Optimized Case Finding of tuberculosis among key populations in Ukraine. A follow up study.

INTRODUCTION: In 2021, there were 4 million tuberculosis (TB) cases that were not detected by health systems, globally. Many of those cases are among hard-to-reach populations or key population groups. An Optimized Case Finding (OCF) strategy was introduced in Ukraine to enhance case detection and identify those "missing" cases. OCF included screening of up to eight referred household and social network contacts of an index TB case. Following the OCF project implementation, TB detection and characteristics of index cases and contacts were assessed. METHODOLOGY: A cohort study using project data (July 2018 - April 2022) was conducted. RESULTS: In total 7,976 close contacts were engaged in the project from 1,028 index TB cases. Among the contacts, 507 were diagnosed with TB. The TB case detection was 6,356/100,000 and the number needed to investigate was 16. Multiple factors were identified as associated with TB detection including smoking, HIV, poverty, etc. About 90% of cases were identified at the initial screening of the contacts. OCF was proven to be 5.8 times more effective than the standard active case finding using household surveys and 106 times more effective than passive case finding in the general public. CONCLUSIONS: Our study demonstrated the effectiveness of OCF in detecting cases among key population groups and their social networks. We encourage adaptation and use of OCF by civil society organizations that already work with key vulnerable populations around the globe.

CRISPR generation of CSF1R-G795A human microglia for robust microglia replacement in a chimeric mouse model.

Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been challenging. Here, we present a protocol to introduce the G795A point mutation using CRISPR-Cas9 into the CSF1R locus of human pluripotent stem cells. We also describe an optimized microglial differentiation technique for transplantation into newborn or adult recipients. We then detail pharmacological paradigms to achieve widespread and near-complete engraftment of human microglia. For complete details on the use and execution of this protocol, please refer to Chadarevian et al. (2023).1.

Novel Vaccine against Pathological Pyroglutamate-Modified Amyloid Beta for Prevention of Alzheimer's Disease.

Post-translationally modified N-terminally truncated amyloid beta peptide with a cyclized form of glutamate at position 3 (pE3Aß) is a highly pathogenic molecule with increased neurotoxicity and propensity for aggregation. In the brains of Alzheimer's Disease (AD) cases, pE3Aß represents a major constituent of the amyloid plaque. The data show that pE3Aß formation is increased at early pre-symptomatic disease stages, while tau phosphorylation and aggregation mostly occur at later stages of the disease. This suggests that pE3Aß accumulation may be an early event in the disease pathogenesis and can be prophylactically targeted to prevent the onset of AD. The vaccine (AV-1986R/A) was generated by chemically conjugating the pE3Aß3-11 fragment to our universal immunogenic vaccine platform MultiTEP, then formulated in AdvaxCpG adjuvant. AV-1986R/A showed high immunogenicity and selectivity, with endpoint titers in the range of 105-106 against pE3Aß and 103-104 against the full-sized peptide in the 5XFAD AD mouse model. The vaccination showed efficient clearance of the pathology, including non-pyroglutamate-modified plaques, from the mice brains. AV-1986R/A is a novel promising candidate for the immunoprevention of AD. It is the first late preclinical candidate which selectively targets a pathology-specific form of amyloid with minimal immunoreactivity against the full-size peptide. Successful translation into clinic may offer a new avenue for the prevention of AD via vaccination of cognitively unimpaired individuals at risk of disease.

Engineering an inhibitor-resistant human CSF1R variant for microglia replacement.

Hematopoietic stem cell transplantation (HSCT) can replace endogenous microglia with circulation-derived macrophages but has high mortality. To mitigate the risks of HSCT and expand the potential for microglia replacement, we engineered an inhibitor-resistant CSF1R that enables robust microglia replacement. A glycine to alanine substitution at position 795 of human CSF1R (G795A) confers resistance to multiple CSF1R inhibitors, including PLX3397 and PLX5622. Biochemical and cell-based assays show no discernable gain or loss of function. G795A- but not wildtype-CSF1R expressing macrophages efficiently engraft the brain of PLX3397-treated mice and persist after cessation of inhibitor treatment. To gauge translational potential, we CRISPR engineered human-induced pluripotent stem cell-derived microglia (iMG) to express G795A. Xenotransplantation studies demonstrate that G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand in the presence of PLX3397, replacing endogenous microglia to fully occupy the brain. In sum, we engineered a human CSF1R variant that enables nontoxic, cell type, and tissue-specific replacement of microglia.

