Major Subpopulations Research Articles

Single-cell RNA sequencing reveals the cellular heterogeneity of aneurysmal infrarenal abdominal aorta.

The artery contains numerous cell types which contribute to multiple vascular diseases. However, the heterogeneity and cellular responses of these vascular cells during abdominal aortic aneurysm (AAA) progression have not been well characterized. Single-cell RNA sequencing was performed on the infrarenal abdominal aortas (IAAs) from C57BL/6J mice at Days 7 and 14 post-sham or peri-adventitial elastase-induced AAA. Unbiased clustering analysis of the transcriptional profiles from >4500 aortic cells identified 17 clusters representing nine-cell lineages, encompassing vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells, immune cells (macrophages, T cells, B cells, and dendritic cells), and two types of rare cells, including neural cells and erythrocyte cells. Seurat clustering analysis identified four smooth muscle cell (SMC) subpopulations and five monocyte/macrophage subpopulations, with distinct transcriptional profiles. During AAA progression, three major SMC subpopulations were proportionally decreased, whereas the small subpopulation was increased, accompanied with down-regulation of SMC contractile markers and up-regulation of pro-inflammatory genes. Another AAA-associated cellular response is immune cell expansion, particularly monocytes/macrophages. Elastase exposure induced significant expansion and activation of aortic resident macrophages, blood-derived monocytes and inflammatory macrophages. We also identified increased blood-derived reparative macrophages expressing anti-inflammatory cytokines suggesting that resolution of inflammation and vascular repair also persist during AAA progression. Our data identify AAA disease-relevant transcriptional signatures of vascular cells in the IAA. Furthermore, we characterize the heterogeneity and cellular responses of VSMCs and monocytes/macrophages during AAA progression, which provide insights into their function and the regulation of AAA onset and progression.

Detection and Differentiation of Bacterial and Fungal Infection of Neutrophils from Peripheral Blood Using Raman Spectroscopy.

Neutrophils are important cells of the innate immune system and the major leukocyte subpopulation in blood. They are responsible for recognizing and neutralizing invading pathogens, such as bacteria or fungi. For this, neutrophils are well equipped with pathogen recognizing receptors, cytokines, effector molecules, and granules filled with reactive oxygen species (ROS)-producing enzymes. Depending on the pathogen type, different reactions are triggered, which result in specific activation states of the neutrophils. Here, we aim to establish a label-free method to indirectly detect infections and to identify the cause of infection by spectroscopic characterization of the neutrophils. For this, isolated neutrophils from human peripheral blood were stimulated in an in vitro infection model with heat-inactivated Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacterial pathogens as well as with heat-inactivated and viable fungi (Candida albicans). Label-free and nondestructive Raman spectroscopy was used to characterize neutrophils on a single cell level. Phagocytized fungi could be visualized in a few high-resolution false color images of individual neutrophils using label-free Raman spectroscopic imaging. Using a high-throughput screening Raman spectroscope (HTS-RS), Raman spectra of more than 2000 individual neutrophils from three different donors were collected in each treatment group, yielding a data set of almost 20 000 neutrophil spectra. Random forest classification models were trained to differentiate infected and noninfected cells with high accuracy (90%). Among the neutrophils challenged with pathogens, even the cause of infection, bacterial or fungal, was predicted correctly with 92% accuracy. Therefore, Raman spectroscopy enables reliable neutrophil phenotyping and infection diagnosis in a label-free manner. In contrast to the microbiological diagnostic standard, where the pathogen is isolated in time-consuming cultivation, this Raman-based method could potentially be blood-culture independent, thus saving precious time in bloodstream infection diagnostics.

