Impact of collaborative actions of Leishmania (Viannia) braziliensis subpopulations on the infection profile.

This study focuses on the role of the population structure of Leishmania spp. on the adaptive capacity of the parasite. Herein, we investigate the contribution of subpopulations of the L. (V.) braziliensis Thor strain (Thor03, Thor10 and Thor22) in the profile of murine macrophages infection. Infection assays were performed with binary combinations of these subpopulations at stationary phases. The initial interaction time showed major effects on the combination assays, as demonstrated by the significant increase in the infection rate at 5 h. Based on the endocytic index (EI), Thor10 (EI = 563.6) and Thor03 (EI = 497) showed a higher infection load compared to Thor22 (EI = 227.3). However, the EI decreased in Thor03 after 48 h (EI = 447) and 72 h (EI = 388.3) of infection, and showed changes in the infection level in all Thor10/Thor22 combinations. Assays with CellTrace CFSE-labelled Thor22 promastigotes indicated an increase (~1.5 fold) in infection by this subpopulation in the presence of Thor10 when compared to the infection profile of Thor03/Thor22 combinations in the same proportions. In addition, the potential of these subpopulations, alone or in binary combinations, to modulate the expression of cytokines and nitric oxide (NO) in vitro was investigated. Lower NO and tumour necrosis factor-α production levels were observed for all Thor10/Thor22 combinations at 24 h compared to these subpopulations alone. In contrast, Thor03/Thor22 combination assays increased IL-10 production at this time. Collectively, these results provide in vitro evidence on the potential of L. (V.) braziliensis population structure to play a relevant role in a host infection by this parasite.

In the Acute Phase of Trypanosoma cruzi Infection, Liver Lymphoid and Myeloid Cells Display an Ambiguous Phenotype Combining Pro- and Anti-Inflammatory Markers.

Multiple cell populations, cellular biochemical pathways, and the autonomic nervous system contribute to maintaining the immunological tolerance in the liver. This tolerance is coherent because the organ is exposed to high levels of bacterial pathogen-associated molecular pattern (PAMP) molecules from the intestinal microbiota, such as lipopolysaccharide endotoxin (LPS). In the case of Trypanosoma cruzi infection, although there is a dramatic acute immune response in the liver, we observed intrahepatic cell populations combining pro- and anti-inflammatory markers. There was loss of fully mature Kupffer cells and an increase in other myeloid cells, which are likely to include monocytes. Among dendritic cells (DCs), the cDC1 population expanded relative to the others, and these cells lost both some macrophage markers (F4/80) and immunosuppressive cytokines (IL-10, TGF-ß1). In parallel, a massive T cell response occured with loss of naïve cells and increase in several post-activation subsets. However, these activated T cells expressed both markers programmed cell death protein (PD-1) and cytokines consistent with immunosuppressive function (IL-10, TGF-ß1). NK and NK-T cells broadly followed the pattern of T cell activation, while TCR-γδ cells appeared to be bystanders. While no data were obtained concerning IL-2, several cell populations also synthesized IFN-γ and TNF-α, which has been linked to host defense but also to tissue injury. It therefore appears that T. cruzi exerts control over liver immunity, causing T cell activation via cDC1 but subverting multiple populations of T cells into immunosuppressive pathways. In this way, T. cruzi engages a mechanism of hepatic T cell tolerance that is familiar from liver allograft tolerance, in which activation and proliferation are followed by T cell inactivation.

Experimental Combination Therapy with Amiodarone and Low-Dose Benznidazole in a Mouse Model of Trypanosoma cruzi Acute Infection.

Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately 6 to 7 million people in Latin America, with cardiomyopathy being the clinical manifestation most commonly associated with patient death during the acute phase. The etiological treatment of CD is restricted to benznidazole (Bz) and nifurtimox (Nif), which involve long periods of administration, frequent side effects, and low efficacy in the chronic phase. Thus, combined therapies emerge as an important tool in the treatment of CD, allowing the reduction of Bz dose and treatment duration. In this sense, amiodarone (AMD), the most efficient antiarrhythmic drug currently available and prescribed to CD patients, is a potential candidate for combined treatment due to its known trypanocidal activity. However, the efficacy of AMD during the acute phase of CD and its interaction with Bz or Nif are still unknown. In the present study, using a well-established murine model of the acute phase of CD, we observed that the Bz/AMD combination was more effective in reducing the peak parasitemia than both monotherapy treatments. Additionally, the Bz/AMD combination reduced (i) interleukin-6 (IL-6) levels in cardiac tissue, (ii) P-wave duration, and (iii) frequency of arrhythmia in infected animals and (iv) restored gap junction integrity in cardiac tissue. Therefore, our study validates AMD as a promising candidate for combined therapy with Bz, reinforcing the strategy of combined therapy for CD. IMPORTANCE Chagas disease affects approximately 6 to 7 million people worldwide, with cardiomyopathy being the clinical manifestation that most commonly leads to patient death. The etiological treatment of Chagas disease is limited to drugs (benznidazole and nifurtimox) with relatively high toxicity and therapeutic failures. In this sense, amiodarone, the most effective currently available antiarrhythmic drug prescribed to patients with Chagas disease, is a potential candidate for combined treatment due to its known trypanocidal effect. In the present study, we show that combined treatment with benznidazole and amiodarone improves the trypanocidal effect and reduces cardiac damage in acutely T. cruzi-infected mice.

Serum 1,3-beta-D-glucan as a noninvasive test to predict histologic activity in patients with inflammatory bowel disease.

BACKGROUND: 1,3-beta-D-glucan (BG) is a ubiquitous cell wall component of gut micro-organisms. We hypothesized that the serum levels of BG could reflect active intestinal inflammation in patients with inflammatory bowel disease. AIM: To determine whether the serum BG concentrations correlate with intestinal inflammation. METHODS: A prospective observational study was performed in a tertiary referral center, from 2016 to 2019, in which serum BG was determined in 115 patients with Crohn's disease (CD), 51 with ulcerative colitis (UC), and 82 controls using a photometric detection kit. Inflammatory activity was determined by ileocolonoscopy, histopathology, magnetic resonance enterography, and biomarkers, including fecal calprotectin (FC), C-reactive protein, and a panel of cytokines. The ability of BG to detect active vs inactive disease was assessed using the area under the receiver operating characteristic curve. In subgroup analysis, serial BG was used to assess the response to therapeutic interventions. RESULTS: The serum BG levels were higher in CD patients than in controls (P = 0.0001). The BG levels paralleled the endoscopic activity in CD patients and histologic activity and combined endoscopic and histologic activity in both CD and UC patients. The area under the curve (AUC) in receiver operating characteristic analysis to predict endoscopic activity was 0.694 [95% confidence interval (CI): 0.60-0.79; P = 0.001] in CD, and 0.662 (95%CI: 0.51-0.81; P = 0.066) in UC patients. The AUC in receiver operating characteristic analysis to predict histologic activity was 0.860 (95%CI: 0.77-0.95; P < 0.001) in CD, and 0.786 (95%CI: 0.57-0.99; P = 0.015) in UC patients. The cut-off values of BG for both endoscopic and histologic activity were 60 µg/mL in CD, and 40 µg/mL in UC patients. Performance analysis showed that the results based on BG of 40 and 60 µg/mL were more specific for predicting endoscopic activity (71.8% and 87.2% for CD; and 87.5% and 87.5% for UC, respectively) than FC (53.3% and 66.7% for CD; and 20% and 80% for UC, respectively); and also histologic activity (60.5% and 76.3% for CD; and 90.0% and 95.0% for UC, respectively) than FC (41.7% and 50.0% for CD; and 25% and 50% for UC, respectively). Regarding the clinical, endoscopic, and histologic activities, the BG levels were reduced following therapeutic intervention in patients with CD (P < 0.0001) and UC (P = 0.003). Compared with endoscopic (AUC: 0.693; P = 0.002) and histologic (AUC: 0.868; P < 0.001) activity, no significant correlation was found between serum BG and transmural healing based on magnetic resonance enterography (AUC: 0.576; P = 0.192). Positive correlations were detected between BG and IL-17 in the CD (r: 0.737; P = 0.001) and the UC group (r: 0.574; P = 0.005), and between BG and interferon-gamma in the CD group (r: 0.597; P = 0.015). CONCLUSION: Serum BG may represent an important novel noninvasive approach for detecting mucosal inflammation and therapeutically monitoring inflammatory bowel diseases, particularly in CD.

