Search

AbstractAimsWHO declared that mental health care should be considered one essential health service to be maintained during the coronavirus disease 2019 (COVID-19) pandemic. This study aims to describe the effect of lockdown and restrictions due to the COVID-19 pandemic in Italy on mental health services’ utilisation, by considering psychiatric diagnoses and type of mental health contacts.MethodsThe study was conducted in the Verona catchment area, located in the Veneto region (northeastern Italy). For each patient, mental health contacts were grouped into: (1) outpatient care, (2) social and supportive interventions, (3) rehabilitation interventions, (4) multi-professional assessments, (5) day care. A ‘difference in differences’ approach was used: difference in the number of contacts between 2019 and 2020 on the weeks of lockdown and intermediate restrictions was compared with the same difference in weeks of no or reduced restrictions, and such difference was interpreted as the effect of restrictions. Both a global regression on all contacts and separate regressions for each type of service were performed and Incidence Rate Ratios (IRRs) were calculated.ResultsIn 2020, a significant reduction in the number of patients who had mental health contacts was found, both overall and for most of the patients’ characteristics considered (except for people aged 18–24 years for foreign-born population and for those with a diagnosis of schizophrenia. Moreover, in 2020 mental health contacts had a reduction of 57 096 (−33.9%) with respect to 2019; such difference remained significant across the various type of contacts considered, with rehabilitation interventions and day care showing the greatest reduction. Negative Binomial regressions displayed a statistically significant effect of lockdown, but not of intermediate restrictions, in terms of reduction in the number of contacts. The lockdown period was responsible of a 32.7% reduction (IRR 0.673; p-value <0.001) in the overall number of contacts. All type of mental health contacts showed a reduction ascribable to the lockdown, except social and supportive interventions.ConclusionsDespite the access to community mental health care during the pandemic was overall reduced, the mental health system in the Verona catchment area was able to maintain support for more vulnerable and severely ill patients, by providing continuity of care and day-by-day support through social and supportive interventions.

AbstractMental disorders are one of the largest contributors to the burden of disease globally, this holds also for children and adolescents, especially in low- and middle-income countries. The prevalence and severity of these disorders are influenced by social determinants, including exposure to adversity. When occurring early in life, these latter events are referred to as adverse childhood experiences (ACEs).In this editorial, we provide an overview of the literature on the role of ACEs as social determinants of mental health through the lenses of global mental health. While the relation between ACEs and mental health has been extensively explored, most research was centred in higher income contexts. We argue that findings from the realm of global mental health should be integrated into that of ACEs, e.g. through preventative and responsive psychosocial interventions for children, adolescents and their caregivers. The field of global mental health should also undertake active efforts to better address ACEs in its initiatives, all with the goal of reducing the burden of mental disorders among children and adolescents globally.

The establishment of Leishmania in mammals depends on the transformation of metacyclic promastigotes into amastigotes within macrophages. The kinetics of this process was examined using mouse macrophages infected with metacyclic promastigotes of L. amazonensis. The appearance of amastigote characteristics, including large lysosome-like organelles called megasomes, stage-specific antigens, high cysteine protease activity and sensitivity to L-leucine methyl ester, was followed over a 5-day period. Megasomes were observed at 48h but probable precursors of these organelles were detected at 12h p.i. The promastigote-specific molecules examined were down-regulated within 5 to 12h after phagocytosis whereas the amastigote-specific antigens studied were detectable from 2 to 12–24h. An increase in the cysteine protease activity and in sensitivity to L-leucine methyl ester of the parasites was detected from 24h. The data indicate that at 48h p.i., parasites exhibit several amastigote features but that complete differentiation requires at least 5 days. The appearance of megasomes or of megasome precursors and the rise in cysteine protease activity correlate quite well with the capacity of parasites to internalize and very likely degrade host MHC molecules. The fact that internalization by the parasites of host cell molecules occurs very early during the differentiation process argues for a role of this mechanism in parasite survival.

