Manganese homeostasis modulates fungal virulence and stress tolerance in Candida albicans.

Due to the scarcity of transition metals within the human host, fungal pathogens have evolved sophisticated mechanisms to uptake and utilize these micronutrients at the infection interface. While considerable attention was turned to iron and copper acquisition mechanisms and their importance in fungal fitness, less was done regarding either the role of manganese (Mn) in infectious processes or the cellular mechanism by which fungal cells achieve their Mn-homeostasis. Here, we undertook transcriptional profiling in the pathogenic fungus Candida albicans experiencing both Mn starvation and excess to capture biological processes that are modulated by this metal. We uncovered that Mn scarcity influences diverse processes associated with fungal fitness including invasion of host cells and antifungal sensitivity. We show that Mn levels influence the abundance of iron and zinc emphasizing the complex crosstalk between metals. The deletion of SMF12, a member of Mn Nramp transporters, confirmed its contribution to Mn uptake. smf12 was unable to form hyphae and damage host cells and exhibited sensitivity to azoles. We found that the unfolded protein response (UPR), likely activated by decreased glycosylation under Mn limitation, was required to recover growth when cells were shifted from an Mn-starved to an Mn-repleted medium. RNA-seq profiling of cells exposed to Mn excess revealed that UPR was also activated. Furthermore, the UPR signaling axis Ire1-Hac1 was required to bypass Mn toxicity. Collectively, this study underscores the importance of Mn homeostasis in fungal virulence and comprehensively provides a portrait of biological functions that are modulated by Mn in a fungal pathogen. IMPORTANCE: Transition metals such as manganese provide considerable functionality across biological systems as they are used as cofactors for many catalytic enzymes. The availability of manganese is very limited inside the human body. Consequently, pathogenic microbes have evolved sophisticated mechanisms to uptake this micronutrient inside the human host to sustain their growth and cause infections. Here, we undertook a comprehensive approach to understand how manganese availability impacts the biology of the prevalent fungal pathogen, Candida albicans. We uncovered that manganese homeostasis in this pathogen modulates different biological processes that are essential for host infection which underscores the value of targeting fungal manganese homeostasis for potential antifungal therapeutics development.

How to improve issuing, transfusion and follow-up of blood components in Southern and Eastern Mediterranean countries? A benchmark assessment.

To determine the existence of guidelines regarding the appropriate clinical use of blood and blood components, transfusion requests, and blood issuing/reception documents and procedures. The different bedside transfusion organizations/processes and hemovigilance are also analyzed. The ultimate objective is to identify safe potential options in order to improve blood safety at the lowest cost. Data emanating from eight Arabic eastern/southern Mediterranean countries who responded to five surveys were collected and tabulated. National recommendations for the clinical use of blood components especially for hemoglobinopathies are lacking in some countries. In matter of good practices in the prescription, issuing and reception of BCs, efforts were made either on national or local basis. Procedures regarding patient information and ethical issues are still lacking. Almost all Mediterranean countries apply two blood testing procedures on each patient sample. Only Morocco, Tunisia and Algeria perform bed side blood group testing; Egypt and Lebanon perform antibody screen and antiglobulin cross matching universally. Automation for blood testing is insufficiently implemented in almost all countries and electronic release is almost absent. National hemovigilance policy is implemented in Tunisia, Morocco, and Lebanon but the reporting system remains inoperative. Insufficient resources severely hinders the implementation of expensive procedures and programs; however, the present work identifies safe procedures that might save resources to improve other parts in the transfusion process (e.g. electronic release to improve safety in issuing). Moreover, setting up regulations regarding ethics in transfusing recipients along with local transfusion committees are crucially needed to implement hemovigilance in transfusion practice.

First Report of the HEV Seroprevalence and the Risk Factor Assessment in the West Bank, Palestine, during the Period of 2015-2017.

