High mobility group box 1, ATP, lipid mediators, and tissue factor are elevated in COVID-19 patients: HMGB1 as a biomarker of worst prognosis.

The severe acute respiratory syndrome coronavirus 2, the agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, has spread worldwide since it was first identified in November 2019 in Wuhan, China. Since then, progress in pathogenesis linked severity of this systemic disease to the hyperactivation of network of cytokine-driven pro-inflammatory cascades. Here, we aimed to identify molecular biomarkers of disease severity by measuring the serum levels of inflammatory mediators in a Brazilian cohort of patients with COVID-19 and healthy controls (HCs). Critically ill patients in the intensive care unit were defined as such by dependence on oxygen supplementation (93% intubated and 7% face mask), and computed tomography profiles showing ground-glass opacity pneumonia associated to and high levels of D-dimer. Our panel of mediators included HMGB1, ATP, tissue factor, PGE2 , LTB4 , and cys-LTs. Follow-up studies showed increased serum levels of every inflammatory mediator in patients with COVID-19 as compared to HCs. Originally acting as a transcription factor, HMGB1 acquires pro-inflammatory functions following secretion by activated leukocytes or necrotic tissues. Serum levels of HMGB1 were positively correlated with cys-LTs, D-dimer, aspartate aminotransferase, and alanine aminotransferase. Notably, the levels of the classical alarmin HMGB1 were higher in deceased patients, allowing their discrimination from patients that had been discharged at the early pulmonary and hyperinflammatory phase of COVID-19. In particular, we verified that HMGB1 levels above 125.4 ng/ml is the cutoff that distinguishes patients that are at higher risk of death. In conclusion, we propose the use of serum levels of HMGB1 as a biomarker of severe prognosis of COVID-19.

COVID-19 diverse outcomes: Aggravated reinfection, type I interferons and antibodies.

SARS-CoV-2 infection intrigued medicine with diverse outcomes ranging from asymptomatic to severe acute respiratory syndrome (SARS) and death. After more than two years of pandemic, reports of reinfection concern researchers and physicists. Here, we will discuss potential mechanisms that can explain reinfections, including the aggravated ones. The major topics of this hypothesis paper are the disbalance between interferon and antibodies responses, HLA heterogeneity among the affected population, and increased proportion of cytotoxic CD4+ T cells polarization in relation to T follicular cells (Tfh) subtypes. These features affect antibody levels and hamper the humoral immunity necessary to prevent or minimize the viral burden in the case of reinfections.

B-1 lymphocytes are able to produce IL-10, but is not pathogenic during Leishmania (Leishmania) amazonensis infection.

Over the years research has found an association between B lymphocytes and pathogenesis during Leishmania sp. infections. Recently we demonstrated that B-2 lymphocytes are the main producers of IL-10 during L. amazonensis infection, and that the disease severity in BALB/c mice was attributed to these IL-10-producing B-2 lymphocytes. Here, we aim to understand the role of peritoneal B-1 lymphocytes in the pathogenesis of L. amazonensis infection. We found that infection resulted in a decrease in the number of B-1a lymphocytes and increase in B-1b lymphocytes in the peritoneal cavity of WT BALB/c mice but not in B lymphocyte deficient mice (BALB/Xid) mice. In vitro interaction between B-1 lymphocytes and L. amazonensis showed that the amastigote form of the parasite was able to induce higher levels of IL-10 in B-1 lymphocytes derived from infected BALB/c mice than the promastigote. Moreover, B-1 lymphocytes derived from infected mice produced more IL-10 than B-1 lymphocytes derived from naïve mice under amastigote interaction. However, the repopulation of BALB/Xid mice with B-1 lymphocytes from WT BALB/c mice did not affect the lesion development. Together, these results suggest that although B-1 lymphocytes are able to produce IL-10 during in vitro interaction with L. amazonensis, they are not directly related to pathogenesis in vivo.

CD43 sialoglycoprotein modulates cardiac inflammation and murine susceptibility to Trypanosoma cruzi infection.

CD43 (leukosialin) is a large sialoglycoprotein abundantly expressed on the surface of most cells from the hematopoietic lineage. CD43 is directly involved in the contact between cells participating in a series of events such as signaling, adherence and host parasite interactions. In this study we examined the role of CD43 in the immune response against Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, a potential life-threatening illness endemic in 21 Latin American countries according to the WHO. The acute stage of infection is marked by intense parasitemia and cardiac tissue parasitism, resulting in the recruitment of inflammatory cells and acute damage to the heart tissue. We show here that CD43-/- mice were more resistant to infection due to increased cytotoxicity of antigen specific CD8+ T cells and reduced inflammatory infiltration in the cardiac tissue, both contributing to lower cardiomyocyte damage. In addition, we demonstrate that the induction of acute myocarditis involves the engagement of CD43 cytoplasmic tripeptide sequence KRR to ezrin-radixin-moiesin cytoskeletal proteins. Together, our results show the participation of CD43 in different events involved in the pathogenesis of T. cruzi infection, contributing to a better overall understanding of the mechanisms underlying the pathogenesis of acute chagasic cardiomyopathy.