In their amastigote stage, Leishmania are obligatory intracellular parasites of mammalian macrophages, residing and multiplying within phagolysosomal compartments called parasitophorous vacuoles (PV). These organelles have properties similar to those described for the MHC class II compartments of antigen-presenting cells, sites where peptide-class II molecule complexes are formed before their expression at the cell surface. After infection with Leishmania amazonensis or L. mexicana, endocytosis and degradation of class II molecules by intracellular amastigotes have also been described, suggesting that these parasites have evolved mechanisms to escape the potentially hazardous antigen-presentation process. To determine whether these events extend to other molecules of the antigen-presentation machinery, we have now studied the fate of the MHC molecule H-2M in mouse macrophages infected with Leishmania amastigotes. At least for certain class II alleles, H-2M is an essential cofactor, which catalyses the release of the invariant chain-derived CLIP peptide from the peptide-binding groove of class II molecules and facilitates the binding of antigenic peptides. H-2M was detected in PV of mouse macrophages infected with various Leishmania species including L. amazonensis, L. mexicana, L. major and L. donovani. PV thus contain all the molecules required for the formation of peptide-class II molecule complexes and especially of complexes with parasite peptides. The present data indicate, however, that if this process occurs, it does not lead to a clear increase of SDS-stable compact (alpha)(beta) dimers of class II. In PV that contained L. amazonensis or L. mexicana, both class II and H-2M molecules often colocalized at the level where amastigotes bind to the PV membrane, suggesting that these molecules are physically associated, directly or indirectly, and possibly interact with parasite components. Furthermore, as class II molecules, H-2M molecules were internalized by amastigotes of these Leishmania species and reached parasite compartments that also contained class II molecules. Immunostaining of H-2M within parasites was increased by treatment of infected macrophages with the cysteine protease inhibitors Z-Phe-AlaCHN2 or Z-Phe-PheCHN2 or by incubation of the parasites with the same inhibitors before infection. These data thus support the idea that amastigotes of certain Leishmania species capture and degrade some of the molecules required for antigen presentation. To examine whether endocytosis of class II molecules by the parasites occurs through interactions with parasite components involving their peptide-binding groove, we made use of the fact that a large fraction of the class II molecules of H-2M(alpha) knock-out H-2(b) mice are occupied by the peptide CLIP and are unable to bind other peptides. We found that, in Leishmania-infected macrophages of these mutant mice, class II-CLIP complexes reached PV and were internalized by amastigotes. These results thus prove that endocytosis of class II molecules by amastigotes (1) is H-2M-independent and (2) does not necessarily involve the peptide-binding pocket of these molecules. Altogether, these data are compatible with an endocytic mechanism based on general properties shared by classical and non-classical class II molecules.