Hepatitis E virus is emerging viral hepatitis with hyperendemicity in many countries. Data on the burden of disease is not available in Palestine. This study aims to determine the seroprevalence and the risk factors of the HEV among the general population of the West Bank, Palestine. In this cross-sectional study, a total of 432 sera samples from 40 localities in the eleven districts of the West Bank and Jerusalem, Palestine, during the period of March 2015 to March 2017, were tested for HEV-IgG. A structured questionnaire was used to collect data of the participants' demographics and disease risk factors. The overall seroprevalence was 3.7%. Level of education was significantly inversely associated with HEV seropositivity (P=0.04). Purely spatial analysis did not detect any significant cluster related to the distribution of HEV-IgG cases; however, living in the southern West Bank is shown to be significantly associated with HEV. Age was also associated with HEV seropositivity. The young (<19 years) and adults (>40 years) had the highest prevalence, compared to those between 20 to 39 years old (P=0.12). Furthermore, males and those in contact with animals were associated with HEV seropositivity (P=0.1 and 0.3, respectively). In conclusion, the seroprevalence of HEV IgG in the West Bank, Palestine is low. Several well-investigated risk factors cannot be supported by our results due to the small number of the positive HEV-IgG samples. Finally, this study is useful for providing a first look into the seroepidemiology of HEV in Palestine.

Genetic diversity, haplotype analysis, and risk factor assessment of hepatitis A virus isolates from the West Bank, Palestine during the period between 2014 and 2016.

BACKGROUND: Hepatitis A virus (HAV) infection is one of the major causes of acute viral hepatitis. HAV genotypes and its genetic diversity is rarely investigated in our region as well as worldwide. AIMS: The aims of the present study were to determine the HAV genotypes and its risk factors and to investigate the genetic diversity of the HAV isolates in the West Bank, Palestine. STUDY DESIGN: A cohort of 161 clinically and laboratory-confirmed HAV (IgM-positive) cases and 170 apparently healthy controls from all the districts of the West Bank, Palestine during the period of 2014 to 2016 were tested for HAV infection using IgM antibodies, RT-PCR and sequence analysis of the VP3/VP1 junction region of the HAV genome. Phylogenetic analysis, genetic diversity and haplotypes analysis were used to characterize the VP3/VP1 sequences. RESULTS: All the 34 sequences of the HAV were found to be of HAV-IB sub-genotype. The phylogenetic analysis showed four main clusters with cluster III exclusively consisting of 18 Palestinian isolates (18/23-78%), but with weak bootstrap values. A high haplotype diversity (Hd) and low nucleotide diversity (π) were observed. Cluster III showed high number of haplotypes (h = 8), but low haplotype (gene) diversity (Hd = 0.69). A total of 28 active haplotypes with some consisting of more than one sequence were observed using haplotype network analysis. The Palestinian haplotypes are characterized by closely related viral haplotypes with one SNV away from each other which ran parallel to cluster III in the phylogenetic tree. A smaller Palestinian haplotype (4 isolates) was three SNVs away from the major haplotype cluster (n = 10) and closer to others haplotypes from Iran, Spain, and South Africa. Young age, low level of parent's education, infrequent hand washing before meals, and drinking of un-treated water were considered the major HAV risk factors in the present study. CONCLUSION: Haplotype network analysis revealed haplotype variation among the HAV Palestinian sequences despite low genetic variation and nucleotide diversity. In addition, this study reconfirmed that age and parent's level of education as HAV risk factors, while hand washing and treating drinking water as protective factors.

Quality and safety measures in transfusion practice: The experience of eight southern/eastern Mediterranean countries.

BACKGROUND AND OBJECTIVES: Blood transfusion is inherently associated with risks, and little is known regarding the available quality and safety measures in developing countries. No studies or census has been carried out, and therefore, no data on this compelling issue are available. MATERIALS AND METHODS: Data emanating from eight Arabic eastern/southern Mediterranean countries who responded to five surveys were collected and tabulated. RESULTS: Asepsis during phlebotomy, screening for serological and immuno-haematological parameters and appropriate storage conditions are maintained across all countries. Variations in blood component processing exist. Universal leucoreduction is systematically applied in Lebanon. Nucleic acid testing is only performed in Egypt. Aphaeresis procedure, leucoreduction and quality control for blood components are virtually inexistent in Mauritania. Written donor questionnaire is absent in Algeria and Tunisia. Most donor deferral periods for infectious agents are inconsistent with international standards. CONCLUSION: Gaps in the processing and in the quality/safety measures applied to the manufacture of blood components are quite evident in most eastern/southern Mediterranean countries. The decision of establishing an effective collaboration network and an independent body - aside from WHO - composed of specialists that oversees all transfusion activities in these countries is certainly a crucial step towards ensuring an optimum level of blood safety.