Input dependent modulation of olfactory bulb activity by HDB GABAergic projections

Basal forebrain modulation of central circuits is associated with active sensation, attention, and learning. While cholinergic modulations have been studied extensively the effect of non-cholinergic basal forebrain subpopulations on sensory processing remains largely unclear. Here, we directly compare optogenetic manipulation effects of two major basal forebrain subpopulations on principal neuron activity in an early sensory processing area, i.e. mitral/tufted cells (MTCs) in the olfactory bulb. In contrast to cholinergic projections, which consistently increased MTC firing, activation of GABAergic fibers from basal forebrain to the olfactory bulb leads to differential modulation effects: while spontaneous MTC activity is mainly inhibited, odor-evoked firing is predominantly enhanced. Moreover, sniff-triggered averages revealed an enhancement of maximal sniff evoked firing amplitude and an inhibition of firing rates outside the maximal sniff phase. These findings demonstrate that GABAergic neuromodulation affects MTC firing in a bimodal, sensory-input dependent way, suggesting that GABAergic basal forebrain modulation could be an important factor in attention mediated filtering of sensory information to the brain.

1973-P: Single-Nuclei Transcriptomics of Human Adipose Tissue Identify Distinct Adipocyte Progenitor Subpopulations in Type 2 Diabetes

Altered adipocyte function underlies type 2 diabetes (DM) pathogenesis. We hypothesized that adipocyte dysfunction in visceral adipose tissue (VAT) in DM originates from distinct adipocyte progenitor cell (APC) subpopulations. VAT was collected from bariatric surgery patients with (HbA1c>6.5) and without DM (NDM, HbA1c<5.7). Single-nuclei RNA sequencing was performed in VAT nuclei (n= 1 NDM, 2 DM) using 10X Genomics. Transcriptionally distinct cell subpopulations were identified by DEG: mature adipocytes (ADIPOQ, LEP), endothelial cells (VWR), immune cells (F13A1, MSR1), and two major APC subpopulations (S1: FABP4, ADIRF, ITLN1 and MSLN; S2: APOD, CFD and WISP2). S2 was uniquely identified by the upregulation of extracellular matrix accumulation and fibrosis markers: LUM, DCN, FBN1, and COL1A1, 3A1, 6A1 and 6A3. S2 had higher expression of APC and committed preadipocyte markers, such as PLIN1, GNAI1, CXCL14, CP, and CRISPLD2, while S1 had higher mesothelial-like cell markers GPM6A, PKHD1L1, LRRN4, CXADR, SLPI, and UPK3B. We FACS-isolated S1 and S2 from VAT CD31-CD45- cells (n= 3 NDM, 5 DM) using the surface marker TM4SF1, which was abundantly expressed in S1, but not S2. TM4SF1 expression (qPCR) was 50% higher in S1 (TM4SF1+) than in S2 (TM4SF1-). TM4SF1 immunostaining of human SVF confirmed distinct APC subpopulations. S1 corresponded to 15 ± 3% and S2 to 69 ± 2% of CD31-CD45- cells. In DM compared to NDM, S2 fraction was increased by 12% while S1 was decreased by 45%. We tested the metabolic function of S1 and S2 sorted from 5 DM VAT. Both subpopulations differentiated into lipid-filled adipocytes upon adipogenic induction in vitro. However, S2 compared to S1, manifested decreased proliferation and increased intracellular lipid accumulation and lipolytic response, consistent with a pro-hypertrophic, lipolytic adipocyte phenotype in DM. Our data suggest that distinct APC subpopulations in human obese VAT contribute to adipose tissue dysfunction in DM. Disclosure C. Strieder-Barboza: None. C.G. Flesher: None. L.M. Geletka: None. R.W. ORourke: None. C.N. Lumeng: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK115190, DK090262)

The Role of Human γδ T Cells in Anti-Tumor Immunity and Their Potential for Cancer Immunotherapy.