In their amastigote stage, Leishmania live in mammalian macrophages within parasitophorous vacuoles (PV), organelles of phagolysosomal origin that, in macrophages activated with IFN-gamma, contain major histocompatibility complex (MHC) class II molecules apparently devoid of invariant chains. We have now studied the fate of PV-associated class II molecules in mouse bone marrow-derived macrophages infected with L. amazonensis amastigotes using immunocytochemical and biochemical approaches. We have found that at least a part of these class II molecules was internalized by amastigotes and reached structures very often located in their posterior poles. This process was much more obvious if infected macrophages were incubated with protease inhibitors like antipain, chymostatin, Z-Phe-AlaCHN2 and Z-Phe-PheCHN2, or if amastigotes were pre-treated with the irreversible cysteine protease inhibitor Z-Phe-AlaCHN2 before infection, clearly indicating that amastigotes also degraded the internalized class II molecules. Study of infected macrophage cryosections by immuno-electron microscopy allowed the identification of the class II-positive structures in amastigotes as the lysosome-like organelles known as megasomes. Other PV membrane components like the prelysosomal/lysosomal glycoproteins Igp110, Igp120 and macrosialin could not be detected in megasomes of amastigotes even after treatment of macrophages with protease inhibitors, suggesting the involvement of some specific mechanism(s) for the internalization of class II molecules. Interestingly, after treatment of infected macrophages with various protease inhibitors (antipain, leupeptin, E-64, Z-Phe-AlaCHN2, Z-Phe-PheCHN2), PV membrane as well as megasomes of amastigotes become positive for invariant chains. A quantitative analysis of amastigote-associated class II molecules based on enzyme immunoassays showed that: (a) amastigotes extracted from macrophages treated with both IFN-gamma and antipain or Z-Phe-AlaCHN2 contained a much greater amount of class II than amastigotes extracted from macrophages treated with IFN-gamma alone; (b) class II molecules associated with the former were mainly intracellular and, at least some of them, were complexed with invariant chains or fragments of invariant chains; (c) amastigotes pre-incubated with Z-Phe-AlaCHN2 before infection accumulated a greater amount of intracellular class II than amastigotes pre-incubated without inhibitor, clearly indicating that the blockade of parasite cysteine proteases was sufficient to enhance the pool of these molecules within megasomes. On the whole, these data are consistent with the idea that class II molecules reaching PV are newly synthesized and still complexed with intact invariant chains or with partially degraded invariant chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Leishmania-infected macrophages are potential antigen-presenting cells for CD4+ T lymphocytes, which recognize parasite antigens bound to major histocompatibility complex class II molecules (Ia). However, the intracellular sites where Ia and antigens may interact are far from clear, since parasites grow within the modified lysosomal compartment of the host cell, whereas Ia molecules seem to be targeted to endosomes. To address this question, the expression and fate of Ia molecules were studied by immunocytochemistry in Leishmania amazonensis-infected murine macrophages stimulated with gamma interferon. In uninfected macrophages, Ia molecules were localized on the plasma membrane and in perinuclear vesicles, but they underwent a dramatic redistribution after infection, since most of the intracellular staining was then associated with the periphery of the parasitophorous vacuoles (p.v.) and quite often polarized towards amastigote-binding sites. The Ii invariant chain, which is transiently associated with Ia during their intracellular transport, although well expressed in infected macrophages, apparently did not reach the p.v. Similar findings were observed with macrophages from mice either resistant or highly susceptible to Leishmania infection. In order to determine the origin of p.v.-associated Ia, the fate of plasma membrane, endosomal, and lysosomal markers, detected with specific antibodies, was determined after infection. At 48 h after infection, p.v. was found to exhibit a membrane composition typical of mature lysosomes. Overall, these data suggest that (i) Ia located in p.v. originate from secondary lysosomes involved in the biogenesis of this compartment or circulate in several endocytic organelles, including lysosomes and (ii) p.v. could play a role in antigen processing and presentation. Alternatively, the presence of high amounts of Ia in p.v. could be due to a Leishmania-induced mechanism by means of which this organism may evade the immune response.

In mammalian hosts, Leishmania amastigotes are obligatory intracellular parasites of macrophages and multiply within parasitophorous vacuoles of phagolysosomal origin. To understand how they escape the harmful strategies developed by macrophages to kill ingested microorganisms, it is important to obtain information on the functional state of parasitophorous vacuole. For this purpose, we studied the intracellular distribution and activity of host lysosomal proteases in rat bone marrow-derived macrophages infected with Leishmania amazonensis amastigotes. Localization of cathepsins B, H, L, and D was investigated by using specific immunoglobulins. In uninfected macrophages, these enzymes were located in perinuclear granules (most of them were probably secondary lysosomes) which, after infection, disappeared progressively. In infected macrophages, cathepsins were detected mainly in the parasitophorous vacuoles, suggesting that the missing secondary lysosomes had fused with these organelles. Biochemical assays of various proteases (cathepsins B, H, and D and dipeptidyl peptidases I and II) showed that infection was accompanied by a progressive increase of all activities tested, except that of dipeptidyl peptidase II, which remained constant. No more than 1 to 10% of these activities could be attributed to amastigotes. These data indicate that (i) Leishmania infection is followed by an increased synthesis and/or a reduced catabolism of host lysosomal proteases, and (ii) amastigotes grow in a compartment rich in apparently fully active proteases. Unexpectedly, it was found that infected and uninfected macrophages degraded endocytosed proteins similarly. The lack of correlation in infected macrophages between increase of protease activities and catabolism of exogenous proteins could be linked to the huge increase in volume of the lysosomal compartment.