Epidemiology of antimicrobial resistance in bacteria isolated from inpatient and outpatient samples, Ecuador, 2018 / Características epidemiológicas de la resistencia antimicrobiana en cepas bacterianas aisladas en pacientes de servicios hospitalarios y ambulatorios de Ecuador, 2018 / Epidemiologia da resistência aos antimicrobianos em bactérias isoladas de amostras hospitalares e ambulatoriais no Equador, 2018

ABSTRACT Objective. To compare the epidemiology of antimicrobial resistance in bacteria isolated from inpatient and outpatient samples in Ecuador. Methods. A secondary analysis was done of data on bacteria isolated from inpatient and outpatient samples. Data were taken from the 2018 national antimicrobial resistance surveillance database of the National Reference Center for Antimicrobial Resistance. The variables included were: age, sex, inpatient versus outpatient setting, type of specimen, bacterial species identified, pattern of resistance to antibiotics, and geographic area. Results. Data from 57 305 bacterial isolates were included in the study: 48.8% were from hospitalized patients, 55.7% were from women, and 60.1% were from patients older than 45 years. Urine (42.9%) and blood (12.4%) were the most common clinical samples. Overall, 77.1% of bacterial isolates were gram-negative (83% and 71% in outpatients and inpatients, respectively). The most common gram-positive and gram-negative species were Staphylococcus aureus and Escherichia coli, respectively. Antimicrobial resistance levels were high (up to 80% for some antimicrobial drugs), and were higher in hospitalized patients compared with outpatients. A variety of carbapenemases were found to confer resistance to carbapenems (antibiotics of last resort) in gram-negative bacteria. Conclusions. The study findings provide an important baseline on antimicrobial resistance in Ecuador. This will allow the strengthening of guidelines of the surveillance system, the creation of public policies for standardization of laboratory methodologies, the proper handling of information, and the development of empirical therapy guidelines based on local epidemiology.

RESUMEN Objetivo. Comparar las características epidemiológicas de la resistencia a los antimicrobianos en cepas bacterianas aisladas de muestras de pacientes de servicios hospitalarios y ambulatorios en Ecuador. Métodos. Se realizó un análisis secundario de los datos sobre cepas bacterianas aisladas en muestras de pacientes de servicios hospitalarios y ambulatorios. Se recogieron los datos de la base de datos nacional del 2018 para la vigilancia de la resistencia a los antimicrobianos del Centro de Referencia Nacional para la Resistencia a los Antimicrobianos. Las variables incluidas fueron: edad, sexo, entorno hospitalario frente a entorno ambulatorio, tipo de muestra, especies bacterianas detectadas, patrón de resistencia a los antibióticos y zona geográfica. Resultados. En el estudio se incluyeron datos de 57 305 cepas aislamientos bacterianos: 48,8% fueron de pacientes hospitalizados, 55,7% fueron de mujeres y 60,1% fueron de pacientes mayores de 45 años. La orina (42,9%) y la sangre (12,4%) fueron las muestras clínicas más comunes. En general, 77,1% de las cepas bacterianas aisladas fueron gramnegativas (83% y 71% en pacientes de servicios ambulatorios y hospitalarios, respectivamente). Las especies grampositivas y gramnegativas más comunes fueron Staphylococcus aureus y Escherichia coli, respectivamente. Los niveles de resistencia a los antimicrobianos fueron elevados (hasta 80% en el caso de algunos fármacos antimicrobianos) y fueron más elevados en los pacientes de servicios hospitalarios en comparación con los pacientes de servicios ambulatorios. Se encontró que una variedad de carbapenemasas confiere resistencia a los carbapenémicos (antibióticos de último recurso) en bacterias gramnegativas. Conclusiones. Los resultados del estudio proporcionan una línea de base importante sobre la resistencia a los antimicrobianos en Ecuador, que permitirá el fortalecimiento de las directrices del sistema de vigilancia, la creación de políticas públicas para la estandarización de los métodos de laboratorio, una adecuada gestión de la información y la elaboración de orientaciones de tratamiento empírico basadas en las características epidemiológicas locales.

