Virulence of invasive Salmonella Typhimurium ST313 in animal models of infection.

Salmonella Typhimurium sequence type (ST) 313 produces septicemia in infants in sub-Saharan Africa. Although there are known genetic and phenotypic differences between ST313 strains and gastroenteritis-associated ST19 strains, conflicting data about the in vivo virulence of ST313 strains have been reported. To resolve these differences, we tested clinical Salmonella Typhimurium ST313 and ST19 strains in murine and rhesus macaque infection models. The 50% lethal dose (LD50) was determined for three Salmonella Typhimurium ST19 and ST313 strains in mice. For dissemination studies, bacterial burden in organs was determined at various time-points post-challenge. Indian rhesus macaques were infected with one ST19 and one ST313 strain. Animals were monitored for clinical signs and bacterial burden and pathology were determined. The LD50 values for ST19 and ST313 infected mice were not significantly different. However, ST313-infected BALB/c mice had significantly higher bacterial numbers in blood at 24 h than ST19-infected mice. ST19-infected rhesus macaques exhibited moderate-to-severe diarrhea while ST313-infected monkeys showed no-to-mild diarrhea. ST19-infected monkeys had higher bacterial burden and increased inflammation in tissues. Our data suggest that Salmonella Typhimurium ST313 invasiveness may be investigated using mice. The non-human primate results are consistent with clinical data, suggesting that ST313 strains do not cause diarrhea.

Turning defense into offense: defensin mimetics as novel antibiotics targeting lipid II.

We have previously reported on the functional interaction of Lipid II with human alpha-defensins, a class of antimicrobial peptides. Lipid II is an essential precursor for bacterial cell wall biosynthesis and an ideal and validated target for natural antibiotic compounds. Using a combination of structural, functional and in silico analyses, we present here the molecular basis for defensin-Lipid II binding. Based on the complex of Lipid II with Human Neutrophil peptide-1, we could identify and characterize chemically diverse low-molecular weight compounds that mimic the interactions between HNP-1 and Lipid II. Lead compound BAS00127538 was further characterized structurally and functionally; it specifically interacts with the N-acetyl muramic acid moiety and isoprenyl tail of Lipid II, targets cell wall synthesis and was protective in an in vivo model for sepsis. For the first time, we have identified and characterized low molecular weight synthetic compounds that target Lipid II with high specificity and affinity. Optimization of these compounds may allow for their development as novel, next generation therapeutic agents for the treatment of Gram-positive pathogenic infections.

In vivo antiprostate tumor potential of Vernonia guineensis Benth. (Asteraceae) tuber extract (VGDE) and the cytotoxicity of its major compound pentaisovaleryl sucrose.

ETHNOPHARMACOLOGICAL RELEVANCE: Vernonia guineensis Benth. (Asteraceae) root decoction is used in folk medicine in Cameroon to treat some ailments including prostate cancer. The aim of this study was to validate the claimed antiprostate cancer activity of Vernonia guineensis Benth. in vivo and to investigate the cytotoxicity of a pentaisovaleryl sucrose isolated from Vernonia guineensis on some cancer cell lines. MATERIALS AND METHODS: A crude dichloromethane extract of Vernonia guineensis (VGDE) was used for this study. For in vivo antiprostate cancer efficacy, nude mice (n=16) were injected subcutaneously with prostate cancer PC-3 cells. Upon the formation of the xenograft tumors, the mice were divided into two equal groups with approximately the same mean tumor volume per group. One group was treated with VGDE orally (500 mg/kg) and the other with a vehicle control for 30 days. Body weight and tumor volumes were measured 2× a week and on the 33rd day, the mice were euthanized and tumors harvested and weighed. For the cytotoxicity study, the WST-1 assay was used to determine the activity of pentaisovaleryl sucrose previously isolated from VGDE. The cancer cell lines used in the cytotoxicity study included breast, colon, leukemia, lung, melanoma, ovarian and prostate. RESULTS: Prostate cancer (PC-3) xenograft tumors treated with VGDE showed a significant decrease in tumor size (P=0.0295) compared to control. Pentaisovaleryl sucrose also demonstrated cytotoxicity against various cancer cell lines with IC50 values as follows: MDA-MD-231-6.66µM; MCF-7-7.50 µM; HCT116-14.12 µM; A549-5.76 µM; HL60-6.43 µM; A375-8.64 µM; OVCAR3-9.53 µM; Capan1-7.13 µM; Mia-Paca 6.47 µM. CONCLUSION: VGDE does possess in vivo activity against prostate tumor and has potential for development into a natural product for the treatment of prostate cancer. This study thus provides preliminary validation for the folk use of Vernonia guineensis against prostate conditions. Further in vivo studies are however required to confirm these results and to understand the mechanism of action of VGDE and the in vivo efficacy of pentaisovaleryl sucrose.

