Alterations in monocyte subsets and cytokine production after TLR activation in American Cutaneous Leishmaniasis.

Phenotypic and functional aspects of monocytes from Localized Cutaneous Leishmaniasis (LCL) patients were evaluated. The frequencies of monocyte subsets and TLR2/TLR4 expression were evaluated in fresh peripheral blood whereas cytokine production was evaluated in whole blood cell cultures stimulated with TLR agonists or Leishmania braziliensis antigen (Ag). CD16+ monocytes frequency was increased in patients compared with controls. A TLR4 agonist (LPS) induced expression of TNF and IL-10 in monocyte subsets of patients and controls. The CD14+ CD16+ monocytes expressed higher levels of these cytokines than CD14+ CD16- cells. The levels of secreted TNF were higher in whole blood cell cultures from patients than controls after LPS/TLR4 or Ag stimulation. Whereas in controls there was a positive correlation between TNF and IL-10 levels, this was not observed in stimulated cell cultures from patients. The high levels of LPS-induced TNF were associated with the number of lesions and the percentages of CD14hi CD16+ monocytes. The levels of TLR2-induced TNF were also associated with number of lesions. All monocyte subsets from patients expressed higher levels of TLR2 and TLR4 than controls. Data suggest that systemically activated monocytes contribute for an imbalance in pro- and anti-inflammatory cytokine production during LCL, participating in the immunopathogenesis of the disease.

Human Interleukin-32γ Plays a Protective Role in an Experimental Model of Visceral Leishmaniasis in Mice.

Visceral leishmaniasis (VL) is a chronic parasitic disease caused by Leishmania infantum in the Americas. During VL, several proinflammatory cytokines are produced in spleen, liver, and bone marrow. However, the role of interleukin-32 (IL-32) has not been explored in this disease. IL-32 can induce production of proinflammatory cytokines in innate immune cells and polarize the adaptive immune response. Herein, we discovered that L. infantum antigens induced expression of mRNA mainly for the IL-32γ isoform but also induced low levels of the IL-32ß transcript in human peripheral blood mononuclear cells. Furthermore, infection of human IL-32γ transgenic mice (IL-32γTg mice) with L. infantum promastigote forms increased IL-32γ expression in the spleen and liver. Interestingly, IL-32γTg mice harbored less parasitism in the spleen and liver than wild-type (WT) mice. In addition, IL-32γTg mice showed increased granuloma formation in the liver compared to WT mice. The protection against VL was associated with increased production of nitric oxide (NO), interferon gamma (IFN-γ), IL-17A, and tumor necrosis factor alpha by splenic cells restimulated ex vivo with L. infantum antigens. In parallel, there was an increase in the number of Th1 and Th17 T cells in the spleens of IL-32γTg mice infected with L. infantum IL-32γ induction of IFN-γ and IL-17A expression was found to be essential for NO production by splenic cells of infected animals. These data indicate that IL-32γ potentiates the Th1/Th17 immune response during experimental VL, thus contributing to the control of L. infantum infection.

TLR4 and TLR2 activation is differentially associated with age during Parkinson's disease.

Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by loss of dopaminergic neurons associated with neuroinflammation. Toll-like receptors (TLRs) are expressed in peripheral blood leukocytes and also in neurons and glial cells mediating inflammation. This study aimed to investigate the peripheral blood leukocyte response to TLR2 and TLR4 agonists in young and elderly PD patients. Two groups of patients with PD were evaluated (≤ 55 years old and ≥ 65 years old), age-matched with healthy controls (n = 26). Severity of PD was evaluated by Unified Parkinson's Disease Rating Scale (UPDRS). Whole blood cultures were stimulated with lipopolysaccharide (LPS), a TLR4 agonist or Pam3Cys (Pam), a TLR2 agonist. Tumor necrosis factor alpha (TNFα) and interleukin 10 (IL-10) were measured by immunoenzimatic assay. 6 h-TNFα production was increased after TLR4 stimulation, mainly in young PD patients, whereas TLR2-induced TNFα and IL-10 levels were decreased in PD patients independent of age (p < 0.05). A reverse correlation between LPS-induced TNFα production and age was observed in PD patients and controls, but TNFα induced by TLR2 agonist was not associated with age of PD patients or controls. TNFα production induced by TLR4 but not by TLR2 was reversely associated with the age at PD onset and disease duration. No associations between UPDRS scores and cytokine levels were detected. In conclusion, TLR4 and TLR2 responses seem to be differentially affected during PD. Data suggest that TLR2 deficiency in periphery is independent of age of the patients, age at PD onset, or PD duration.