RESUMO Objetivo. Comparar a epidemiologia da resistência aos antimicrobianos em bactérias isoladas de amostras hospitalares e ambulatoriais no Equador. Métodos. Foi feita uma análise secundária de dados sobre bactérias isoladas de amostras hospitalares e ambulatoriais. Os dados foram obtidos do banco de dados nacional de vigilância da resistência aos antimicrobianos de 2018 do Centro Nacional de Referência para a Resistência aos Antimicrobianos. As variáveis incluídas foram: idade, sexo, ambiente hospitalar versus ambiente ambulatorial, tipo de espécime, espécies bacterianas identificadas, padrão de resistência a antibióticos e área geográfica. Resultados. Foram incluídos no estudo os dados de 57 305 isolados bacterianos: 48,8% eram de pacientes hospitalizados, 55,7% eram de mulheres e 60,1% eram de pacientes com mais de 45 anos. As amostras clínicas mais comuns foram urina (42,9%) e sangue (12,4%). No total, 77,1% dos isolados bacterianos eram gram-negativos (83% e 71% em pacientes ambulatoriais e pacientes internados, respectivamente). As espécies gram-positivas e gram-negativas mais comuns foram Staphylococcus aureus e Escherichia coli, respectivamente. Os níveis de resistência aos antimicrobianos foram elevados (até 80% para alguns antimicrobianos) e foram mais elevados em pacientes hospitalizados em comparação com pacientes ambulatoriais. Foram encontradas várias carbapenemases que conferem resistência aos carbapenêmicos (antibióticos de último recurso) em bactérias gram-negativas. Conclusões. Os resultados do estudo fornecem uma importante linha de base sobre a resistência aos antimicrobianos no Equador. Isto permitirá o fortalecimento das diretrizes do sistema de vigilância, a criação de políticas públicas para padronização de metodologias laboratoriais, o manejo adequado de informações e o desenvolvimento de diretrizes para a antibioticoterapia empírica com base na epidemiologia local.

Exosome loaded immunomodulatory biomaterials alleviate local immune response in immunocompetent diabetic mice post islet xenotransplantation.

Foreign body response (FBR) to biomaterials compromises the function of implants and leads to medical complications. Here, we report a hybrid alginate microcapsule (AlgXO) that attenuated the immune response after implantation, through releasing exosomes derived from human Umbilical Cord Mesenchymal Stem Cells (XOs). Upon release, XOs suppress the local immune microenvironment, where xenotransplantation of rat islets encapsulated in AlgXO led to >170 days euglycemia in immunocompetent mouse model of Type 1 Diabetes. In vitro analyses revealed that XOs suppressed the proliferation of CD3/CD28 activated splenocytes and CD3+ T cells. Comparing suppressive potency of XOs in purified CD3+ T cells versus splenocytes, we found XOs more profoundly suppressed T cells in the splenocytes co-culture, where a heterogenous cell population is present. XOs also suppressed CD3/CD28 activated human peripheral blood mononuclear cells (PBMCs) and reduced their cytokine secretion including IL-2, IL-6, IL-12p70, IL-22, and TNFα. We further demonstrate that XOs mechanism of action is likely mediated via myeloid cells and XOs suppress both murine and human macrophages partly by interfering with NFκB pathway. We propose that through controlled release of XOs, AlgXO provide a promising new platform that could alleviate the local immune response to implantable biomaterials.

Effectiveness and safety of fully oral modified shorter treatment regimen for multidrug-resistant tuberculosis in Georgia, 2019-2020.