After Experimental Trypanosoma cruzi Infection, Dying Hepatic CD3+TCRαß+B220+ T Lymphocytes Are Rescued from Death by Peripheral T Cells and Become Activated.

The unusual phenotype of CD3+ T lymphocyte expressing B220, a marker originally attributed to B lymphocytes, was first observed in the liver of Fas/Fas-L-deficient mice as a marker of apoptotic T lymphocytes. However, other CD3+B220+ T lymphocyte populations were later described in the periphery as functional cytotoxic or regulatory cells, for example. Then, in this work, we studied whether hepatic CD3+B220+ T lymphocytes could play a role in experimental Trypanosoma cruzi infection. In control and infected mice, we observed two subpopulations that could be discerned based on CD117 expression, which were conventional apoptotic CD3+B220+(CD117-) and thymus-independent CD3+B220+CD117+ T lymphocytes. Regardless of CD117 expression, most B220+ T lymphocytes were 7AAD+, confirming this molecule as a marker of dying T cells. However, after infection, we found that around 15% of the CD3+B220+CD117+ hepatic population became B220 and 7AAD negative, turned into CD90.2+, and upregulated the expression of CD44, CD49d, and CD11a, a phenotype consistent with activated T lymphocytes. Moreover, we observed that the hepatic CD3+B220+CD117+ population was rescued from death by previously activated peripheral T lymphocytes. Our results extend the comprehension of the hepatic CD3+B220+ T lymphocyte subpopulations and illustrate the complex interactions that occur in the liver.

Induced Pluripotent Stem Cells: Hope in the Treatment of Diseases, including Muscular Dystrophies.

Induced pluripotent stem (iPS) cells are laboratory-produced cells that combine the biological advantages of somatic adult and stem cells for cell-based therapy. The reprogramming of cells, such as fibroblasts, to an embryonic stem cell-like state is done by the ectopic expression of transcription factors responsible for generating embryonic stem cell properties. These primary factors are octamer-binding transcription factor 4 (Oct3/4), sex-determining region Y-box 2 (Sox2), Krüppel-like factor 4 (Klf4), and the proto-oncogene protein homolog of avian myelocytomatosis (c-Myc). The somatic cells can be easily obtained from the patient who will be subjected to cellular therapy and be reprogrammed to acquire the necessary high plasticity of embryonic stem cells. These cells have no ethical limitations involved, as in the case of embryonic stem cells, and display minimal immunological rejection risks after transplant. Currently, several clinical trials are in progress, most of them in phase I or II. Still, some inherent risks, such as chromosomal instability, insertional tumors, and teratoma formation, must be overcome to reach full clinical translation. However, with the clinical trials and extensive basic research studying the biology of these cells, a promising future for human cell-based therapies using iPS cells seems to be increasingly clear and close.

In the presence of Trypanosoma cruzi antigens, activated peripheral T lymphocytes retained in the liver induce a proinflammatory phenotypic and functional shift in intrahepatic T lymphocyte.

In secondary lymphoid organs, pathogen-derived and endogenous danger molecules are recognized by pattern recognition receptors, leading to adaptive proinflammatory immune responses. This conceptual rule does not apply directly to the liver, as hepatic immune cells tolerate gut-derived bacterial molecules from the flora. Therefore, the recognition of danger and proinflammatory stimuli differs between the periphery and the liver. However, the tolerant nature of the liver must be overcome in the case of infections or cancer, for example. The central paradigm is the basis for danger recognition and the balance between inflammation and tolerance in the liver. Here, we observed functional integration, with activated peripheral T lymphocytes playing a role in the induction of a proinflammatory environment in the liver in the presence of Trypanosoma cruzi antigens. When only parasite extract was orally administered, it led to the up-regulation of hepatic tolerance markers, but oral treatment plus adoptively transferred activated splenic T lymphocytes led to a proinflammatory response. Moreover, treated/recipient mice showed increased levels of TNF, IFN-γ, IL-6, and CCL2 in the liver and increased numbers of effector and/or effector memory T lymphocytes and F4/80+ cells. There was a reduction in FoxP3+ Treg cells, NKT cells, and γδ T lymphocytes with increased liver damage in the presence of activated peripheral T cells. Our results show that the induction of a proinflammatory liver response against T. cruzi danger molecules is at least partially dependent on cooperation with activated peripheral T cells.

Chloroquine and Sulfadoxine Derivatives Inhibit ZIKV Replication in Cervical Cells.

