S-adenosylmethionine blocks tumorigenesis and with immune checkpoint inhibitor enhances anti-cancer efficacy against BRAF mutant and wildtype melanomas.

Despite marked success in treatment with immune checkpoint inhibitor (CPI), only a third of patients are responsive. Thus, melanoma still has one of the highest prevalence and mortality rates; which has led to a search for novel combination therapies that might complement CPI. Aberrant methylomes are one of the mechanisms of resistance to CPI therapy. S-adenosylmethionine (SAM), methyl donor of important epigenetic processes, has significant anti-cancer effects in several malignancies; however, SAM's effect has never been extensively investigated in melanoma. We demonstrate that SAM modulates phenotype switching of melanoma cells and directs the cells towards differentiation indicated by increased melanogenesis (melanin and melanosome synthesis), melanocyte-like morphology, elevated Mitf and Mitf activators' expression, increased antigen expression, reduced proliferation, and reduced stemness genes' expression. Consistently, providing SAM orally, reduced tumor growth and progression, and metastasis of syngeneic BRAF mutant and wild-type (WT) melanoma mouse models. Of note, SAM and anti-PD-1 antibody combination treatment had enhanced anti-cancer efficacy compared to monotherapies, showed significant reduction in tumor growth and progression, and increased survival. Furthermore, SAM and anti-PD-1 antibody combination triggered significantly higher immune cell infiltration, higher CD8+ T cells infiltration and effector functions, and polyfunctionality of CD8+ T cells in YUMMER1.7 tumors. Therefore, SAM combined with CPI provides a novel therapeutic strategy against BRAF mutant and WT melanomas and provides potential to be translated into clinic.

Mechanisms of human FoxP3+ Treg cell development and function in health and disease.

Regulatory T (Treg ) cells represent an essential component of peripheral tolerance. Given their potently immunosuppressive functions that is orchestrated by the lineage-defining transcription factor forkhead box protein 3 (FoxP3), clinical modulation of these cells in autoimmunity and cancer is a promising therapeutic target. However, recent evidence in mice and humans indicates that Treg cells represent a phenotypically and functionally heterogeneic population. Indeed, both suppressive and non-suppressive Treg cells exist in human blood that are otherwise indistinguishable from one another using classical Treg cell markers such as CD25 and FoxP3. Moreover, murine Treg cells display a degree of plasticity through which they acquire the trafficking pathways needed to home to tissues containing target effector T (Teff ) cells. However, this plasticity can also result in Treg cell lineage instability and acquisition of proinflammatory Teff cell functions. Consequently, these dysfunctional CD4+ FoxP3+ T cells in human and mouse may fail to maintain peripheral tolerance and instead support immunopathology. The mechanisms driving human Treg cell dysfunction are largely undefined, and obscured by the scarcity of reliable immunophenotypical markers and the disregard paid to Treg cell antigen-specificity in functional assays. Here, we review the mechanisms controlling the stability of the FoxP3+ Treg cell lineage phenotype. Particular attention will be paid to the developmental and functional heterogeneity of human Treg cells, and how abrogating these mechanisms can lead to lineage instability and Treg cell dysfunction in diseases like immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, type 1 diabetes, rheumatoid arthritis and cancer.

Toxoplasma gondii reorganizes the host cell architecture during spontaneous cyst formation in vitro.

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a 'cage' around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection.

Development of dual fluorescent stage specific reporter strain of Toxoplasma gondii to follow tachyzoite and bradyzoite development in vitro and in vivo.

Toxoplasma gondii is a protozoan that infects 30% of humans as intermediate hosts. T Sexual reproduction can occur only within the intestinal tract of felines, however, infection in other mammals and birds is associated with asexual replication and interconversion between the tachyzoite and bradyzoite stages. Bradyzoites are slow growing forms found in tissue cysts in latent infection. Recently, our group described the biological behavior of the EGS strain that forms thick walled cysts spontaneously in tissue culture, constituting a useful tool for examining the developmental biology of T. gondii. To further improve the usefulness of this model, we constructed genetically modified EGS parasites that express fluorescent tags under the control of stage specific promoters. The promoter regions for SAG-1 (tachyzoite specific), BAG-1 and LDH-2 (bradyzoite specific) were amplified by PCR and plasmids were constructed with mCherry (redT) and sfGFP (greenB) sequences, respectively. Strains of parasites were selected using FACS to arrive at single fluorescent and dual fluorescent strains of EGS expressing tags in a stage specific manner. In cell cultures, vacuoles labeled by immunofluorescence assay using anti-CST-1 a marker for T. gondii cyst wall contained parasites that were positive for BAG1-GFP and negative for SAG1-mCherry. Tachyzoites and bradyzoites harvested from the mice expressed stage specific mCherry and GFP proteins, respectively. These new dual fluorescent transgenic EGS strains are a promising tool to elucidate the mechanisms of T. gondii differentiation both in vitro and in vivo.

Spontaneous cystogenesis in vitro of a Brazilian strain of Toxoplasma gondii.