Tuberculosis treatment is challenging, especially among people with drug-resistant forms of tuberculosis. The introduction of fully oral modified short treatment regimen has a great potential to shorten duration of treatment, improve safety and ultimately increase treatment success rate. In 2019 Georgia has piloted the modified fully oral shorter treatment regimen in a routine programmatic condition. Our study aimed to evaluate effectiveness and safety of the modified shorter treatment regimen in Georgia among the first 25 consecutively enrolled patients with rifampicin-resistant tuberculosis with proven sensitivity to fluoroquinolone and without prior exposure to second-line tuberculosis drugs. Regimen consisted of 9-month daily administration of bedaquilline, linezolid, levofloxacin, clofazimine and cycloserine. Study patients were enrolled between March-August 2019. We used a national electronic surveillance system, medical records and active TB drug-safety monitoring and management database to extract study related data. The mean age of the study participants was 48 years, 68% were male, 8% were HIV positive, 16% had diabetes and 12% tested positive for hepatitis C infection. The median time to culture conversion among 16 patients who were culture positive at treatment initiation was 1.0 (95% CI: 1.0-2.0) month. Of those, by the end of treatment 15 patients converted to negative. Out of the 25 patients in the study cohort 22 (88%) had successful treatment outcome, one patient (4%) died and two (8%) were lost to follow up. The regimen was largely well tolerated. Three patients (12%) experienced serious adverse events, of which in two cases were possibly related to TB drugs in the regimen. Seven patients developed adverse events of interest in eight instances, including musculoskeletal (twice), psychiatric, gastrointestinal disorders, hepatotoxicity, peripheral neuropathy, cardiotoxicity and myelosuppression (once each). In four patients (16%) the duration of the treatment was extended beyond nine months due to insufficient radiological improvements. Our findings demonstrate that good treatment outcomes are achievable in people with fluoroquinolone-sensitive tuberculosis within routine programmatic conditions using fully oral modified short treatment regimen. The extensive use of fully oral modified shorter treatment regimen in Georgia and other high priority countries in the World Health Organization European Region is warranted.

People with high HIV viral load within risk networks: who are these people and who refers them best?

Introduction: Viral load is one of the most important determinants for HIV transmission. Identification of people with high viral load (PHVL) can be effective in limiting onward HIV transmission. In order to improve the identification of these individuals within risk networks, we determined a) the number of PHVL recruited through risk networks b) their socio-demographic, behavioural and clinical characteristics and c) the characteristics of individuals who referred these PHVL to the study. Methodology: From November 2013 to March 2016, in Odessa, Ukraine, Transmission Reduction Intervention Project (TRIP) was implemented to identify people recently infected with HIV within the risk networks of "seeds" and "venues" where they engaged in risk behaviour. Results: TRIP identified 53 PHVL, of whom 32 (60%) injected drugs; 42 (79%) were unaware of their HIV status; 25 (47%) had more than one sex partner, and only 14 (26%) were using condoms. There were 164 people who referred individuals into the study; 33 of them (20%) referred PHVL. In terms of referrers, those with lower than secondary level of education, not living with a sex partner, and reporting regular condom use were significantly more likely (p < 0.05) to refer PHVL. Most PHVL (38, 72%) and their referrers (27, 82%) were found through venues. Conclusions: In Odessa city, PHVL are at high risk of transmitting HIV as the majority inject drugs, do not know their HIV status, and have unprotected sex and/or multiple partners. Targeting these individuals for HIV prevention, harm reduction and initiation of antiretroviral treatment (ART) is urgent.

Development of a Chimeric Model to Study and Manipulate Human Microglia In Vivo.

iPSC-derived microglia offer a powerful tool to study microglial homeostasis and disease-associated inflammatory responses. Yet, microglia are highly sensitive to their environment, exhibiting transcriptomic deficiencies when kept in isolation from the brain. Furthermore, species-specific genetic variations demonstrate that rodent microglia fail to fully recapitulate the human condition. To address this, we developed an approach to study human microglia within a surrogate brain environment. Transplantation of iPSC-derived hematopoietic-progenitors into the postnatal brain of humanized, immune-deficient mice results in context-dependent differentiation into microglia and other CNS macrophages, acquisition of an ex vivo human microglial gene signature, and responsiveness to both acute and chronic insults. Most notably, transplanted microglia exhibit robust transcriptional responses to Aß-plaques that only partially overlap with that of murine microglia, revealing new, human-specific Aß-responsive genes. We therefore have demonstrated that this chimeric model provides a powerful new system to examine the in vivo function of patient-derived and genetically modified microglia.

