Characterization of Actinobacteria strains in Lebanese soil with an emphasis on investigating their antibacterial activity.

With the alarming rise of drug resistant pathogens, the quest for new bioactive compounds from natural habitats has increased. Actinobacteria are Gram-positive bacteria, considered prominent natural antibiotic synthesizers. This study aimed at isolating Actinobacteria from agricultural soil samples of Tamnine El Tahta and Haddatha, with an emphasis on the physicochemical soil characteristics. It also aimed at screening and identifying the antibacterial-producing Actinobacteria, with a determination of the chemical composition of the extract. Forty-six Actinobacteria were isolated from six soil samples. Actinobacteria load exhibited a positive correlation with moisture content, and a negative correlation with pH, salinity, and organic matter content. Primary screening for antibacterial activity was performed against various Gram-positive and Gram-negative bacteria by cross-streak method. Fourteen Actinobacteria isolates were potent against the test microorganisms, and the most effective isolate (T25) was selected for identification, and extract preparation. The antibacterial activity of the extract was tested using secondary screening, in addition to minimal inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) determination. T25 isolate exhibited a 92% similarity with Micrococcus luteus/lylae. MIC recorded was 12.5 mg/ml and the MBC was higher than 100 mg/ml against all test microorganisms. Total phenol content was estimated to be 18.5 ± 0.0015 mg GAE/g dry weight using Folin-Ciocalteu method, and total flavonoid content recorded 2.3 ± 0.02 mg RE/g dry weight using aluminum nitrate colorimetric method. This study revealed that the physicochemical parameters in soils impact the distribution of Actinobacteria. Moreover, it focuses on Micrococcus luteus/lylae strain, considered a promising antibacterial resource for further potential clinical investigations.

HAS 1: A natural product from soil-isolated Streptomyces species with potent activity against cutaneous leishmaniasis caused by Leishmania tropica.

Cutaneous Leishmaniasis (CL) is a neglected tropical disease, classified by the World Health Organization (WHO) as one of the most unrestrained diseases. The Syrian war and the significant displacement of refugees aggravated the spread of this ailment into several neighboring countries in the Eastern Mediterranean Region (EMR). In Syria, Leishmania tropica is identified as one of the most aggressive and endemic identified species, causing localized or generalized lesions, often chronic or relapsing. Pentavalent antimonial drugs are currently used as first line treatment against CL. Nonetheless, these drugs exhibit several limitations, including the repetitive painful injections, high cost, poor availability, and mainly systemic toxicity. Besides, the emergence of acquired parasitic resistance hinders their potency, stressing the need for new therapies to combat CL. Natural products (NPs) epitomize a valuable source in drug discovery. NPs are secondary metabolites (SMs) produced by plants, sponges, or a wide variety of organisms, including environmental microorganisms. The EMR is characterized by its immense biodiversity, yet it remains a relatively untapped area in drug discovery. NPs of the region were explored over the last 2 decades, but their discoveries lack biogeographical diversity and are limited to the Red Sea. Here, we isolated previously uncultured environmental soil-dwelling Streptomyces sp. HAS1, from Hasbaya region in southeast Lebanon. When fermented in one of our production media named INA, HAS1 produced a crude extract with significant potency against a clinical Leishmania tropica isolate. Using bio-guided fractionation, the bioactive compound was purified and the structure was elucidated by NMR and LC-HRMS. Our findings establish NPs as strong candidates for treating Leishmania tropica and further dwells on the importance of these natural sources to combat microbial infections.

EAPB0503, an Imidazoquinoxaline Derivative Modulates SENP3/ARF Mediated SUMOylation, and Induces NPM1c Degradation in NPM1 Mutant AML.

