Jackson-Thompson BM et al. (2018) PLoS Negl Trop Dis "Brugia malayi infection in ferrets - A small mammal model of lymphatic filariasis."

Jaiswal S, Jackson-Thompson BM, Dardzinski BJ, Scott JR, Fite JJ, Laurie K, Kim SY, Jones SR, Mitre E, Morris CP, Hoy AR

[PLoS Negl Trop Dis, 2018]

BACKGROUND: The lack of effective short-course therapies for treatment of the adult stage of filarial worms is a major limitation in the global effort to eliminate lymphatic filariasis. Studies using current small mammal models of lymphatic filariasis are limited by difficulties in quantifying adult worm numbers and in assessing lymphatic anatomy and function. METHODOLOGY/PRINCIPAL FINDINGS: Here, we re-established Brugia malayi infection of ferrets as a model for lymphatic filariasis and demonstrated parasitological, immunological, and histological parallels with human infection. Subcutaneous injection of L3 larvae into a hind-footpad resulted in a mean of 18 adult worms recovered 16 weeks post-infection, primarily from the draining inguinal and femoral lymphatics of the injected limb. Infected ferrets developed microfilaremia, with patency lasting from 12-26 weeks post-infection. Quantitative PCR assessing cytokine transcription by antigen-stimulated lymph node cells demonstrated a mixed Th1/Th2 response occurring during early infection. Immunoregulation with production of down-regulatory cytokine IL-10 occurred just prior to peak microfilaremia. Histological analysis revealed progressive inflammation of the lymphatic vessel walls, with intimal thickening and disorganization of collagen fibers. Inflammation was observed as early as 8 weeks post-infection and extended into the perivascular and subcutaneous tissues by 16 weeks post-infection. Finally, we developed a novel ferret PET/CT lymphoscintigraphy method demonstrating substantial changes in lymphatic anatomy and function as early as 3 weeks post-infection, with progression over the course of infection. CONCLUSIONS/SIGNIFICANCE: B. malayi infection of ferrets is a robust model of human lymphatic filariasis that can be utilized to study efficacy of novel antifilarial agents against adult worms residing within lymphatic vessels. In conjunction with PET/CT lymphoscintigraphy, this model can also be used to investigate pathogenesis of lymphatic dysfunction in lymphatic filariasis and efficacy of medications aimed at reversing lymphatic dysfunction after clearance of adult worms.

Jackson-Thompson BM et al. (2020) J Transl Autoimmun "Axenic Caenorhabditis elegans antigen protects against development of type-1 diabetes in NOD mice."

Long J, Torrero M, Mitre BK, Mitre E, Jackson-Thompson BM, Packiam M

[J Transl Autoimmun, 2020]

Studies in humans and animals have demonstrated that infection with helminths (parasitic worms) is protective against a range of hyperinflammatory diseases. A number of factors limit translation into clinical use, including: potential contamination of helminths obtained from infected humans or animals, lack of batch to batch stability, and potential pathological risks derived from live worm infections. To overcome these limitations we tested whether an antigen homogenate of the non-pathogenic nematode <i>Caenorhabditis elegans</i> confers protection against type 1 diabetes mellitus (T1D) using the Non Obese Diabetic (NOD) mouse model. Our study demonstrates that twice weekly intraperitoneal injections of axenically cultured <i>C.elegans</i> antigen (aCeAg) confers substantial protection against type 1 diabetes in NOD mice. Whereas 80% of control mice (PBS-injected) developed clinical disease, only 10% of aCeAg-treated mice became diabetic. Additionally, aCeAg treated mice had significantly greater numbers of insulin-producing pancreatic islets and greater numbers of islets negative for lymphocyte infiltration. Immunological changes observed in aCeAg treated mice included increases in total IgE and total IgG1, consistent with induction of a type 2 immune response similar to that typically seen in parasitic worm infection. Although evidence suggests that helminth infections induce strong immunoregulatory signals, we did not observe significant changes in regulatory T cell numbers or in production of the regulatory cytokines TGF and IL-10. The lack of a regulatory response may be due to our time point of observation, or perhaps the mechanism of aCeAg efficacy may differ from that of helminth infection. Discovery that antigens obtained from a non-parasitic environmental nematode replicate the protective phenotype induced by parasitic worm infections may accelerate our ability to develop nematode-derived therapies for allergy and autoimmune diseases.

