Corrigendum: Clinical validation of circulating immune complexes for use as a diagnostic marker of canine leishmaniosis.

[This corrects the article DOI: 10.3389/fvets.2024.1368929.].

Clinical validation of circulating immune complexes for use as a diagnostic marker of canine leishmaniosis.

Introduction: Canine leishmaniosis (CanL) is a systemic disease that affects dogs. When multiplication of the parasite cannot be controlled, dogs consistently show high levels of antigen and IgG antibodies, which lead to the formation of circulating immune complexes (CIC). Timely intervention to reduce the parasite load and CIC levels is crucial for preventing irreversible organ damage. However, a diagnostic test to quantify CIC levels is currently lacking. Methods: In this real-world study, we aimed to examine the performance of a new ELISA to measure CIC levels in dogs naturally infected with Leishmania infantum. Thirty-four dogs were treated according to their clinical condition and followed for 360 days. Before (day 0) and after treatment (days 30, 90, 180, 270, and 360), all dogs underwent a physical examination, and blood samples were obtained for CBC, biochemical profile, serum protein electrophoresis and IFAT. Serum PEG-precipitated CIC were determined by ELISA. Results: Our results indicate higher CIC levels in dogs in advanced disease stages showing higher antibody titres (p < 0.0001, r = 0.735), anemia (p < 0.0001), dysproteinemia (p < 0.0001), and proteinuria (p = 0.004). Importantly, dogs responding well to treatment exhibited declining CIC levels (p < 0.0001), while in poor responders and those experiencing relapses, CIC were consistently elevated. CIC emerged as a robust discriminator of relapse, with an area under the curve (AUC) of 0.808. The optimal cut-off to accurately identify relapse was an optical density of 1.539. Discussion: Our findings suggest that declining CIC levels should be expected in dogs showing a favorable treatment response. Conversely, in dogs displaying a poor response and recurrent clinical relapses, CIC levels will be high, emphasizing the need for vigilant monitoring. These findings suggest that CIC could serve as a valuable biomarker for disease progression, treatment efficacy, and relapse detection in CanL. Our study contributes to enhancing diagnostic approaches for CanL and underscores the potential of CIC as a complementary tool in veterinary practice. As we move forward, larger studies will be essential to confirm these findings and establish definitive cut-offs for clinical application.

Laboratory validation of an ELISA method to measure circulating immune complexes levels in canine visceral leishmaniasis.

Susceptible dogs suffering from canine leishmaniasis (CanL) develop an ineffective humoral immune response that leads to the formation of circulating immune complexes (CIC). These CIC are aggregates of Leishmania proteins and anti-Leishmania immunoglobulins. Their deposition in different tissues is considered the main cause of mortality. For this reason, CIC have been suggested as an excellent CanL biomarker for measuring the progression of the disease and the effectiveness of specific treatments. The present study aims to perform a laboratory validation of a Leishmania-specific method to isolate and quantify CIC in dog serum samples. CIC isolated from serum samples of infected dogs, grouped according to the LeishVet classification, were quantified following a PEG-ELISA procedure. The validation established a cut-off of 0.274 OD. All the parameters analyzed (including linearity, specificity, precision, and robustness) fulfilled the defined criteria, confirmed by statistical analyses. The results also proved the reproducibility and reliability of the method when samples were tested under the same conditions, and the consistency and usefulness of the method for an optimal staging of infected dogs. In conclusion, the laboratory validated method offers a potent tool to clinicians for a proper CanL management and to measure the progression of the disease.

Role of Circulating Immune Complexes in the Pathogenesis of Canine Leishmaniasis: New Players in Vaccine Development.

During canine visceral leishmaniasis (CanL), due to Leishmania infantum (L. infantum), uncontrolled infection leads to a strong humoral immune response. As a consequence of the production of high antibody levels and the prolonged presence of parasite antigens, circulating immune complexes (CIC) are formed, which can be deposited in certain organs and tissues, inducing vasculitis, uveitis, dermatitis and especially glomerulonephritis and renal failure. A method to detect CIC and quantify their levels in serum samples from dogs infected with L. infantum has been recently described. It allowed demonstration of a correlation between CIC levels and disease severity. Thus, CIC measurement may be useful for diagnosis, assessment of disease progression and monitoring response to treatment. This is an interesting finding, considering that there remains an urgent need for identification of novel biomarkers to achieve a correct diagnosis and for optimal disease staging of dogs suffering from Leishmania infection. The objective of the present review is to shed light on the role of CIC in CanL, as well as to highlight their potential use not only as diagnostic and prognostic biomarkers but also as a valuable tool in vaccine development and new immunotherapy strategies to prevent or control disease outcome.

Specific Dermatophagoides farinae extract for canine immunotherapy.