Nucleophosmin-1 (NPM1) is a pleiotropic protein involved in numerous cellular processes. NPM1 shuttles between the nucleus and the cytoplasm, but exhibits a predominant nucleolar localization, where its fate and functions are exquisitely controlled by dynamic post-translational modifications (PTM). Sentrin/SUMO Specific Peptidase 3 (SENP3) and ARF are two nucleolar proteins involved in NPM1 PTMs. SENP3 antagonizes ARF-mediated NPM1 SUMOylation, to promote ribosomal biogenesis. In Acute Myeloid Leukemia (AML), NPM1 is frequently mutated, and exhibits an aberrant cytoplasmic localization (NPM1c). NPM1c mutations define a separate AML entity with good prognosis in some AML patients, rendering NPM1c as a potential therapeutic target. SENP3-mediated NPM1 de-SUMOylation induces resistance to therapy in NPM1c AML. Here, we demonstrate that the imidazoquinoxaline EAPB0503 prolongs the survival and results in selective reduction in the leukemia burden of NPM1c AML xenograft mice. Indeed, EAPB0503 selectively downregulates HDM2 expression and activates the p53 pathway in NPM1c expressing cells, resulting in apoptosis. Importantly, we unraveled that NPM1c expressing cells exhibit low basal levels of SUMOylation paralleled with high SENP3 and low ARF basal levels. EAPB0503 reverted these molecular players by inducing NPM1c SUMOylation and ubiquitylation, leading to its proteasomal degradation. EAPB0503-induced NPM1c SUMOylation is concurrent with SENP3 downregulation and ARF upregulation in NPM1c expressing cells. Collectively, these results provide a strong rationale for testing therapies modulating NPM1c post-translational modifications in the management of NPM1c AML.

Microsatellite Instability in Near East Sebaceous Neoplasms: Toward Improved Prediction.

Sebaceous neoplasms (SN) comprise a heterogeneous spectrum of tumors with different biological behaviors. In the Near-East Region (NER), microsatellite instability (MSI) in SN's development, and its correlation with the clinicopathologic features of tumors is not well elucidated. A cohort of 225 SN patients (40 benign SNs and 185 sebaceous carcinomas) from the NER was retrospectively reviewed. Clinical variables and available follow-up information were recorded. MSI proteins (MLH1, MSH2, MSH6, and PMS2) as well as P53, P16, EMA, CD8, and PDL-1 expressions were examined by immunohistochemistry. Detection of human papilloma virus was determined by polymerase chain reaction. Microscopic features such as mitotic count and tumor-infiltrating lymphocytes were documented. A minority of SNs from benign (n=2) or malignant (n=3) tumors in the NER exhibit MSI (2.2%). MSI is exclusively found in patients with extraocular lesions (back, n=5) and presented a poor outcome. Among these, PMS2 protein was mostly lost (average=80%, n=4). SN with MSI exhibited a significant increase in p53 expression, (average=62.10%, P=0.002). There was no significant correlation between MSI status and any of the following: PD-L1, CD8, p16, and human papilloma virus infection. Microscopically, SN with MSI show significantly higher mitotic count, cystic changes and increased tumor-infiltrating lymphocytes. MSI is rarely found in NER's SN. When detected, it is exclusively in extraocular SNs with minimal predicative microscopic features and worse outcome.

Comprehensive Overview of Toxoplasma gondii-Induced and Associated Diseases.

Toxoplasma gondii (T. gondii) is a prevalent protozoan parasite of medical and veterinary significance. It is the etiologic agent of toxoplasmosis, a neglected disease in which incidence and symptoms differ between patients and regions. In immunocompetent patients, toxoplasmosis manifests as acute and chronic forms. Acute toxoplasmosis presents as mild or asymptomatic disease that evolves, under the host immune response, into a persistent chronic disease in healthy individuals. Chronic toxoplasmosis establishes as latent tissue cysts in the brain and skeletal muscles. In immunocompromised patients, chronic toxoplasmosis may reactivate, leading to a potentially life-threatening condition. Recently, the association between toxoplasmosis and various diseases has been shown. These span primary neuropathies, behavioral and psychiatric disorders, and different types of cancer. Currently, a direct pre-clinical or clinical molecular connotation between toxoplasmosis and most of its associated diseases remains poorly understood. In this review, we provide a comprehensive overview on Toxoplasma-induced and associated diseases with a focus on available knowledge of the molecular players dictating these associations. We will also abridge the existing therapeutic options of toxoplasmosis and highlight the current gaps to explore the implications of toxoplasmosis on its associated diseases to advance treatment modalities.

P18 (SRS35/TgSAG4) Plays a Role in the Invasion and Virulence of Toxoplasma gondii.

