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Coibacins A D, Antileishmanial Marine Cyanobacterial Polyketides with Intriguing Biosynthetic Origins
Get PDFFour unsaturated polyketide lactone derivatives, coibacins A-D, were isolated from a Panamanian marine cyanobacterium, cf. Oscillatoria sp. The two different types of termini observed in these co-occurring metabolites, either a methyl cyclopropyl ring as seen in curacin A or a methyl vinyl chloride similar to that observed in the jamaicamides, suggest an intriguing flexibility in the “beta branch” forming biosynthetic process. The coibacins possess selective antileishmanial activity as well as potent anti-inflammatory activity.Four unsaturated polyketide lactone derivatives, coibacins A-D, were isolated from a Panamanian marine cyanobacterium, cf. Oscillatoria sp. The two different types of termini observed in these co-occurring metabolites, either a methyl cyclopropyl ring as seen in curacin A or a methyl vinyl chloride similar to that observed in the jamaicamides, suggest an intriguing flexibility in the “beta branch” forming biosynthetic process. The coibacins possess selective antileishmanial activity as well as potent anti-inflammatory activity
Differential responses of osteoblast lineage cells to nanotopographically-modified, microroughened titaniumealuminumevanadium alloy surfaces
Get PDFSurface structural modifications at the micrometer and nanometer scales have driven improved success rates of dental and orthopaedic implants by mimicking the hierarchical structure of bone. However, how initial osteoblast-lineage cells populating an implant surface respond to different hierarchical surface topographical cues remains to be elucidated, with bone marrow mesenchymal stem cells (MSCs) or immature osteoblasts as possible initial colonizers. Here we show that in the absence of any exogenous soluble factors, osteoblastic maturation of primary human osteoblasts (HOBs) but not osteoblastic differentiation of MSCs is strongly influenced by nanostructures superimposed onto a microrough Ti6Al4V (TiAlV) alloy. The sensitivity of osteoblasts to both surface microroughness and nanostructures led to a synergistic effect on maturation and local factor production. Osteoblastic differentiation of MSCs was sensitive to TiAlV surface microroughness with respect to production of differentiation markers, but no further enhancement was found when cultured on micro/nanostructured surfaces. Superposition of nanostructures to microroughened surfaces affected final MSC numbers and enhanced production of vascular endothelial growth factor (VEGF) but the magnitude of the response was lower than for HOB cultures. Our results suggest that the differentiation state of osteoblast-lineage cells determines the recognition of surface nanostructures and subsequent cell response, which has implications for clinical evaluation of new implant surface nanomodifications.Surface structural modifications at the micrometer and nanometer scales have driven improved success rates of dental and orthopaedic implants by mimicking the hierarchical structure of bone. However, how initial osteoblast-lineage cells populating an implant surface respond to different hierarchical surface topographical cues remains to be elucidated, with bone marrow mesenchymal stem cells (MSCs) or immature osteoblasts as possible initial colonizers. Here we show that in the absence of any exogenous soluble factors, osteoblastic maturation of primary human osteoblasts (HOBs) but not osteoblastic differentiation of MSCs is strongly influenced by nanostructures superimposed onto a microrough Ti6Al4V (TiAlV) alloy. The sensitivity of osteoblasts to both surface microroughness and nanostructures led to a synergistic effect on maturation and local factor production. Osteoblastic differentiation of MSCs was sensitive to TiAlV surface microroughness with respect to production of differentiation markers, but no further enhancement was found when cultured on micro/nanostructured surfaces. Superposition of nanostructures to microroughened surfaces affected final MSC numbers and enhanced production of vascular endothelial growth factor (VEGF) but the magnitude of the response was lower than for HOB cultures. Our results suggest that the differentiation state of osteoblast-lineage cells determines the recognition of surface nanostructures and subsequent cell response, which has implications for clinical evaluation of new implant surface nanomodifications
