A Case of Concurrent Disseminated Coccidioidomycosis and Embryonal Carcinoma When Lice and Fleas Coexist.

Coccidioidomycosis (CM) is a fungal infection endemic to the southwestern United States with a wide range of clinical presentations depending on the infected organ systems. Most infections are asymptomatic. Coccidioidomycosis causes a primary pulmonary infection and when symptoms occur, they most often resemble community-acquired pneumonia. One percent of cases disseminate, typically via hematogenous or lymphatic spread. It is in these cases that more severe symptoms may present and potentially overlap with those characteristics of other systemic illnesses. This is a case of CM disseminated to lymph nodes in a 24-year-old man with concomitant metastatic embryonal carcinoma. It is difficult to identify the primary etiology for many components of this patient's presentation, including diffuse lymphadenopathy and multiple pulmonary nodules. Furthermore, the relationship between these 2 concurrent disease processes is not entirely clear. Factors that may contribute include the well-known phenomenon of locus minoris resistentiae (LMR) or potentially a shared immune failure between infectious organisms and malignant cells.

Immunopotentiation by Lymph-Node Targeting of a Malaria Transmission-Blocking Nanovaccine.

A successful malaria transmission blocking vaccine (TBV) requires the induction of a high antibody titer that leads to abrogation of parasite traversal of the mosquito midgut following ingestion of an infectious bloodmeal, thereby blocking the cascade of secondary human infections. Previously, we developed an optimized construct UF6b that elicits an antigen-specific antibody response to a neutralizing epitope of Anopheline alanyl aminopeptidase N (AnAPN1), an evolutionarily conserved pan-malaria mosquito midgut-based TBV target, as well as established a size-controlled lymph node targeting biodegradable nanoparticle delivery system that leads to efficient and durable antigen-specific antibody responses using the model antigen ovalbumin. Herein, we demonstrate that co-delivery of UF6b with the adjuvant CpG oligodeoxynucleotide immunostimulatory sequence (ODN ISS) 1018 using this biodegradable nanoparticle vaccine delivery system generates an AnAPN1-specific immune response that blocks parasite transmission in a standard membrane feeding assay. Importantly, this platform allows for antigen dose-sparing, wherein lower antigen payloads elicit higher-quality antibodies, therefore less antigen-specific IgG is needed for potent transmission-reducing activity. By targeting lymph nodes directly, the resulting immunopotentiation of AnAPN1 suggests that the de facto assumption that high antibody titers are needed for a TBV to be successful needs to be re-examined. This nanovaccine formulation is stable at -20°C storage for at least 3 months, an important consideration for vaccine transport and distribution in regions with poor healthcare infrastructure. Together, these data support further development of this nanovaccine platform for malaria TBVs.

Transcriptomic Analysis of Ovine Hepatic Lymph Node Following Fasciola hepatica Infection - Inhibition of NK Cell and IgE-Mediated Signaling.

Fasciola hepatica is a trematode parasite responsible for major economic losses in livestock production, and is also a food-borne zoonotic agent in developing rural regions. For years, the immunoregulatory mechanisms employed by the parasite have hampered efforts to develop a successful vaccine candidate. Given that a comprehensive understanding of the immune response to infection is needed, we investigated the gene expression changes in ovine hepatic lymph nodes after experimental infection with F. hepatica. Lymph nodes from uninfected and infected animals were processed for RNA sequencing (RNA-seq) at 16 weeks post-infection. Comparison of groups revealed 5,132 differentially-expressed genes (DEGs). An inhibition of pro-inflammatory pathways, which has previously been described during fasciolosis, was evident in our data. However, other signals previously identified in ruminant peripheral blood mononuclear cells (PBMC) or liver tissue, such as activation of TGF-ß or apoptosis-related pathways were not detected. We found inhibition of some key immunological pathways, including natural killer (NK) cell activity and IgE-mediated signaling. These may point to additional some as yet unrecognized mechanisms employed by the parasite to evade the host immune response. Understanding these, and leveraging information from this and other omics studies, will be important for the development of future vaccine prototypes against this parasite.