Arumugam S et al. (2016) PLoS Negl Trop Dis "Vaccination of Gerbils with Bm-103 and Bm-RAL-2 Concurrently or as a Fusion Protein Confers Consistent and Improved Protection against Brugia malayi Infection."

Bottazzi ME, Klei TR, Wei J, Abraham D, Arumugam S, Liu Z, Lustigman S, Zhan B, Hotez PJ, Bell A

[PLoS Negl Trop Dis, 2016]

BACKGROUND: The Brugia malayi Bm-103 and Bm-RAL-2 proteins are orthologous to Onchocerca volvulus Ov-103 and Ov-RAL-2, and which were selected as the best candidates for the development of an O. volvulus vaccine. The B. malayi gerbil model was used to confirm the efficacy of these Ov vaccine candidates on adult worms and to determine whether their combination is more efficacious. METHODOLOGY AND PRINCIPLE FINDINGS: Vaccine efficacy of recombinant Bm-103 and Bm-RAL-2 administered individually, concurrently or as a fusion protein were tested in gerbils using alum as adjuvant. Vaccination with Bm-103 resulted in worm reductions of 39%, 34% and 22% on 42, 120 and 150 days post infection (dpi), respectively, and vaccination with Bm-RAL-2 resulted in worm reductions of 42%, 22% and 46% on 42, 120 and 150 dpi, respectively. Vaccination with a fusion protein comprised of Bm-103 and Bm-RAL-2 resulted in improved efficacy with significant reduction of worm burden of 51% and 49% at 90 dpi, as did the concurrent vaccination with Bm-103 and Bm-RAL-2, with worm reduction of 61% and 56% at 90 dpi. Vaccination with Bm-103 and Bm-RAL-2 as a fusion protein or concurrently not only induced a significant worm reduction of 61% and 42%, respectively, at 150 dpi, but also significantly reduced the fecundity of female worms as determined by embryograms. Elevated levels of antigen-specific IgG were observed in all vaccinated gerbils. Serum from gerbils vaccinated with Bm-103 and Bm-RAL-2 individually, concurrently or as a fusion protein killed third stage larvae in vitro when combined with peritoneal exudate cells. CONCLUSION: Although vaccination with Bm-103 and Bm-RAL-2 individually conferred protection against B. malayi infection in gerbils, a more consistent and enhanced protection was induced by vaccination with Bm-103 and Bm-RAL-2 fusion protein and when they were used concurrently. Further characterization and optimization of these filarial vaccines are warranted.

McIntyre DC et al. (2023) Development "Niche cells regulate primordial germ cell quiescence in response to basement membrane signaling."

McIntyre DC, Nance J

[Development, 2023]

Stem cell quiescence, proliferation and differentiation are controlled by interactions with niche cells and a specialized extracellular matrix called basement membrane (BM). Direct interactions with adjacent BM are known to regulate stem cell quiescence; however, it is less clear how niche BM relays signals to stem cells that it does not contact. Here, we examine how niche BM regulates C. elegans primordial germ cells (PGCs). BM regulates PGC quiescence even though PGCs are enwrapped by somatic niche cells and do not contact the BM; this can be demonstrated by depleting laminin, which causes normally quiescent embryonic PGCs to proliferate. We show that following laminin depletion, niche cells relay proliferation-inducing signals from the gonadal BM to PGCs via integrin receptors. Disrupting the BM proteoglycan perlecan blocks PGC proliferation when laminin is depleted, indicating laminin functions to inhibit a proliferation-inducing signal originating from perlecan. Reducing perlecan levels in fed larvae hampers germ line growth, suggesting BM signals regulate germ cell proliferation under physiological conditions. Our results reveal how BM signals can regulate stem cell quiescence indirectly, by activating niche cell integrin receptors.

Farghaly AM et al. (2016) J Egypt Soc Parasitol "DEVELOPMENT OF AN IN VITRO RNAI TO INVESTIGATE GENE FUNCTION IN BRUGIA MALAYI."