Despite the severe morbidity caused by Zika fever, its specific treatment is still a challenge for public health. Several research groups have investigated the drug repurposing of chloroquine. However, the highly toxic side effect induced by chloroquine paves the way for the improvement of this drug for use in Zika fever clinics. Our aim is to evaluate the anti-Zika virus (ZIKV) effect of hybrid compounds derived from chloroquine and sulfadoxine antimalarial drugs. The antiviral activity of hybrid compounds (C-Sd1 to C-Sd7) was assessed in an in-vitro model of human cervical and Vero cell lines infected with a Brazilian (BR) ZIKV strain. First, we evaluated the cytotoxic effect on cultures treated with up to 200 µM of C-Sds and observed CC50 values that ranged from 112.0 ± 1.8 to >200 µM in cervical cells and 43.2 ± 0.4 to 143.0 ± 1.3 µM in Vero cells. Then, the cultures were ZIKV-infected and treated with up to 25 µM of C-Sds for 48 h. The treatment of cervical cells with C-Sds at 12 µM induced a reduction of 79.8% ± 4.2% to 90.7% ± 1.5% of ZIKV-envelope glycoprotein expression in infected cells as compared to 36.8% ± 2.9% of infection in vehicle control. The viral load was also investigated and revealed a reduction of 2- to 3-logs of ZIKV genome copies/mL in culture supernatants compared to 6.7 ± 0.7 × 108 copies/mL in vehicle control. The dose-response curve by plaque-forming reduction (PFR) in cervical cells revealed a potent dose-dependent activity of C-Sds in inhibiting ZIKV replication, with PFR above 50% and 90% at 6 and 12 µM, respectively, while 25 µM inhibited 100% of viral progeny. The treatment of Vero cells at 12 µM led to 100% PFR, confirming the C-Sds activity in another cell type. Regarding effective concentration in cervical cells, the EC50 values ranged from 3.2 ± 0.1 to 5.0 ± 0.2 µM, and the EC90 values ranged from 7.2 ± 0.1 to 11.6 ± 0.1 µM, with selectivity index above 40 for most C-Sds, showing a good therapeutic window. Here, our aim is to investigate the anti-ZIKV activity of new hybrid compounds that show highly potent efficacy as inhibitors of ZIKV in-vitro infection. However, further studies will be needed to investigate whether these new chemical structures can lead to the improvement of chloroquine antiviral activity.

Impact of autologous whole blood administration upon experimental mouse models of acute Trypanosoma cruzi infection

Background: Autologous whole blood (AWB) administration is described as alternative/complementary medical practice widely employed in medical and veterinary therapy against infections, chronic pathologies and neoplasias. Our aim is to investigate in vivo biological effect of AWB using healthy murine models under the course of Trypanosoma cruzi acute infection. Methods: The first set of studies consisted of injecting different volumes of AWB and saline (SAL) into the posterior region of quadriceps muscle of healthy male Swiss mice under distinct therapeutic schemes evaluating: animal behavior, body and organ weight, hemogram, plasmatic biochemical markers for tissue damage and inflammatory cytokine levels and profile. To assess the impact on the experimental T. cruzi infection, different schemes (prior and post infection) and periods of AWB administration (from one up to 10 days) were conducted, also employing heterologous whole blood (HWB) and evaluating plasma cytokine profile. Results: No major adverse events were observed in healthy AWB-treated mice, except gait impairment in animals that received three doses of 20 L AWB in the same hind limb. AWB and SAL triggered an immediate polymorphonuclear response followed by mononuclear infiltrate. Although SAL triggered an inflammatory response, the kinetics and intensity of the histological profile and humoral mediator levels were different from AWB, the latter occurring earlier and more intensely with concomitant elevation of plasma IL-6. Inflammatory peak response of SAL, mainly composed of mononuclear cells with IL-10, was increased at 24 h. According to the mouse model of acute T. cruzi infection, only minor decreases ( 30%) in the parasitemia levels were produced by AWB and HWB given before and after infection, without protecting against mortality. Rises in IFN-gamma, TNF-alpha and...