Relationships between specific antibody responses and clinical signs of dogs living in Tunisian endemic areas of canine leishmaniasis caused by Leishmania infantum.

The first step of the diagnostic process of canine leishmaniasis (CanL) is initiated by veterinarians and relies on their assessment of a high number of clinical signs common to other infectious diseases. We investigated herein the relationship between the clinical profile of 64 domestic dogs living in Tunisian endemic areas and their serological immune status with the aim to identify leishmanial serological markers of diagnosis and disease staging. Seven clinical signs were examined and a total clinical score that describes the number (TCS1) and the number plus the intensity of the clinical signs (TCS2) were determined. Laboratory tests consisted of parasitological examination (PE) of Giemsa-stained popliteal lymph node smears, indirect fluorescent antibody test (IFAT), IgG-, IgG1-, IgG2-Enzyme-Linked-Immunosorbent-Assay (ELISA), and IgG1-, IgG2- Western blotting (WB). Dogs' categorization according to the results of routine diagnostic tests, the TCS1 and TCS2, and the relative IgG1 and IgG2 specific reactivity allowed us to show that active CanL is characterized by an increased reactivity of the IgG2 specific antibodies. Interestingly, the IgG1 levels increased in parallel with the TCS1 and especially with the TCS2, indicating that this isotype is a better marker of dogs' health deterioration. PE & IFAT positive dogs which presented the highest TCS2 and IgG1 reactivity demonstrated significantly more severe weight loss and paleness of the mucosal membranes, suggesting that these signs characterize the latest stages of the disease. WB analysis showed that threeleishmanial polypeptides merit attention and further investigations. The antigens with MWs 32kDa reacting with IgG1 and 37kDa reacting withIgG2 antibodies were found associated with the results of diagnostic tests and late CanL stages, whereas the 24kDa antigen reacting with the IgG2 isotype and associated with low TCS2 seems to be a marker of the early stages.

Systems Biology Analysis of the Radiation-Attenuated Schistosome Vaccine Reveals a Role for Growth Factors in Protection and Hemostasis Inhibition in Parasite Survival.

In spite of several decades of research, an effective vaccine against schistosomiasis remains elusive. The radiation-attenuated (RA) cercarial vaccine is still the best model eliciting high protection levels, although the immune mechanisms have not yet been fully characterized. In order to identify genes and pathways underlying protection we investigated patterns of gene expression in PBMC and skin draining Lymph Nodes (LN) from mice using two exposure comparisons: vaccination with 500 attenuated cercariae versus infection with 500 normal cercariae; one versus three doses. Vaccinated mice were challenged with 120 normal parasites. Integration of PBMC and LN data from the infected group revealed early up-regulation of pathways associated with Th2 skewing and polarization of IgG antibody profiles. Additionally, hemostasis pathways were downregulated in infected mice, correlating with platelet reduction, potentially a mechanism to assist parasite migration through capillary beds. Conversely, up regulation of such mechanisms after vaccination may explain parasite blockade in the lungs. In contrast, a single exposure to attenuated parasites revealed early establishment of a Th1 bias (signaling of IL-1, IFN-γ; and Leishmania infection). Genes encoding chemokines and their receptors were more prominent in vaccinated mice, indicating an enhanced capacity for inflammation, potentially augmenting the inhibition of intravascular migration. Increasing the vaccinations from one to three did not dramatically elevate protection, but there was a clear shift towards antibody-mediated effectors. However, elements of the Th1 bias were still evident. Notable features after three vaccinations were markers of cytotoxicity (including IL-6 and NK cells) together with growth factors and their receptors (FGFR/VEGF/EGF) and the apoptosis pathway. Indeed, there is evidence for the development of anergy after three vaccinations, borne out by the limited responses detected in samples after challenge. We infer that persistence of a Th1 response puts a limit on expression of antibody-mediated mechanisms. This feature may explain the failure of multiple doses to drive protection towards sterile immunity. We suggest that the secretions of lung stage parasites would make a novel cohort of antigens for testing in protection experiments.

Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue.

BACKGROUND: Alveolar echinococcosis (AE) is caused by metacestode larva of the tapeworm Echinococcus multilocularis. AE diagnostics currently rely on imaging techniques supported by serology, but unequivocal detection of AE is difficult. Although polymerase chain reaction (PCR)-based methods to detect tapeworm DNA in biopsies have been suggested for several species, no validated protocol adhering to accepted guidelines has so far been presented for AE diagnostics. We herein established a PCR protocol for metacestode biopsies and technically evaluated the method using isolated parasite DNA and cells, biopsies of clinically relevant material, and formalin fixed paraffin-embedded (FFPE) human tissue blocks. We compared the results with an immunochemical (IHC) approach using the monoclonal antibody Em2G11 specific for the antigen Em2 of E. mulitlocularis. METHODOLOGY/PRINCIPAL FINDINGS: Based on tapeworm 12S rDNA sequences we established and validated a PCR protocol for robust detection of as little as 50 parasite cells per specimen and report 127 cases of positive identification of Echinococcus species in samples from humans and animals. For further validation, we analyzed 45 liver, heart, brain, and soft tissue samples as well as cytological probes of aspirates of FFPE-material from 18 patients with clinically confirmed AE. Of each patient we analyzed (i) fully viable lesions with laminated layer; (ii) tissue with mAbEm2G11-positive small particles of E. multilocularis (spems); (iii) mAbEm2G11-negative tissue adjacent to the main lesion; and (iv) lymph node tissue with mAbEm2G11-positive spems. To identify the areas for the PCR-based approach, we performed IHC-staining with the monoclonal antibody Em2G11. Micro-dissected tissue of these areas was then used for PCR-analysis. 9 of 15 analyzed samples with viable E. multilocularis lesions with laminated layer were positive by PCR. Of this group, all samples preserved for less than 6 years (6/6) were tested positive. 11 of 15 samples of spems and 7 of 9 samples of the control group mAbEm2G11-negative tissue were negative by PCR. We further show that all probes from lymph nodes with spems are PCR negative. CONCLUSIONS/SIGNIFICANCE: We present a sensitive PCR method for the detection of E. multilocularis in human tissue, particularly in fresh biopsy material and tissue blocks stored for less than 5 years. While the diagnostic sensitivity of material containing only spems was higher using IHC, PCR detection was possible in IHC negative liver tissue and in patients with negative serology. Our results support the view that spems do not contain parasitic DNA or viable cells of the parasite. spems thus most probably do not directly contribute to metastasis formation during AE.

Cytokine profile and parasite load in lymph nodes of dogs naturally infected with Leishmania infantum from distinct epidemiological scenarios in São Paulo State, Brazil.

Visceral leishmaniasis (VL) is an important zoonotic vector-borne disease and domestic dogs are considered the main domiciliary and peri-domiciliary reservoir of Leishmania (Leishmania) infantum in South America. Distinct eco-epidemiological scenarios associated to the prevalence of the disease, clusters of parasite genotypes and chemotypes of vectors population are described in Brazil, especially in the state of São Paulo (SP). In this context, the purpose of the present study is to evaluate the clinical signs, histopathological lesions, parasite load and cytokine profile by immunohistochemistry (IHC) in popliteal lymph nodes of canines naturally infected with L. infantum, from different municipalities of the state of SP. Eighty-three dogs with VL, 61 from northwest SP (NWSP) and 22 from southeast SP (SESP), were clinically classified in stage II, with no babesiosis and ehrlichiosis. Subcapsular inflammatory infiltration and histiocytosis were significantly higher in the SESP group (p = 0.0128; 0.0077, respectively). On the other hand, dogs from NWSP revealed 4.6-fold significantly higher parasite burden (p = 0.0004) and higher IHC scores of IL-1ß (p = 0.0275) and IL-4 (p = 0.0327) in the popliteal lymph node tissues, which may be associated with the susceptibility and progression of the disease in these dogs. Differences in immune response profile associated with higher parasite load in dogs can also contribute to explain the distinct eco-epidemiological patterns of VL in specific geographic regions.