An attenuated Lassa vaccine in SIV-infected rhesus macaques does not persist or cause arenavirus disease but does elicit Lassa virus-specific immunity.

BACKGROUND: Lassa hemorrhagic fever (LHF) is a rodent-borne viral disease that can be fatal for human beings. In this study, an attenuated Lassa vaccine candidate, ML29, was tested in SIV-infected rhesus macaques for its ability to elicit immune responses without instigating signs pathognomonic for arenavirus disease. ML29 is a reassortant between Lassa and Mopeia viruses that causes a transient infection in non-human primates and confers sterilizing protection from lethal Lassa viral challenge. However, since the LHF endemic area of West Africa also has high HIV seroprevalence, it is important to determine whether vaccination could be safe in the context of HIV infection. RESULTS: SIV-infected and uninfected rhesus macaques were vaccinated with the ML29 virus and monitored for specific humoral and cellular immune responses, as well as for classical and non-classical signs of arenavirus disease. Classical disease signs included viremia, rash, respiratory distress, malaise, high liver enzyme levels, and virus invasion of the central nervous system. Non-classical signs, derived from profiling the blood transcriptome of virulent and non-virulent arenavirus infections, included increased expression of interferon-stimulated genes (ISG) and decreased expression of COX2, IL-1ß, coagulation intermediates and nuclear receptors needed for stress signaling. All vaccinated monkeys showed ML29-specific antibody responses and ML29-specific cell-mediated immunity. CONCLUSION: SIV-infected and uninfected rhesus macaques responded similarly to ML29 vaccination, and none developed chronic arenavirus infection. Importantly, none of the macaques developed signs, classical or non-classical, of arenavirus disease.

Cytotoxic sesquiterpene lactones from the leaves of Vernonia guineensis Benth. (Asteraceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Vernonia guineensis Benth. (Asteraceae) preparations are used in folk medicine in Cameroon to treat a number of ailments, including prostate cancer and malaria, and is used as an anthelmintic, adaptogen and antidote. The aim of this study was to continue the validation of the activity of Vernonia guineensis Benth. extracts and isolated molecules against cancer cell lines following the previous isolation of an anti-prostate cancer sugar ester from the root extract. MATERIALS AND METHODS: Acetone extracts of Vernonia guineensis Benth. leaves were tested for activity against 10 cancer cell lines (Breast-MDA-MB-231, Breast-MCF-7, Colon-HCT-116, Leukemia-HL-60, Lung-A549, Melanoma-A375, Ovarian-OVCAR3, Pancreas-Mia-paca, Prostate-PC-3 and Prostate-DU-145). The acetone extract was subjected to bioactivity guided fractionation. Anti-proliferation and clonogenic activity of the isolated compounds were tested. The WST-1 assay was used for the anti-proliferation activity, while the standard clonogenic test was used to determine the clonogenic activity. RESULTS: The acetone extract of Vernonia guineensis Benth. demonstrated in vitro activity ranging from IC50 4-26µg/mL against the 10 cell lines. Activity guided fractionation of this extract yielded two sesquiterpene lactones, isolated for the first time from the genus Vernonia. The compounds were characterized using spectroscopic experiments, including a combination of 1D and 2D NMR data. Vernopicrin (1) and Vernomelitensin (2) demonstrated in vitro activity against human cancer cell lines with IC50 ranging from 0.35-2.04µM (P<0.05) and 0.13-1.5µM (P<0.05), respectively, between the most and least sensitive cell lines for each compound. Vernopicrin was most active against the human melanoma (A375) cell line and least active against the lung cancer (A549) cell line, while Vernomelitensin was also most active against the human melanoma (A375) cell line and least active against the breast cancer (MCF-7) cell line. Both compounds also demonstrated anticlonogenic activity. CONCLUSION: The cytotoxicity demonstrated by the crude extract and isolated sesquiterpenes against cancer cell lines highlights the medicinal potential of V. guineensis. The selective anti-proliferation and dose dependent anticlonogenic activities suggest that the identified sesquiterpenes could be potential antitumor agents.