Leishmania amastigotes are intracellular protozoan parasites of mononuclear phagocytes which reside within parasitophorous vacuoles of phagolysosomal origin. The pH of these compartments was studied with the aim of elucidating strategies used by these microorganisms to evade the microbicidal mechanisms of their host cells. For this purpose, rat bone marrow-derived macrophages were infected with L. amazonensis amastigotes. Intracellular acidic compartments were localized by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine as a probe. This indicator, which can be detected by light microscopy by using immunocytochemical methods, mainly accumulated in perinuclear lysosomes of uninfected cells, whereas in infected cells, it was essentially localized in parasitophorous vacuoles, which thus appeared acidified. Phagolysosomal pH was estimated quantitatively in living cells loaded with the pH-sensitive endocytic tracer fluoresceinated dextran. After a 15- to 20-h exposure, the tracer was mainly detected in perinuclear lysosomes and parasitophorous vacuoles of uninfected and infected macrophages, respectively. Fluorescence intensities were determined from digitized video images of single cells after processing and automatic subtraction of background. We found statistically different mean pH values of 5.17 to 5.48 for lysosomes and 4.74 to 5.26 for parasitophorous vacuoles. As for lysosomes of monensin-treated cells, the pH gradient of parasitophorous vacuoles collapsed after monensin was added. This very likely indicates that these vacuoles maintain an acidic internal pH by an active process. These results show that L. amazonensis amastigotes are acidophilic and opportunistic organisms and suggest that these intracellular parasites have evolved means for survival under these harsh conditions and have acquired plasma membrane components compatible with the environment.

The natural resistance-associated macrophage protein (Nramp1) regulates macrophage activation. One of its pleiotropic effects on macrophage function is to regulate expression of major histocompatibility class II molecules. In this study macrophages stably transfected with the wild-type (infection-resistant) or the natural mutant (infection-susceptible) allele of the Nramp1 gene were used to study class II expression and processing and presentation of recombinant protein antigens to CD4+ T-cell hybridomas. As demonstrated previously for macrophages from Nramp1-resistant and -susceptible congenic mouse strains, transfected macrophage clones carrying the wild-type allele showed enhanced upregulation of class II molecules in response to gamma interferon compared to that shown by macrophage clones carrying an endogenous mutant allele or transfected with the mutant allele expressed under a viral long terminal repeat promoter. The wild-type allele-transfected macrophage clones also demonstrated an enhanced, lipopolysaccharide-dependent ability to process the recombinant leishmanial antigen LACK-delta 1 (the Leishmania homolog of receptors for activated C kinase) for presentation to LACK-specific CD4+ T cells. An influence on antigen processing must therefore be added to the growing list of pleiotropic effects of the Nramp1 gene potentially contributing to its role in infectious and autoimmune disease susceptibility. These results also have important implications for analysis of T-cell responses to vaccination, especially where antigens are presented to the immune system using live Salmonella species or Mycobacterium bovis BCG as a vaccine vehicle.

The natural resistance-associated macrophage protein (Nramp1) regulates macrophage activation. One of its pleiotropic effects on macrophage function is to regulate expression of major histocompatibility class II molecules. In this study macrophages stably transfected with the wild-type (infection-resistant) or the natural mutant (infection-susceptible) allele of the Nramp1 gene were used to study class II expression and processing and presentation of recombinant protein antigens to CD4+ T-cell hybridomas. As demonstrated previously for macrophages from Nramp1-resistant and -susceptible congenic mouse strains, transfected macrophage clones carrying the wild-type allele showed enhanced upregulation of class II molecules in response to gamma interferon compared to that shown by macrophage clones carrying an endogenous mutant allele or transfected with the mutant allele expressed under a viral long terminal repeat promoter. The wild-type allele-transfected macrophage clones also demonstrated an enhanced, lipopolysaccharide-dependent ability to process the recombinant leishmanial antigen LACK-delta 1 (the Leishmania homolog of receptors for activated C kinase) for presentation to LACK-specific CD4+ T cells. An influence on antigen processing must therefore be added to the growing list of pleiotropic effects of the Nramp1 gene potentially contributing to its role in infectious and autoimmune disease susceptibility. These results also have important implications for analysis of T-cell responses to vaccination, especially where antigens are presented to the immune system using live Salmonella species or Mycobacterium bovis BCG as a vaccine vehicle.