New species, phylogeny, host-associations and geographic distribution of genus Cryptosporella (Gnomoniaceae, Diaporthales)
Get PDFThe phylogeny of Cryptosporella is revised to include recently discovered species. Eight species new to science are described and two new combinations are proposed, raising the total number of species accepted in Cryptosporella to 19. The species delimitation and phylogeny for Cryptosporella are determined based on analyses of DNA sequences from three genes (btubulin, ITS and tef1-a), comparative morphology of sexual structures on their host substrate, and host associations. The inferred phylogeny suggests that Cryptosporella has speciated primarily on Betulaceae with 16 species occurring on hosts in that plant family. The host range of most species seems to be narrow with nine species reported from a single host species or subspecies and seven species occurring on plants within a single host genus. A key to species is provided. The known distribution of Cryptosporella is expanded to mountain cloud forests of the provinces of Chiriquı´ in Panama and Tucuma´n in Argentina.The phylogeny of Cryptosporella is revised to include recently discovered species. Eight species new to science are described and two new combinations are proposed, raising the total number of species accepted in Cryptosporella to 19. The species delimitation and phylogeny for Cryptosporella are determined based on analyses of DNA sequences from three genes (btubulin, ITS and tef1-a), comparative morphology of sexual structures on their host substrate, and host associations. The inferred phylogeny suggests that Cryptosporella has speciated primarily on Betulaceae with 16 species occurring on hosts in that plant family. The host range of most species seems to be narrow with nine species reported from a single host species or subspecies and seven species occurring on plants within a single host genus. A key to species is provided. The known distribution of Cryptosporella is expanded to mountain cloud forests of the provinces of Chiriquı´ in Panama and Tucuma´n in Argentina
Plasmodium falciparum field isolates use complement receptor 1 (CR1) as a receptor for invasion of erythrocytes
Get PDFAmajorityofPlasmodiumfalciparumstrainsinvadeerythrocytesthroughinteractionswithsialicacid(SA) on glycophorins. However, we recently reported that complement receptor 1 (CR1) is a SA-independent invasionreceptorofmanylaboratorystrainsofP.falciparum.TodeterminetheroleofCR1inerythrocyte invasion among P. falciparum field isolates, we tested eight isolates obtained from children in Kenya. All the parasites examined were capable of invading in a SA-independent manner, and invasion of neuraminidase-treatederythrocyteswasnearlycompletelyblockedbyanti-CR1andsolubleCR1(sCR1). Inaddition,anti-CR1andsCR1partiallyinhibitedinvasionofintacterythrocytesinamajorityofisolates tested. Sequencing of the hypervariable region of P. falciparum AMA-1 showed considerable diversity among all the isolates. These data demonstrate that CR1 mediates SA-independent erythrocyte invasion in P. falciparum field isolates.AmajorityofPlasmodiumfalciparumstrainsinvadeerythrocytesthroughinteractionswithsialicacid(SA) on glycophorins. However, we recently reported that complement receptor 1 (CR1) is a SA-independent invasionreceptorofmanylaboratorystrainsofP.falciparum.TodeterminetheroleofCR1inerythrocyte invasion among P. falciparum field isolates, we tested eight isolates obtained from children in Kenya. All the parasites examined were capable of invading in a SA-independent manner, and invasion of neuraminidase-treatederythrocyteswasnearlycompletelyblockedbyanti-CR1andsolubleCR1(sCR1). Inaddition,anti-CR1andsCR1partiallyinhibitedinvasionofintacterythrocytesinamajorityofisolates tested. Sequencing of the hypervariable region of P. falciparum AMA-1 showed considerable diversity among all the isolates. These data demonstrate that CR1 mediates SA-independent erythrocyte invasion in P. falciparum field isolates
Credneramides A and B: Neuromodulatory Phenethylamine and Isopentylamine Derivatives of a Vinyl Chloride-Containing Fatty Acid from cf. Trichodesmium sp. nov.
