Microfilaria-dependent thoracic pathology associated with eosinophilic and fibrotic polyps in filaria-infected rodents.

BACKGROUND: Pulmonary manifestations are regularly reported in both human and animal filariasis. In human filariasis, the main known lung manifestations are the tropical pulmonary eosinophilia syndrome. Its duration and severity are correlated with the presence of microfilariae. Litomosoides sigmodontis is a filarial parasite residing in the pleural cavity of rodents. This model is widely used to understand the immune mechanisms that are established during infection and for the screening of therapeutic molecules. Some pulmonary manifestations during the patent phase of infection with L. sigmodontis have been described in different rodent hosts more or less permissive to infection. METHODS: Here, the permissive Mongolian gerbil (Meriones unguiculatus) was infected with L. sigmodontis. Prevalence and density of microfilariae and adult parasites were evaluated. Lungs were analyzed for pathological signatures using immunohistochemistry and 3D imaging techniques (two-photon and light sheet microscopy). RESULTS: Microfilaremia in gerbils was correlated with parasite load, as amicrofilaremic individuals had fewer parasites in their pleural cavities. Fibrotic polypoid structures were observed on both pleurae of infected gerbils. Polyps were of variable size and developed from the visceral mesothelium over the entire pleura. The larger polyps were vascularized and strongly infiltrated by immune cells such as eosinophils, macrophages or lymphocytes. The formation of these structures was induced by the presence of adult filariae since small and rare polyps were observed before patency, but they were exacerbated by the presence of gravid females and microfilariae. CONCLUSIONS: Altogether, these data emphasize the role of host-specific factors in the pathogenesis of filarial infections.

Measurement and immunophenotyping of pleural fluid EpCAM-positive cells and clusters for the management of non-small cell lung cancer patients.

OBJECTIVES: A malignant pleural effusion (MPE) is a common complication in non-small cell lung cancer (NSCLC) with important staging and prognostic information. Patients with MPEs are often candidates for advanced therapies, however, the current gold standard, cytological analysis of pleural fluid samples, has limited sensitivity. We aimed to demonstrate the feasibility of non-invasive enumeration and immunophenotyping of EpCAM-positive cells in pleural fluid samples for the diagnosis of a MPE in NSCLC patients. MATERIALS AND METHODS: Pleural fluid specimens were prospectively collected from patients with NSCLC and the CellSearch® technology was utilized for the enumeration of pleural EpCAM-positive cells (PECs) and determination of PD-L1 expression on PECs from pleural fluid samples. The diagnostic performance of the enumeration of single PECs and PEC clusters was assessed using receiver operating characteristic (ROC) curves. The Kaplan-Meier method and Cox proportional hazards model was used to assess the impact of PECs and PEC clusters on overall survival (OS). RESULTS: 101 NSCLC patients were enrolled. The median number of PECs was significantly greater in the malignant (n = 84) versus non-malignant group (n = 17) (730 PECs/mL vs 1.0 PEC/mL, p < 0.001). The area under the ROC curve was 0.91. A cutoff value of 105 PECs/mL had a sensitivity and specificity of 73% and 100% for the diagnosis of a MPE, respectively. Among 69 patients with a pathology-confirmed MPE and tissue immunohistochemistry (IHC) results, 15 (22%) had greater than 50% PD-L1+ PECs. Overall concordance between tissue and PEC PD-L1 expression was 76%. Higher numbers of pleural effusion single PECs were associated with inferior overall survival (Cox adjusted HR 1.8, 95% CI: 1.02-3.05 p = 0.043). CONCLUSION: Non-invasive measurement of PECs in NSCLC patients, using an automated, clinically available approach, may improve the diagnostic accuracy of a MPE, allow for immunophenotyping of PECs, and provide prognostic information.

Management for hydatid cysts of the lung ruptured in the pleural cavity.

INTRODUCTION: Our purpose is to report our experience in the management of pulmonary hydatid cysts ruptured in the pleura. Materiel and methods: We collected all records of patients with a ruptured hydatid cyst of the lung in the pleura who underwent surgery for this in the department of thoracic surgery of the CHU Hassan II of Fes during the 6-year period that started in 2010. RESULTS: The study included 20 men and 14 women with an average age of 30.44 ± 18.4 years. Radiological findings showed a hydropneumothorax in 21 cases, hydrothorax in 10, pachypleuritis in 29, and a floating membrane in 13 cases. In all cases, pleuropulmonary decortication was associated with pericystectomy in 20 cases and parenchymal resection in 3 cases. A hydatid membrane bathing in the pleural cavity was found in 32 cases. The postoperative course was uneventful in 28 cases. CONCLUSION: Long-term follow-up should be established to detect possible recurrences or pleural dissemination, which appear to be prevented by long-term use of anthelmintic agents.