Natural resistance to worms exacerbates bovine tuberculosis severity independently of worm coinfection.

Pathogen interactions arising during coinfection can exacerbate disease severity, for example when the immune response mounted against one pathogen negatively affects defense of another. It is also possible that host immune responses to a pathogen, shaped by historical evolutionary interactions between host and pathogen, may modify host immune defenses in ways that have repercussions for other pathogens. In this case, negative interactions between two pathogens could emerge even in the absence of concurrent infection. Parasitic worms and tuberculosis (TB) are involved in one of the most geographically extensive of pathogen interactions, and during coinfection worms can exacerbate TB disease outcomes. Here, we show that in a wild mammal natural resistance to worms affects bovine tuberculosis (BTB) severity independently of active worm infection. We found that worm-resistant individuals were more likely to die of BTB than were nonresistant individuals, and their disease progressed more quickly. Anthelmintic treatment moderated, but did not eliminate, the resistance effect, and the effects of resistance and treatment were opposite and additive, with untreated, resistant individuals experiencing the highest mortality. Furthermore, resistance and anthelmintic treatment had nonoverlapping effects on BTB pathology. The effects of resistance manifested in the lungs (the primary site of BTB infection), while the effects of treatment manifested almost entirely in the lymph nodes (the site of disseminated disease), suggesting that resistance and active worm infection affect BTB progression via distinct mechanisms. Our findings reveal that interactions between pathogens can occur as a consequence of processes arising on very different timescales.

Antileishmanial efficacy and tolerability of combined treatment with non-ionic surfactant vesicle formulations of sodium stibogluconate and paromomycin in dogs.

Infection with Leishmania infantum causes the disease visceral leishmaniasis (VL), which is a serious clinical and veterinary problem. The drugs used to treat canine leishmaniasis (CanL) do not cause complete parasite clearance; they can be toxic, and emerging drug resistance in parasite populations limits their clinical utility. Therefore, in this study we have evaluated the toxicity and efficacy of joint treatment with a 1:1 mixture of sodium stibogluconate-NIV (SSG-NIV, 10 mg Sbv/day) and paromomycin-NIV (PMM-NIV, 10 mg PMM/kg/day), given intravenously daily for seven days from day 270 post-infection, to nine-month-old female beagle dogs (n = 6) experimentally infected with Leishmania infantum. Treatment significantly improved the clinical symptoms of VL infection in all the treated dogs, reduced parasite burdens in lymph nodes and bone marrow, and all symptomatic treated dogs, were asymptomatic at 90 days post-treatment. Treatment was associated with a progressive and significant decrease in specific IgG anti-Leishmania antibodies using parasite soluble antigen (p < 0.01) or rK39 (p < 0.01) as the target antigen. In addition, all dogs were classified as parasite negative based on Leishmania nested PCR and quantitative real time PCR tests and as well as an inability to culture of promastigote parasites from lymph nodes and bone marrow tissue samples taken at day 90 post-treatment. However, treatment did not cure the dogs as parasites were detected at 10 months post-treatment, indicating that a different dosing regimen is required to cause long term cure or prevent relapse.

Early immune responses and parasite tissue distribution in mice experimentally infected with oocysts of either archetypal or non-archetypal genotypes of Toxoplasma gondii.