BACKGROUND: Canine atopic dermatitis (cAD) is a pruritic allergic skin disease most often caused by Dermatophagoides farinae. Differences in the sensitization profile to D. farinae have been reported between people and dogs. However, allergic dogs traditionally have been treated with extracts intended for human immunotherapy. HYPOTHESIS/OBJECTIVES: To develop a specific allergen immunotherapy for veterinary practice enriched in canine major allergens and to demonstrate its in vitro efficacy. ANIMALS: Twenty privately owned dogs, clinically diagnosed with cAD, and three healthy dogs. METHODS AND MATERIALS: A veterinary D. farinae allergen extract was manufactured and characterized compared to D. farinae extract used for human immunotherapy. The protein profile was analysed by SDS-PAGE and size exclusion chromatography and Der f 15 and Der f 18 allergens quantified by mass spectrometry. The allergenic profile was studied by immunoblot and the biological potency by enzyme-linked immunosorbent assay-inhibition assays. The extract's capacity to induce cytokine production [interleukin (IL)-10, interferon (IFN)-Ɣ] by peripheral blood mononuclear cells also was evaluated. RESULTS: The veterinary extract showed a higher content of high molecular weight proteins, preferentially recognized by atopic dog sera. The fold-increases in Der f 15 and Der f 18 with respect to the human extract were 2.07 ± 0.32 and 1.63 ± 0.15, respectively. The veterinary extract showed higher biological potency (0.062 versus 0.132 µg required for 50% inhibition of dogs sera) compared to the human extract and induced significantly higher levels of IL-10 (1,780 pg/mL) and IFN-Ɣ (50.4 pg/mL) with respect to the negative control. CONCLUSIONS AND CLINICAL IMPORTANCE: A veterinary D. farinae extract with a higher content of dog major allergens was developed and in vitro efficacy demonstrated by immunological parameters.

Vaccination with LetiFend® reduces circulating immune complexes in dogs experimentally infected with L. infantum.

Domestic dogs constitute the main reservoir of Leishmania infantum and play a key role in transmission to humans. The main tool for controlling infection spread is a safe and effective vaccine, as successful immunization of dogs could significantly reduce the incidence of human visceral leishmaniosis (VL) and is the most cost-effective control strategy. The factors that determine disease progression in canine leishmaniosis (CanL) remain poorly understood, though a previous study in naturally infected dogs has demonstrated a clear relationship between the presence of circulating immune complexes (CIC) in the blood and disease progression. Thus, the aim of this study was to compare CIC levels in serum samples from dogs vaccinated or unvaccinated with LetiFend®, a new vaccine containing recombinant Protein Q, and experimentally infected with L. infantum. CIC were isolated from vaccinated or unvaccinated dogs after experimental infection with L. infantum and their levels measured by ELISA. Furthermore, reverse phase-liquid chromatography-mass spectrometry (RP-LC-MS/MS) analysis was used to investigate the protein composition of precipitated CIC. At all the time points analyzed after infection, the amount of CIC was lower in the vaccinated group compared to the placebo group. Furthermore, there were differences in the protein composition of precipitated CIC between the vaccinated and unvaccinated groups. In conclusion, administration of LetiFend® was able to reduce CIC elicited after experimental infection with L. infantum in a dog model in a process that may be related to complement system activation.

Circulating immune complexes levels correlate with the progression of canine leishmaniosis in naturally infected dogs.

Dogs are the main domestic reservoir of Leishmania infantum, and in cases of uncontrolled infection, a strong humoral immune response is elicited, which is inefficient against the parasites. Previous studies have suggested that an adequate antigen/antibody ratio, with a moderate prevalence of antigens with respect to the antibodies, could result in the formation of circulating immune complexes (CIC) in canine leishmaniosis (CanL). Deposition of these complexes in tissues has been associated with vasculitis, uveitis, arthritis, dermatitis and especially glomerulonephritis and renal failure. However, little is known about the relationship between the presence of CIC and disease progression. The aim of this study was to evaluate serum CIC level and its correlation with disease severity in infected dogs with different stages of disease and non-infected animals as a control. A total of 60 dogs were included in the study, classified according to the proposed LeishVet classification criteria: healthy non-infected (n = 13); healthy infected (n = 12); sick stage I (n = 9); sick stage II (n = 17); sick stage III (n = 8); and sick stage IV (n = 1). CIC were isolated from serum samples using a modified polyethylene glycol precipitation method, and their levels measured by ELISA and bicinchoninic acid protein assay. A nanoparticle tracking analysis was performed to investigate the relationship between the molecular size distribution of the CIC and disease progression. In conclusion, the results confirmed a positive association between CIC levels, their molecular size and disease progression that suggests a potential use of CIC as biomarkers of CanL.

Analysis of calcium-induced conformational changes in calcium-binding allergens and quantitative determination of their IgE binding properties.