IL-6 is required for protective immune responses against early filarial infection.

IL-6 has a wide range of biological activities that includes anti- and pro-inflammatory aspects. In this study, we investigated the role of IL-6 in immune responses to the rodent filarial nematode Litomosoides sigmodontis, a model for human filarial infections. IL-6-/- mice had a significantly increased worm burden after natural infection compared with wild type controls at early time points p.i. Given that the worm burden in IL-6-/- mice was already increased at the time point the infective larvae reached the pleural cavity, immune responses that may facilitate the migration from the site of infection (skin) via the lymphatics to the pleural cavity were analysed. Increased vascular permeability may facilitate larval migration, but blocking of histamine receptors had no effect on worm burden and vascular permeability was similar between IL-6-/- mice and wild type controls. In contrast, blocking mast cell degranulation reduced the worm burden in IL-6-/- mice partially, suggesting that release of mast cell-derived mediators improves larval migration to some degree. Protective immune responses within the skin were involved, as bypassing the skin barrier by inoculating infective L3s subcutaneously resulted in a comparable worm recovery in both mouse strains. Analysis of the cellular composition by flow cytometry and PCR array in the skin after exposure to filarial extract or L3s, respectively, indicate that the absence of IL-6 results in a delayed recruitment of neutrophils and macrophages to the site of initial infection. These results demonstrate that IL-6 is essentially involved in protective immune responses within the skin that impair migration of infective L3s.

Myeloid cell recruitment versus local proliferation differentiates susceptibility from resistance to filarial infection.

Both TH2-dependent helminth killing and suppression of the TH2 effector response have been attributed to macrophages (MΦ) activated by IL-4 (M(IL-4)). To investigate how M(IL-4) contribute to diverse infection outcomes, the MΦ compartment of susceptible BALB/c mice and more resistant C57BL/6 mice was profiled during infection of the pleural cavity with the filarial nematode, Litomosoides sigmodontis. C57BL/6 mice exhibited a profoundly expanded resident MΦ (resMΦ) population, which was gradually replenished from the bone marrow in an age-dependent manner. Infection status did not alter the bone-marrow derived contribution to the resMΦ population, confirming local proliferation as the driver of resMΦ expansion. Significantly less resMΦ expansion was observed in the susceptible BALB/c strain, which instead exhibited an influx of monocytes that assumed an immunosuppressive PD-L2+ phenotype. Inhibition of monocyte recruitment enhanced nematode killing. Thus, the balance of monocytic vs. resident M(IL-4) numbers varies between inbred mouse strains and impacts infection outcome.

IL-4/5 signalling plays an important role during Litomosoides sigmodontis infection, influencing both immune system regulation and tissue pathology in the thoracic cavity.

Approximately 100 million people suffer from filarial diseases including lymphatic filariasis (elephantiasis), onchocerciasis (river blindness) and loiasis. These diseases are amongst the most devastating of the neglected tropical diseases in terms of social and economic impact. Moreover, many infection-induced immune mechanisms in the host, their relationship to disease-related symptoms and the development of pathology within the site of infection remain unclear. To improve on current drug therapies or vaccines, further studies are necessary to decipher the mechanisms behind filaria-driven immune responses and pathology development, and thus the rodent model of Litomosoides sigmodontis can be used to unravel host-filaria interactions. Interestingly, BALB/c mice develop a patent state (release of microfilariae, the transmission life-stage, into the periphery) when exposed to L. sigmodontis. Thus, using this model, we determined levels of host inflammation and pathology development during a L. sigmodontis infection in vivo for the first known time. Our study reveals that after 30days p.i., inflammation and pathology began to develop in infected wild type BALB/c mice between the lung and diaphragm, close to the site of infection - the thoracic cavity. Interestingly, infected IL-4Rα/IL-5-/- BALB/c mice had accentuated inflammation of the pleural lung and pleural diaphragm, and higher parasite burdens. Corresponding to the pleural inflammation, levels of IP-10, MIP-1α, MIP-1ß, MIP-2 and RANTES were significantly elevated in the thoracic cavity fluid of infected IL-4Rα/IL-5-/- mice compared with wild type controls. Moreover, upon L. sigmodontis antigen stimulation, IFN-γ and IL-17A secretions by cells isolated from draining lymph nodes of IL-4Rα/IL-5-/- mice were significantly elevated, whereas secretion of IL-5, IL-13 and IL-10 was reduced. Elevated filaria-specific IFN-γ secretion was also observed in spleen-derived CD4+ T cell co-cultures from IL-4Rα/IL-5-/- mice. In summary, this study unravels the essential role of IL-4/IL-5 signalling in controlling immunity against filarial infections and demonstrates the requirement of this pathway for the host to control ensuing pathology and inflammation.