Abd El-Raouf Taha A, Abd El-Hai Afify H, Mostafa E, Farghaly AM

[J Egypt Soc Parasitol, 2016]

The RNA interference (RNAi) has the ability to turn off individual gene expression. So, it affords a remarkably specific tool for studying the effects of genes. It is regarded as a direct approach for determining such gene/genes functions and offers a valuable tool for modern drug discovery. The study aimed to develop in vitro RNAil in Brugia malayi with particular interest to study the function of Brugia malayi avr-14 (Bm-avr-14) and Brugia malayi f-tubulin (Bm-fi-tubulin) genes. Bm-avr-14 is a gene encoding glutamate gated chloride channel (GluCl) which binds ivermectin and Bm--tubulin is a gene encoding -tubulin which binds albendazole. Adult female worms were soaked in heterogeneous short interfering RNA (hsiRNA) with interest to study the role of two genes Bm-avr-14 and Bm--tubulin. Then, we assessed the knock down effects of target genes using worminator system and real time PCR. We found that worms treated with hsiRNA of Bm-avr-14 had a significant reduction in microfilariae (mf) production in comparison with untreated worms or worms treated with hsiRNA of green fluorescent protein (GFP). No significant reduction in mf production with Bm--tubulin gene was obtained. There were no changes in the movement of adults treated with either Bm-avr-14 or Bm- -tubulin hsiRNAs. Inconsistent RNAi mediated suppression was achieved with Bm-avr-14 and Bm-- tubulin using real time PCR. The data may be helpful in assessment of drug target potential of genes.

Matus DQ et al. (2014) Nat Commun "Cell division and targeted cell cycle arrest opens and stabilizes basement membrane gaps."

Chi Q, Makohon-Moore SC, Matus DQ, Sherwood DR, Chang E, Hagedorn MA

[Nat Commun, 2014]

Large gaps in basement membrane (BM) occur during organ remodelling and cancer cell invasion. Whether dividing cells, which temporarily reduce their attachment to BM, influence these breaches is unknown. Here we analyse uterine-vulval attachment during development across 21 species of rhabditid nematodes and find that the BM gap that forms between these organs is always bounded by a non-dividing vulval cell. Through cell cycle manipulation and live cell imaging in Caenorhabditis elegans, we show that actively dividing vulval cells facilitate enlargement of this breach by promoting BM movement. In contrast, targeted cell cycle arrest halts BM movement and limits gap opening. Further, we demonstrate that the BM component laminin accumulates at the BM gap edge and promotes increased integrin levels in non-dividing vulval cells, stabilizing gap position. Together, these studies reveal that cell division can be used as a mechanism to regulate BM breaches, thus controlling the exchange of cells between tissues.

McClatchey ST et al. (2016) Elife "Boundary cells restrict dystroglycan trafficking to control basement membrane sliding during tissue remodeling."

Shen W, Wang Z, Chen A, Sherwood DR, Chi Q, Linden LM, Hastie EL, Wang L, McClatchey ST

[Elife, 2016]

Epithelial cells and their underlying basement membranes (BMs) slide along each other to renew epithelia, shape organs, and enlarge BM openings. How BM sliding is controlled, however, is poorly understood. Using genetic and live cell imaging approaches during uterine-vulval attachment in C. elegans, we have discovered that the invasive uterine anchor cell activates Notch signaling in neighboring uterine cells at the boundary of the BM gap through which it invades to promote BM sliding. Through an RNAi screen, we found that Notch activation upregulates expression of ctg-1, which encodes a Sec14-GOLD protein and member of the Sec14 phosphatidylinositol-transfer protein superfamily that is implicated in vesicle trafficking. Through photobleaching, targeted knockdown, and cell-specific rescue, our results suggest that CTG-1 restricts BM adhesion receptor DGN-1 (dystroglycan) trafficking to the cell-BM interface, which promotes BM sliding. Together, these studies reveal a new morphogenetic signaling pathway that controls BM sliding to remodel tissues.

Kushwaha S et al. (2012) Microbes Infect "Recombinant trehalose-6-phosphate phosphatase of Brugia malayi cross-reacts with human Wuchereria bancrofti immune sera and engenders a robust protective outcome in mice."