Get PDFCredneramides A (1) and B (2), two vinyl chloride-containing metabolites, were isolated from a Papua New Guinea collection of cf. Trichodesmium sp. nov. and expand a recently described class of vinyl chloride-containing natural products. The precursor fatty acid, credneric acid (3), was isolated from both the aqueous and organic fractions of the parent fraction as well as from another geographically and phylogenetically distinct cyanobacterial collection (Panama). Credneramides A and B inhibited spontaneous calcium oscillations in murine cerebrocortical neurons at low micromolar concentrations (1, IC50 4.0 μM; 2, IC50 3.8 μM).Credneramides A (1) and B (2), two vinyl chloride-containing metabolites, were isolated from a Papua New Guinea collection of cf. Trichodesmium sp. nov. and expand a recently described class of vinyl chloride-containing natural products. The precursor fatty acid, credneric acid (3), was isolated from both the aqueous and organic fractions of the parent fraction as well as from another geographically and phylogenetically distinct cyanobacterial collection (Panama). Credneramides A and B inhibited spontaneous calcium oscillations in murine cerebrocortical neurons at low micromolar concentrations (1, IC50 4.0 μM; 2, IC50 3.8 μM)
The effects of combined micron-/submicron-scale surface roughness and nanoscale features on cell proliferation and differentiation
Get PDFTitanium (Ti) osseointegration is critical for the success of dental and orthopedic implants. Previous studies have shown that surface roughness at the micro- and submicro-scales promotes osseointegration by enhancing osteoblast differentiation and local factor production. Only relatively recently have the effects of nanoscale roughness on cell response been considered. The aim of the present study was to develop a simple and scalable surface modification treatment that introduces nanoscale features to the surfaces of Ti substrates without greatly affecting other surface features, and to determine the effects of such superimposed nano-features on the differentiation and local factor production of osteoblasts. A simple oxidation treatment was developed for generating controlled nanoscale topographies on Ti surfaces, while retaining the starting micro-/submicro-scale roughness. Such nano-modified surfaces also possessed similar elemental compositions, and exhibited similar contact angles, as the original surfaces, but possessed a different surface crystal structure. MG63 cells were seeded on machined (PT), nano-modified PT (NMPT), sandblasted/acid-etched (SLA), and nano-modified SLA (NMSLA) Ti disks. The results suggested that the introduction of such nanoscale structures in combination with micro-/submicro-scale roughness improves osteoblast differentiation and local factor production, which, in turn, indicates the potential for improved implant osseointegration in vivoTitanium (Ti) osseointegration is critical for the success of dental and orthopedic implants. Previous studies have shown that surface roughness at the micro- and submicro-scales promotes osseointegration by enhancing osteoblast differentiation and local factor production. Only relatively recently have the effects of nanoscale roughness on cell response been considered. The aim of the present study was to develop a simple and scalable surface modification treatment that introduces nanoscale features to the surfaces of Ti substrates without greatly affecting other surface features, and to determine the effects of such superimposed nano-features on the differentiation and local factor production of osteoblasts. A simple oxidation treatment was developed for generating controlled nanoscale topographies on Ti surfaces, while retaining the starting micro-/submicro-scale roughness. Such nano-modified surfaces also possessed similar elemental compositions, and exhibited similar contact angles, as the original surfaces, but possessed a different surface crystal structure. MG63 cells were seeded on machined (PT), nano-modified PT (NMPT), sandblasted/acid-etched (SLA), and nano-modified SLA (NMSLA) Ti disks. The results suggested that the introduction of such nanoscale structures in combination with micro-/submicro-scale roughness improves osteoblast differentiation and local factor production, which, in turn, indicates the potential for improved implant osseointegration in viv
