Progress 03/01/91 to 06/30/12
Outputs
OUTPUTS: OBJECTIVES: 1. To characterize the cellular immune mechanisms responsible for the induction and regulation of lymphatic lesions associated with filariasis. A Brujia-jird model will be used and is uniquely suited for this purpose. 2. To study the endosymbiosis between Brugia malayi and its bacteria endosymbiont-Wolbachia. APPROACH: The approaches inclued the characterization of the pattern of cytokine geneexpression during the induction, maintenance and down regulation of the granulomatous inflammatory response to filarial antigen, measurement of the induction of filariae associated granulomatous inflammation and cytokine expression in jirds primed to possess a dominant Th1 cytokine profile and conversely the effect of a Th1 inducing agent on these inflammatory and cytokine responses during a chronic filarial infection, and measurement of the effect of exogenous cytokines on the induction and regulation of granulomatous inflammatory responses and cytokine expression during a primary filarial infection. Filarial nematodes harbor Wolbachia bacteria and depletion of Wolbachia in filarial nematodes results in defects in nematode development, fertility and viability. Induction of apoptosis in Brugia malayi upon Wolbachia depletion was shown and thus suggested the involvement of factors originating from Wolbachia (Landmann et al., 2011). To further confirm that apoptosis occurring during anti-filarial drug treatment is caused by Wolbachia's depletion, we compared two types of drugs: a macrofilaricidal drug which doesn't target Wolbachia such as flubendazole and gentamycin, an antibiotic known to be ineffective in killing Wolbachia vs. tetracycline, which targets Wolbachia. Adult B. malayi worms were cultured in vitro and treated with 40 μg/ml of tetracycline, 20 μg/ml of flubendazole or 40 μg/ml of gentamycin. As a control for effects of death, we used untreated adult worms that were killed by freezing. Drug treated worms were embedded in parafin and sectioned and stained for Wolbachia using anti-WSP antibodies and a TUNNEL assay was performed to study the extent of apoptosis in drug treated B. malayi worms. PARTICIPANTS: Dr.Thomas Klei, Dr. Sridhar Arumugam, Danielle Ward, Dr. Krishna P. Shakya, Dr. Rao, Drs. Enright, Elzer, Horohov, and O'Reily, Dr.Chirgwin SR, Coleman SU, Dr. Porthouse KH, Nowling JM, Punkosdy GA, Nassare, C, Horohov, D.W, Mai, Z, Kranhenbuhl, J, McVay, C S, M Bakeer, V A Dennis, Farrar, R G, R C Montelaro, M D West, C S McVay, Lin, D S. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
To measure the immune response in mongolian gerbils, reagents and assays for gerbil (jird) was generated. Genes for gerbil IL-2, IL-4,5,10, IFN-y and HPRT was cloned and quautitative PCR assay was developed to measure these cytokines. Using this assay, initial experiments demonstrated that a primary infection of B. pahangi induces a shift in cytokine expression to one dominated by IL-4 and IL-10. Activation of macrophages and TNF production have been shown to correlate with the kinetics of parasite induced granulation inflammatory responses. Brugia infection induces the expression of IFN gamma in peritoneal exudate cells but not in lymph nodes or the spleen. Data also indicated the cytokine profile of gerbils with chronic infection is dominated by IL-4 and IL-10. This project also investigated the effect of IFN gamma on the alteration of Brugia pahangi induced cytokine profiles and establishment of infections in the peritoneal cavity of gerbils. This project also brought insight into the role of intra cellular symbiont Wolbachia on the pathogenesis and survival of the Brugia pahangi. Also the migration pattern of B.pahangi L3 larvae was studied in this project and characterized the genes encoding for troponin, hemolysin, abundant larval transcript-2 and cytochrome oxidase and their potential role in larval migration on day 3 post-infection (3 DPI. Filarial infective larval migration is central to parasite establishment and the development potential development of vaccines. The utilization of an in vitro assay would facilitate this process. The data collected to date suggest that larval migration through the skin may be such a system. This project also focused on gene expression in L3 stages of the parasite which are important targets of vaccines must consider potential differences in the effects of specific sites within the host and in vivo parasite activity. co-infection of Brugia pahangi and Helicobacter pylori was also studied in this project and results revealed that the Mongolian gerbil model proved to be useful in assessing the long-term effect of Brugia filariasis on Hp-induced gastritis . Effect of anti-filarial drugs on B. malayi was studied invitro and to confirm that apoptosis occurring during anti-filarial drug treatment is caused by Wolbachia's depletion, we compared two types of drugs: a macrofilaricidal drug which doesn't target Wolbachia such as flubendazole and gentamycin, an antibiotic known to be ineffective in killing Wolbachia vs. tetracycline, which targets Wolbachia. Tetracycline and flubendazole killed B. malayi adult worms on day 5 and day 6 of treatment, respectively where as with gentamycin treated worms survived for 10 days and untreated worms survived for more 2 weeks. Treated worms were harvested at the point of death or alive and stained for Wolbachia and a Tunnel assay was performed to detect apoptosis. Our results show that induction of apoptosis following anti-filarial drug treatment is mainly caused by worm death due to the drug's activity and its toxicity and is not necessarily due to the depletion of Wolbachia alone, as proposed earlier.