Performance of two immunochromatographic tests for diagnosis of visceral leishmaniasis in patients coinfected with HIV.

Because of visceral leishmaniasis (VL) urbanization and spreading of the human immunodeficiency virus (HIV) infection to rural areas, coinfection has become more common. Here, we compared the accuracy of Kalazar Detect® (KD), an rK39-based immunochromatographic (IC) test, and OrangeLife® (OL), an rK39 + rK28 IC test, for diagnosing VL in patients coinfected with HIV in an endemic area in Brazil. Seventy-six VL patients and 40 patients with other diseases, of which 31 and 21 patients, respectively, were infected with HIV, were examined. The sensitivity of OL and KD tests was 88.89 and 95.45%, respectively, in patients without HIV. The sensitivity dropped to 67.74 and 61.29%, respectively, in coinfected patients. The decrease in sensitivity was not related to a decrease in the production of Leishmania-specific IgG. Because of the low sensitivity of rk39 test in HIV-infected patients, we suggest that patients with negative rK39 results should undergo further investigation with additional serological tests that are not based only on the rK39 antigen and examination of bone marrow aspirates.

The cytotoxic activity of miltefosine against Leishmania and macrophages is associated with dynamic changes in plasma membrane proteins.

In this study, we combined electron paramagnetic resonance (EPR) spectroscopy with an analysis of biophysical cellular parameters to study the mechanisms underlying the in vitro anti-leishmanial activity of miltefosine (MT). A thiol-specific spin label attached to membrane-bound proteins of Leishmania amazonensis and peritoneal macrophages indicated that MT may bind to plasma membrane proteins in large quantities via a detergent-like action and cause structural changes associated with a marked increase in dynamics and exposure to an aqueous environment. EPR spectra of a spin-labeled stearic acid indicated strong interactions between the probe and membrane proteins and a marked increase in the membrane fluidity of MT-treated cells. The cytotoxicity of MT was found to depend on the cell concentration used in the assay. This dependence was described by an equation involving the 50% inhibitory concentrations of MT in the aqueous medium (cw50) and the cell membrane (cm50) and the membrane-aqueous medium partition coefficient of MT (K). With a cw50 of 8.7µM, macrophages were less sensitive to MT than amastigotes and promastigotes of Leishmania, which had cw50 values of 2.4-3.1µM. The estimated cm50 of MT for Leishmania was 1.8M, which appears sufficient to cause ruptures or formation of pores in the plasma membrane. Additionally, we demonstrated that the changes in the plasma membrane detected by EPR spectroscopy occurred at cytotoxic concentrations of MT, as assessed through in vitro assays.

Leishmania (Viannia) braziliensis amastigotes induces the expression of TNFα and IL-10 by human peripheral blood mononuclear cells in vitro in a TLR4-dependent manner.