Screening and evaluation of antiparasitic and in vitro anticancer activities of Panamanian endophytic fungi
Get PDFParasitic infections are major causes of human chronic diseases in most countries of the tropics. The parasites include protozoa and helminths, infect billions of people, and the resulting diseases cause debilitating injuries such as blind ness and disfigurement, or death in millions of people. According to World Health Organization (WHO) estimates, 25% of the human population is infected with parasitic worms. However, attempts to develop vaccines against these pathogens have been frustrated by the difficulty of cultivating the parasites in the laboratory, the complexity of their multicellular organization and—in many species—their multistage development, in addition to their impressive antigenic variability [http://www.who.int/vaccine_research/diseases/ soa_parasitic/en/index.html]. Malaria is the most dangerous parasitic disease, as evidenced by the high rates of complications and mortality caused by the most fatal species, Plasmodium falciparum [15]. Chagas disease, or American trypanosomiasis, is a potentially life-threatening two-phase illness caused by the protozoan Trypanosoma cruzi. The acute phase persists for about two months after infection; symptoms are absent or mild and can include fever, headache, enlarged lymph glands, pallor, muscle pain, difficulty in breathing, swelling, and abdominal or chest pain. In the chronic phase, the parasites reside mainly in the heart and digestive muscle, resulting in cardiac disorders in up to 30% of patients and digestive, neurological, or mixed pathologies in up to 10%. Eventually, the infection can lead to sudden death or heart failure, caused by progressive destruction of cardiac muscle [10,15]. Leishmaniasis, a worldwide disease, is caused by several species of the flagellated protozoan parasite Leishmania. In its more severe forms, the disease causes serious disfigurement and may be fatal. The WHO estimates a worldwide prevalence of leishmaniasis of approximately 12 million cases, with an annual mortality of about 60,000 and approximately 350 million people at risk. The expansion of leishmaniasis and the alarming rise in the number of cases has been attributed to environmental changes, such as deforestation, dam construction, new irrigation schemes, and the migration of non-immune individuals to endemic areas [10,15]. At the same time, the frequency of drug-resistant parasites has greatly increased and most treatments involve highly toxic drugs. In addition, the chemotherapeutic agents used in patients with these diseases have lacked effectiveness. Thus, there is an urgent need to search for novel drugs from previously unexplored sources, including natural products, to combat the global health problems posed by parasitic infections. Cancer is another major cause of mortality worldwide; in 2008, it accounted for 7.6 million deaths. According to WHO forecasts, an increase to 11 million deaths annually is expected by 2030. The prevalence is higher in low and middle-income countries. As a part of the on-going research activities, the Panamanian International Cooperative Biodiversity Group (ICBG) [17] recently decided to explore endophytic fungi as a source of molecules with antiparasitic and anticancer bioactivities [18,21,22]. Within the ICBG program, we have assayed the antiparasitic and in vitro anticancer activities of 25 isolates, while also analyzing the effect of the culture medium on the production of secondary metabolites by Panamanian endophytic fungi. The results of these studies are reported and discussed hereinParasitic infections are major causes of human chronic diseases in most countries of the tropics. The parasites include protozoa and helminths, infect billions of people, and the resulting diseases cause debilitating injuries such as blind ness and disfigurement, or death in millions of people. According to World Health Organization (WHO) estimates, 25% of the human population is infected with parasitic worms. However, attempts to develop vaccines against these pathogens have been frustrated by the difficulty of cultivating the parasites in the laboratory, the complexity of their multicellular organization and—in many species—their