Publications
- Sridhar Arumugam, Danielle Ward, Sara Lustigman, Thomas R. Klei. (2012). Filaricidal drugs induce apoptosis in the filarial nematode-Brugia malayi and this effect is not primarily due to Wolbachia depletion. 7th International Wolbachia Conference, Oleron, France.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: n select Helicobacter pylori-infected populations with low gastric cancer, nematode coinfections are common and both helicobacter gastritis and filariasis are modeled in gerbils. We evaluated gastritis, worm counts, tissue cytokine gene expression levels and Th1/Th2-associated antibody responses in H. pylori and Brugia pahangi mono- and coinfected gerbils. H. pylori-associated gastritis indices were significantly lower 21 weeks post-infection in coinfected gerbils (p < or = 0.05) and were inversely proportional to worm counts (r(2) = -0.62, p < 0.003). Additionally, IFN-gamma, IL-1 beta, CXCL1, IL-4 and IL-10 mRNA levels in the gastric antrum reflected a significant host response to gastric H. pylori and as well as systemic filariasis (p < or = 0.05). Despite increasing worm burden (p < 0.05), gastritis progressed in coinfected gerbils (p < 0.03) becoming equivalent to H. pylori-infected gerbils at 42 weeks (p = 0.7). Pro- and anti-inflammatory mediator mRNA levels were notably downregulated in B. pahangi infected gerbils below uninfected control values, suggesting hyporesponsiveness to B. pahangi. Consistent with an increasing Th1 response to H. pylori, IgG2a (p < 0.01), IL-1 beta (p = 0.04) and CXCL1 (p = 0.006) responses significantly increased and IL-4 (p = 0.05) and IL-10 (p = 0.04) were decreased in coinfected gerbils at 42 weeks. Initial systemic responses to B. pahangi resulted in attenuated gastritis in coinfected gerbils, but subsequent filarid-associated hyporesponsiveness appears to have promoted H. pylori gastritis. PARTICIPANTS: The project principle investigator is Prof. Thomas Klei. Dr. Krishna P. Shakyais worked as postdoctoral researcher. Collaborators Dr.James G. Fox, Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139, USA TARGET AUDIENCES: Scientists in the field of immunology, bacteriology and parasitology. PROJECT MODIFICATIONS: There are no major changes or modifications in research approach.
Impacts
The Mongolian gerbil model proved to be useful in assessing the long-term effect of Brugia filariasis on Hp-induced gastritis
Publications
- Martin HR, Shakya KP, Muthupalani S, Ge Z, Klei TR, Whary MT, Fox JG.Brugia filariasis differentially modulates persistent Helicobacter pylori gastritis in the gerbil model.Microbes Infect. 2010 Sep;12(10):748-58. Epub 2010 May 31.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Brugia malayi (Bm) and B. pahangi (Bp) are closely related filarial species that can be maintained in jirds and Aedes aegypti. Previous reports have identified developmental and biochemical differences between vector-derived infective larvae (vL3) from the two species. The purpose of this study was to compare gene expression profiles present in Bm vL3 and Bp vL3 for 15, 412 Brugia elements in the v2 filarial microarray. Eight hybridizations were performed for each 65-mer element on the array. Expression signals were detected for 4,788 elements with one or more L3 types. 330 elements up-regulated in Bm vL3 relative to Bp vL3 included genes that encode enzymes for energy metabolism, various proteases (cathepsins) and protease inhibitors (chymotrypsin/elastase), antioxidants (GST, oxidoreductase), signaling molecules, and immunomodulators (TGF-b, cystatins, ES-62 homolog, endochitinase, larval allergen) and many novel proteins. Bm vL3 had dramatically higher (> 100 fold) expression of genes encoding NADH dehydrogenase and cytochrome subunits. 321 elements up-regulated in Bp vL3 relative to Bm vL3 included genes related to energy metabolism, antioxidant-GPX, and immunomodulators (serpin, CPI-1). KEGG analysis showed that L3 from both species had strong expression of genes related to alanine and aspartate metabolism and to regulation of actin cytoskeleton. Mitochondrial energy metabolism and carbohydrate metabolism were enriched in Bm vL3. In contrast, nitrogen metabolism, metabolism of co-factors, and biosynthesis of secondary metabolites were enriched in Bp vL3. These differences suggest that the two species may have different metabolic adaptations and approaches to parasitism. Gene ontology analysis also suggested biological functions related to energy use were enriched in Bm vL3 while cellular physiological processes (protein polymerization, amino acid and nucleotide biosynthesis) were enriched in Bp vL3. Homologues of 18% of Bm vL3 and 21% of Bp vL3 up-regulated genes have severe RNAi phenotypes in C. elegans. vL3 gene expression is likely to be important for establishment of infection and immune evasion. Differences in Bm and Bp vL3 gene expression may be related to differences in host preference, tissue tropism, and pathogenicity observed between these species. These reslts were presented at the Am Soc Trop Med and Hyg conference. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The results of these experiments demonstrated the poential gentic differences between these closely related zoopnotic filarial parasites. They may in part explaine differences in the life cycles seen in experimental infections of laboratory andimals and man.