While the role of Toll-like receptors (TLRs) has been investigated in murine models of tegumentary leishmaniasis caused by Leishmania (Viannia) braziliensis, the interaction between TLRs and Leishmania sp. has not been investigated in human cells. The aim of this study was to evaluate the involvement of TLR4 in cytokine production of human peripheral blood mononuclear cells (PBMCs) induced by L. braziliensis, and whether the parasite alters the expression of TLR4 on monocytes/macrophages. Amastigote forms were obtained from mice lesions and PBMCs were isolated from healthy donors. PBMCs were cultured in absence or presence of IFNγ, TLR4 neutralizing antibodies, natural antagonist of TLR4 (Bartonella LPS), TLR4 agonist (E. coli LPS), and amastigote forms. The concentrations of tumor necrosis factor (TNFα) and interleukin 10 (IL-10) were assayed by ELISA and TLR4 expression by flow cytometry. Amastigotes forms of L. braziliensis induced TNFα and IL-10 production only in IFNγ-primed PBMCs. The TNFα and IL-10 production was inhibited by TLR4 neutralization, both with anti-TLR4 antibodies and Bartonella LPS. Interestingly, addition of E. coli LPS further increased TNFα but not IL-10 production induced by L. braziliensis amastigotes. Amastigotes of L. braziliensis strongly reduced membrane TLR4 expression on monocytes/macrophages, apparently by internalization after the infection. The present study reveals that TLR4 drives the production of TNFα and IL-10 induced by L. braziliensis amastigotes and that the parasites decrease TLR4 expression on monocyte surface.

Decreased Toll-Like Receptor 2 and Toll-Like Receptor 7/8-Induced Cytokines in Parkinson's Disease Patients.

OBJECTIVES: Toll-like receptors (TLRs) are expressed in several immune cells including blood monocytes and resident macrophages, such as microglia in the central nervous system. TLRs recognize pathogen- or damage-associated molecular patterns, leading to the release of inflammatory and toxic molecules, which can contribute to neuroinflammation associated with Parkinson's disease (PD). The aim of this study was to compare the potential of peripheral blood cells from PD patients or healthy subjects to produce cytokines after exposure to TLR agonists, and to investigate TLR2 and TLR4 expression on monocyte subsets. METHODS: Twenty-one patients and 21 healthy controls were recruited. Patients were evaluated according to the Unified Parkinson's Disease Rating Scale, and Hoehn and Yahr stage. Cytokines were measured in supernatants of whole blood cultures after incubation with TLR2, TLR4, or TLR7/8 agonists, by cytometric bead array. Expression of CD14, CD16, TLR2, and TLR4 was analyzed by cytometry. RESULTS: Patient blood cells produced lower levels of cytokines in response to TLR2 and also after TLR7/8/R848 activation than controls. Percentages of CD14+CD16+ or CD14+CD16- monocytes and TLR2 and TLR4 expression were similar between patients and controls. CONCLUSIONS: Blood leukocyte TLR2 and TLR7/8 responses are impaired in PD. This was neither associated with imbalance in monocyte subsets nor with TLR2/TLR4 expression on these cells. The association between a decreased TLR response in periphery and damage of brain in PD must be further investigated.

Miltefosine increases lipid and protein dynamics in Leishmania amazonensis membranes at concentrations similar to those needed for cytotoxicity activity.

Miltefosine (MT) is a membrane-active alkylphospholipid licensed for the topical treatment of breast cancer skin metastases and the oral treatment of leishmaniasis, although its mechanism of action remains unclear. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled lipid and a thiol-specific spin label in the plasma membrane of Leishmania promastigotes showed that MT causes dramatic increases in membrane dynamics. Although these alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Cell lysis was also detected by analyzing the supernatants of centrifuged samples for the presence of spin-labeled membrane fragments and cytoplasmic proteins. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment under the same range of MT concentrations that cause cell growth inhibition. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ∼70% cell death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. After this initial impact on the number of viable parasites, the processes of cell death and growth continued during the first 4 h of incubation. The EPR spectra of spin-labeled membrane-bound proteins were consistent with more expanded and solvent-exposed protein conformations, suggesting a detergent-like action. Thus, MT may form micelle-like structures around polypeptide chains, and proteins with a higher hydrophobicity may induce the penetration of hydrophilic groups of MT into the membrane, causing its rupture.