Toxoplasmosis is a prevalent parasitic disease caused by Toxoplasma gondii (T. gondii). Under the control of the host immune system, T. gondii persists as latent bradyzoite cysts. Immunosuppression leads to their reactivation, a potentially life-threatening condition. Interferon-gamma (IFN-γ) controls the different stages of toxoplasmosis. Here, we addressed the role of the parasite surface antigen P18, belonging to the Surface-Antigen 1 (SAG-1) Related Sequence (SRS) family, in a cyst-forming strain. Deletion of P18 gene (KO P18) impaired the invasion of parasites in macrophages and IFN-γ-mediated activation of macrophages further reduced the invasion capacity of this KO, as compared to WT strain. Mice infected by KO P18, showed a marked decrease in virulence during acute toxoplasmosis. This was consequent to less parasitemia, accompanied by a substantial recruitment of dendritic cells, macrophages and natural killer cells (NK). Furthermore, KO P18 resulted in a higher number of bradyzoite cysts, and a stronger inflammatory response. A prolonged survival of mice was observed upon immunosuppression of KO P18 infected BALB/c mice or upon oral infection of Severe Combined Immunodeficiency (SCID) mice, with intact macrophages and natural killer (NK) cells. In stark contrast, oral infection of NSG (NOD/Shi-scid/IL-2Rγnull) mice, defective in macrophages and NK cells, with KO P18, was as lethal as that of the control strain showing that the conversion from bradyzoites to tachyzoites is intact and, suggesting a role of P18 in the response to host IFN-γ. Collectively, these data demonstrate a role for P18 surface antigen in the invasion of macrophages and in the virulence of the parasite, during acute and chronic toxoplasmosis.

Sebaceous neoplasms: prevalence of HPV infection and relation to immunohistochemical surrogate markers.

BACKGROUND: Sebaceous neoplasms (SNs) and carcinomas (SCs) represent rare skin adnexal tumours. OBJECTIVES: To establish the prevalence of HPV in SNs, assess the relationship between HPV, p16 and p53 expression, and further elucidate the carcinogenetic course of SCs. MATERIALS & METHODS: A total of 113 resected SNs (five sebaceous adenomas, 10 sebaceomas and 98 SCs) from the Near-East were reviewed. Clinical information (age, gender, size and anatomical location), microscopic variables, and expression of several immunohistochemical markers (EMA, CK5/6, p63, p40, AR, p16 and p53) were documented. Cases were evaluated by fluorescently labelled PCR for HPV detection, followed by DNA microarray hybridization for subtype detection. RESULTS: HPV infection was detected in 9.4% of SNs: 28.6% sebaceous adenomas (HPV-16 and HPV-66), 9.1% sebaceomas (HPV-18) and 8.1% SCs. High-risk HPV types (HPV-16, -18, -52 and -66) constituted 90.9% of HPV infections. Histologically, HPV-positive SCs showed significantly milder cytologic atypia and patchy cellular necrosis. p16 was expressed in SNs irrespective of HPV status (20.0%, 33.3% and 65.5% of HPV-negative sebaceous adenomas, sebaceomas, and SCs, respectively), and p53 was abnormally expressed in 95.5% of HPV-negative SCs and all HPV-positive SCs. CONCLUSION: HPV infection is significantly present in benign and malignant SNs. HPV-positive SCs exhibit less cytologic atypia and necrosis than HPV-negative cases. p16 is not a surrogate marker of HPV infection in the SN setting. Further elucidation of various carcinogenic mechanisms in SCs will allow clinicians to single out the various populations at risk, optimize possible preventive strategies and develop targeted therapies.

Expanded genome-wide comparisons give novel insights into population structure and genetic heterogeneity of Leishmania tropica complex.

Leishmania tropica is one of the main causative agents of cutaneous leishmaniasis (CL). Population structures of L. tropica appear to be genetically highly diverse. However, the relationship between L. tropica strains genomic diversity, protein coding gene evolution and biogeography are still poorly understood. In this study, we sequenced the genomes of three new clinical L. tropica isolates, two derived from a recent outbreak of CL in camps hosting Syrian refugees in Lebanon and one historical isolate from Azerbaijan to further refine comparative genome analyses. In silico multilocus microsatellite typing (MLMT) was performed to integrate the current diversity of genome sequence data in the wider available MLMT genetic population framework. Single nucleotide polymorphism (SNPs), gene copy number variations (CNVs) and chromosome ploidy were investigated across the available 18 L. tropica genomes with a main focus on protein coding genes. MLMT divided the strains in three populations that broadly correlated with their geographical distribution but not populations defined by SNPs. Unique SNPs profiles divided the 18 strains into five populations based on principal component analysis. Gene ontology enrichment analysis of the protein coding genes with population specific SNPs profiles revealed various biological processes, including iron acquisition, sterols synthesis and drug resistance. This study further highlights the complex links between L. tropica important genomic heterogeneity and the parasite broad geographic distribution. Unique sequence features in protein coding genes identified in distinct populations reveal potential novel markers that could be exploited for the development of more accurate typing schemes to further improve our knowledge of the evolution and epidemiology of the parasite as well as highlighting protein variants of potential functional importance underlying L. tropica specific biology.