Publications
- U. Rao, Thomas R. Klei, Yuefang Huang, Krishna P. Shakya, Michael Heinz, Sahar Abubucker, Makedonka Mitreva, Gary J. Weil, 2009. Comparative analysis of gene expression profiles in infective larva of Brugia malayi and Brugia pahangi Am Soc Trop Med Hyg, Washington DC
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Information form the studies completed to date have been disseminated by presentations to the scientific and medical communities involved in basic filariasis research at the annual meeting of the American Society of Tropical Medicine and Hygiene in 2008. PARTICIPANTS: Drs. Enright, Elzer, Horohov, and O'Reily are no longer involved in this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Third stage infective filarial larvae (L3) are the transition stage from the insect vector to the mammalian host. Changes in gene expression that accompany this transition may shed light on parasite molecules and adaptations required for invasion and for survival and development in the mammalian environment. These events were studied by comparing gene expression profiles for post-infectious Brugia pahangi L3 (ipL3 and idL3, each collected three days after injection into jirds) with L3 maintained for three days in culture (cL3) and L3 freshly isolated from mosquitoes (vL3). We used the Version 2 Filarial Microarray with 15, 412, 65-mer B. malayi oligonucleotide elements to perform two-way comparisons with these different L3 types. Expression signals were detected for 4980 (32%) elements with one or more L3 types. Eight hybridizations were performed for each element on the array; genes with 2-fold differences in expression signals with P < 0.01 were considered to be differentially expressed. 601, 415 and 522 array elements were differentially expressed in ipL3, idL3, and cL3, respectively, relative to vL3. 104 genes had increased expression in all 3 types of post-infective L3 relative to vL3. These included genes that encode muscle and cytoskeletal proteins, immunomodulators, antioxidants, proteins needed for protein synthesis and folding, and many novel genes. These genes may be required for parasite migration, growth, and development. 129 genes were down-regulated in all three post-infectious L3 types relative to vL3. These encode proteases and protease inhibitors (cathepsins, serpin), allergens, ALT's, and novel proteins. These proteins may be involved in invasion, and their rapid down-regulation may be important for evading the host's immune system. It was interesting that while 46 genes were differentially regulated between ipL3 and idL3, only 49 and 4 genes were differentially regulated in ipL3 and idL3 relative to cL3, respectively. These preliminary results suggest that B. pahangi vL3 undergo dramatic changes in gene transcription shortly after their transition from mosquitoes into conditions that support growth and molting. Gene expression patterns in idL3 and cL3 were more similar to each other than either was to ipL3; some genes may have site-specific expression. This study has provided interesting clues regarding gene products that might be required for invasion, adaptation, and development of filarial
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
The studies conducted on gene expersion of infective larvae resualted in data that were analyzed and presented at meetings and in the form of a paper.