Interleukin 32γ (IL-32γ) is highly expressed in cutaneous and mucosal lesions of American Tegumentary Leishmaniasis patients: association with tumor necrosis factor (TNF) and IL-10.

BACKGROUND: The interleukin 32 (IL-32) is a proinflammatory cytokine produced by immune and non-immune cells. It can be induced during bacterial and viral infections, but its production was never investigated in protozoan infections. American Tegumentary Leishmaniasis (ATL) is caused by Leishmania protozoan leading to cutaneous, nasal or oral lesions. The aim of this study was to evaluate the expression of IL-32 in cutaneous and mucosal lesions as well as in peripheral blood mononuclear cells (PBMC) exposed to Leishmania (Viannia) braziliensis. METHODS: IL-32, tumour necrosis factor (TNF) and IL-10 protein expression was evaluated by immunohistochemistry in cutaneous, mucosal lesions and compared to healthy specimens. The isoforms of IL-32α, ß, δ, γ mRNA, TNF mRNA and IL-10 mRNA were assessed by qPCR in tissue biopsies of lesions and healthy skin and mucosa. In addition, PBMC from healthy donors were cultured with amastigotes of L. (V.) braziliensis. In lesions, the parasite subgenus was identified by PCR-RFLP. RESULTS: We showed that the mRNA expression of IL-32, in particular IL-32γ was similarly up-regulated in lesions of cutaneous (CL) or mucosal (ML) leishmaniasis patients. IL-32 protein was produced by epithelial, endothelial, mononuclear cells and giant cells. The IL-32 protein expression was associated with TNF in ML but not in CL. IL-32 was not associated with IL-10 in both CL and ML. Expression of TNF mRNA was higher in ML than in CL lesions, however levels of IL-10 mRNA were similar in both clinical forms. In all lesions in which the parasite was detected, L. (Viannia) subgenus was identified. Interestingly, L. (V.) braziliensis induced only IL-32γ mRNA expression in PBMC from healthy individuals. CONCLUSIONS: These data suggest that IL-32 plays a major role in the inflammatory process caused by L. (Viannia) sp or that IL-32 is crucial for controlling the L. (Viannia) sp infection.

Heterologous Booster with BNT162b2 Induced High Specific Antibody Levels in CoronaVac Vaccinees.

Immune responses after COVID-19 vaccination should be evaluated in different populations around the world. This study compared antibody responses induced by ChAdOx1 nCoV-19, CoronaVac, and BNT162b2 vaccines. Blood samples from vaccinees were collected pre- and post-vaccinations with the second and third doses. The study enrolled 78 vaccinees, of whom 62.8% were women, with the following median ages: 26 years-ChAdOx1 nCoV-19; 40 years-CoronaVac; 30 years-BNT162b2. Serum samples were quantified for anti-RBD IgG and anti-RBD IgA and anti-spike IgG by ELISA. After two vaccine doses, BNT162b2 vaccinees produced higher levels of anti-RBD IgA and IgG, and anti-spike IgG compared to ChAdOx1 nCoV-19 and CoronaVac vaccinees. The third dose booster with BNT162b2 induced higher levels of anti-RBD IgA and IgG, and anti-spike IgG in CoronaVac vaccinees. Individuals who reported a SARS-CoV-2 infection before or during the study had higher anti-RBD IgA and IgG production. In conclusion, two doses of the studied vaccines induced detectable levels of anti-RBD IgA and IgG and anti-spike IgG in vaccinees. The heterologous booster with BNT162b2 increased anti-RBD IgA and IgG and anti-spike IgG levels in CoronaVac vaccinees and anti-RBD IgA levels in ChAdOx1 nCoV-19 vaccinees. Furthermore, SARS-CoV-2 infection induced higher anti-RBD IgA and IgG levels in CoronaVac vaccinees.

Improvements in obtaining New World Leishmania sp from mucosal lesions: notes on isolating and stocking parasites.