In most of the world Toxoplasma gondii is comprised of archetypal types (types I, II and III); however, South America displays several non-archetypal strains. This study used an experimental mouse model to characterize the immune response and parasite kinetics following infection with different parasite genotypes. An oral inoculation of 50 oocysts per mouse from T. gondii M4 type II (archetypal, avirulent), BrI or BrIII (non-archetypal, virulent and intermediate virulent, respectively) for groups (G)2, G3 and G4, respectively was used. The levels of mRNA expression of cytokines, immune compounds, cell surface markers and receptor adapters [interferon gamma (IFNγ), interleukin (IL)-12, CD8, CD4, CD25, CXCR3 and MyD88] were quantified by SYBR green reverse transcription-quantitative polymerase chain reaction. Lesions were characterized by histology and detection by immunohistochemistry established distribution of parasites. Infection in G2 mice was mild and characterized by an early MyD88-dependent pathway. In G3, there were high levels of expression of pro-inflammatory cytokines IFNγ and IL-12 in the mice showing severe clinical symptoms at 8­11 days post infection (dpi), combined with the upregulation of CD25, abundant tachyzoites and tissue lesions in livers, lungs and intestines. Significant longer expression of IFNγ and IL-12 genes, with other Th1-balanced immune responses, such as increased levels of CXCR3 and MyD88 in G4, resulted in survival of mice and chronic toxoplasmosis, with the occurrence of tissue cysts in brain and lungs, at 14 and 21 dpi. Different immune responses and kinetics of gene expression appear to be elicited by the different strains and non-archetypal parasites demonstrated higher virulence.

Immunohistological detection of small particles of Echinococcus multilocularis and Echinococcus granulosus in lymph nodes is associated with enlarged lymph nodes in alveolar and cystic echinococcosis.

BACKGROUND: Alveolar (AE) and cystic echinococcosis (CE) in humans are caused by the metacestode of the tapeworms Echinococcus multilocularis and Echinococcus granulosus sensu lato (s.l.). Immunohistochemistry with the monoclonal antibodies (mAb) Em2G11, specific for AE, and the mAb EmG3, specific for AE and CE, is an important pillar of the histological diagnosis of these two infections. Our aim was to further evaluate mAb EmG3 in a diagnostic setting and to analyze in detail the localization, distribution, and impact of small particles of Echinococcus multilocularis (spems) and small particles of Echinococcus granulosus s.l. (spegs) on lymph nodes. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the mAb EmG3 in a cohort of formalin-fixed, paraffin embedded (FFPE) specimens of AE (n = 360) and CE (n = 178). These samples originated from 156 AE-patients and 77 CE-patients. mAb EmG3 showed a specific staining of the metacestode stadium of E. multilocularis and E. granulosus s.l. and had a higher sensitivity for spems than mAb Em2G11. Furthermore, we detected spegs in the surrounding host tissue and in almost all tested lymph nodes (39/41) of infected patients. 38/47 lymph nodes of AE showed a positive reaction for spems with mAb EmG3, whereas 29/47 tested positive when stained with mAb Em2G11. Spegs were detected in the germinal centers, co-located with CD23-positive follicular dendritic cells, and were present in the sinuses. Likewise, lymph nodes with spems and spegs in AE and CE were significantly enlarged in size in comparison to the control group. CONCLUSIONS/SIGNIFICANCE: mAb EmG3 is specific for AE and CE and is a valuable tool in the histological diagnosis of echinococcosis. Based on the observed staining patterns, we hypothesize that the interaction between parasite and host is not restricted to the main lesion since spegs are detected in lymph nodes. Moreover, in AE the number of spems-affected lymph nodes is higher than previously assumed. The enlargement of lymph nodes with spems and spegs points to an immunological interaction with the small immunogenic particles (spems and spegs) of Echinococcus spp.

New features on the survival of human-infective Trypanosoma rangeli in a murine model: Parasite accumulation is observed in lymphoid organs.