Lenalidomide in Combination with Arsenic Trioxide: an Effective Therapy for Primary Effusion Lymphoma.

Primary effusion lymphoma (PEL) is a rare aggressive subset of non-Hodgkin B cell lymphoma. PEL is secondary to Kaposi sarcoma herpes virus (KSHV) and predominantly develops in serous cavities. Conventional chemotherapy remains the treatment of choice for PEL and yields high response rates with no significant comorbidities. Yet, chemotherapy often fails in achieving or maintaining long-term remission. Lenalidomide (Lena), an immunomodulatory drug, displayed some efficacy in the treatment of PEL. On the other hand, arsenic trioxide (ATO) in combination with other agents effectively treated a number of blood malignancies, including PEL. In this study, we present evidence that the combination of ATO/Lena significantly enhanced survival of PEL mice, decreased the volume of exacerbated ascites in the peritoneum, and reduced tumor infiltration in organs of treated animals. In ex vivo treated PEL cells, ATO/Lena decreased the proliferation and downregulated the expression of KSHV latent viral proteins. This was associated with decreased NF-κB activation, resulting in reactivation of viral replication, downregulation of interleukin-6 (IL-6) and IL-10, inhibition of vascular endothelial growth factor, and apoptosis. Our results elucidate the mechanism of action of ATO/Lena and present it as a promising targeted therapeutic modality in PEL management, which warrants further clinical investigation.

Cutaneous leishmaniasis: an evolving disease with ancient roots.

BACKGROUND: Cutaneous leishmaniasis (CL) remains a prioritized neglected tropical disease. CL novel presentations call for updating its features. METHODS: A multiregional cohort of 396 patients with confirmed CL was reviewed. Lesion's clinical stage and eruption type were assigned. Disease was considered as extensive if numerous (≥5), large (>3 cm), disfiguring, threatening vital sensory organs, and/or older than 12 months. Microscopically, Ackerman's inflammatory pattern, Ridley's pattern (RP), and parasitic index (PI) were recorded. Microscopic variables pertaining to the organisms, epidermis, and host's inflammatory response were also assessed. All cases were confirmed and speciated molecularly. RESULTS: In our region, 71.8% of cases showed extensive disease with 15.7% exceeding 12 months duration. Leishmania tropica accounted for 91.3% of cases while Leishmania major constituted 8.7% and presented solely as dry lesions. The dominant inflammatory composite consisted of plasma cells, lymphocytes, and histiocytes. Granulomatous inflammation was present in 55.5%. Most cases showed interface changes (72.7%), spongiosis (75.3%), and marked epidermal hyperplasia (63.9%). Transepidermal elimination of organisms was present in 29.2% of cases. None of traditional classification patterns (clinical stage, microscopic pattern, and RP) showed the predicted linear correlation with lesion age. High and low PI levels correlated with early and healing microscopic patterns, respectively, but did not correlate with the corresponding RPs. PI was bimodal with peaks at 3-6 and 9-12 months. CONCLUSION: Cutaneous leishmaniasis is an evolving disease defying the traditional prediction classifications. Our study sets the ground for adopting updated clinical courses, microscopic presentation, and species mapping.

EAPB0503: An Imiquimod analog with potent in vitro activity against cutaneous leishmaniasis caused by Leishmania major and Leishmania tropica.