Tegumentary leishmaniasis is an endemic protozoan disease that, in Brazil, is caused by parasites from Viannia or Leishmania complex. The clinical forms of cutaneous disease comprise localized, disseminated, mucosal or mucocutaneous, and diffuse leishmaniasis. Viannia complex parasites are not easy to isolate from patient lesions, especially from mucosal lesions, and they are difficult to culture. The aim of the present study was to compare the efficiency of ex vivo (culture) and in vivo (IFNγ-deficient mice) parasite isolation methods to improve the isolation rate and storage of stocks of New World Leishmania sp that cause cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). Biopsy fragments from cutaneous or mucosal lesions were inoculated into culture medium or mouse footpads. We evaluated 114 samples (86 CL, 28 ML) using both methods independently. Samples from CL patients had a higher isolation rate in ex vivo cultures than in mice (34.1% vs. 18.7%, P<0.05). Nevertheless, almost twice the number of isolates from ML lesions was isolated using the mouse model compared to ex vivo cultures (mouse, 6/25; culture, 3/27). The overall rates of isolation were 40.2% for CL samples and 29.6% for ML samples. Of the 43 isolations, we successfully stocked 35 isolates (81.4%; 27 CL, 8 ML). Contaminations were more frequently detected in cultures of ML than CL lesions. For comparison, the use of both methods simultaneously was performed in 74 samples of CL and 25 samples of ML, and similar results were obtained. Of the eight ML isolates, five were isolated only in mice, indicating the advantage of using the in vivo method to obtain ML parasites. All parasites obtained from in vivo isolation were cryopreserved, whereas only 68% of ex vivo isolations from CL lesions were stocked. In conclusion, the use of genetically modified mice can improve the isolation of parasites from ML. Isolation and stocking of New World Leishmania parasites, especially those from ML that are almost absent in laboratory stocks, are critical for evaluating parasite genetic diversity as well as studying host-parasite interactions to identify biological markers of Leishmania. In this paper, we also discuss some of the difficulties associated with isolating and stocking parasites.

Ivermectin and curcumin cause plasma membrane rigidity in Leishmania amazonensis due to oxidative stress.

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to study the mechanisms of action of ivermectin and curcumin against Leishmania (L.) amazonensis promastigotes. EPR spectra showed that treatment of the parasites with both compounds results in plasma membrane rigidity due to oxidative processes. With the IC50 and EPR measurements for assays using different parasite concentrations, estimations could be made for the membrane-water partition coefficient (KM/W), and the concentration of the compound in the membrane (cm50) and in the aqueous phase (cw50), which inhibits cell growth by 50%. The KM/W values indicated that ivermectin has a greater affinity than curcumin for the parasite membrane. Therefore, the activity of ivermectin was higher for experiments with low cell concentrations, but for concentrations greater than 1.5 × 108 parasites/mL the compounds did not show significantly different results. The cm50 values indicated that the concentration of compound in the membrane leading to growth inhibition or membrane alteration is approximately 1 M for both ivermectin and curcumin. This high membrane concentration suggests that many ivermectin molecules per chlorine channel are needed to cause an increase in chlorine ion influx.

Plasma membrane rigidity effects of 4-hydroxy-2-nonenal in Leishmania, erythrocyte and macrophage.

4-hydroxy-2-nonenal (HNE) is a reactive aldehyde produced by cells under conditions of oxidative stress, which has been shown to react with proteins and phosphatidylethanolamine in biological membranes. Using electron paramagnetic resonance (EPR) spectroscopy of a spin label it was demonstrated that 2 h of treatment with HNE causes membrane rigidity in promastigotes of Leishmania (L.) amazonensis, J774.A1 macrophages and erythrocytes. Remarkable fluidity-reducing effects on the parasite membrane were observed at HNE concentrations approximately 4-fold lower than in the case of erythrocyte and macrophage membranes. Autofluorescence of the parasites in PBS suspension (1 × 107 cell/mL) with excitation at 354 nm showed a linear increase of intensity in the range of 400 to 600 nm over 3 h after treatment with 30 µM HNE. Parasite ghosts prepared after this period of HNE treatment showed a high degree of membrane rigidity. Bovine serum albumin (BSA) in PBS treated with HNE for 2 h showed an increase in molecular dynamics and suffered a decrease in its ability to bind a lipid probe. In addition, the antiproliferative activity of L. amazonensis promastigotes, macrophage cytotoxicity and hemolytic potential were assessed for HNE. An IC50 of 24 µM was found, which was a concentration > 10 times lower than the cytotoxic and hemolytic concentrations of HNE. These results indicate that the action of HNE has high selectivity indices for the parasite as opposed to the macrophage and erythrocyte.