Endothelial cells release soluble factors that support the long-term survival of filarial worms in vitro.

The inability to maintain filarial nematodes in long-term in vitro culture greatly limits research into the basic biology of these parasites and hinders in vitro screening of novel anti-filarial agents. In this study, we sought to characterize nutrients that promote the long-term survival of filarial worms in vitro. Using microfilariae (MF) obtained from gerbils infected with Litomosoides sigmodontis, a filarial parasite of rodents, we found that Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) resulted in MF survival of only 5 days. However, co-culturing MF with a mouse endothelial cell line (EOMA) enabled survival for 40 days. Culturing EOMA cells in transwell plates extended MF survival to the same degree as direct co-culture, suggesting that the factors microfilariae require are soluble in nature. Heat inactivation of EOMA conditioned media at 56 °C reduced MF survival by approximately 50%, and heat inactivation at 100 °C reduced survival to 3 days, demonstrating that both heat labile and heat stable factors are involved. EOMA cells require FBS to produce these factors, as conditioned media collected from EOMA cells grown in the absence of FBS failed to prolong survival. The removal of lipids also abrogated survival, indicating MF are likely utilizing lipid factors released by EOMA cells. Dialysis experiments demonstrate that at least some of the required factors are between 0.1 and 1 kDa in size. Importantly, L. sigmodontis adult worms also show significantly extended survival when cultured in EOMA conditioned media. Together, these results suggest that EOMA-produced factors include lipid-containing molecules, heat labile molecules (likely a protein), and micronutrients between 0.1 and 1 kDa in size. These studies have established a cell-free approach to maintaining MF and adult stage filarial worms in long-term in vitro culture and have taken important steps towards biochemically characterizing host-derived nutrients required for parasite survival.

Pleural Myiasis Associated With Pleural Angiosarcoma.

Myiasis refers to a parasitic infestation of vertebrate mammals by dipterous larvae (maggots) of higher flies. Infections in humans typically occur in tropical and subtropical regions, regions with limited medical access, and areas with poor hygiene and living conditions. Infestations in humans have been described in subcutaneous, nasal, ocular, oropharyngeal, and orotracheal cases; however, reports of pulmonary myiasis in humans in the United States and other developed countries are extremely rare. We describe a patient with recently diagnosed primary pleural angiosarcoma who presented to our clinic for the management of a thoracostomy tube and was diagnosed with pleural myiasis.

Pleural cavity type 2 innate lymphoid cells precede Th2 expansion in murine Litomosoides sigmodontis infection.

Recently, a family of innate cells has been identified that respond to IL-25 and IL-33 in murine intestinal helminths. Termed Type 2 innate lymphoid cells (ILC2s) they facilitate the development of Th2 responses responsible for helminth clearance. We evaluated these cells in a tissue-invasive helminth model. Using Litomosides sigmodontis (a strong Th2 polarizing filarial infection) we observed a robust Th2 response in the pleural cavity, where adult worms reside, marked by increased levels of IL-5 and IL-13 in infected mice. In parallel, ILC2s were expanded in the pleural cavity early in the infection, peaking during the pre-patent period. L. sigmodontis also elicits a strong systemic Th2 response, which includes significantly increased levels of IgG1, IgE and IL-5 in the plasma of infected mice. Although ILC2s were expanded locally, they were not expanded in the spleen, blood, or mediastinal lymph nodes in response to L. sigmodontis infection, suggesting that ILC2s function primarily at the site of infection. The increase in ILC2s in the pleural cavity and the expansion in Th2 responses indicates a probable role for these cells in initiating and maintaining the Th2 response and highlights the importance of these cells in helminth infections and their role in Th2 immunity.