γδ T cells are a distinct subset of T cells whose T cell receptors consist of γ chains and δ chains, different from conventional αβ T cells. γδ T cells are considered as a member of the innate immunity because of their non-MHC restricted antigen recognition, rapid response to invading pathogens and sense early changes of malignant cells. Upon activation, they can further promote the activation of adaptive immune cells, such as T cells and B cells, by secreting various cytokines. Thus, γδ T cells are regarded as a bridge between innate immunity and acquired immunity. γδ T cells are involved in a variety of immune response processes, including immune defense and immune surveillance against infection and tumorigenesis. γδ T cells recognize multiple tumor-associated antigens or molecules in T cell receptors (TCRs)-dependent and natural killer cell receptors (NKRs)-dependent ways. γδ T cells not only display a direct killing capacity on a variety of tumors, but also exert anti-tumor immune responses indirectly by facilitating the function of other immune cells, such as dendritic cells (DCs), B cells and CD8+ T cells. In this review, we summarize the major subpopulations, the tumor recognition mechanisms, and the anti-tumor effects of human γδ T cells, particularly the potential of γδ T cells for cancer immunotherapy.

Circulating Low Density Neutrophils In Advanced Lung Cancer Patients Exhibit Unique Immune Signatures and affect prognosis

Abstract The role of innate immunity, in especially neutrophils, in supporting anti-tumor immunity in lung cancer is understudied. Neutrophil "flavors", such as low-density neutrophils (LDN) and high-density neutrophils (HDNs), have been described in cancer patients and associated with tumor-supportive properties. Nevertheless, HDN and LDN phenotypic properties and their cancer-driven modulations are still largely unknown. Here, we aimed to evaluate the clinical relevance and significance of circulating LDN in lung cancer patients, and by using data-driven artificial intelligence supported analysis tools evaluated phenotypic states of circulating neutrophils subsets during lung cancer progression. COPD patients and healthy volunteers served as controls. Using mass cytometry (CyTOF), we aimed to identify major sub-populations within circulating LDN and HDN subsets and determine a possible change in phenotype due to cancer. LDN were highly enriched in advanced lung cancer patients (12.9±2.4%), but not in early stage patients (1.2±0.2%), healthy individuals (1.3±1.1%), or stable COPD patients (2.2±0.6%). Elevated LDN (>10% of LDF) showed a remarkable correlation with poorer prognosis in advanced patients. Our CyTOF analysis revealed the presence of three main neutrophil subsets with a great variability in the presence of CD10+ vs. CD10− LDNs between patients. We further demonstrated that both HDN and LDN retain a degree of inherent spontaneous plasticity. Deep characterization of cancer-related circulating neutrophils, as well as their modulation along tumor progression, will assist in suggesting new biomarkers and novel targets to improve response to current conventional therapies and immunotherapies.

Multilocus sequence typing of Enterocytozoon bieneusi in crab-eating macaques (Macaca fascicularis) in Hainan, China

BackgroundEnterocytozoon bieneusi is one of common intestinal pathogens in humans and animals including non-human primates (NHPs). Many zoonotic pathogens including E. bieneusi have been found in these animals. However, there are few studies on the population structure of E. bieneusi in NHPs. To infer the gene diversity and population genetics of E. bieneusi, we selected 88 E. bieneusi-positive samples from crab-eating macaques for multilocus characterizations in this study.MethodsThe E. bieneusi isolates examined belonged to three common genotypes with different host ranges by sequence analysis of the ribosomal internal transcribed spacer (ITS): Type IV (n = 44), Macaque3 (n = 24) and Peru8 (n = 20). They were further characterized by sequence analysis at four microsatellite and minisatellite loci (MS1, MS3, MS4 and MS7). DnaSP, Arlequin and LIAN were used to analyze the sequence data together with those from the ITS locus to infer the population genetics. Subpopulation structure was inferred using phylogenetic and STRUCTURE analyses.ResultsSeventy-two (81.8%), 71 (80.7%), 76 (86.4%) and 79 (89.8%) samples were amplified and sequenced successfully at the MS1, MS3, MS4 and MS7 loci, respectively, with 53 having sequence data at all five MLST loci including ITS. Altogether, 33 multilocus genotypes (MLGs) were produced based on concatenated sequences from the 53 samples. In phylogenetic analyses of sequences and allelic data, four major subpopulations (SPs) were observed with different ITS genotypes in each of them: Type IV and Peru8 in SP1 and SP2; Type IV, Macaque3 and Peru8 in SP3; and Type IV and Macaque3 in SP4. SP3 and SP4 were phylogenetically related and might be NHP-specific based on the fact that Macaque3 is mostly found in NHPs. A strong linkage disequilibrium (LD) was observed among the multilocus sequences and allelic data.ConclusionsThe significant LD in the multilocus sequence analysis indicated the presence of an overall clonal population structure of E. bieneusi in crab-eating macaques. The inconsistent segregation of MLGs among ITS genotypes suggested some occurrence of genetic recombination. These observations should improve our understanding of the population genetics of E. bieneusi in NHPs.