Deletion of a Csf1r enhancer selectively impacts CSF1R expression and development of tissue macrophage populations.

The proliferation, differentiation and survival of mononuclear phagocytes depend on signals from the receptor for macrophage colony-stimulating factor, CSF1R. The mammalian Csf1r locus contains a highly conserved super-enhancer, the fms-intronic regulatory element (FIRE). Here we show that genomic deletion of FIRE in mice selectively impacts CSF1R expression and tissue macrophage development in specific tissues. Deletion of FIRE ablates macrophage development from murine embryonic stem cells. Csf1rΔFIRE/ΔFIRE mice lack macrophages in the embryo, brain microglia and resident macrophages in the skin, kidney, heart and peritoneum. The homeostasis of other macrophage populations and monocytes is unaffected, but monocytes and their progenitors in bone marrow lack surface CSF1R. Finally, Csf1rΔFIRE/ΔFIRE mice are healthy and fertile without the growth, neurological or developmental abnormalities reported in Csf1r-/- rodents. Csf1rΔFIRE/ΔFIRE mice thus provide a model to explore the homeostatic, physiological and immunological functions of tissue-specific macrophage populations in adult animals.

Strategies for active detection of tuberculosis in Ukraine: Comparative effectiveness amongst key populations (2014-2018).

INTRODUCTION: Ukraine has gaps in Tuberculosis (TB) service coverage, especially in key populations (KPs). We compared effectiveness of three different strategies for active TB detection among KPs and their linkage to TB treatment during three time periods. METHODOLOGY: The KPs included people who inject drugs (PWID), sex workers (SW), men who have sex with men (MSM) and groups at-risk of TB (ex-prisoners, Roma and homeless). The active case finding included decentralized symptom screening and specimen collection (2014, strategy-1), decentralized screening with patient referred for specimen collection (2015-2017, strategy-2) and strategy-2 plus GeneXpert (2018, strategy-3). RESULTS: In total 680,760 KPs were screened, of whom 68% were PWID. TB case detection per 100,000 populations was 1,191 in strategy-1, 302 in strategy-2, and 235 in strategy-3. The number needed to screen (NNS) to identify one case was respectively 84, 332, and 425. TB detection was highest among homeless (range: 1,839-2,297 per 100,000 population). The lowest detection was among the MSM and SW. Between 2014 and 2018, 82-94% of all diagnosed TB patients in KPs started TB treatment. CONCLUSIONS: The active case finding in KPs increased detection of TB cases in Ukraine, and the majority of diagnosed KPs initiated TB treatment. Centralization of diagnosis reduced the effectiveness of TB screening. Each region in Ukraine should assess the composition and the needs of KPs which will allow for adoption of specific strategies to detect TB among KPs with high TB prevalence.

Development and validation of a simplified method to generate human microglia from pluripotent stem cells.

BACKGROUND: Microglia, the principle immune cells of the brain, play important roles in neuronal development, homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease, we, and others, have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology, labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore, we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia. RESULTS: iPSCs are first differentiated toward a mesodermal, hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43+ hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF, IL-34, and TGFß-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation, complex media formulation, FACS sorting, or co-culture, thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia, we performed RNA-sequencing, functional testing, and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGFß signaling (IDE1) can be used to replace recombinant TGFß1, further reducing costs, we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach, although transcriptional differences do occur that should be considered. CONCLUSION: We anticipate that this new and greatly simplified protocol will enable many interested labs, including those with little prior stem cell or flow cytometry experience, to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation, the field will continue to improve our understanding of microglial biology and their important roles in human development, homeostasis, and disease.

MultiTEP platform-based DNA vaccines for alpha-synucleinopathies: preclinical evaluation of immunogenicity and therapeutic potency.