Kushwaha S, Soni VK, Misra-Bhattacharya S, Gupta J, Singh PK

[Microbes Infect, 2012]

Trehalose-6-phosphate phosphatase of Brugia malayi (Bm-TPP) represents an attractive vaccine candidate because it is present in all the major life stages of parasite, but is absent in mammals. We have previously cloned, purified and biochemically characterized Bm-TPP. In the present study, we investigated the cross-reactivity of recombinant Bm-TPP (r-Bm-TPP) with the sera of human bancroftian patients belonging to different disease categories. In silico study using bioinformatics tool demonstrated that Bm-TPP is highly immunogenic in nature. BALB/c mice administered with r-Bm-TPP alone or in combination with Freund's complete adjuvant (FCA) generated a strong IgG response. Further investigations on the antibody isotypes showed generation of a mixed T helper cell response which was marginally biased towards Th1 phenotype. r-Bm-TPP with or without adjuvant lead to significantly increased accumulation of CD4+ and CD8+ T cells in the spleen of infected mice and increased the activation of peritoneal macrophages. Additionally, r-Bm-TPP enhanced the production of both proinflammatory (IL-2, IFN-) and anti-inflammatory (IL-4, IL-10) cytokines and mice immunized with r-Bm-TPP alone or in combination with FCA showed 54.5% and 67% protection respectively against B. malayi infective larvae challenge. Taken together, our findings suggest that Bm-TPP is protective in nature and might be a potential candidate for development of vaccine against lymphatic filarial infections.

Kelley, Laura C et al. (2013) International Worm Meeting "Understanding the Role of MMPs In Basement Membrane Breaching In Vivo."

Kelley, Laura C, Chi, Qiuyi, Sherwood, David R, Matus, David Q

[International Worm Meeting, 2013]

Basement membrane (BM) is a dense sheet-like extracellular matrix that encapsulates and separates tissue compartments. The invasive ability of cells to cross BM barriers is required for normal and disease processes. Matrix metalloproteinases (MMPs) are overexpressed in cells responsible for tissue remodeling, wound healing, and cancer and are hypothesized to enzymatically facilitate BM removal. Due to the high number of MMPs expressed in vertebrates, the relevance and potential function MMPs in movement through BM is unclear. Our laboratory has combined high-resolution 4D imaging with the visually accessible and genetically tractable model of anchor cell (AC) invasion in C. elegans to analyze BM breaching in vivo. We have previously shown that the c-fos oncogene homologue, FOS-1A, promotes BM penetration during AC invasion (Sherwood and Sternberg, 2005). In fos-1a mutants, the AC extends invasive protrusions that are blocked at the BM, indicating that FOS-1A regulates the transcription of genes that mediate BM removal. Recently, we identified that FOS-1A regulates the expression of three MMPs in the AC during the time of invasion. In addition, two of the three remaining MMPs in the C. elegans genome are expressed in the tissues surrounding the invasive cell. Individual knockdown of these MMPs fails to cause defects in invasion suggesting that they act redundantly to promote invasion. To test this we genetically derived animals that lack all five MMPs expressed within the gonad prior to, and during the invasive process. Unexpectedly, AC invasion persists in the quintuple-MMP mutant animals, indicating that these proteases are not absolutely required for BM removal. Live-cell imaging of the BM, however, revealed distinct differences in the BM dynamics and architecture in MMP-deficient animals. In the MMP (-) mutants BM removal under the AC occurred at a slower rate. Further, BM clearing was disorganized and less uniform in the absence of MMPs. Thus, it appears that MMPs play a modulatory role during the invasive process, functioning to ensure more precise and rapid breaching of BM. These results also suggest that other transcriptional targets of FOS-1A must play a role alongside the MMPs in clearing BM.

Hagedorn EJ et al. (2011) Curr Opin Cell Biol "Cell invasion through basement membrane: the anchor cell breaches the barrier."

Hagedorn EJ, Sherwood DR

[Curr Opin Cell Biol, 2011]

Cell invasion through basement membrane (BM) is a specialized cellular behavior critical to many normal developmental events, immune surveillance, and cancer metastasis. A highly dynamic process, cell invasion involves a complex interplay between cell-intrinsic elements that promote the invasive phenotype, and cell-cell and cell-BM interactions that regulate the timing and targeting of BM transmigration. The intricate nature of these interactions has made it challenging to study cell invasion in vivo and model in vitro. Anchor cell invasion in Caenorhabditis elegans is emerging as an important experimental paradigm for comprehensive analysis of BM invasion, revealing the gene networks that specify invasive behavior and the interactions that occur at the cell-BM interface.