Gefitinib plus tremelimumab combination in refractory non-small cell lung cancer patients harbouring EGFR mutations: The GEFTREM phase I trial.

INTRODUCTION: A phase I open-label multicentre study was initiated to evaluate the association of tremelimumab with gefitinib in EGFR-mutant NSCLC patients who progressed after first-generation EGFR-TKI. Here we provide the efficacy data from the entire cohort. MATERIAL AND METHODS: Patients with advanced EGFR-mutant NSCLC with progression after response to EGFR-TKI were enrolled. Study treatment was gefitinib 250 mg daily and tremelimumab at 3 dose levels: 3, 6 and 10 mg/kg IV Q4W for 6 cycles followed by Q12W until progression or unacceptable toxicity. The primary objective was safety and tolerability, and to establish a RP2D. RESULTS: Between January 2014 and July 2015, 27 patients (21 in the escalating dose cohort and 6 in expansion cohort) received at least one dose of tremelimumab. DLTs occurred in 4 patients: 1 at 3 mg/kg (one grade 3 diarrhoea), 1 at 6 mg/kg (one grade 3 diarrhoea) and 2 at 10 mg/kg (one grade 3 diarrhoea and one grade 3 AST/ALT increase) of tremelimumab. Grade 3 TRAE occurred in 22 patients (81%), most frequently diarrhoea (30%) and ALT/AST increase (15%). Stable disease was the best overall response in 72% patients, with median PFS of 2.2 months (95% CI, 1.8-4.2). All patients discontinued treatment, most frequently due to disease progression (63% of patients). CONCLUSION: The recommended dose of tremelimumab in combination with gefitinib in EGFR-mutant NSCLC patients was 3 mg/kg. The gastrointestinal toxicity and the limited efficacy data prevented further evaluation of this combination. (GEFTREM; clinical trial number NCT02040064).

Clinical Utility and Outcomes Impact of Crystal Digital PCR of Sensitizing and Resistance EGFR Mutations in Patients With Advanced Non-Small Cell Lung Cancer.

BACKGROUND: EGFRm represent 15% of advanced NSCLC in European patients. LB for molecular profiling offers a non-invasive alternative to tissue. cdPCR is a high-sensitive and low-cost LB to detect molecular alterations. We aimed to describe cdPCR clinical utility for EGFRm detection in advanced NSCLC. METHODS: Prospective blood sample collection in patients with advanced NSCLC harbouring EGFRm either at diagnosis, under response or at PD between January 16 and September 20 at Gustave Roussy. LB was performed by cdPCR (Stilla): sensitizing (exon19; exon21 [p.L858R]) and exon 20 p.T790M resistance EGFRm. We defined high tumour burden (high-TB) as >2 metastatic sites. We analysed EGFRm detection by cdPCR and its correlation with progression-free and overall survival (PFS, OS). RESULTS: A total of 252 blood samples were collected in 140 patients. At baseline (n=25), sensitizing EGFRm were detected in 64% of samples, 88% in patients with high-TB (n=8) and 40% among those with intracranial/intrathoracic isolated lesions (n=5). At PD to tyrosine-kinase inhibitors (TKI) (n=117), detection rate (sensitizing EGFRm) was 56%; 30% in patients with intracranial/thoracic isolated lesions (n=37) vs. 67% in those with high-TB (n=63). At PD to first/second generation TKI (n=81), the p.T790M mutation was found in 22% (18/81); detection rate was 9% for intracranial/thoracic (n=23) vs. 32% for high-TB (n=41) cases. The clearance of EGFRm allelic frequency was correlated with radiological response. The absence of EGFRm detection at TKI-failure was associated with longer OS (39.1 vs. 18.4 months; P=.02). CONCLUSIONS: cdPCR is a sensitive LB for sensitizing and resistance EGFRm detection. cdPCR positivity was more likely observed in systemic PD cases with high-TB. It is a low-cost EGFRm detecting approach to guide treatment in NSCLC, however metastatic profile should be taken into account.