Trypanosoma rangeli is a non-pathogenic protozoan parasite that infects mammals, including humans, in Chagas disease-endemic areas of South and Central America. The parasite is transmitted to a mammalian host when an infected triatomine injects metacyclic trypomastigotes into the host's skin during a bloodmeal. Infected mammals behave as parasite reservoirs for several months and despite intensive research, some major aspects of T. rangeli-vertebrate interactions are still poorly understood. In particular, many questions still remain unanswered, e.g. parasite survival and development inside vertebrates, as no parasite multiplication sites have yet been identified. The present study used an insect bite transmission strategy to investigate whether the vector inoculation spot in the skin behave as a parasite-replication site. Histological data from the skin identified extracellular parasites in the dermis and hypodermis of infected mice in the first 24 hours post-infection, as well as the presence of inflammatory infiltrates in a period of up to 7 days. However, qPCR analyses demonstrated that T. rangeli is eliminated from the skin after 7 days of infection despite being still consistently found on circulating blood and secondary lymphoid tissues for up to 30 days post-infection. Interestingly, significant numbers of parasites were found in the spleen and mesenteric lymph nodes of infected mice during different periods of infection and steady basal numbers of flagellates are maintained in the host's bloodstream, which might behave as a transmission source to insect vectors. The presence of parasites in the spleen was confirmed by fluorescent photomicrography of free and cell-associated T. rangeli forms. Altogether our results suggest that this organ could possibly behave as a T. rangeli maintenance hotspot in vertebrates.

Efficacy of histamine H1 receptor antagonists azelastine and fexofenadine against cutaneous Leishmania major infection.

Current drug therapies for cutaneous leishmaniasis are often difficult to administer and treatment failure is an increasingly common occurrence. The efficacy of anti-leishmanial therapy relies on a combination of anti-parasite activity of drugs and the patient's immune response. Previous studies have reported in vitro antimicrobial activity of histamine 1-receptor antagonists (H1RAs) against different pathogens. We used an ex vivo explant culture of lymph nodes from mice infected with Leishmania major to screen H1RAs compounds. Azelastine (AZ) and Fexofenadine (FX) showed remarkable ex vivo efficacy (EC50 = 0.05 and 1.50 µM respectively) and low in vitro cytotoxicity yielding a high therapeutic index. AZ significantly decreased the expression of H1R and the proinflammatory cytokine IL-1ẞ in the ex vivo system, which were shown to be augmented by histamine addition. The anti-leishmanial efficacy of AZ was enhanced in the presence of T cells from infected mice suggesting an immune-modulatory mechanism of parasite suppression. L. major infected BALB/c mice treated per os with FX or intralesionally with AZ showed a significant reduction of lesion size (FX = 69%; AZ = 52%). Furthermore, there was significant parasite suppression in the lesion (FX = 82%; AZ = 87%) and lymph nodes (FX = 81%; AZ = 36%) with no observable side effects. AZ and FX and potentially other H1RAs are good candidates for assessing efficacy in larger studies as monotherapies or in combination with current anti-leishmanial drugs to treat cutaneous leishmaniasis.

A natural cell-penetrating nanopeptide combined with pentavalent antimonial as experimental therapy against cutaneous leishmaniasis.

The inadequacy of available treatments for leishmaniasis has presented up to 40% therapeutic failure. This fact suggests an urgency in the discovery of new drugs or alternative approaches for treating this disease. The objective of this study was to evaluate the antileishmanial activity of combined therapy between crotamine (CTA) from Crotalus durissus terrificus and the pentavalent antimonial Glucantime® (GLU). The assays were in vitro performed measuring the inhibition of Leishmania amazonensis amastigotes, followed by the evaluation of cellular production of cytokines and nitrites. After that, analytical methods were performed in order to characterize the molecules involved in the study by Mass Spectrometry, molecular affinity through an in silico assay and Surface Plasmon Resonance. In vivo experiments with BALB/c mice were performed by analyzing parasitemia, lesion size and immunological mediators. In the in vitro experiments, the pharmacological association improved the inhibition of the amastigotes, modulated the production of cytokines and nitric oxide. The therapy improved the effectiveness of the GLU, demonstrating a decreased parasitemia in the infected tissues. Altogether, the results suggest that the combined approach with CTA and GLU may be a promising alternative for the treatment of cutaneous leishmaniasis.