Cutaneous Leishmaniasis (CL) is a parasitic infection classified by the WHO as one of the most uncontrolled spreading neglected diseases. Syria is endemic for Leishmania tropica and Leishmania major, causing CL in the Eastern Mediterranean. The large-scale displacement of Syrian refugees exacerbated the spread of CL into neighboring countries. Therapeutic interventions against CL include local, systemic and physical treatments. The high risk for drug-resistance to current treatments stresses the need for new therapies. Imiquimod is an immunomodulatory drug with a tested efficacy against L. major species. Yet, Imiquimod efficacy against L. tropica and the molecular mechanisms dictating its potency are still underexplored. In this study, we characterized the effect of Imiquimod against L. tropica and L. major, and characterized the molecular mechanisms dictating its anti-leishmanial efficacy against both strains. We also investigated the potency and molecular mechanisms of an Imiquimod analog, EAPB0503, against these two strains. We have tested the effect of Imiquimod and EAPB0503 on macrophages infected with either L. major, L. tropica strains, or patient-derived freshly isolated L. tropica parasites. The anti-amastigote activity of either drugs was assessed by quantitative real time PCR (RT-PCR) using kinetoplast specific primers, confocal microscopy using the Glycoprotein 63 (Gp63) Leishmania amastigote antibody or by histology staining. The mechanism of action of either drugs on the canonical nuclear factor kappa- B (NF-κB) pathway was determined by western blot, and confocal microscopy. The immune production of cytokines upon treatment of infected macrophages with either drugs was assessed by ELISA. Both drugs reduced amastigote replication. EAPB0503 proved more potent, particularly on the wild type L. tropica amastigotes. Toll-Like Receptor-7 was upregulated, mainly by Imiquimod, and to a lesser extent by EAPB0503. Both drugs activated the NF-κB canonical pathway triggering an immune response and i-NOS upregulation in infected macrophages. Our findings establish Imiquimod as a strong candidate for treating L. tropica and show the higher potency of its analog EAPB0503 against CL.

Acute disseminated encephalomyelitis and reactivation of cerebral toxoplasmosis in a child: Case report.

Toxoplasma gondii is an opportunistic parasite that infects a broad range of hosts including humans. The chronic latent phase of the disease manifests as intra-neuronal cerebral cysts tightly controlled by the host immune system. In immunocompromised patients, reactivation of cerebral toxoplasmosis can have severe neurological outcomes that may sometimes lead to death. Despite the efficient prophylactic and treatment measures taken against the rare reactivation of cerebral toxoplasmosis, many reports including several recent ones revealed the still occurrence of this spectrum of disease. We present the case of a 4 years-6 months old apparently immunocompetent child whose premortem clinical presentation and investigations were highly consistent with severe acute disseminated encephalomyelitis (ADEM). The patient received all appropriate medications with initial improvement followed by rapid deterioration and death. Postmortem brain autopsy revealed a wide reactivation of cerebral toxoplasmosis. This is a peculiar case presentation as such medical treatment for ADEM (i.e. steroids) may worsen the Toxoplasma infection with ominous consequences. This case highlights the importance to rule out the possibility of such infections in apparently immunocompetent hosts by performing the appropriate investigations to prevent complications.

Fatal Visceral Leishmaniasis Caused by Leishmania infantum, Lebanon.

Visceral leishmaniasis, a fatal disease if not treated, is caused by Leishmania parasites. This disease might be overlooked in the Middle East because of limited awareness and low incidence. We report 5 patients who died of visceral leishmaniasis in Lebanon and make recommendations to improve faster diagnosis and treatment.

Toxoplasma gondii autophagy-related protein ATG9 is crucial for the survival of parasites in their host.

Autophagy is a conserved, life-promoting, catabolic process involved in the recycling of nonessential cellular components in response to stress. The parasite Toxoplasma gondii is an early-diverging eukaryote in which part of the autophagy machinery is not exclusively involved in a catabolic process but instead has been repurposed for an original function in organelle inheritance during cell division. This function, depending essentially on protein TgATG8 and its membrane conjugation system, is crucial for parasite survival and prevented an in depth study of autophagy in the mutants generated so far in Toxoplasma. Thus, in order to decipher the primary function of canonical autophagy in the parasites, we generated a cell line deficient for TgATG9, a protein thought to be involved in the early steps of the autophagy process. Although the protein proved to be dispensable for the development of these obligate intracellular parasites in vitro, the absence of TgATG9 led to a reduced ability to sustain prolonged extracellular stress. Importantly, depletion of the protein significantly reduced parasites survival in macrophages and markedly attenuated their virulence in mice. Altogether, this shows TgATG9 is important for the fate of Toxoplasma in immune cells and contributes to the overall virulence of the parasite, possibly through an involvement in a canonical autophagy pathway.