Toxicity, antimicrobial and anthelmintic activities of Vernonia guineensis Benth. (Asteraceae) crude extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: This study examined the antibacterial, antifungal, and anthelmintic properties of extracts obtained from the plant Vernonia guineensis, a plant commonly used in traditional Cameroonian medicine. MATERIALS AND METHODS: For in vitro studies, 10 g of leaf and tuber powder from V. guineensis was extracted separately using dichloromethane, methanol and distilled water. The extracts were dried in vacuo and used for antimicrobial and anthelmintic activity studies. In the antimicrobial assay, extracts were tested against bacterial and fungal organisms including; Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii, Aspergillus fumigatus, Candida albicans and Trichophyton mentagrophytes. In the anthelmintic assay, larval and adult stages of the hookworm Ancylostoma ceylanicum and the mouse nematode Trichuris muris were used. For the acute toxicity test, male and female rats of 150-200 g body weight were used in the experiment. The aqueous extract of V. guineensis tubers was administered in 4 doses of 500, 1000, 2000 and 4000 mg/kg per group (n=6), respectively, and the control group received distilled water. RESULTS: The crude extracts exhibited weak antibacterial and antifungal activity except for the dichloromethane extract, which showed moderate activity against A. fumigatus (MIC=200 µg/ml). In the anthelmintic assay, the organic extracts of the tubers had 100% killing efficacy against T. muris at 2mg/ml in 48 h, while the aqueous extract showed no activity. The organic leaf extracts demonstrated potent activity killing 100% of the adult worms 1mg/ml in 24h. The aqueous leaf extract was active at 2mg/ml in 72 h, killing 100% of the adult worms. In the acute toxicity test, V. guineensis did not produce any toxic signs or death at the maximum concentration of 4000 mg/kg. CONCLUSION: Crude extracts from V. guineensis possess anthelmintic activity against T. muris with only weak antibiotic activity. Acute administration of aqueous extract from V. guineensis tubers did not produce toxic effects in rats. The absence of acute toxicity at the highest concentration tested indicates that the tea decoction from V. guineensis extract is safe at concentrations ≤ 4000 mg/kg.