Sonographic findings of pinpoint hyperechoic foci in the small intestine, liver, and mesenteric lymph nodes are indicative of canine Heterobilharzia americana infection.

Canine schistosomiasis, caused by the trematode Heterobilharzia americana, can pose a diagnostic challenge due to nonspecific symptoms. The aim of this multicenter, retrospective, descriptive study was to compare the prevalence and extent of sonographic changes associated with schistosomiasis between affected and infection-free dogs. Medical records of two referral centers were searched for dogs with confirmed schistosomiasis that had undergone an abdominal ultrasound. Fifty-five cases fulfilled the inclusion criteria, and a contemporaneous control group was derived from dogs that tested negative for H. americana. Two blinded reviewers evaluated the images. The majority of Heterobilharzia-infected (further termed H-pos) dogs (82%) had ultrasonographic abnormalities in the small intestine ± liver. Abnormal layering of the small intestine was noted in 38 of 54 H-pos dogs, compared to six of 54 control dogs (P < .0001). Pinpoint hyperechoic foci were noted in the small intestinal submucosa or muscularis layers in 25 of 54 H-pos dogs, but only three controls (P < .0001). Heterogeneity of the hepatic parenchyma and pinpoint hyperechoic foci were more prevalent in H-pos dogs (65% vs 40%; P = .0213 and 44% vs 18%; P = .0068, respectively). Pinpoint hyperechoic foci within mesenteric lymph nodes were noted in seven H-pos dogs and none of the controls (P = .0128). The combination of heterogeneous small intestine wall layering and pinpoint hyperechoic foci in small intestine, liver, or mesenteric lymph nodes was the most reliable indication of infection (P = .0001; odds ratio = 36.87), with positive predictive value of 94%, yet modest sensitivity for the detection of infection (58%). Observing these sonographic features suggests schistosomiasis and should prompt further testing.

Microautophagy upregulation in cutaneous lymph nodes of dogs naturally infected by Leishmania infantum.

This is the first study showing an in vivo microautophagy upregulation by Leishmania infantum in dogs. Both Leishmania amastigotes and promastigotes were detected in the cytoplasm of many professional and nonprofessional phagocytic cells of popliteal lymph node of three dogs suffering from chronic cutaneous leishmaniasis. Ultrastructurally, parasites appeared to be wrapped by lysosomes and/or multivesicular bodies. Neither phagophores nor double-membraned vacuoles consistent with autophagosomes were observed. Transcription factor EB (TFEB), a key factor involved in lysosome biogenesis, showed a statistically significant increase in the total component when examined by western blot in samples from leishmaniotic dogs compared with samples from healthy dogs. Instead, phosphorylated TFEB showed unmodified expression levels both in leishmaniotic and healthy dogs. Furthermore, Hsc70 and endosomal sorting complex required for transport (ESCRT)-I, which are known to play a role in microautophagy, showed no variation in expression levels both in diseased and healthy animals. Vps4A/B, an evolutionary conserved ATPase responsible for ESCRT-I complex disassembly and MVB maturation, was statistically significantly overexpressed in lymph nodal samples from leishmaniotic dogs. Bag3 was downregulated in diseased dogs whereas CHIP, p62, and LC3-II did not show any variation in expression levels. The altered expression profile of Bag3 suggested an altered interaction of Bag3 with Hsc70 and CHIP, which usually form a molecular complex involved in autophagosome-lysosome pathways. Ultrastructural and molecular findings suggested that the microautophagy pathway is upregulated in lymph nodes of dogs suffering from a chronic natural infection by Leishmania infantum.

Visceral leishmaniasis: a practical strategy for quantitative molecular diagnosis in naturally infected dogs.