Co-infection restrains Litomosoides sigmodontis filarial load and plasmodial P. yoelii but not P. chabaudi parasitaemia in mice.

Infection with multiple parasite species is clearly the norm rather than the exception, in animals as well as in humans. Filarial nematodes and Plasmodium spp. are important parasites in human public health and they are often co-endemic. Interactions between these parasites are complex. The mechanisms underlying the modulation of both the course of malaria and the outcome of filarial infection are poorly understood. Despite increasing activity in recent years, studies comparing co- and mono-infections are very much in their infancy and results are contradictory at first sight. In this study we performed controlled and simultaneous co-infections of BALB/c mice with Litomosoides sigmodontis filaria and with Plasmodium spp. (Plasmodium yoelii 17 XNL or Plasmodium chabaudi 864VD). An analysis of pathological lesions in the kidneys and lungs and a parasitological study were conducted at different times of infection. Whatever the plasmodial species, the filarial recovery rate was strongly decreased. The peak of parasitaemia in the plasmodial infection was decreased in the course of P. yoelii infection but not in that of P. chabaudi. Regarding pathological lesions, L. sigmodontis can reverse lesions in the kidneys due to the presence of both Plasmodium species but does not modify the course of pulmonary lesions. The filarial infection induces granulomas in the lungs.

Eotaxin-1 is involved in parasite clearance during chronic filarial infection.

Eosinophil migration as key feature of helminth infection is increased during infection with filarial nematodes. In a mouse model of filariasis, we investigated the role of the eosinophil-attracting chemokine Eotaxin-1 on disease outcome. BALB/c and Eotaxin-1(-/-) mice were infected with the rodent filaria Litomosoides sigmodontis, and parasitic parameters, cellular migration to the site of infection, and cellular responsiveness were investigated. We found increased parasite survival but unaffected eosinophil migration to the site of infection in Eotaxin-1(-/-) mice. Expression of CD80 and CD86 was reduced on eosinophils from Eotaxin-1(-/-) mice after in vitro TLR2 stimulation and exposure to filarial antigen, respectively, suggesting a potential reduced activation state of eosinophils in Eotaxin-1 deficient mice. We further demonstrated that macrophages from Eotaxin-1(-/-) mice produce decreased amounts of IL-6 in vitro, a cytokine found to be associated with parasite containment, suggesting possible mechanisms by which Eotaxin-1 regulates activation of inflammatory cells and thus parasite survival.

Differential tissular distribution of Litomosoides sigmodontis microfilariae between microfilaremic and amicrofilaremic mice following experimental infection.

Filariases are caused by onchocercid nematodes that are transmitted by arthropod vectors. More than 180 million people are infected worldwide. Mass drug administration has been set up in many endemic areas to control the parasite burden. Although very successful in limiting microfilarial load, transmission has not been completely interrupted in such areas. A proportion of infected patients with lymphatic filariasis or loiasis are known to be amicrofilaremic, as they do not present microfilariae in their bloodstream despite the presence of adult worms. A mirror status also exists in CBA/Ca mice infected with Litomosoides sigmodontis, the well-established model of filariasis. Using this model, the goal of this study was to determine if the kinetics of blood clearance of microfilariae differed between amicrofilaremic CBA/Ca mice and microfilaremic BALB/c mice. For this purpose, a qPCR approach was devised to detect microfilariae in different tissues, after a controlled inoculation of microfilariae. We showed that the rapid clearance of microfilariae from the pleural cavity or from the bloodstream of CBA/Ca mice was associated with a massive accumulation of first stage larvae in the lungs, liver and spleen.

Litomosoides pardinasi n. sp. (Nematoda, Onchocercidae) from two species of cricetid rodents in northern Patagonia, Argentina.

Seventy-seven cricetid rodents, corresponding to six different species, deposited at the Mammal Collection at Centro Nacional Patagónico, Puerto Madryn, Chubut, were examined for parasites. Litomosoides pardinasi n. sp. is described parasitizing the pleural and abdominal cavities of Phyllotis xanthopygus (Phyllotini) and the abdominal cavity of Oligoryzomys longicaudatus (Oryzomynii) in the northern Patagonia, Argentina. The new species is included in the carinii group, characterized by the presence of a right spicule with a dorsal heel and a terminal cap well cuticularized; left spicule composed with a handle longer or as long as the blade, and the blade constituted by a cuticularized distal filament. Litomosoides pardinasi n. sp. possesses a buccal capsule tubular with an anterior enlargement, and a smooth cavity; four labial and two dorsal cephalic papillae; vulva posterior to the esophagus-intestinal junction; male tail with three to five pairs of symmetric postcloacal papillae; female tail slender; vagina globular and small. This is the first record for a filarioid in a Phyllotini rodent and the southernmost record of any filarioid species worldwide.