Mycoplasmal lipid-associated membrane proteins and Mycoplasma arthritidis mitogen recognition by serum antibodies from patients with rheumatoid arthritis.

Mycoplasmal lipid-associated membrane proteins (LAMPs) and Mycoplasma arthritidis mitogen (MAM superantigen) are potent stimulators of the immune system. The objective of this work was to detect antibodies to MAM and LAMPs of Mycoplasma hominis and M. fermentans in the sera of patients affected by rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) to identify mycoplasmal products that can be involved in the etiopathogenesis of these autoimmune diseases. Serum samples from female RA and SLE patients and controls, recombinant MAM, and LAMPs of M. hominis PG21 and M. fermentans PG18 were used in Western blot assays. A similar frequency of sera from patients and controls reactive to MAM was detected. A larger number of M. hominis and M. fermentans LAMPs were recognized by sera from RA patients than controls, but no differences were detected between sera from SLE patients and controls. Among the LAMPs recognized by IgG antibodies from RA patients, proteins of molecular masses in a range of <49 and ≥20 KDa (M. hominis) and <102 and ≥58 KDa (M. fermentans) were the most reactive. These preliminary results demonstrate the strong reactivity of antibodies of RA patients with some M. hominis and M. fermentans LAMPs. These LAMPs could be investigated as mycoplasmal antigens that can take part in the induction or amplification of human autoimmune responses.

Comparative EPR spectroscopy analysis of amphotericin B and miltefosine interactions with Leishmania, erythrocyte and macrophage membranes.

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to study the interactions of amphotericin B (AmB) with the plasma membrane of Leishmania (L.) amazonensis promastigotes, human erythrocytes and J774.A1 murine macrophages, in comparison with reported and novel data for miltefosine (MIL). One of the objectives of this work is to look for the relationships between the activities of these two drugs in the Leishmania parasite with their changes in the cell membrane. A spin-labeled stearic acid inserted into the cell membranes showed strong interactions with putative AmB/sterol complexes, characterized by reductions in molecular dynamics. The concentration of the drugs in the plasma membrane that reduced the cell population by 50%, and the membrane-water partition coefficient of the drugs, were assessed. These biophysical parameters enabled estimates of possible therapeutic concentrations of these two drugs in the interstitial fluids of the tissues to be made. AmB displayed higher affinity for the plasma membrane of L. amazonensis than for that of the macrophage and erythrocyte, denoting a preference for a membrane that contains ergosterol. AmB also demonstrated higher hemolytic potential than MIL for measurements on erythrocytes in both PBS and whole blood. For MIL, the EPR technique detected membrane changes induced by the drug in the same concentration range that inhibited the growth of parasites, but in the case of AmB, an 8-fold higher concentration of the IC50 was necessary to observe a reduction in membrane fluidity, suggesting a better localized effect of AmB on the membrane. Taken together, the results demonstrate that the antiproliferative and cytotoxic effects of both drugs are associated with changes in cell membranes.

New world Leishmania spp. infection in people living with HIV: Concerns about relapses and secondary prophylaxis.