Impact of autologous whole blood administration upon experimental mouse models of acute Trypanosoma cruzi infection

Autologous whole blood (AWB) administration is described as alternative/complementary medical practice widely employed in medical and veterinary therapy against infections, chronic pathologies and neoplasias. Our aim is to investigate in vivo biological effect of AWB using healthy murine models under the course of Trypanosoma cruzi acute infection. Methods: The first set of studies consisted of injecting different volumes of AWB and saline (SAL) into the posterior region of quadriceps muscle of healthy male Swiss mice under distinct therapeutic schemes evaluating: animal behavior, body and organ weight, hemogram, plasmatic biochemical markers for tissue damage and inflammatory cytokine levels and profile. To assess the impact on the experimental T. cruzi infection, different schemes (prior and post infection) and periods of AWB administration (from one up to 10 days) were conducted, also employing heterologous whole blood (HWB) and evaluating plasma cytokine profile. Results: No major adverse events were observed in healthy AWB-treated mice, except gait impairment in animals that received three doses of 20 µL AWB in the same hind limb. AWB and SAL triggered an immediate polymorphonuclear response followed by mononuclear infiltrate. Although SAL triggered an inflammatory response, the kinetics and intensity of the histological profile and humoral mediator levels were different from AWB, the latter occurring earlier and more intensely with concomitant elevation of plasma IL-6. Inflammatory peak response of SAL, mainly composed of mononuclear cells with IL-10, was increased at 24 h. According to the mouse model of acute T. cruzi infection, only minor decreases (< 30%) in the parasitemia levels were produced by AWB and HWB given before and after infection, without protecting against mortality. Rises in IFN-gamma, TNF-alpha and IL-6 were detected at 9 dpi in all infected animals as compared to uninfected mice but only Bz displayed a statistically significant diminution (p= 0.02) in TNF-alpha levels than infected and untreated mice. Conclusions: This study revealed that the use of autologous whole blood (AWB) in the acute model employed was unable to reduce the parasitic load of infected mice, providing only a minor decrease in parasitemia levels (up to 30%) but without protecting against animal mortality. Further in vivo studies will be necessary to elucidate the effective impact of this procedure.(AU)

Chemotaxis and Immunoregulatory Function of Cardiac γδ T Cells in Dystrophin-Deficient Mice.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by mutations in the dystrophin gene that lead to degeneration of skeletal and cardiac muscles and to chronic inflammation. Despite the importance of γδ T cells in many diseases, this cellular subpopulation has not been described in DMD patients or in mdx mice, a widely used mouse model for studying DMD. Therefore, in this study, we aimed to evaluate the migration of γδ T cells to the cardiac muscle of mdx mice and to characterize their phenotype and functional activity. We observed no migration of γδ T cells to skeletal muscles, but these cells were found in the hearts of mdx mice during the study period, reaching a peak in 12-wk-old mice. These cells migrate primarily owing to CCL2 and CCL5 chemokines produced by cardiac tissue, and they are Vγ1+/CD27+ and thus produce high levels of IFN-γ. In vivo depletion of the γδ T cells revealed γδ T cell-dependent cardiac inflammatory immunoregulation, with increased numbers of CD3+CD4+, CD3+CD8+, and, in particular, F4/80+ cells in the heart and increased cardiac damage in mdx mice. We also observed in vitro that purified cardiac Γδ T cells are cytotoxic against adherent endomysial cardiac cells, mostly macrophages, but not against peritoneal cells, in a perforin/granzyme-dependent manner. Our present data indicate that γδ T cells exert protective effects on the hearts of mdx mice, possibly by selectively killing pathogenic macrophages, and this function may be important for the late onset of cardiac damage in DMD.

Caracterização do ambiente inflamatório cardíaco e estudo de interações células T/endotélio em camundongos mdx/mdx / Characterization of cardiac inflammatory environment interactions and study T cell / endothelium in mice mdx / mdx