Dietary Vitamin D3 Deficiency Increases Resistance to Leishmania (Leishmania) amazonensis Infection in Mice.

The leishmaniases are a group of diseases caused by Leishmania parasites, which have different clinical manifestations. Leishmania (Leishmania) amazonensis is endemic in South America and causes cutaneous leishmaniasis (CL), which can evolve into a diffuse form, characterized by an anergic immune response. Since the leishmaniases mainly affect poor populations, it is important to understand the involvement of immunonutrition, how the immune system is modulated by dietary nutrients and the effect this has on Leishmania infection. Vitamin D3 (VitD) is an immunonutrient obtained from diet or endogenously synthesized, which suppresses Th1 and Th17 responses by favoring T helper (Th) 2 and regulatory T cell (Treg) generation. Based on these findings, this study aims to evaluate dietary VitD influence on L. (L.) amazonensis experimental infection in C57BL/6 and BALB/c mice. Thus, C57BL/6 and BALB/c VitD deficient (VDD) mice were generated through dietary VitD restriction 45 days prior to infection. Both strains of VDD mice showed a more controlled lesion development compared to mice on a regular diet (Ctrl). There were no differences in serum levels of anti-Leishmania IgG1 and IgG2a, but there was a decrease in IgE levels in BALB/c VDD mice. Although CD4+ T cell number was not changed, the CD4+ IFN-y+ T cell population was increased in both absolute number and percentage in C57BL/6 and BALB/c VDD mice compared to Ctrl mice. There was also no difference in IL-4 and IL-17 production, however, there was reduction of IL-10 production in VDD mice. Together, our data indicate that VitD contributes to murine cutaneous leishmaniasis susceptibility and that the Th1 cell population may be related to the resistance of VDD mice to L. (L.) amazonensis infection.

The role of TLR9 on Leishmania amazonensis infection and its influence on intranasal LaAg vaccine efficacy.

Leishmania (L.) amazonensis is one of the etiological agents of cutaneous leishmaniasis (CL) in Brazil. Currently, there is no vaccine approved for human use against leishmaniasis, although several vaccine preparations are in experimental stages. One of them is Leishvacin, or LaAg, a first-generation vaccine composed of total L. amazonensis antigens that has consistently shown an increase of mouse resistance against CL when administered intranasally (i.n.). Since Toll-like receptor 9 (TLR9) is highly expressed in the nasal mucosa and LaAg is composed of TLR9-binding DNA CpG motifs, in this study we proposed to investigate the role of TLR9 in both L. amazonensis infection and in LaAg vaccine efficacy in C57BL/6 (WT) mice and TLR9-/- mice. First, we evaluated, the infection of macrophages by L. amazonensis in vitro, showing no significant difference between macrophages from WT and TLR9-/- mice in terms of both infection percentage and total number of intracellular amastigotes, as well as NO production. In addition, neutrophils from WT and TLR9-/- mice had similar capacity to produce neutrophil extracellular traps (NETs) in response to L. amazonensis. L. amazonensis did not activate dendritic cells from WT and TLR9-/- mice, analysed by MHCII and CD86 expression. However, in vivo, TLR9-/- mice were slightly more susceptible to L. amazonensis infection than WT mice, presenting a larger lesion and an increased parasite load at the peak of infection and in the chronic phase. The increased TLR9-/- mice susceptibility was accompanied by an increased IgG and IgG1 production; a decrease of IFN-γ in infected tissue, but not IL-4 and IL-10; and a decreased number of IFN-γ producing CD8+ T cells, but not CD4+ T cells in the lesion-draining lymph nodes. Also, TLR9-/- mice could not control parasite growth following i.n. LaAg vaccination unlike the WT mice. This protection failure was associated with a reduction of the hypersensitivity response induced by immunization. The TLR9-/- vaccinated mice failed to respond to antigen stimulation and to produce IFN-γ by lymph node cells. Together, these results suggest that TLR9 contributes to C57BL/6 mouse resistance against L. amazonensis, and that the TLR9-binding LaAg comprising CpG motifs may be important for intranasal vaccine efficacy against CL.

Probiotic treatment during neonatal age provides optimal protection against experimental asthma through the modulation of microbiota and T cells.

The incidence of allergic diseases, which increased to epidemic proportions in developed countries over the last few decades, has been correlated with altered gut microbiota colonization. Although probiotics may play a critical role in the restoration of gut homeostasis, their efficiency in the control of allergy is controversial. Here, we aimed to investigate the effects of probiotic treatment initiated at neonatal or adult ages on the suppression of experimental ovalbumin (OVA)-induced asthma. Neonatal or adult mice were orally treated with probiotic bacteria and subjected to OVA-induced allergy. Asthma-like symptoms, microbiota composition and frequencies of the total CD4+ T lymphocytes and CD4+Foxp3+ regulatory T (Treg) cells were evaluated in both groups. Probiotic administration to neonates, but not to adults, was necessary and sufficient for the absolute prevention of experimental allergen-induced sensitization. The neonatally acquired tolerance, transferrable to probiotic-untreated adult recipients by splenic cells from tolerant donors, was associated with modulation of gut bacterial composition, augmented levels of cecum butyrate and selective accumulation of Treg cells in the airways. Our findings reveal that a cross-talk between a healthy microbiota and qualitative features inherent to neonatal T cells, especially in the Treg cell subset, might support the beneficial effect of perinatal exposure to probiotic bacteria on the development of long-term tolerance to allergens.