multistage development, in addition to their impressive antigenic variability [http://www.who.int/vaccine_research/diseases/ soa_parasitic/en/index.html]. Malaria is the most dangerous parasitic disease, as evidenced by the high rates of complications and mortality caused by the most fatal species, Plasmodium falciparum [15]. Chagas disease, or American trypanosomiasis, is a potentially life-threatening two-phase illness caused by the protozoan Trypanosoma cruzi. The acute phase persists for about two months after infection; symptoms are absent or mild and can include fever, headache, enlarged lymph glands, pallor, muscle pain, difficulty in breathing, swelling, and abdominal or chest pain. In the chronic phase, the parasites reside mainly in the heart and digestive muscle, resulting in cardiac disorders in up to 30% of patients and digestive, neurological, or mixed pathologies in up to 10%. Eventually, the infection can lead to sudden death or heart failure, caused by progressive destruction of cardiac muscle [10,15]. Leishmaniasis, a worldwide disease, is caused by several species of the flagellated protozoan parasite Leishmania. In its more severe forms, the disease causes serious disfigurement and may be fatal. The WHO estimates a worldwide prevalence of leishmaniasis of approximately 12 million cases, with an annual mortality of about 60,000 and approximately 350 million people at risk. The expansion of leishmaniasis and the alarming rise in the number of cases has been attributed to environmental changes, such as deforestation, dam construction, new irrigation schemes, and the migration of non-immune individuals to endemic areas [10,15]. At the same time, the frequency of drug-resistant parasites has greatly increased and most treatments involve highly toxic drugs. In addition, the chemotherapeutic agents used in patients with these diseases have lacked effectiveness. Thus, there is an urgent need to search for novel drugs from previously unexplored sources, including natural products, to combat the global health problems posed by parasitic infections. Cancer is another major cause of mortality worldwide; in 2008, it accounted for 7.6 million deaths. According to WHO forecasts, an increase to 11 million deaths annually is expected by 2030. The prevalence is higher in low and middle-income countries. As a part of the on-going research activities, the Panamanian International Cooperative Biodiversity Group (ICBG) [17] recently decided to explore endophytic fungi as a source of molecules with antiparasitic and anticancer bioactivities [18,21,22]. Within the ICBG program, we have assayed the antiparasitic and in vitro anticancer activities of 25 isolates, while also analyzing the effect of the culture medium on the production of secondary metabolites by Panamanian endophytic fungi. The results of these studies are reported and discussed herei
Cytotoxic Veraguamides, Alkynyl Bromide-containing cyclic Depsipeptides from the marine Cyanobacterium cf. Osciallatoria margaritifera. EmilyCytotoxic Veraguamides, Alkynyl Bromide-containing cyclic Depsipeptides from the marine Cyanobacterium cf. Osciallatoria margaritifera.
No full textA family of cancer cell cytotoxic cyclodepsipeptides, veraguamides A-C( 1-3) and H-L( 4-8), were isolated from a collection of cf. Oscillatoria margaritifera obtained from the Coiba National Park, Panama, as part of the Panama International Cooperation Biodiversity Group program. The planar structure of veraguamide A (1) was deduced by 2D NMR spectroscopy and mass spectrometry, whereas the structures of 2-8 were mainly determinedbyacombinationof 1HNMRandMS2/MS3techniques. These new compounds are analogous to the mollusk-derived kulomo’opunalidenaturalproducts,withtwooftheveraguamides(Cand H) containing the same terminal alkyne moiety. However, four veraguamides, A, B, K, and L, also feature an alkynyl bromide, a functionality that has been previously observed in only one other marine natural product, jamaicamide A. Veraguamide A showed potent cytotoxicity to the H-460 human lung cancer cell line (LD50 = 141 nM).A family of cancer cell cytotoxic cyclodepsipeptides, veraguamides A-C( 1-3) and H-L( 4-8), were isolated from a collection of cf. Oscillatoria margaritifera obtained from the Coiba National Park, Panama, as part of the Panama International