Dromedary camel CD14high MHCIIhigh monocytes display inflammatory properties and are reduced in newborn camel calves

BackgroundIn human and different animal species, blood monocytes are classified based on their expression pattern of different monocytic markers into phenotypically and functionally different subsets. In the current study, we used flow cytometry and monoclonal antibodies to CD172a, CD14, CD163 and MHCII to identify monocyte subsets in peripheral blood of dromedary camels.ResultsBased on CD14, CD163 and MHCII expression, camel CD172a + monocytes were divided into three subsets: The major subpopulation of camel monocytes (mo-I) showed high expression of CD14 and CD163, but low expression of MHCII. A second subset of monocytes (mo-II) expressed highly all three markers, CD14, CD163 and MHCII. A third monocyte subset (mo-III) displayed low expression of CD14 and CD163 with high MHCII expression. While the two MHCIIhigh subsets (mo-II and mo-III) showed higher expression of CD11a in comparison to the MHCIIlow subset (mo-I), CD18 and CD11b were highest expressed on the two CD14high subsets (mo-I and mo-II). Bacterial stimulation of camel leukocytes identified mo-II cells as an antimicrobial monocyte subset with the highest phagocytic and ROS production capacity. The comparison of monocyte counts and phenotype between newborn calves and adult camels revealed significantly reduced numbers of mo-II cells in newborn animals. Monocytes of newborns expressed significantly more CD172a and CD163 molecules but less CD14 and MHCII molecules than monocytes of adult camels.ConclusionsCamel monocyte subsets, mo-I, mo-II and mo-III are counterparts of bovine classical, intermediate and non-classical monocytes respectively. The distribution of camel monocyte subsets is influenced by age.

Circulating neutrophil subsets in advanced lung cancer patients exhibit unique immune signature and relate to prognosis.

The accumulation of circulating low-density neutrophils (LDN) has been described in cancer patients and associated with tumor-supportive properties, as opposed to the high-density neutrophils (HDN). Here we aimed to evaluate the clinical significance of circulating LDN in lung cancer patients, and further assessed its diagnostic vs prognostic value. Using mass cytometry (CyTOF), we identified major subpopulations within the circulating LDN/HDN subsets and determined phenotypic modulations of these subsets along tumor progression. LDN were highly enriched in the low-density (LD) fraction of advanced lung cancer patients (median 7.0%; range 0.2%-80%, n=64), but not in early stage patients (0.7%; 0.05%-6%; n=35), healthy individuals (0.8%; 0%-3.5%; n=15), or stable chronic obstructive pulmonary disease (COPD) patients (1.2%; 0.3%-7.4%, n=13). Elevated LDN (>10%) remarkably related with poorer prognosis in late stage patients. We identified three main neutrophil subsets which proportions are markedly modified in cancer patients, with CD66b+ /CD10low /CXCR4+ /PDL1inter subset almost exclusively found in advanced lung cancer patients. We found substantial variability in subsets between patients, and demonstrated that HDN and LDN retain a degree of inherent spontaneous plasticity. Deep phenotypic characterization of cancer-related circulating neutrophils and their modulation along tumor progression is an important advancement in understanding the role of myeloid cells in lung cancer.