Coinfection with the human immunodeficiency virus (HIV) and Leishmania impairs immune responses, increases treatment failure and relapse rates in patients with American tegumentary leishmaniasis (ATL), as well as visceral leishmaniasis (VL). There is insufficient data on the treatment, relapse, and secondary prophylaxis in patients coinfected with HIV/Leishmania in Brazil. This study investigated patients with HIV/ATL and HIV/VL to describe the outcome of leishmaniasis in patients assisted at a referral hospital of Brazilian midwestern region. Patients with HIV/ATL (n = 21) mainly presented cutaneous diseases (76.2%) with an overall relapse rate of 28.57% after treatment, whereas HIV/VL (n = 28) patients accounted for 17.5% of the cases. The counts of CD4+ T cells and CD8+ T cells and the CD4+/CD8+ cell ratios at diagnosis or relapses were not significantly different between relapsing and non-relapsing patients. Patients with HIV/ATL or HIV/VL showed high levels of activation markers in CD4+ and CD8+ T cells. The regular use of highly active antiretroviral therapy (HAART) and viral load at the time of diagnosis did not influence the relapse rates. Relapses occurred in 36.4% (4/11) of the patients with HIV/VL receiving secondary prophylaxis and in 5.9% (1/17) of the patients who did not receive secondary prophylaxis (p = 0.06). These data are relevant for the therapeutic management of the patients coinfected with HIV/Leishmania.

A Multicenter Analysis of the Outcome of Cancer Patients with Neutropenia and COVID-19 Optionally Treated with Granulocyte-Colony Stimulating Factor (G-CSF): A Comparative Analysis.

BACKGROUND: Approximately 15% of patients infected by SARS-CoV-2 develop a distress syndrome secondary to a host hyperinflammatory response induced by a cytokine storm. Myelosuppression is associated with a higher risk of infections and mortality. There are data to support methods of management for neutropenia and COVID-19. We present a multicenter experience during the first COVID-19 outbreak in neutropenic cancer patients infected by SARS-CoV-2. METHODS: Clinical retrospective data were collected from neutropenic cancer patients with COVID-19. Comorbidities, tumor type, stage, treatment, neutropenia severity, G-CSF, COVID-19 parameters, and mortality were analyzed. A bivariate analysis of the impact on mortality was carried out. Additionally, we performed a multivariable logistic regression to predict respiratory failure and death. RESULTS: Among the 943 cancer patients screened, 83 patients (11.3%) simultaneously had neutropenia and an infection with COVID-19. The lungs (26%) and breasts (22%) were the primary locations affected, and most patients had advanced disease (67%). In the logistic model, as adjusted covariates, sex, age, treatment (palliative vs. curative), tumor type, and the lowest level of neutrophils were used. A significant effect was obtained for the number of days of G-CSF treatment (OR = 1.4, 95% CI [1,1,03,92], p-value = 0.01). CONCLUSIONS: Our findings suggest that a prolonged G-CSF treatment could be disadvantageous for these cancer patients with infections by COVID-19, with a higher probability of worse outcome.

In vitro sensitivity of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Brazilian isolates to meglumine antimoniate and amphotericin B.

Resistance of Leishmania parasites to specific chemotherapy has become a well-documented problem in the Indian subcontinent in recent years but only a few studies have focused on the susceptibility of American Leishmania isolates. Our susceptibility assays to meglumine antimoniate were performed against intracellular amastigotes after standardizing an in vitro model of macrophage infection appropriate for Leishmania (Viannia) braziliensis isolates. For the determination of promastigote susceptibility to amphotericin B, we developed a simplified MTT-test. The sensitivity in vitro to meglumine antimoniate and amphotericin B of 13 isolates obtained from Brazilian patients was determined. L. (V.) braziliensis isolates were more susceptible to meglumine antimoniate than Leishmania (Leishmania) amazonensis. EC(50), EC(90) and activity indexes (calculated over the sensitivity of reference strains), suggested that all isolates tested were susceptible in vitro to meglumine antimoniate, and did not show association with the clinical outcomes. Isolates were also uniformly susceptible in vitro to amphotericin B.