Cooperation Biodiversity Group program. The planar structure of veraguamide A (1) was deduced by 2D NMR spectroscopy and mass spectrometry, whereas the structures of 2-8 were mainly determinedbyacombinationof 1HNMRandMS2/MS3techniques. These new compounds are analogous to the mollusk-derived kulomo’opunalidenaturalproducts,withtwooftheveraguamides(Cand H) containing the same terminal alkyne moiety. However, four veraguamides, A, B, K, and L, also feature an alkynyl bromide, a functionality that has been previously observed in only one other marine natural product, jamaicamide A. Veraguamide A showed potent cytotoxicity to the H-460 human lung cancer cell line (LD50 = 141 nM)
Anti-lipid antibody in M. tuberculosis infection--basis for a new biomarker-based test to monitor treatment response
Get PDFTuberculosis (TB) faces difficult challenges for its treatment and control. Both the diagnosis of TB and Mycobacterium tuberculosis (M. tuberculosis) drug susceptibility testing take weeks and clinicians often do not know if the patient is taking an appropriate set of drugs until complications or even death occur. Consequently, early determination of a successful drug therapy response in individuals infected with M. tuberculosis is urgently needed. Since the M. tuberculosis cell wall is comprised of a diverse repertoire of lipids, we examined the possible role of these lipids as antigens for serologic response during M. tuberculosis infection. This dissertation is focused on the examination of lipid-antibody response as a potential biomarker used to monitor treatment response in M. tuberculosis infected hosts. Briefly, Chapter 1 describes the current pitfalls of monitoring tuberculosis treatment with current methods, including acid-fast bacilli (AFB) smear and culture conversion. Chapter 1 also covers the definition of biomarkers and the rationale to use M. tuberculosis cell wall lipids to develop an anti-lipid-antibody based test. Evidence from a similar biomarker-based test for syphilis is presented. The chapter also discusses the biological basis which guided the lipid-antibody biomarker search and discovery.
Chapter 2 describes the use of a serum bank from patients with pulmonary TB provided by the World Health Organization-Tropical Diseases Research consortium (WHO-TDR) to identify M. tuberculosis lipid candidates as targets of antibody response. These samples were used to look for an antibody response to multiple mycobacterial lipids resolved by thin-layer chromatography immunoblot (TLC-I). This approach allowed us to identify M. tuberculosis cardiolipin by mass spectrometry and we determined that the IgM antibody response to cardiolipin can be used as a biomarker of infection.
In Chapter 3, we investigate the biological evidence behind the production of anti-phospholipid IgM antibody during TB infection and anti-TB treatment. For this purpose, we used the Cornell mouse model of infection to monitor the change in IgM antibody response against four phospholipids including cardiolipin (CL), phosphatidyl choline (PTC), phosphatidyl ethanolamine (PE) and phosphatidyl inositol (PI) over the course of M. tuberculosis infection and treatment. We separated BALB/c mice into three groups including acute infection (AI), chronic infection (CI), and healthy control (HC). Both AI and CI groups were infected via the aerosol route with M. tuberculosis strain H37Rv at day 0. The AI group was treated from 4 to 12 weeks post-infection, while the CI group was treated from 20 to 28 weeks post-infection. We also measured the levels of pro-inflammatory cytokines, IL-5 and MCP-1. We observed that in treated AI mice, anti-phospholipid IgM antibody levels decreased compared to those of healthy mice at all time points. Anti-PTC IgM antibodies remained significantly higher in CI mice than in AI mice at all time points post-infection. The anti-PTC IgM antibody levels in CI mice decreased to levels similar to those of AI and HC mice at 32 weeks post-infection. The anti-phospholipid IgM antibody levels correlated with the bacterial load in the lungs, with treated mice showing fewer M. tuberculosis colony-forming units (CFU) after eight weeks of treatment. Furthermore, IL-5 was mainly produced by the site of infection in the lung and decreased with anti-tuberculosis treatment within the CI mice group.