Expression of leukosialin (CD43) defines a major intrahepatic T cell subset associated with protective responses in visceral leishmaniasis.

BACKGROUND: Leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania protozoa that are transmitted to mammalian hosts by infected sand flies. Infection is associated with distinct clinical manifestations that include cutaneous, mucocutaneous and visceral lesions. Visceral leishmaniasis (VL) is the most severe form of the disease and is considered second in terms of mortality and fourth in terms of morbidity among tropical diseases. IFN-γ-producing T cells are involved in protection against the disease. METHODS: CD43⁺/⁺ and CD43⁻/⁻ mice on a C57BL/6 background were intravenously injected with 5 × 10 7 amastigotes of Leishmania (L.) infantum chagasi, and 30 days after infection the clinical signs of disease were examined; the splenocytes were isolated and assayed for cytokine production; and the livers were removed for phenotypic analysis of T cell subsets by flow cytometry. RESULTS: We report that mice lacking CD43 display increased susceptibility to infection by Leishmania (L.) infantum chagasi, with higher parasite burdens than wild-type mice. The increased susceptibility of CD43⁻/⁻ mice were associated with a weakened delayed hypersensitivity response and reduced levels of IgG2a antibodies to leishmania antigens. We further showed that expression of CD43 defines a major intrahepatic CD4⁺ and CD8⁺ T cell subsets with pro-inflammatory phenotypes and leads to increased levels of IFN-γ secretion by activated splenocytes. CONCLUSIONS: Our findings point to a role of CD43 in the development of host resistance to visceral leishmaniasis.

Antigenicity of a whole parasite vaccine as promising candidate against canine leishmaniasis.

Human visceral leishmaniasis, one of the most important zoonoses, is caused by the protozoa Leishmania chagasi (syn. L. infantum) and is present as a fatal disease common in South America and Europe where dogs and wild canids are the main reservoirs. A vaccine against visceral leishmaniasis would be an important tool in the control of this disease in dogs. Although the current strategies for vaccination against leishmaniasis are based on the use of recombinant antigens, killed vaccines are still attractive in terms of stability of their biochemical composition and antigenicity, cost, and safety. Here we evaluate the immunogenicity of a whole parasite vaccine as a promising candidate against canine leishmaniasis, demonstrated by cellular reactivity, changes in the cellular profile of the peripheral blood and by the differential production of immunoglobulins. Our results showed that immunization elicited mainly a strong cellular reactivity and increase in T-lymphocytes, particularly the subpopulation CD8(+) that would be related to the control of tissue parasitism. In addition, a higher production of anti-Leishmania total IgG, characterized by mixed isotypes profile (IgG1 and IgG2), was demonstrated.

Immunogenicity in dogs of three recombinant antigens (TSA, LeIF and LmSTI1) potential vaccine candidates for canine visceral leishmaniasis.

Control of canine visceral leishmaniasis (VL) remains a difficult and serious problem mostly because there is no reliable and effective vaccine available to prevent this disease. A mixture of three recombinant leishmanial antigens (TSA, LeIF and LmSTI1) encoded by three genes highly conserved in the Leishmania genus have been shown to induce excellent protection against infection in both murine and simian models of cutaneous leishmaniasis. A human clinical trial with these antigens is currently underway. Because of the high degree of conservation, these antigens might be useful vaccine candidates for VL as well. In the present study, using the dog model of the visceral disease, we evaluated the immunogenicity of these three antigens formulated with two different adjuvants, MPL-SE and AdjuPrime. The results were compared with a whole parasite vaccine formulated with BCG as the adjuvant. In order to investigate if sensitization with the recombinant antigens would result in recognition of the corresponding native parasite antigens upon infection, the animals were exposed for four weeks after the termination of the immunization protocol with the recombinant antigens to a low number of L. chagasi promastigotes, an etiological agent of VL. Immune response was evaluated by quantitative ELISA in the animal sera before and after exposure to the viable parasites. Both antigen specific IgG1 and IgG2 antibody levels were measured. Immunization of dogs with the recombinant antigens formulated in either MPL-SE or AdjuPrime resulted in high antibody levels particularly to LmSTI1. In addition, this immunization although to low levels, resulted in the development of antibody response to the whole parasite lysate. Importantly, experimental exposure with low numbers of culture forms of L. chagasi promastigotes caused a clear boost in the immune response to both the recombinant antigens and the corresponding native molecules. The boost response was predominantly of the IgG2 isotype in animals primed with the recombinant antigens plus MPL-SE. In contrast, animals primed with the recombinant antigens formulated in AdjuPrime as well as animals vaccinated with crude antigen preparation responded with mixed IgG1/IgG2 isotypes. These results point to the possible use of this antigen cocktail formulated with the adjuvant MPL-SE in efficacy field trials against canine VL.