Impacts
Events occurring during early filarial nematode migrations are central to parasite establishment but rarely studied. Brugia pahangi larvae injected intradermal (ID) into the hind limb of the gerbil (Meriones unguiculatus) can be recovered from the popliteal lymph node (POP) at 3 days post infection (DPI). They have been designated migrating larvae (IDL3). Alternatively, L3 recovered at 3 DPI from the peritoneal cavity (IPL3) do not migrate. Subtracted cDNA libraries using IDL3 and IPL3 revealed distinct gene profiles between IDL3 and IPL3. Troponin-c was significantly upregulated in IDL3, while Cathepsin L was significantly increased in IPL3. Differences in mRNA levels were also observed with these and other genes between IDL3, IPL3 and L3 isolated from mosquitoes (VL3). These data indicate a change in knowledge and indicate that migratory activity, exposure to potentially different host environments and/or host location may be important external factors in influencing
larval gene expression.
Publications
- Chirgwin SR, Coleman SU, Klei TR. 2008.Brugia pahangi: in vivo tissue migration of early L3 alters gene expression. Exp Parasitol.118(1):89-95.
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Progress 01/01/06 to 12/31/06
Outputs
Events which occur during early filarial nematode migrations are central to parasite establishment but yet rarely studied including in vivo gene expression. Brugia pahangi larvae injected intradermal (ID) into the hind limb of the gerbil (Meriones unguiculatus) actively migrate and are recovered from the popliteal lymph node (POP) at 3 days post infection (DPI). They have been designated migrating larvae (IDL3). Alternatively, L3 recovered at 3 DPI from the peritoneal cavity (IPL3) do not migrate. Subtracted cDNA libraries using IDL3 and IPL3 revealed the two larval populations showed distinct gene profiles. Troponin-c was significantly up regulated in IDL3, while Cathepsin L was significantly increased in IPL3. Differences in mRNA levels were also observed with these and other genes between IDL3, IPL3 and L3 isolated from mosquitoes (VL3). These data suggest that migratory activity, exposure to potentially different host environments and/or host location may be an
important external factor in influencing larval gene expression and should be considered in future studies of host induced gene expression.
Impacts
The data collected indicate that studies focused on gene expression in L3 stages of the parasite which are important targets of vaccines must consider potential differences in the effects of specific sites within the host and in vivo parasite activity.
Publications
- Chirgwin SR, Coleman SU, Porthouse KH, Klei TR. 2006. Tissue migration capability of larval and adult Brugia pahangi. J Parasitol. 92(1):46-51.
- Porthouse KH, Chirgwin SR, Coleman SU, Taylor HW, Klei TR. 2006. Inflammatory responses to migrating Brugia pahangi third-stage larvae. Infect Immun.74(4):2366-72.
- Rao RU, Klei TR. 2006.Cytokine profiles of filarial granulomas in jirds infected with Brugia pahangi. Filaria J.16;5:3.
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Progress 01/01/05 to 12/31/05
Outputs
Mosquito borne third stage infective larvae (L3) of filarial nematodes emerge from the mosquito labella into a drop of haemolymph during mosquito feeding. They reportedly migrate into the host through the vector produced wound and then through various connective tissues of the skin to a lymph or blood vessel. Earlier studies have demonstrated the capacity of Brugia to penetrate a variety of tissue types rapidly Nothing is known regarding the mechanisms associated with this process. An in vitro model was developed attempting to quantify this early prelymphatic migration in order to define the parasite molecules involved in this process. Skin from gerbils (Meriones unguiculatus) was cut into 1cm circles and inserted into the top of a blind well chamber and sealed. Different procedures were used to prepare the surface of the skin. These included; clipping, puncturing, shaving, tape striping and combinations of each. RPMI 1640 without sera was used to fill the bottom of
the chambers and 200L3 were placed in 100ul of media on the top of the chamber. Chambers were incubated for 1-3 hrs at 37 C in 5% CO2. Larvae were counted in the upper and lower chambers after incubation. The total number that penetrated the skin was calculated. Large numbers (80%) of L3 rapidly(1hr) migrated through the punctured skin and this system was discounted. L3 also migrated into and through the skin prepared in all other methods. Depending on the initial preparation this resulted in 70-30% entry and 17-14% complete migration of L3 through the skin. Histologic examination of the skin showed larvae in all tissues. It appears that larvae penetrate minimally disrupted epidermis and in some cases hair follicles. Initial attempts to alter this migration with anti-L3 excretory secretory antibodies will be described.
Impacts
Filarial infective larval migration is central to parasite establishment and the development potential development of vaccines. The utilization of an in vitro assay would facilitate this process. The data collected to date suggest that larval migration through the skin may be such a system.
Publications
- Chirgwin SR, Rao UR, Mai Z, Coleman SU, Nowling JM, Klei TR 2005. Kinetics of T cell cytokine gene expression in gerbils after a primary subcutaneous Brugia pahangi infection. J Parasitol. 91(2):264-8.