Finally, in Chapter 4, we examine the use of anti-phospholipid IgM antibody changes as a biomarker for treatment response in patients with smear positive pulmonary TB. Serum samples were obtained from pulmonary TB patients at the start and end of the intensive phase of treatment (40 doses of anti-TB combination therapy) enrolled from Kampala, Uganda in a CDC-TB Trials Consortium randomized clinical trial. The samples were screened for IgM antibody levels against five commercially available phospholipids by an in-house ELISA assay. The lipid antigens included CL, PI, PE, PTC, and sphingolipid (SL). IgM antibody levels to CL, PE, PI, PTC and SL significantly decreased following anti-TB drug treatment in patients without lung cavities on their baseline chest radiograph. In contrast, patients with cavitary TB showed an overall increase in the anti-phospholipid IgM antibody response following anti-TB drug treatment, notably with a significant increase in anti-PE antibody levels. Thus, anti-lipid IgM response appears to be a useful biomarker for treatment response, especially in those with non-cavitary disease.
Chapter 5 summarizes the conclusions in support of using the anti-phospholipid IgM antibody response as a useful biomarker for monitoring TB treatment response. This novel biomarker test would greatly facilitate TB management in resource-poor settings. The development of a point of care (POC) test based on anti-phospholipid IgM antibody will be an affordable and highly sensitive alternative to microscopy or culture testing for monitoring treatment response in individuals with TB. Chapter 5 also gives examples of three platforms that might be used for the development of such a POC test. However, we note that it is necessary to explore the specificity of this assay further by testing patients with HIV infection, latent TB infection, and non-TB pulmonary diseases.Tuberculosis (TB) faces difficult challenges for its treatment and control. Both the diagnosis of TB and Mycobacterium tuberculosis (M. tuberculosis) drug susceptibility testing take weeks and clinicians often do not know if the patient is taking an appropriate set of drugs until complications or even death occur. Consequently, early determination of a successful drug therapy response in individuals infected with M. tuberculosis is urgently needed. Since the M. tuberculosis cell wall is comprised of a diverse repertoire of lipids, we examined the possible role of these lipids as antigens for serologic response during M. tuberculosis infection. This dissertation is focused on the examination of lipid-antibody response as a potential biomarker used to monitor treatment response in M. tuberculosis infected hosts. Briefly, Chapter 1 describes the current pitfalls of monitoring tuberculosis treatment with current methods, including acid-fast bacilli (AFB) smear and culture conversion. Chapter 1 also covers the definition of biomarkers and the rationale to use M. tuberculosis cell wall lipids to develop an anti-lipid-antibody based test. Evidence from a similar biomarker-based test for syphilis is presented. The chapter also discusses the biological basis which guided the lipid-antibody biomarker search and discovery.
Chapter 2 describes the use of a serum bank from patients with pulmonary TB provided by the World Health Organization-Tropical Diseases Research consortium (WHO-TDR) to identify M. tuberculosis lipid candidates as targets of antibody response. These samples were used to look for an antibody response to multiple mycobacterial lipids resolved by thin-layer chromatography immunoblot (TLC-I). This approach allowed us to identify M. tuberculosis cardiolipin by mass spectrometry and we determined that the IgM antibody response to cardiolipin can be used as a biomarker of infection.
In Chapter 3, we investigate the biological evidence behind the production of anti-phospholipid IgM antibody during TB infection and anti-TB treatment. For this purpose, we used the Cornell mouse model of infection to monitor the change in IgM antibody response against four phospholipids including cardiolipin (CL), phosphatidyl choline (PTC), phosphatidyl ethanolamine (PE) and phosphatidyl inositol (PI) over the course of M. tuberculosis infection and treatment. We separated BALB/c mice into three groups including acute infection (AI), chronic infection (CI), and healthy control (HC). Both AI and CI groups were infected via the aerosol route with M. tuberculosis strain H37Rv at day 0. The AI group was treated from 4 to 12 weeks post-infection, while the CI group was treated from 20 to 28 weeks post-infection. We also measured the levels of pro-inflammatory cytokines, IL-5 and MCP-1. We observed that in treated AI mice, anti-phospholipid IgM antibody levels decreased compared to those of healthy mice at all time points. Anti-PTC IgM antibodies remained significantly higher in CI mice than in AI mice at all time points post-infection. The anti-PTC IgM antibody levels in CI mice decreased to levels similar to those of AI and HC mice at 32 weeks post-infection. The anti-phospholipid IgM antibody levels correlated with the bacterial load in the lungs, with treated mice showing fewer M. tuberculosis colony-forming units (CFU) after eight weeks of treatment. Furthermore, IL-5 was mainly produced by the site of infection in the lung and decreased with anti-tuberculosis treatment within the CI mice group.