A Distrofia muscular de Duchenne (DMD) é uma doença recessiva ligada ao cromossomo X que afeta 1 a cada 3500 meninos nascidos vivos e é causada por mutações no gene da distrofina. A ausência da distrofina leva à degeneração progressiva dos músculos esqueléticos e cardíaco assim como à inflamação crônica. O camundongo mdx/mdx é um dos modelos mais utilizados para estudo da DMD e apresenta muitas características da doença, embora a progressão da patologia seja mais branda e não letal. Este trabalho visa avaliar a migração de células T para o coração de camundongos mdx/mdx e possíveis alterações na expressão de moléculas de adesão que possam modular esse processo. Leucócitos sanguíneos, incluindo células T de camundongos mdx/mdx de 6 semanas de idade, são CD62L (mais) , mas sofrem uma modulação negativa nos animais de 12 semanas, com apenas 40(por cento) dos linfócitos T mantendo a expressão dessa molécula. Nossos resultados apontam para uma clivagem de CD62L dependente de P2X7 (com altos níveis de CD62L no soro) que reduz a competência dos linfócitos T sanguíneos de camundongos mdx/mdx de 12 semanas aderirem aos vasos sanguíneos cardíacos in vitro. In vivo, nós observamos que mesmo após a infecção com Trypanosoma cruzi, um conhecido indutor de miocardite linfóide, essas células raramente são encontradas no co ração. Quando camundongos mdx/mdx são tratados com BBG, um bloqueador de P2X7, esses linfócitos sanguíneos mantém a expressão de CD62L e são capazes de migrar para o coração. Esses resultados fornecem novas informações sobre os mecanismos de infiltração in flamatória e regulação imune na DMD.

Caracterização de populações inflamatórias cardíacas e avaliação de interações leucócitos/endotélio na distrofia muscular tipo Duchenne / Characterization of cardiac inflammatory populations and evaluation of interactions leukocytes/endothelium evaluation in the Duchenne type muscular distrophy

Distrofia muscular do tipo Duchenne (DMD) é uma miopatia inflamatória recessiva fatal ligada ao cromossomo X (locus Xp21) que afeta 1 a cada 3500 meninos nascidos vivos. A maioria dos pacientes apresenta deleção e/ou ampla mutação no gene dmd, o que determina a não expressão ou a expressão de uma molécula truncada não funcional da isoforma 427kDa da proteína distrofina nas células musculares esqueléticas e outros tipos celulares. As alterações clínicas são evidentes ainda na primeira infância, nas crianças com quatro a cinco anos de idade. A perda da capacidade de deambular ocorre entre oito a dez anos de idade e a evolução para o óbito é em torno da segunda década de vida, geralmente devido à insuficiência cardíaca ou respiratória. O camundongo mdx, um mutante natural que não expressa a distrofina, é amplamente utilizado como modelo animal da DMD e apresenta as alterações histológicas características da doença: mionecrose (6 semanas), regeneração (12 semanas) e fibrose (24 e 48 semanas). Dados anteriores mostram que os diferentes músculos esqueléticos, e cardíaco, se comportam de forma peculiar de acordo com o perfil de sub-populações de células inflamatórias encontradas e cinética de migração. Este trabalho visa avaliar a migração de sub-populações celulares para o músculo cardíaco com possíveis funções efetoras/reguladoras relacionadas à progressão da doença. Os tecidos foram coletados de animais com 2, 6, 12, 24 e 48 semanas de idade e a análise histopatológica mostrou principalmente macrófagos e fibroblastos nos infiltrados inflamatórios em todas as idades. Com relação aos mastócitos, encontramos células principalmente do fenótipo mucoso (MMC) no pericárdio de animais com 2 semanas. Porém, esses mastócitos aparentemente migram para o endocárdio enquanto assumem o fenótipo de mastócitos de tecido conjuntivo (CTMC). Com relação aos linfócitos de sangue, em animais de 6 semanas observamos um fenótipo compatível com a literatura, sendo...

Presence of the P2X(7) purinergic receptor on immune cells that invade the rat endometrium during oestrus.

Eosinophils, macrophages and other leucocytes invade the uterine endometrium during oestrus and play a role in the tissue remodeling and immune responses that occur prior to implantation of the fertilized ovum. Adenosine 5'-triphosphate (ATP) and its metabolites influence uterine function via ATP receptors. In this study, we investigated the presence and localisation of the P2X(7) nucleotide receptor in the cells that infiltrate the uterine endometrium of adult female rats during oestrus at the electron microscope level, using gold-silver pre-embedding immunocytochemical techniques. P2X(7) receptor expression was found in the cytoplasm and the cell membrane of eosinophils, macrophages and fibroblasts in the endometrium during oestrus. These results suggest that ATP-mediated responses may be important in uterine preparation and remodeling before implantation and that this may involve several types of cells. In particular, the presence of P2X(7) receptors on endometrial stromal cells may indicate their involvement in apoptosis and immune and inflammatory responses.