Litomosoides sigmodontis: a simple method to infect mice with L3 larvae obtained from the pleural space of recently infected jirds (Meriones unguiculatus).

Litomosoides sigmodontis is a filarial nematode that is used as a mouse model for human filarial infections. The life cycle of L. sigmodontis comprises rodents as definitive hosts and tropical rat mites as alternate hosts. Here, we describe a method of infecting mice with third stage larvae (L3) extracted from the pleural space of recently infected jirds (Meriones unguiculatus). This method enables infection of mice with a known number of L3 larvae without the time-consuming dissection of L3 larvae from mites and results in higher worm recovery and patency rates than conventional methods. Additionally, this method allows for geographical separation of the facility maintaining the L. sigmodontis life cycle from the institution at which mice are infected.

Ultrasonography in filaria-infected rodents: detection of adult Litomosoides sigmodontis and Brugia malayi filariae.

OBJECTIVE: To evaluate the usefulness of ultrasonography (USG) in the detection of adult filariae in rodents. Wuchereria bancrofti are frequently detected using USG in humans, whereas adult Brugia malayi have not been so far. METHODS: A Meriones unguiculatus with Litomosoides sigmodontis infection was examined to visualize adult filariae of a similar length as W. bancrofti. Similarly, three Mastomys coucha, infected with B. malayi, were examined using USG to verify whether the adult worms, which are far smaller than W. bancrofti and L. sigmodontis, can be located using USG in the animals. RESULTS: Adult L. sigmodontis were detected using USG in the pleural cavity of M. unguiculatus, and in M. coucha adult B. malayi were visualized in the hearts, lungs, axillary lymph nodes and scrotum. Ultrasound findings were verified by dissection of the rodents. CONCLUSIONS: Although adult B. malayi are far smaller than L. sigmodontis and W. bancrofti, they can be detected using USG in rodents. USG may serve as an adjunctive tool to support parasitological examinations and can add information on filarial infections at any time point of an observation period, particularly in cryptic infections and without the need for invasive measures or killing of the rodent. Thus, USG can support the early detection of macrofilaricidal activities of new compounds and can be used to determine the location of adult worms in the animals. It is possible to give a rough estimate of the number of adult worms, but determination of the exact numbers of adult filariae in various locations is impossible with USG.

Vaccination against filarial nematodes with irradiated larvae provides long-term protection against the third larval stage but not against subsequent life cycle stages.

Sustainable control of human filariasis would benefit enormously from the development of an effective vaccine. The ability to vaccinate experimental animals, with reductions in worm burden of over 70%, suggests this aim is possible. However, in experimental vaccinations the challenge is usually administered 2 weeks after the immunisation phase and thus the protection obtained is likely to be biased by persisting inflammation. Using the murine model Litomosoides sigmodontis, we increased the time between immunisation with irradiated larvae and challenge with fully infective L3 to 5 months. Significant protection was achieved (54-58%) and the reduced worm burden was observed by 10 days p.i. The developmental stage targeted was the L3, since no nematodes died once they reached the pleural cavity of vaccinated mice, as has been previously shown in short-term protocols. However, larval developmental rate was faster in vaccinated than in primary-infected mice. Immunological assessments were made prior to challenge and then from 6 h to 34 days post-challenge. Samples were taken from the subcutaneous tissue where the larvae were inoculated, the lymph nodes through which they migrate and the pleural cavity in which they establish. Eosinophils were still present although scarce in the subcutaneous tissue of vaccinated mice before challenge. Cytokine and specific antibody production of vaccinated and challenged mice were L3-specific and Th2-biased and greatly exceeded the response of primary-infected mice. The heightened Th2 response may explain the faster development of the filarial worms in vaccinated mice. Thus, long-term vaccination protocols generated a strong memory response that led to significant but incomplete protection that was limited to the infective larval stage suggesting alternative vaccination strategies are needed.