- Chirgwin SR, Rao UR, Coleman SU, Klei TR 2005. Profiling the cellular immune response to multiple Brugia pahangi infections in a susceptible host. J Parasitol. 91 (4):822-829.
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Progress 01/01/04 to 12/31/04
Outputs
Studies continue on L3 migration. Under natural conditions, Brugia larvae are deposited on the skin of a human by an infected mosquito, and must then migrate through the various skin layers to establish infection within the lymphatics. This migration has been mimicked in our laboratory with the development of an intradermal (ID) inoculation model using the gerbil and B. pahangi. Experiments investigating this migration model have shown that at 3 days post infection (DPI) a majority of the larvae inoculated ID are present in the popliteal lymph node (PLN). Following 3 DPI, most of the larvae have left the PLN. We have classified larvae isolated from the PLN at 3 DPI as migrating larvae (IDL3). In contrast, larvae that are injected intraperitoneally (IP) do not migrate away from the peritoneal cavity and have been classified as non-migrating larvae (IPL3). Total RNA was extracted from 3 day IDL3 and IPL3, and suppressive subtractive hybridization was conducted. Analysis
of the resulting subtracted cDNA showed the presence of 4 strongly expressed bands in IDL3. These bands have been identified as encoding troponin, hemolysin, abundant larval transcript-2 and cytochrome oxidase 1. Preliminary data using semi-quantitative and quantitative RT-PCR suggests these genes are up-regulated in IDL3, compared to IPL3. These data suggest that larvae inoculated into different sites within a host show altered gene expression profiles. Whether this is due to differences in larval activity, such as migration versus no migration, or is a result of varying host responses directed at larvae within each site is currently unknown.
Impacts
These data on early migrations impact our understanding of parasite establishment and the induction of immunoregulatory events. It is likly these determine the subsequent disease conditions of infected individuals.
Publications
- Klei TR, and Chirgwin SR. 2004 The interaction of Wolbachia, filarial nematodes and their hosts. Vet Parasitol 125;147-151.
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Progress 01/01/03 to 12/31/03
Outputs
Studies continue on the host - parasite interaction during early filarial nematode migration. A model of early cutaneous filarid migration using Brugia pahangi in the jird host has been developed and the histologic and cytokine response has been examined during this period. Male jirds ere inoculated intradermally in the hind limb with 100 B. pahangi L3. Jirds were necropsied at 3 hours, 24 hours, 3 days, 7 days, and 28 days post-infection (p.i.). Larvae were recovered and counted and tissues collected for histology and cytokine measurement. At 3 hours p.i., almost all larvae were recovered from the tissue associated with the infection site. Significant migration away from the infection site occurred within 24 hours, and by 7 days p.i., larvae were well dispersed. Larvae were identified on histologic exam in the dermis, muscle interstitium, renal lymph nodes and lymphatics at all time points. The prototypical TH1/TH2 cytokines IL-4 and IFN gamma, and TNF were measured
in the spleen and popliteal and renal lymph nodes by RT-PCR. IL-4 remains low in all tissues through 7 days and rises by 28 days. IFN gamma levels show earlier peaks with the highest levels at 3 hours in the renal nodes and 24 hours in the spleen. Levels in the popliteal node were low at all time points with a peak at 24 hours. In all tissues, TNF levels were highest at 3 hours with a decline to consistently low levels by 24 hours. These results indicate the ability of filarid L3 to rapidly migrate through host tissue and support the use of intradermal jird infection as a model for early filarid infection. Cytokine analysis was compatible with the classically described development of a TH 2 immune response to filarid infection over time (IL-4) and suggests the presence of a proinflammatory host environment acutely after infection (TNF and IFN gamma).
Impacts
These data on early migrations impact our understanding of the induction of immunoregulatory events. It is likly these determine the subsequent disease conditions of infected individuals.
Publications
- Chirgwin SR, Coleman SU, Porthouse KH, Nowling JM, Punkosdy GA, Klei TR*. 2003. Removal of Wolbachia from Brugia pahangi is closely linked to worm death and fecundity but does not result in altered lymphatic lesion formation in Mongolian gerbils (Meriones unguiculatus). Infect and Immun. 2003 71:6986-94.
- Chirgwin, S.R., Nowling, J.M., Coleman, S.U., and Klei, T.R.* 2003 Brugia pahangi and Wolbachia: The Kinetics of Bacteria Elimination,Worm Viability and Host Responses Following Tetracycline Treatment Exp Parasitol. 103: 16-26.