ABO Blood Groups are Efficient in Delineating Relationships across Linguistic Subpopulations of Pakistan

Background: Varying frequencies of ABO blood groups serve objectively to characterize populations just as the traditional characters are observed and measured by anthropologist. The study aimed to delineate genealogical relationships across the five major linguistic subpopulations of Pakistan using ABO blood groups phenotype and gene frequencies. Methods: This was a prospective multiple cohort study design. The study was conducted during 2009-2014. In Karachi. Cohorts were formed because of mother tongue and termed as linguistic subpopulations. There were five major cohorts namely Balochi, Punjabi, Pashto, Sindhi and Urdu. Punjabi, Sindhi and Urdu belong to Indo Aryan class while Balochi and Pashto belong to Indo Iranian class of languages. Blood samples were collected from 2,327 unrelated individuals chosen through random sampling from different sampling units. ABO blood groups were determined using antisera. ABO gene frequencies were estimated through Bernstein methods. Cluster and Correspondence analyses were performed to explore genealogical relationships between the five linguistic subpopulations. Results: Gene B was the most common across all the subpopulations except Balochis which showed relatively higher frequency of gene A. Dendrogram of cluster analysis and bi-plot of correspondence analysis showed closeness of three Indo Aryan linguistic subpopulations namely Punjabi, Sindhi and Urdu, while Indo Iranian linguistic subpopulations Balochi and Pashto stood out from Indo Aryan cluster. Conclusion: ABO blood groups efficiently delineated genealogical relationship between the five linguistic subpopulations of Pakistan. Randomization, un-relatedness and a larger sample size enhanced the reliability of the results. Inclusion of more genetic markers may further elaborate the relationships between the subpopulations.

Pervasive within-host recombination and epistasis as major determinants of the molecular evolution of the foot-and-mouth disease virus capsid.

Although recombination is known to occur in foot-and-mouth disease virus (FMDV), it is considered only a minor determinant of virus sequence diversity. Analysis at phylogenetic scales shows inter-serotypic recombination events are rare, whereby recombination occurs almost exclusively in non-structural proteins. In this study we have estimated recombination rates within a natural host in an experimental setting. African buffaloes were inoculated with a SAT-1 FMDV strain containing two major viral sub-populations differing in their capsid sequence. This population structure enabled the detection of extensive within-host recombination in the genomic region coding for structural proteins and allowed recombination rates between the two sub-populations to be estimated. Quite surprisingly, the effective recombination rate in VP1 during the acute infection phase turns out to be about 0.1 per base per year, i.e. comparable to the mutation/substitution rate. Using a high-resolution map of effective within-host recombination in the capsid-coding region, we identified a linkage disequilibrium pattern in VP1 that is consistent with a mosaic structure with two main genetic blocks. Positive epistatic interactions between co-evolved variants appear to be present both within and between blocks. These interactions are due to intra-host selection both at the RNA and protein level. Overall our findings show that during FMDV co-infections by closely related strains, capsid-coding genes recombine within the host at a much higher rate than expected, despite the presence of strong constraints dictated by the capsid structure. Although these intra-host results are not immediately translatable to a phylogenetic setting, recombination and epistasis must play a major and so far underappreciated role in the molecular evolution of the virus at all scales.

Vaccination in children with chronic severe neutropenia – review of recommendations and a practical approach

While the management of childhood neutropenia associated with a modifiable factor should be appropriate for the primary cause, there are misconceptions regarding the management of severe congenital neutropenia, immune neutropenia and cases classified as “idiopathic”. Antibiotic prophylaxis or granulocyte-colony stimulating factor (G-CSF) are prescribed by specialists in pediatric hematology or immunology, whereas immunization may be conducted by primary care physicians should clear recommendations by provided. There is a belief that severe neutropenia, as an immunodeficiency, is associated with compromised effectiveness and increased rate of complications of immunization. The immunization might be delayed or omitted, increasing the risk of unnecessary infection. We discuss the available data and recommendations regarding vaccination of children with chronic severe neutropenia. While there are virtually no studies addressing the safety and effectiveness of vaccination in neutropenia, expert opinions provide information on immunization policy in “phagocytic cells defects” or explicitly neutropenia. There are no contraindications for inactivated vaccines in neutropenia. Live bacterial vaccines are contraindicated. While in general the vaccination with live viral vaccines is encouraged, occasionally neutropenia might be associated with defects of adaptive immunity, which would preclude the administration of such vaccines. Although this should be easily phenotypically identified, we propose assessing immunoglobulin levels and performing a low-cost flow cytometry test for major lymphocyte subpopulations to exclude significant defects in adaptive immunity before administration of live viral vaccines to such patients. This can improve the adherence of patients’ guardians and physicians to proposed vaccination policy and the professional and legal safety associated with the procedure.