We have previously demonstrated that anti-beta amyloid DNA vaccine (AV-1959D) based on our proprietary MultiTEP platform technology is extremely immunogenic in mice, rabbits, and monkeys. Importantly, MultiTEP platform enables development of vaccines targeting pathological molecules involved in various neurodegenerative disorders. Taking advantage of the universality of MultiTEP platform, we developed DNA vaccines targeting 3 B-cell epitopes (amino acids [aa]85-99, aa109-126, and aa126-140) of human alpha-synuclein (hα-Syn) separately or all 3 epitopes simultaneously. All 4 DNA vaccines (1) generate high titers of anti-hα-Syn antibodies and (2) induce robust MultiTEP-specific T-helper cell responses without activation of potentially detrimental autoreactive anti-hα-Syn T-helper cells. Generated antibodies recognize misfolded hα-Syn produced by neuroblastoma cells, hα-Syn in the brain tissues of transgenic mouse strains and in the brain tissues of dementia with Lewy body cases. Based on these results, the most promising vaccine targeting 3 B-cell epitopes of hα-Syn simultaneously (PV-1950D) has been chosen for ongoing preclinical assessment in mouse models of hα-Syn with the aim to translate it to the human clinical trials.

MultiTEP platform-based DNA epitope vaccine targeting N-terminus of tau induces strong immune responses and reduces tau pathology in THY-Tau22 mice.

BACKGROUND: By the time clinical symptoms of Alzheimer's disease (AD) manifest in patients there is already substantial tau pathology in the brain. Recent evidence also suggests that tau pathology can become self-propagating, further accelerating disease progression. Over the last decade several groups have tested the efficacy of protein-based anti-tau immunotherapeutics in various animal models of tauopathy. Here we report on the immunological and therapeutic potency of the first anti-tau DNA vaccine based on the MultiTEP platform, AV-1980D, in THY-Tau22 transgenic mice. METHODS: Starting at 3months of age, mice were immunized intramuscularly with AV-1980D vaccine targeting a tau B cell epitope spanning aa2-18 followed by electroporation (EP). Humoral and cellular immune responses in vaccinated animals were analyzed by ELISA and ELISpot, respectively. Neuropathological changes in the brains of experimental and control mice were then analyzed by biochemical (WB and ELISA) and immunohistochemical (IHC) methods at 9months of age. RESULTS: EP-mediated AV-1980D vaccinations of THY-Tau22 mice induced activation of Th cells specific to the MultiTEP vaccine platform and triggered robust humoral immunity response specific to tau. Importantly, no activation of potentially harmful autoreactive Th cell responses specific to endogenous tau species was detected. The maximum titers of anti-tau antibodies were reached after two immunizations and remained slightly lower, but steady during five subsequent monthly immunizations. Vaccinations with AV-1980D followed by EP significantly reduced total tau and pS199 and AT180 phosphorylated tau levels in the brains extracts of vaccinated mice, but produced on subtle non-significant effects on other phosphorylated tau species. CONCLUSIONS: These data demonstrate that MultiTEP-based DNA epitope vaccination targeting the N-terminus of tau is highly immunogenic and therapeutically potent in the THY-Tau22 mouse model of tauopathy and indicate that EP-mediated DNA immunization is an attractive alternative to protein-based adjuvanted vaccines for inducing high concentrations of anti-tau antibodies.

Human Neural Progenitor Transplantation Rescues Behavior and Reduces α-Synuclein in a Transgenic Model of Dementia with Lewy Bodies.

Synucleinopathies are a group of neurodegenerative disorders sharing the common feature of misfolding and accumulation of the presynaptic protein α-synuclein (α-syn) into insoluble aggregates. Within this diverse group, Dementia with Lewy Bodies (DLB) is characterized by the aberrant accumulation of α-syn in cortical, hippocampal, and brainstem neurons, resulting in multiple cellular stressors that particularly impair dopamine and glutamate neurotransmission and related motor and cognitive function. Recent studies show that murine neural stem cell (NSC) transplantation can improve cognitive or motor function in transgenic models of Alzheimer's and Huntington's disease, and DLB. However, examination of clinically relevant human NSCs in these models is hindered by the challenges of xenotransplantation and the confounding effects of immunosuppressant drugs on pathology and behavior. To address this challenge, we developed an immune-deficient transgenic model of DLB that lacks T-, B-, and NK-cells, yet exhibits progressive accumulation of human α-syn (h-α-syn)-laden inclusions and cognitive and motor impairments. We demonstrate that clinically relevant human neural progenitor cells (line CNS10-hNPCs) survive, migrate extensively and begin to differentiate preferentially into astrocytes following striatal transplantation into this DLB model. Critically, grafted CNS10-hNPCs rescue both cognitive and motor deficits after 1 and 3 months and, furthermore, restore striatal dopamine and glutamate systems. These behavioral and neurochemical benefits are likely achieved by reducing α-syn oligomers. Collectively, these results using a new model of DLB demonstrate that hNPC transplantation can impact a broad array of disease mechanisms and phenotypes and suggest a cellular therapeutic strategy that should be pursued. Stem Cells Translational Medicine 2017;6:1477-1490.