In vitro anti-prostate cancer and ex vivo antiangiogenic activity of Vernonia guineensis Benth. (Asteraceae) tuber extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Prostate cancer is a major problem worldwide and affects most men above the age of forty-five. Vernonia guineensis Benth. (Asteraceae) root decoction is used in folk medicine in Cameroon to treat a number of ailments including prostate cancer. The aim of this study was to provide a preliminary validation of the use of Vernonia guineensis Benth. extracts to treat prostate cancer by evaluating the in vitro activity of its crude extracts and isolated molecules on prostate cancer cells lines and effect on angiogenesis which is essential for growth and metastases of prostate cancer. MATERIALS AND METHODS: Aqueous, dichloromethane and methanol extracts of Vernonia guineensis Benth. tubers were tested for activity against three prostate cancer cell lines (PC-3, DU-145 and AT3B-1). The dichloromethane extract was subjected to bioactivity guided fractionation. Anti-proliferation, clonogenic and antiangiogenic activity of the crude extracts and isolated compound were tested. The WST-1 assay was used for the anti-proliferation activity meanwhile the standard clonogenic test and the rat ring aorta assay were carried out to determine the clonogenic and antiangiogenic activity of tested products respectively. RESULTS: The aqueous and methanol extracts of Vernonia guineensis Benth. demonstrated weak activity against prostate cancer cell lines in vitro with IC(50)>100 µg/mL. The dichloromethane extract was more potent with IC(50) of 56.233±3.630 µg/ml and 67.316±2.452 µg/ml against the DU-145 and PC-3 cell lines respectively. Activity guided fractionation of this extract yielded a Pentaisovalerylsucrose (1) isolated for the first time from a natural source to the best of our knowledge. Compound 1 demonstrated in vitro activity against the human prostate cancer cell lines PC-3 and DU-145 with IC(50) of 5.701±0.142 µM and 4.275±0.710 µM, respectively. The IC(50) of the compound was 5.763±0.425 µM against AT3B-1, a rat prostate cancer cell line expressing P-glycoprotein which is linked to drug resistance in most metastatic cancers. Compared to compound 1, Paclitaxel and Docetaxel were active against AT3B-1 at 2.641±1.253 µM and 0.613±0.251 µM. Paclitaxel showed IC(50) values of 0.004±0.002 µM and 0.003±0.001 µM against DU-145 and PC-3 prostate cancer cell lines respectively. Docetaxel showed IC(50) values of 0.002±0.001 µM and 0.004±0.001 µM against DU-145 and PC-3 prostate cancer cell lines respectively. CONCLUSION: The in vitro anti-prostate cancer and the antiangiogenic activity of Vernonia guineensis Benth. extracts and isolated compound support the use of the tubers of this plant for the treatment of prostate cancer.

The metastatic microenvironment: brain-residing melanoma metastasis and dormant micrometastasis.

Brain metastasis occurs frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. We generated a reproducible melanoma brain metastasis model, consisting of brain-metastasizing variants and local, subdermal variants that originate from the same melanomas thus sharing a common genetic background. The brain-metastasizing variants were obtained by intracardiac inoculation. Brain metastasis variants when inoculated subdermally yielded spontaneous brain dormant micrometastasis. Cultured cells from the spontaneous brain micrometastasis grew very well in vitro and generated subdermal tumors after an orthotopic inoculation. Expression analysis assays indicated that the brain metastasis and micrometastasis cells expressed higher levels of angiopoietin-like 4, prostaglandin-synthesizing enzyme cyclooxygenase-2, matrix metalloproteinase-1 and preferentially expressed antigen in melanoma and lower levels of claudin-1 and cysteine-rich protein 61 than the corresponding cutaneous variants. The reproducible models of human melanoma metastasizing experimentally and spontaneously to the brain will facilitate the identification of novel biomarkers and targets for therapy and contribute to the deciphering of mechanisms underlying melanoma metastasis.

Lymphocytic choriomeningitis virus (LCMV) infection of macaques: a model for Lassa fever.

Arenaviruses such as Lassa fever virus (LASV) and lymphocytic choriomeningitis virus (LCMV) are benign in their natural reservoir hosts, and can occasionally cause severe viral hemorrhagic fever (VHF) in non-human primates and in human beings. LCMV is considerably more benign for human beings than Lassa virus, however certain strains, like the LCMV-WE strain, can cause severe disease when the virus is delivered as a high-dose inoculum. Here we describe a rhesus macaque model for Lassa fever that employs a virulent strain of LCMV. Since LASV must be studied within Biosafety Level-4 (BSL-4) facilities, the LCMV-infected macaque model has the advantage that it can be used at BSL-3. LCMV-induced disease is rarely as severe as other VHF, but it is similar in cases where vascular leakage leads to lethal systemic failure. The LCMV-infected macaque has been valuable for describing the course of disease with differing viral strains, doses and routes of infection. By monitoring system-wide changes in physiology and gene expression in a controlled experimental setting, it is possible to identify events that are pathognomonic for developing VHF and potential treatment targets.