- Chirgwin,S. R., Nowling, J.M., Coleman, S.U., and Klei, T.R.* 2003 Effect of immunostimulatory oligodeoxynucliotides on host responses and the establishment of Brugia phangi in mongolian gerbils (Meriones unguicualtus) J Parasitol 89: 483-489.
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Progress 01/01/02 to 12/31/02
Outputs
Studies on the role of the intra cellular symbiont Wolbachia on the pathogenesis and survival of the parasite have continued. Tetracycline treatment of L4 or adult Brugia pahangi in vivo resulted in Wolbachia clearance. Less tetracycline was required to clear Wolbachia when treatment began at the L4 stage, compared with adults. Female worms died earlier than male worms when tetracycline was administered at the L4 stage. In all cases, Wolbachia clearance was closely associated with worm death. Worm recoveries decreased following the L4-L5 molt, suggesting tetracycline does not interrupt molting in this model system. Despite worm death and the assumed release of both bacterial- and worm-derived molecules, differences in inflammatory cell population and T cell cytokine mRNA profiles were negligible between tetracycline treated and non-treated B. pahangi infected gerbils. These data suggest the contribution of Wolbachia to the in vivo induction of the gerbil immune
response to B. pahangi may be small. Studies have begun to develop an in vivo model of early Brugia migration and its role in the generation of immune responses and pathology.
Impacts
The understanding of the resltonship between Wolbachia and its nematode host Brugia pahangi has been expanded as well as this interaction with the host response to these agents.
Publications
- Chrigwin, SR, Porthouse, KH, Nowling, JM,and Klei, T.R. 2002. The filarial endosymbiont Wolbachia sp. is absent from Setaria equina. J Parasitol. 2002 88:1248-50.
- Chirgwin, S.R., Elzer,P.E., Coleman, S.U., Nowling, J.M., Hagius, S.D., Edmonds, M.D., and Klei, T.R.2002. Infection outcome and cytokine gene expression in Brugia pahangi infected gerbils (Meriones unguiculatus) sensitized with Brucella abortus. Infection and Immunity 11: 5938-5945.
- Rajan, T.V., Ganley, L., Paciorkowski, N., Spencer, L., Klei, T.R., and Shultz, L.D., 2002. Brugian infection in the peritoneal cavities of laboratory mice: Kinetics of infection and cellular responses. Exp. Parasitol. 100:235B247.
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Progress 01/01/01 to 12/31/01
Outputs
Studies have continued to examine the potential role of the endosymbiont Wolbachia in the pathogenesis of lymphatic lesions in the Brugia - jird model. Tetracycline treatment of subcutaneously infected jirds 28 dpi was shown to eliminate Wolbachia as assessed by PCR and concomitantly microfilariae production was also eliminated. Lymphatic lesion severity was measured in treated and nontreated controls quantitatively by counts of parasite associated granulomas in the spermatic cord lymphatics and qualitatively by the histologic character of these lesions. To date significant changes in the severity have not been detected in the group from which Wolbachia has been eliminated. Similarly changes in the levels of the cytokines IFNgamma and IL-4 have not been detected between these groups. Three experiments have been conducted and termainated times which surround the peak of lymphatic granuloma formation, 70,90, and 100 dpi. The results suggest that while Wolbachia may play
some role in lesion induction it is not necessary to sustain this inflammatory response and likely not the major factor involved in its initiation.
Impacts
These studies further our understanding of the pathogenesis of human lymphatic filariasis.
Publications
- Rao, U.R., Williams, S.A., and Klei. T.R. 2002. Quantification of PCR amplification products of Brugia HhaI repeat DNA using a semiautomatic Q-PCR. Molecular and Cellular Probes 16: 13-23
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Progress 01/01/00 to 12/31/00
Outputs
Studies have continued to measure the effect of IFN gamma on the alteration of Brugia pahangi induced cytokine profiles and establishment of infections in the peritoneal cavity of jirds. Both killed Brucella abortus s19 and immunostimulatory bacterial oligonucleotides ,different CpG motifs, have been studied for this purpose and optimal inoculation protocols established. To date both types of experiments promote the induction of IFN gamma by the parasite and reduce but do not eliminate the expression of IL-4 a signature type-2 cytokine. Parasite establishment was either enhanced or decreased depending on the protocol used. These experiments are being repeated. In initial studies on the potential role of the obligate intracellular symbiont of Brugia, Wolachia, where begun. Parasites were eliminated from the worms by treatment of infected jirds with tetracycline. Removal of the bacteria did not reduce the recovery of adult worms but did eliminate the production of
microfilariae. However, the cytokine profiles of renal lymph nodes and spleens and severity of lymphatic lesions was not altered in the treated animalsat 90 dpi. This suggests that the bacteria and microfilariae may not have a significant role in early pathogenesis of these lesions.