Tuberculosis treatment and Smoking, Armenia, 2014-2016.

INTRODUCTION: Tuberculosis and tobacco prove to be increasingly apparent world problems. Armenia is a developing country which is facing issues related to the high rates of tobacco consumption. Moreover, it is among the list of high multi-drug resistant (MDR) Tuberculosis TB burden countries. Treatment success rate in Armenia for sputum smear-positive cases never reached World Health Organization's (WHO) target of 85% in last 15 years. Data from different studies completed across the world suggests that there is an association between smoking and negative treatment outcomes. METHODS: This retrospective study was designed to investigate aforementioned associations between TB treatment outcomes and smoking status of TB patients. Data for the study were derived from the national data available in the electronic database of the Armenian National TB Center. RESULTS: Based on inclusion and exclusion criteria 992 TB patients registered in 2014 were enrolled in this study. All of them are were TB patients in which 387 were smokers and 605 were non-smokers. Notably, adjusted analysis showed that individuals who smoked during TB treatment had 1.61 higher odds of having unsuccessful TB treatment outcome. Additionally, consistent with the literature, statistically significant association was identified between TB treatment outcome and other well factors such as sputum smear status (OR = 2.24, p < 0.01), HIV status (OR, = 1.87, p < 0.01) of patients, etc. CONCLUSIONS: The smoking, HIV positive status, positive sputum smear microscopy test were identified as an important factors associated with the unsuccessful TB treatment outcome in Armenia. It highlights the necessity of having specific restrictions and campaign programs to reduce smoking rates among TB patients in order to improve current TB treatment and care services throughout Armenia.

Targeting TLR-4 with a novel pharmaceutical grade plant derived agonist, Immunomax®, as a therapeutic strategy for metastatic breast cancer.

BACKGROUND: Previously we demonstrated that the resection of primary 4T1 tumors only slightly prolongs mouse survival, but importantly, creates a "window of opportunity" with attenuated suppressor cell and increased activated T cell populations. This suggests that additional activation of the immune system by immunostimulatory agents during this period may enhance anti-tumor immunity and potentially eradicate micro-metastatic disease in this stringent model. We hypothesized that the immunostimulator Immunomax®, which is comprised of a plant-derived polysaccharide, is non-toxic in humans and stimulates immune defense during the infectious diseases treatment, may have also anti-tumor activity and be beneficial in the adjuvant setting when endogenous anti-tumor responses are present and during the "window of opportunity" in post-resection metastatic breast cancer model. Here we provide the initial report that Immunomax® demonstrates the capacity to eliminate micro-metastatic disease in the post-resection, 4T1 mouse model of breast cancer. METHODS: The efficacy of Immunomax® was evaluated by analyzing survival rate and the number of spontaneous clonogenic tumor cells in the lung homogenates of mice. The frequencies of activated NK, CD4(+) and CD8(+) cells as well as myeloid-derived suppressor cells and Treg cells were evaluated using flow cytometry. Highly purified mouse and human dendritic and NK cells were sorted and the effect of Immunomax® on activation status of these cells was assessed by flow cytometry. The property of Immunomax® as TLR-4 agonist was determined by NF-κB/SEAP reporter gene assay, WB, RT-PCR. RESULTS: Immunomax® injections significantly prolonged overall survival and cured 31% of mice. This immunostimulator activates DCs via the TLR-4, which in turn stimulates tumoricidal NK cells and in vitro, completely inhibits growth of 4T1 cells. Incubation of PBMC from healthy donors with Immunomax® activates NK cells via activation of plasmacytoid DC leading significantly higher efficacy in killing of human NK-target cells K562 compared with non-treated cells. CONCLUSION: This is the first demonstration that Immunomax® is a TLR-4 agonist and the first report of a documented role for this pharmaceutical grade immunostimulator in augmenting anti-tumor activity, suggesting that incorporation of Immunomax® into developing breast cancer therapeutic strategies may be beneficial and with less potential toxicity than checkpoint inhibitors.