Conversion of Toxoplasma gondii tachyzoites to the bradyzoite stage and tissue cyst formation in the life cycle of the parasite have crucial roles in the establishment of chronic toxoplasmosis. In this work we investigated the in vitro cystogenesis and behavior of the EGS strain, isolated from human amniotic fluid. We observed that tachyzoites of the EGS strain converted to intracellular cysts spontaneously in LLC-MK2 epithelial cells, HSFS fibroblasts and C6 glial cell lineage. The peak of conversion occurred in the LLC-MK2 cells after 4days of infection, when 72.3±15.9 of the infected cells contained DBA positive cysts. Using specific markers against bradyzoite, tachyzoite and cyst wall components, we confirmed stage conversion and distinguished immature from mature cysts. It was also observed that the deposition of cyst wall components occurred before the total conversion of parasites. Transmission electron microscopy confirmed the fully conversion of parasites presenting the typical characteristics of bradyzoites as the posterior position of the nucleus and the presence of amylopectin granules. A thick cyst wall was also detected. Besides, the scanning microscopy revealed that the intracyst matrix tubules were shorter than those from the parasitophorous vacuole intravacuolar network and were immersed in a granular electron dense material. The EGS strain spontaneously forms high burden of cysts in cell culture without artificial stress conditions, and constitutes a useful tool to study this stage of the T. gondii life cycle.

Fine structure of the biogenesis of Giardia lamblia encystation secretory vesicles.

Synthesis, transport, and assembly of the extracellular cyst wall is the hallmark of Giardia lamblia encystation. Much is known of the biochemical pathways and their regulation. However, from a cell biology point of view, the biogenesis of the encystation specific vesicles (ESVs) that transport cyst wall proteins to the periphery of the cell is poorly understood. Therefore, we exploited a number of complementary ultrastructural approaches to test the hypothesis that the formation of ESVs utilizes a novel regulated secretory pathway. We analyzed parasites at different stages of encystation in vitro by electron microscopy of thin sections, freeze fracture replicas, and three-dimensional reconstruction from serial sections of cells fixed for cytochemical localization of the endoplasmic reticulum (ER) marker, glucose 6-phosphatase. We also used a stereological approach to determine the area occupied by the ER, clefts, ESVs, and cyst wall. Taken together, our kinetic data suggest that some ER cisternae first dilate to form clefts, which enlarge into the ESVs. Living non-encysting and early-encysting trophozoites were labeled around the periphery of both nuclei with C(6)-NBD-ceramide. At 18-21 h, outward migration of some ESVs frequently caused protrusions at the periphery of encysting trophozoites. The presence of lysosome-like peripheral vesicles between the ESV and plasma membrane of the cell was confirmed using acridine orange, an acidic compartment marker. Our data suggest that G. lamblia has a novel secretory pathway in which certain functions of the ER and Golgi co-localize spatially and temporally. These studies will increase understanding of the evolutionary appearance of regulated secretory pathways for assembly of a primitive extracellular matrix in an early diverging eukaryote.

Megasome biogenesis in Leishmania amazonensis: a morphometric and cytochemical study.

Megasomes are large lysosomes found in the amastigote stage of Leishmania species belonging to the mexicana complex. The biogenesis of megasomes was investigated by transmission electron microscopy during the transformation of promastigotes into the amastigote form of L. amazonensis maintained in axenic cultures. Mainly small vacuoles with low electron density were found in the promastigote and early intermediate forms. Morphometrical analysis showed an increase in the volume density of these structures during the transformation process. Cysteine proteinase was localized in this structure by immunocytochemical assay. Membrane-bounded structures filled with electron-dense material were also found in significant amounts from the 2nd day on. These structures were relatively abundant, both in axenic and lesion-derived amastigotes, but not in stable long-term axenic amastigote culture. A three-dimensional reconstruction of lesion-derived amastigotes and axenic amastigotes of L. amazonensis demonstrated that megasomes comprise almost 5% of the total cell volume. In addition, the development of other organelles was examined during the transformation process.

The social climate in chronic haemodialysis units as perceived by patients and nurses.

The purpose of this research was to check if there was a correlation between how patients perceived the atmosphere in dialysis units compared to the nurses' perception. The intention was to examine commonality and differences between the two groups. This would serve as a basis to change the behaviour of the nurses and/or to develop or create settings that are more suitable. Sixteen haemodialysis units in Israel participated in this study, a total of 190 participants of which ninety-three were nurses and ninety-seven were patients. Patients and nurses gave their consent in writing and answered anonymously. The Moos Ward Atmosphere Scale was used using 100 true and false questions. These questions were condensed into six main categories. Our results showed that the patients and staff had significantly different perceptions in the following categories: 1. Openness and sensitivity, 2. Staff attitude, 3. Order and organization, 4. Mutual support, 5. New treatment approaches. The greatest degree of agreement between the two groups was found in only one category: that of the "doctor's attitude" or behaviour. Three studies have been found that have investigated the unit atmosphere as perceived by patients and staff-two in Israel in an oncology and a psychiatric unit. Rhodes did the third study in a haemodialysis unit in the Unites States and his results are compared with the results in this study. The differences found between nurses and patients show that there is a communication problem. It is recommended that interpersonal communication be improved to close the gap in perceptions, thereby improving the unit atmosphere. New strategies should be developed for coping and helping the patient to adjust.

