid-phase peptide synthesis approach. The conformational transition between polyproline type I helix and polyproline type II helix was observed by circular dichroism in branched polyproline building blocks with more than 14 proline residues and in the resulting dendrimers. Both linear and dendritic polyprolines were found to be actively internalized by rat kidney cells. Preliminary results show that the antibiotic ciprofloxacin form complexes with branched polyproline chains in 99.5% propanol.

The cytotoxic prodigiosin induces phosphorylation of p38-MAPK but not of SAPK/JNK.

Prodigiosin (PG) is a red pigment produced by Serratia marcescens, with cytotoxic and immunosuppressive activity. It induces apoptosis in several cancer cell lines, including Jurkat-T cells. Here we examine the role of two stress-stimulated kinase cascades in this induction. Time course experiments using polyclonal antibodies showed that p38-MAPK phosphorylation began at 15 min and lasted for 3 h, whereas JNK was not phosphorylated, although both proteins were present. SB203580, a selective inhibitor of p38-MAPK, blocked its phosphorylation in PG-treated cells. Taken together, these data suggest that the PG induces phosphorylation of p38-MAPK but not of SAPK/JNK and that it increases the expression of both c-jun and c-fos oncoproteins.