The diagnosis of canine visceral leishmaniasis (CVL) has been a problem for public health services due to the variety of clinical signs similar to other diseases and low sensitivity and specificity of available tests. In this sense, our main objective was to develop a simple, rapid, and accurate quantitative real-time PCR (qPCR) diagnosis for CVL. Thus, low-invasive samples from bone marrow (BM), popliteal lymph nodes (PLN), and conjunctival swabs (CS) were selected from negative and VL-positive dogs, using as gold standard, immunological and parasitological tests performed with different tissues. Oligonucleotides for Leishmania infantum kDNA were designed and the limit of quantification and amplification efficiency of the qPCR were determined using tissue-specific standards produced with DNA from those different tissues, mixed with DNA from a known amount of L. infantum promastigotes. Endogenous control was used to validate a comparative Ct method, and tissue parasite concentrations were estimated by comparison with tissue-specific reference standard samples. The overall analysis of the qPCR data suggests the following ranking for tissue choice: PLN > BM > CS. Finally, we have concluded that this molecular approach simplifies and accelerates the quantitative diagnostic process because it is easy to perform, requiring no DNA dosing or standard curve application, and it shows good diagnostic parameters, especially when using popliteal lymph node samples.

Interleukin-33 Induces the Enzyme Tryptophan Hydroxylase 1 to Promote Inflammatory Group 2 Innate Lymphoid Cell-Mediated Immunity.

Group 2 innate lymphoid cells (ILC2s) regulate immunity, inflammation, and tissue homeostasis. Two distinct subsets of ILC2s have been described: steady-state natural ILC2s and inflammatory ILC2s, which are elicited following helminth infection. However, how tissue-specific cues regulate these two subsets of ILC2s and their effector functions remains elusive. Here, we report that interleukin-33 (IL-33) promotes the generation of inflammatory ILC2s (ILC2INFLAM) via induction of the enzyme tryptophan hydroxylase 1 (Tph1). Tph1 expression was upregulated in ILC2s upon activation with IL-33 or following helminth infection in an IL-33-dependent manner. Conditional deletion of Tph1 in lymphocytes resulted in selective impairment of ILC2INFLAM responses and increased susceptibility to helminth infection. Further, RNA sequencing analysis revealed altered gene expression in Tph1 deficient ILC2s including inducible T cell co-stimulator (Icos). Collectively, these data reveal a previously unrecognized function for IL-33, Tph1, and ICOS in promoting inflammatory ILC2 responses and type 2 immunity at mucosal barriers.

Detection of amastigotes and histopathological alterations in the thymus of Leishmania infantum-infected dogs.

INTRODUCTION: In canine visceral leishmaniasis (CVL), lymphopenia, and the disorganization of lymphoid organs such as spleen and lymph nodes have been demonstrated. However, the involvement of thymus in CVL has not been evaluated so far. Herein, we investigated whether the thymus can be colonized by Leishmania infantum in naturally infected dogs. METHODS: Thymus were obtained from 16 of 58 dogs and samples of this organ were submitted to immunohistochemistry for laminin and fibronectin detection, histopathology, in situ hybridization and polymerase chain reaction (PCR) targeting the gene ITS-1 for Leishmania and sequenced. Samples of spleen, skin and popliteal lymph nodes were collected and submitted to immunohistochemistry and parasitological culture followed by multilocus enzyme electrophoresis. RESULTS: L. infantum was identified in all dogs. DNA and amastigote forms of Leishmania were detected in the thymus from 16 dogs by PCR and in eight by immunohistochemistry. Besides thymus, parasites were detected in spleen, lymph nodes, and skin. A granulomatous or pyogranulomatous thymitis was observed in eight dogs associated to intact amastigotes forms of this parasite. Fibronectin deposition in thymus was higher in dogs with more clinical signs. CONCLUSIONS: These results demonstrate that the thymus of dogs can be parasitized by L. infantum, which may generate inflammatory reactions leading to alterations in thymic microarchitecture.