Ultrastructural and biochemical characterization of promastigote and cystic forms of Leptomonas wallacei n. sp. isolated from the intestine of its natural host Oncopeltus fasciatus (Hemiptera: Lygaeidae).

Promastigote forms of a trypanosomatid were isolated from the third and fourth ventricles of the midgut and from the hindgut of the phytophagous hemipteran Oncopeltus fasciatus. Some individuals had adhered to its anterior region, close to the flagellar pocket, or to the flagellum up to four rounded aflagellated forms known as straphangers cysts. Scanning electron microscopy revealed that the flagellated forms presented a twisted cell body and a long flagellum, and the cysts, smaller than the parental promastigote, had a nascent flagellum. Transmission electron microscopy showed that promastigotes were typical, while cystic forms were ovoid dense cells devoid of a cyst wall, but presenting a cell coat, a special subpellicular region limited by a membrane unit, and a condensed cytoplasm. The kinetoplast-DNA fibrils appeared as dense spots and the condensed chromatin was arranged in a labyrinthic structure. Desmosome-like structures, observed in the region of adhesion of the precystic forms to the parental promastigote, could explain how cysts remain attached to the mother cell during the encystation process. Release of membranes from the surface of promastigotes and cysts seems to be correlated with the condensation of the cytoplasm during encystment. Morphological and isozyme analyses indicated that this trypanosomatid belongs to the genus Leptomonas. The molecular karyotype of this isolate was compared with that of a strain of Leptomonas oncopelti obtained from Oncopeltus varicolor by contour-clamped homogeneous electric field (CHEF) electrophoresis and revealed similar DNA banding patterns between 2,200-825 Kb, but not in lower bands (825-225 Kb). This suggested that the isolate from O. fasciatus and that from O. varicolor were not identical. Based on our findings we are describing Leptomonas wallacei n. sp. for our isolate from O. fasciatus.

CD4 mononuclear cell infiltrates and Fas/Fas ligand positive mammary gland cells in breast tissue from a patient with Sjögren's syndrome.

We describe a 49-year-old patient with lip biopsy proven Sjögren's syndrome (SS) and keratoconjunctivitis sicca, who had dental caries, xerostomia, recurrent upper respiratory tract infections, arthritis in her hands, elbows and knees, and recurrent parotid inflammation. She developed bilateral breast nodules in 1988. Right breast nodules were excised in 1993 and 1995, but reappeared in 1996, requiring 2 more excisions. Breast tissue samples showed remarkable intralobular and perilobular mononuclear cell infiltrates that were predominantly CD4+ T cells and expressed bcl-2. A few cells stained CD20+ and CD8+. SS breast glandular epithelial cells stained more intensely for Fas compared to normal cells. CD4+ T cells and Fas mediated cell death may be involved in the mammary gland lesions in SS.

Ultrastructural and biochemical analysis of the relationship of Crithidia deanei with its endosymbiont.

Some protozoa of the Trypanosomatidae family harbor in their cytoplasm bacterial endosymbionts that provide essential nutrients to and induce morphological alterations in the protozoa. In the present study, a close association between endosymbionts and glycosomes, a peroxisome-like organelle where most of the enzymes of the glycolytic pathway are compartmentalized, was identified by conventional transmission electron microscopy in Crithidia deanei. Such an association was further supported by the cytochemical localization of catalase in the glycosome and also confirmed by 3-D reconstruction of the protozoan. The enzymes cytochrome oxidase and succinate dehydrogenase were detected by ultrastructural cytochemistry. A positive reaction was observed in the protozoan mitochondrion but not in the endosymbiont envelope. Enzymatic assays for succinate cytochrome c reductase reinforced these results, as a low enzymatic activity was detected in an endosymbiont-enriched fraction, while high activity was observed in a purified protozoan mitochondrion fraction. We also demonstrated that a purified symbiont fraction was able to hydrolyze ATP. This activity was Mg+2 dependent, since it was highly stimulated by the presence of physiological concentrations of this ion. Taken together, these observations suggest that no electron transporting system is active in the symbionts of Crithidia deanei and that they might obtain energetic molecules derivated from the protozoan glycosomes.

The cell surface of Phytomonas davidi.

Carbohydrates were located on the surface of Phytomonas davidi using ultrastructural cytochemistry, and agglutination induced by lectins which bind to residues of mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid. The surface charge of the cells was analysed by the binding of cationic particles (colloidal iron and cationized ferritin) to the cell surface and by cell electrophoretic mobility (EPM). Based on observations of binding of cationic particles to the cell surface; a decrease in the binding of these particles to the cell surface; a decrease in the mean EPM of the cells after their incubation in the presence of neuraminidase; and detection of N-acetylneuraminic acid by paper and gas-liquid chromatography, it was concluded that sialic acid residues are exposed on the surface of P. davidi. These residues may be glycolipids or are masked on the cell surface since only after brief trypsinization were the cells agglutinated by the lectin from Limulus polyphemus.