Impacts
These data are relevant to the understanding of the pathgenesis of human lymphatic filariasis and its treatment.
Publications
- Rao, U.R., Salinas, G., Kapil, M . And Klei, T.R. 2000. Identification and localization of glutathione S-transferase as a potential target enzyme in Brugia species. Parasitol Res 86: 908-915.
- Babu S, Ganley LM, Klei TR, Shultz LD, Rajan TV.2000. Role of gamma interferon and interleukin-4 in host defense against the human filarial parasite Brugia malayi. Infect Immun.68(5):3034-5.
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Progress 01/01/99 to 12/31/99
Outputs
Studies have continued to characterize cytokine gene expression in Brugia infected gerbils. It has been demonstrated that the expression of the type-1 and type -2 cytokines ( IFN gamma and IL-4 respectively ) differ in different compartments of the immune system. Brugia infection induces the expression of IFN gamma in peritoneal exudate cells but not in lymph nodes or the spleen. This observation differs from reports in other nonpermissive host species which focused on the spleen or in human patients which focus on peripheral blood mononuclear cells and suggest that regulatory mechanisms may occur in these helminth parasite infections other than those currently proposed. Infections of gerbils with different strains of Brucella abortus were conducted so that they might be used as potential experimental modifiers of the immune response prior to and following Brugia infection. The virulent strains 2308 and S19 induced the highest and most prolonged expression of IFN
gamma and may be used in future experiments.
Impacts
These experiments are important the understanding of inflammatory responses induced during lymphatic filariasis in man.
Publications
- Rao, U.R.,Nassare, C., Coleman,S.U.,Horohov, D.W., and Klei, T.R. 1999. Granulomatous inflammatory responses to recombinant filarial proteins of Brugia species. Am. J. Trop Med. and Hyg. 60, 251-254.
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Progress 01/01/98 to 12/31/98
Outputs
Efforts have continued to define cytokine gene expression following infection. Data to date continue to indicate that the early Type 1 cytokine response is induced by some non immune events but is rapidly altered, possibly by the subsequent induction of Type 2 cytokines. Cytokine profile has been shown to be Brugia stage specific in mice. Studies were continued in this regard by measuring the cytokine profile in jirds induced by L3, microfilariae, or adults following IP inoculations. The data suggest that all stages induce similar cytokine profiles in vivo but that the expression varies in different compartments of the immune system. Brugia pahangi infections down regulate peritoneal macrophage activation during the course of infection as well as decreasing systemic granulomatous inflammation to parasite antigen. These events correspond temporally with the increase in IL-4 expression seen in lymph nodes and spleens, as noted above, in the absence of IFN gamma
expression which occurs very early and again very late during the course of the infection. Attempts to investigate the regulation of these events by cytokines has begun using concomitant infections of Brugia and Brucella abortus, a bacterium which induces a strong Type 1 cytokine response.
Impacts
(N/A)
Publications
- Nassare, C., Kranhenbuhl, J., and Klei, T.R., 1998. Down regulation of macrophage activation in Brugia pahangi - infected jirds. Infection and Immunity 66, 1063-1069.
- Mai, Z., Horohov, D.,W., and Klei,T.,R., 1998. Hypoxanthine Phosphoribosyltransferase cDNA in Gerbils (Meriones unguiculatus). Laboratory Animal Science 48, 179-183.