Non-walled spherical Acinetobacter baumannii is an important type of persister upon β-lactam antibiotic treatment

ABSTRACT Bacterial persistence is one of the major causes of antibiotic treatment failure and the step stone for antibiotic resistance. However, the mechanism by which persisters arise has not been well understood. Maintaining a dormant state to prevent antibiotics from taking effect is believed to be the fundamental mechanistic basis, and persisters normally maintain an intact cellular structure. Here we examined the morphologies of persisters in Acinetobacter baumannii survived from the treatment by three major classes of antibiotics (i.e. β-lactam, aminoglycoside, and fluoroquinolone) with microcopy and found that a fraction of enlarged spherical bacteria constitutes a major sub-population of bacterial survivors from β-lactam antibiotic treatment, whereas survivors from the treatment of aminoglycoside and fluoroquinolone were less changed morphologically. Further studies showed that these spherical bacteria had completely lost their cell wall structures but could survive without any osmoprotective reagent. The spherical bacteria were not the viable-but-non-culturable cells and they could revive upon the removal of β-lactam antibiotics. Importantly, these non-walled spherical bacteria also persisted during antibiotic therapy in vivo using Galleria mellonella as the infection model. Additionally, the combinational treatment on A. baumannii by β-lactam and membrane-targeting antibiotic significantly enhanced the killing efficacy. Our results indicate that in addition to the dormant, structure intact persisters, the non-wall spherical bacterium is another important type of persister in A. baumannii. The finding suggests that targeting the bacterial cell membrane during β-lactam chemotherapy could enhance therapeutic efficacy on A. baumannii infection, which might also help to reduce the resistance development of A. baumannii.

Respiratory Health Problem among the Taxi Drivers of Pokhara Metropolitan City, Nepal

Respiratory health problems affect the respiratory tract and lungs. WHO states that four major potentially fatal respiratory problems will account for about one in five deaths worldwide by 2030. Taxi drivers are among the major sub-population at the risk of respiratory problems because of their exposure to polluted environment. Therefore, the present study aimed to find out the respiratory health problems among the taxi drivers of Pokhara metropolitan city of Nepal. A cross sectional study was conducted among 203 taxi drivers of the Pokhara Metropolitan city. Multistage sampling method was used to select the desired number of taxi drivers. Data were entered into EPI-DATA 3.1 and then analyzed in SPSS 20. Percentage, mean, and standard deviation were assessed to describe, and chi square test was used to infer the findings. Ethical approval for this study was obtained from Nepal Health Research Council. All samples were male with mean age of 38.46 ± 7.8 years. Majority of the taxi drivers were educated up to secondary level (54.2%), married (91.6%), were married and 78.8 percent had income of NRs 1000-1500/day. A large proportion of the drivers (96.6%) had to work for more than 10 hours/day and three-quarters (74.4%) of them did not take rest even in weekends. Nearly a quarter (24.1%) of them complained at least one respiratory health problem or symptom. Prevalence of respiratory health problems among the taxi drivers was 24.1 percent. Job duration was significantly associated with the respiratory symptoms.
 Key words : Respiratory problem, preventive practice, taxi driver

Single-Cell Sequencing of Mouse Heart Immune Infiltrate in Pressure Overload-Driven Heart Failure Reveals Extent of Immune Activation.