The polcalcin family is one of the most epidemiologically relevant families of calcium-binding allergens. Polcalcins are potent plant allergens that contain one or several EF-hand motifs and their allergenicity is primarily associated with the Ca(2+)-bound form of the protein. Conformation, stability, as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. We describe a protocol that uses three techniques (SDS-PAGE, circular dichroism spectroscopy, and ELISA) to describe the effects that calcium has on the structural changes in an allergen and its IgE binding properties.

Modulation of the humoral response to Dermatophagoides pteronyssinus allergens in BALB/c mice by extract modification and adjuvant use.

BACKGROUND: Currently, several strategies are being used in order to improve the safety and efficacy of allergen-specific immunotherapy; these strategies include the use of modified hypoallergenic extracts as well as different adjuvants with immunomodulatory properties in combination with native or modified extracts. The objectives of this study were to investigate the humoral response generated in mice to modified Dermatophagoides pteronyssinus extracts in the presence or absence of two different adjuvants. METHODS: BALB/c mice were inoculated either with native, depigmented or depigmented-polymerised D. pteronyssinus without adjuvants or combined with aluminium hydroxide or oligodeoxinucleotides containing CpG motifs. IgE concentration, specific total IgG, IgG1 and IgG2a titres were measured in mice sera and cross-reactivity inhibition experiments were performed. IgG antigenic profiles were obtained by immunoblotting for all formulations. RESULTS: Inoculation of depigmented-polymerised extract induced statistically significant lower IgE levels than the native extract even when adsorbed onto aluminium hydroxide. When this extract was inoculated in the presence of oligodeoxinucleotides containing CpG motifs, it elicited high IgG levels, a high IgG2a/lgG1 ratio and low IgE production. Furthermore, the antigenic profiles observed after extract inoculation showed punctual differences between the depigmented-polymerised extract and the native or depigmented extracts. CONCLUSIONS: Our results suggest that the depigmentation and polymerisation process modifies the native extract's antigenic and immunogenic properties and converts the depigmented-polymerised extract into a better choice for allergen-specific immunotherapy.

Molecular cloning and characterization of Cup a 4, a new allergen from Cupressus arizonica.

Sensitization to Cupressaceae pollen has become one of the most important causes of pollinosis in Western countries during winter and early spring. However, the characterization of the extracts, the allergens involved and the cross-reactivity with other pollen sources still remain poorly studied; in the case of Cupressus arizonica only two allergens have been described so far. A new allergen from C. arizonica pollen, Cup a 4, was cloned and expressed in Escherichia coli as an N-terminally His-tag recombinant protein that was characterized biochemically, immunologically and by circular dichroism spectroscopy. The new allergen has high sequence identity with Prickly Juniper allergen Jun o 4 and contains four EF-hand domains. The recombinant protein has structural similarities with other calcium binding allergens such as Ole e 3, Ole e 8 and Phl p 7. Cup a 4 is expressed in mature pollen grains and shares antigenic properties with the recombinant form. Sera from 9.6% C. arizonica allergic patients contain specific IgE antibodies against recombinant Cup a 4.

A modified protocol for RNA isolation from high polysaccharide containing Cupressus arizonica pollen. Applications for RT-PCR and phage display library construction.

RNA isolation is the first step in the study of gene expression and recombinant protein production. However, the isolation of high quantity and high-quality RNA from tissues containing large amounts of polysaccharides has proven to be a difficult process. Cupressus arizonica pollen, in addition to containing high polysaccharide levels, is a challenging starting material for RNA isolation due to the roughness of the pollen grain's walls. Here, we describe an improved technique for RNA isolation from C. arizonica pollen grains. The protocol includes a special disruption and homogenization process as well as a two-step modified RNA isolation technique which consists of an acid phenol extraction followed by a final cleanup using a commercial kit. Resulting RNA proved to be free of contaminants as determined by UV spectrophotometry. The quality of the RNA was analyzed on a bioanalyzer and showed visible 25S and 18S bands. This RNA was successfully used in downstream applications such as RT-PCR and phage display library construction.

Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice.

In the present work we analyze the antigenicity of Leishmania major ribosomal proteins (LRP) in infected BALB/c mice. We show that BALB/c mice vaccinated with LRP in the presence of CpG oligodeoxynucleotides (CpG-ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load after challenge with L. major. This protection was associated with the induction of an IL-12 dependent specific-IFN-gamma response mediated mainly by CD4(+) T cell, albeit a minor contribution of CD8(+) T cells cannot be ruled out. Induction of Th1 responses against LRP also resulted in a reversion of the Th2 responses associated with susceptibility. A marked reduction of IgG1 antibody titer against parasite antigens besides an impaired IL-4 and IL-10 cytokine production by parasite specific T cells was observed. In addition, we show that the administration of the LRP plus CpG-ODN preparation also conferred protection in the naturally resistant C57BL/6 mice. In this strain protection was associated with a LRP specific IFN-gamma production in lymph nodes draining the challenge site. We believe that these evolutionary conserved proteins, combined with adjuvants that favor Th1 responses, may be relevant components of a pan-Leishmania vaccine.