- Rao, U., R., Nassare, C., Coleman, S.,U., Horohov, D.,W., and Klei, T.,R., 1998. Granulomatous inflammatory response to recombinant filarial proteins of Brugia species. American Journal of Tropical Medicine and Hygiene (IN PRESS)
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Progress 01/01/97 to 12/31/97
Outputs
Studies have continued to characterize the cytokine gene expression in lymphoid tissues of jirds during primary infection of Brugia pahangi. These efforts have been aided by the development of a semiquantitative RT-PCR assay to measure cytokine mRNA using a Perkin Elmer Q-PCR 5000 which became available during the past year. Cytokines examined included IL-2, IL-4, IL-5, IL-10, and IFN-gamma. The housekeeping gene HPRT was used to normalize quantities of mRNA between samples. All jirds were infected by inoculation of third stage larvae (L3) via the subcutaneous route which results in infections of the lymphatic vessels and lymph nodes. Cytokine gene expression was measured at 1,3,7, 14, 28, 56, and >120 days post-infection (DPI) in the renal lymph nodes , spleen and in one experiment in the spermatic cord lymphatics. Responses induced following inoculation of viable L3 and L3 which has been irradiated with 45Krad of gamma radiation were compared. This comparison
allowed the differentiation by correlation of response which resulted in a downregulation of the cellular immune response and a maintenance of susceptibility, induced by viable L3, with those which resulted in a maintenance of the cellular immunity and partial immunity to reinfection, induced by irradiated L3. Viable L3 induced an IL-4 response as early as 1 DPI, indicating a non T-cell source of this cytokine. This IL-4 response was dominant and increased during the course of infection. This increase in IL-4 gene expression corresponds to a down regulation of granulomatous inflammatory responses to parasite antigens, macrophage activation, reduced lymphatic lesions, reduced lymphoproliforation to worm antigen in vitro and an absence of protective resistance. This early dominant IL-4 response was seen follwing incoulation of irradiated L3. However, an IL-4 response did develop at a later time in these animals. These results suggest that an initial contact by a viable L3 is necessary
during the course of infection for the eventual induction of the down regulated state seen in these individuals.
Impacts
(N/A)
Publications
- Nassare, C S, Coleman, U, Rao, U R and Klei, T R. 1997. Brugia pahangi: Differential induction and regulation of jird inflammatory responses by life-cycle stages. Exp. Parasitol. 87, 20-29.
- Klei, T R, McVay, C S, Coleman, S U, Enright, F M and Rao, U R. 1997. Adoptive transfer of granulomatous inflammation to Brugia antigens in jirds. J. Parasitol. 83, 626-629.
- Nasarre, C, Rao, U R, Coleman, S U, and Klei, T R. 1997. Effect of gamma irradiation on Brugia development in vivo and the kinetics of granulomatous inflammation induced by these parasites. J. Parasitol. 83, 1119-1125.
- Nassare, C, Krahanbuhl, J, and Klei, T R. 1998. Down regulation of macrophage activation in Brugia pahangi-infected jirds (Meriones unquiculatus). Infection and Immunity (In Press).
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Progress 01/01/96 to 12/30/96
Outputs
Regeants and assays for gerbil (jird) immune responses have been produced. Genesfor gerbil IL-4,5,10, IFN-y and HPRT have been cloned. Recombinant IL-2 has been expressed in prokaryotic expression systems and shown to be biologically active. A RT-PCR ELISA to determine the mRNA concentration of these cytokines has been devloped. Using this assay, initial experiments demonstrate that a primary infection of B. pahangi induces a shift in cytokine expression to one dominated by IL-4 and IL-10. Activation of macrophages and tNF production have been shown to correlate with the kinetics of parasite induced granulation inflammatory responses. Studies have been completed which experimentally demonstrate that the induction and maintenance of the hyporesponsive cellular immune response is not dependent on microfilariae. Data also indicated the cytokine profile of gerbils with chronic infection is dominated by IL-4 and IL-10. The competitive renewal application for this project
was funded until 2001.
Impacts
(N/A)
Publications
- Rao, U R, C S Nasarre, S U Coleman, M Bakeer, V A Dennis, D W Horohov, and T R Klei. 1996. Cellular immune responses of jirds to extracts of the life cycle stages and adult excretory secretory products during early development of Brugiapah
- Farrar, R G, R C Montelaro, F M Enright, M D West, C S McVay, S U Coleman and T R Klei. 1995. Brugia pahangi: Differential granulomatous reactivity of infected birds (Meriones unguiculatus) to fractions of adult worm antigen. Exp. Parasitol
- Bosshardt, S C, S U Coleman, C S McVay, K L Jones, and T R Klei. 1995. Impact ofmicrofilaremia on the maintenance of a hyporesponsive cellular immune response in Brugia-infected gerbils (Meriones unguiculatus). Infect. & Immunity. 63:940-9
- Lin, D S, S U Coleman, U R Rao, and T R Klei. 1995. Absence of protective resistance to homologous challenge infections in birds with chronic, amicrofilaremic infections of Brugia pahangi. J. Parasitol. 81:640-646.
- Klei, T R, C S McVay, S U Coleman, F M Enright, and U R Rao. 1997. Adoptive transfer of granulomatous inflammation to Brugia antigens in birds. J. Parasitol. (In Press).
- Nassare, C, S U Coleman, U R Rao, and T R Klei. 1997. Brugia pahangi: Differential induction and regulation of bird inflammatory responses by life cycle stages. Exp. Parasitol. (Accepted).
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