Finally, in Chapter 4, we examine the use of anti-phospholipid IgM antibody changes as a biomarker for treatment response in patients with smear positive pulmonary TB. Serum samples were obtained from pulmonary TB patients at the start and end of the intensive phase of treatment (40 doses of anti-TB combination therapy) enrolled from Kampala, Uganda in a CDC-TB Trials Consortium randomized clinical trial. The samples were screened for IgM antibody levels against five commercially available phospholipids by an in-house ELISA assay. The lipid antigens included CL, PI, PE, PTC, and sphingolipid (SL). IgM antibody levels to CL, PE, PI, PTC and SL significantly decreased following anti-TB drug treatment in patients without lung cavities on their baseline chest radiograph. In contrast, patients with cavitary TB showed an overall increase in the anti-phospholipid IgM antibody response following anti-TB drug treatment, notably with a significant increase in anti-PE antibody levels. Thus, anti-lipid IgM response appears to be a useful biomarker for treatment response, especially in those with non-cavitary disease.
Chapter 5 summarizes the conclusions in support of using the anti-phospholipid IgM antibody response as a useful biomarker for monitoring TB treatment response. This novel biomarker test would greatly facilitate TB management in resource-poor settings. The development of a point of care (POC) test based on anti-phospholipid IgM antibody will be an affordable and highly sensitive alternative to microscopy or culture testing for monitoring treatment response in individuals with TB. Chapter 5 also gives examples of three platforms that might be used for the development of such a POC test. However, we note that it is necessary to explore the specificity of this assay further by testing patients with HIV infection, latent TB infection, and non-TB pulmonary diseases
Screening and evaluation of antiparasitic and in vitro anticancer activities of Panamanian endophytic fungi
Get PDFMany compounds produced by fungi have relevant pharmaceutical applications. The purpose of this study was to collect and isolate endophytic fungi from different regions of Panama and then to test their potential therapeutic activities against Leishmania donovani, Plasmodium falciparum, and Trypanosoma cruzi as well as their anticancer activities in MCF-7 cells. Of the 25 fungal isolates obtained, ten of them had good anti-parasitic potential, showing selective activity against L. donovani; four had significant anti-malarial activity; and three inhibited the growth of T. cruzi. Anticancer activity was demonstrated in four isolates. Of the active isolates, Edenia sp. strain F0755, Xylaria sp. strain F1220, Aspergillus sp. strain F1544, Mycoleptodiscus sp. strain F0194, Phomopsis sp. strain F1566, Pycnoporus sp. strain F0305, and Diaporthe sp. strain F1647 showed the most promise based on their selective bioactivity and lack of toxicity in the assays.Many compounds produced by fungi have relevant pharmaceutical applications. The purpose of this study was to collect and isolate endophytic fungi from different regions of Panama and then to test their potential therapeutic activities against Leishmania donovani, Plasmodium falciparum, and Trypanosoma cruzi as well as their anticancer activities in MCF-7 cells. Of the 25 fungal isolates obtained, ten of them had good anti-parasitic potential, showing selective activity against L. donovani; four had significant anti-malarial activity; and three inhibited the growth of T. cruzi. Anticancer activity was demonstrated in four isolates. Of the active isolates, Edenia sp. strain F0755, Xylaria sp. strain F1220, Aspergillus sp. strain F1544, Mycoleptodiscus sp. strain F0194, Phomopsis sp. strain F1566, Pycnoporus sp. strain F0305, and Diaporthe sp. strain F1647 showed the most promise based on their selective bioactivity and lack of toxicity in the assays