How to manage transfusion systems in developing countries: The Experience of Eastern and Southern Mediterranean countries.

OBJECTIVES: To outline and analyse the national organisation, infrastructure and management of transfusion systems in countries sharing common historical, cultural and economic features and to decipher management trends, in order to potentially benchmark. BACKGROUND: Little is known regarding transfusion systems in Eastern/southern Mediterranean at a time international organisations are calling for the establishment of a safe and sustainable blood system. MATERIALS AND METHODS: Data emanating from eight Arabic-speaking Eastern/Southern Mediterranean countries who responded to five surveys were collected and tabulated. RESULTS: While similarities in terms of supervision by national authorities, authorization of blood centres, quality control and management information system are evident, some significant divergence between these countries do exists. Only Lebanon does not possess a national blood establishment or organisation for blood supply. Blood components are fully government-subsidised in Algeria and Mauritania. Algeria, Morocco and Tunisia have a blood supply that relies mainly on Voluntary non-remunerated donors. Plateletpheresis is performed in all countries except Mauritania while plasmapheresis exists only in Algeria and Egypt. Morocco is the sole country outsourcing its plasma for Plasma derived products. CONCLUSION: Despite the various challenges facing these countries, lot of progresses have been made so far in the field of transfusion medicine. Yet, nationally coordinated blood programs overviewed by national regulatory authorities and actively supported by local governments are still needed to ensure the optimum level of blood safety.

Salivary gland epithelial cell exosomes: A source of autoantigenic ribonucleoproteins.

OBJECTIVE: Exosomes are membrane vesicles of endosomal origin that are distinct from apoptotic bodies and are thought to represent an acellular mechanism for antigen transfer to classic antigen-presenting cells, as well as for direct antigen presentation with the capacity to induce immune response or tolerance. Nevertheless, it is not known whether exosomes are involved in the induction or regulation of immune responses against intracellular autoantigens that characterize autoimmune diseases. Exosomes have been shown to be secreted by several types of cells, whereas studies of non-neoplastic epithelial cells are lacking. This study was undertaken to investigate the capacity of non-neoplastic salivary gland epithelial cells (SGECs) to release exosomes, and to determine whether epithelial exosomes contain RNPs, which are major autoantigens in systemic rheumatic diseases. METHODS: Exosomes were isolated by ultracentrifugation from culture supernatants of 26 non-neoplastic SGEC lines established from patients with various rheumatic disorders. They were analyzed by electron microscopy, immunoblotting, or immunoprecipitation. RESULTS: All SGEC lines were found to release comparable and significant amounts of exosomes. Similar to other cell systems, exosome secretion was constitutive and was unrelated to activation or apoptotic processes. SGEC-derived exosomes were found to contain the autoantigenic Ro/SSA, La/SSB, and Sm RNPs, as well as epithelial-specific cytokeratins. CONCLUSION: SGECs constitutively secrete exosomes that contain the major autoantigens Ro/SSA, La/SSB, and Sm. This mechanism may represent a pathway whereby intracellular autoantigens are presented to the immune system with an immunogenic or tolerogenic outcome.

Establishment of a convenient system for the long-term culture and study of non-neoplastic human salivary gland epithelial cells.

Epithelial cells appear to play an important role in the initiation and maintenance of autoimmune lesions in the salivary glands of patients with Sjogren's syndrome. Therefore, the detailed study of immunological function of salivary gland epithelial cells (SGEC) may provide useful information for the understanding of Sjögren's syndrome pathogenesis. In this report we aimed to formulate a protocol for the establishment of human non-neoplastic SGEC lines as a tool for the study of the physiology and pathophysiology of these cells. Pointing towards a practical approach, we sought to establish SGEC lines from quite a limited amount of biopsy tissue obtained during the diagnostic evaluation of patients. Herein, the favorable conditions for the long-term maintenance of human non-neoplastic SGEC lines are presented and involve the successive application of a serum-containing and a serum-free culture medium, supplemented with essential epithelial growth factors. This protocol has been found reliable and convenient, as attested by the reproducible establishment of non-neoplastic SGEC lines. The analysis of SGEC phenotypic features, as well as a coculture system for the study of interactions between epithelial cells and lymphocytes, are also described. Such techniques may provide valuable means for the functional and molecular investigation of human SGEC and particularly for the study of Sjögren's syndrome and other disorders of glandular epithelia.