Primary 4T1 tumor resection provides critical "window of opportunity" for immunotherapy.

It is believed that primary tumor resection modulates host-tumor immune interaction, but this has not been characterized in a stringent breast cancer tumor model. This report, using the 4T1 murine mammary tumor model, characterizes for the first time the dynamic longitudinal changes in immunosuppressive and effector components of the immune system after resection of an established orthotopic primary tumor with a defined natural history of developing lung metastases. More specifically, we analyzed changes of absolute numbers and frequencies of MDSC, regulatory T cells (Treg), as well as activated CD4 and CD8 positive T cells in spleens and, in some studies, lungs of 4T1 tumor-bearing mice and mice after primary tumor resection. Importantly, using mathematical analyses we established that primary resection of an orthotopic tumor had created a "window of opportunity" with decreased tumor-associated immune suppression that existed for approximately 10 days. Although tumor resection did slightly prolong survival, it did not affect the ultimate development of metastatic disease since animals with resected tumors or intact primary tumors eventually died by day 47 and 43, respectively. This window of opportunity likely occurs in humans providing a rationale and parameters for integration and testing of immunotherapeutic strategies in this critical "window of opportunity" to combat the development of metastatic disease.

Epitope-based DNA vaccine for Alzheimer's disease: translational study in macaques.

BACKGROUND: Clinical trials with passive and active Alzheimer's disease (AD) vaccines suggest that early interventions are needed for improvement of cognitive and/or functional performance in patients, providing impetus for the development of safe and immunologically potent active vaccines targeting amyloid ß (Aß). The AN-1792 trial has indicated that Aß-specific T cells may be unsafe for humans; therefore, other vaccines based on small Aß epitopes are undergoing preclinical and clinical testing. METHODS: Humoral and cellular immune responses elicited in response to a novel DNA epitope-based vaccine (AV-1955) delivered to rhesus macaques using the TriGrid electroporation device were evaluated. Functional activities of anti-Aß antibodies generated in response to vaccination were assessed in vitro. RESULTS: AV-1955 generates long-term, potent anti-Aß antibodies and cellular immune responses specific to foreign T-helper epitopes but not to self-Aß. CONCLUSIONS: This translational study demonstrates that a DNA-based epitope vaccine for AD could be appropriate for human clinical testing.

A transgenic Alzheimer rat with plaques, tau pathology, behavioral impairment, oligomeric aß, and frank neuronal loss.

Alzheimer's disease (AD) is hallmarked by amyloid plaques, neurofibrillary tangles, and widespread cortical neuronal loss (Selkoe, 2001). The "amyloid cascade hypothesis" posits that cerebral amyloid sets neurotoxic events into motion that precipitate Alzheimer dementia (Hardy and Allsop, 1991). Yet, faithful recapitulation of all AD features in widely used transgenic (Tg) mice engineered to overproduce Aß peptides has been elusive. We have developed a Tg rat model (line TgF344-AD) expressing mutant human amyloid precursor protein (APPsw) and presenilin 1 (PS1ΔE9) genes, each independent causes of early-onset familial AD. TgF344-AD rats manifest age-dependent cerebral amyloidosis that precedes tauopathy, gliosis, apoptotic loss of neurons in the cerebral cortex and hippocampus, and cognitive disturbance. These results demonstrate progressive neurodegeneration of the Alzheimer type in these animals. The TgF344-AD rat fills a critical need for a next-generation animal model to enable basic and translational AD research.