Inflammation is a key component of cardiac disease, with macrophages and T lymphocytes mediating essential roles in the progression to heart failure. Nonetheless, little insight exists on other immune subsets involved in the cardiotoxic response. Here, we used single-cell RNA sequencing to map the cardiac immune composition in the standard murine nonischemic, pressure-overload heart failure model. By focusing our analysis on CD45+ cells, we obtained a higher resolution identification of the immune cell subsets in the heart, at early and late stages of disease and in controls. We then integrated our findings using multiparameter flow cytometry, immunohistochemistry, and tissue clarification immunofluorescence in mouse and human. We found that most major immune cell subpopulations, including macrophages, B cells, T cells and regulatory T cells, dendritic cells, Natural Killer cells, neutrophils, and mast cells are present in both healthy and diseased hearts. Most cell subsets are found within the myocardium, whereas mast cells are found also in the epicardium. Upon induction of pressure overload, immune activation occurs across the entire range of immune cell types. Activation led to upregulation of key subset-specific molecules, such as oncostatin M in proinflammatory macrophages and PD-1 in regulatory T cells, that may help explain clinical findings such as the refractivity of patients with heart failure to anti-tumor necrosis factor therapy and cardiac toxicity during anti-PD-1 cancer immunotherapy, respectively. Despite the absence of infectious agents or an autoimmune trigger, induction of disease leads to immune activation that involves far more cell types than previously thought, including neutrophils, B cells, Natural Killer cells, and mast cells. This opens up the field of cardioimmunology to further investigation by using toolkits that have already been developed to study the aforementioned immune subsets. The subset-specific molecules that mediate their activation may thus become useful targets for the diagnostics or therapy of heart failure.

MORPHO-FUNCTIONAL CHANGES OF THYMUS AND CONTENTS OF BLOOD LYMPHOCYTE SUBPOPULATIONS IN FEMALE WISTAR RATS WITH DIFFERENT RESISTANCE TO HYPOXIA IN SYSTEMIC INFLAMMATORY RESPONSE

Hypoxia and immune reactions are closely interrelated at molecular, cellular and organism levels, and the individuals differ in resistance to oxygen deficiency. Animals with high and low resistance to hypoxia have different adaptive capabilities and predisposition to the development of inflammatory diseases. Data on the individual characteristics of hypoxia resistance in female laboratory animals and humans, and its relationship to immune system reactions in both normal conditions and inflammatory diseases are not available in the literature. It is known, however, that acute infectious and inflammatory diseases develop at lesser rates and are less severe in women and female laboratory animals than in males, which can be explained by higher resistance of females to hypoxia. The aim of our study is to reveal the features of morpho-functional thymus changes, and subpopulation of peripheral blood lymphocytes in systemic inflammatory response induced by LPS administration to female Wistar rats with different resistance to hypoxia. Resistance of mature female Wistar rats to hypoxia was determined as a survival period in a ventilated lowpressure chamber simulating high altitude condition (11 500 m). The rats with a lifetime “at high altitude” of > 180 s have been classified as highly resistant to hypoxia, and the animals surviving for < 20 seconds were designated low-resistant. One month after determining the hypoxia resistance, the females were injected intraperitoneally with E. coli O26:B6 lipopolysaccharide (LPS) at a dose of 1.5 mg/kg during the dioestrus phase. The animals were withdrawn from the experiment by i/m Zoletyl injection (15 mg/kg) one day after LPS administration. The relative volume fractions of thymic cortex and medulla were evaluated; the areas of necrosis were determined in the liver, and the number of neutrophils in the interalveolar septa was counted in the lungs. The serum contents of corticosterone, testosterone, TGF-β were determined. A flow cytometry evaluation of the relative and absolute numbers was performed for major subpopulations of lymphocytes in peripheral blood. The number of apoptotically dying cells of the thymus was assessed. For statistical processing of the obtained data, the Statistica 8.0 software was applied, using criteria of multiple comparisons by Kruskal–Wallis and Dann. The differences were considered statistically significant at p < 0.05.In both high- and low-resistant to hypoxia females, the development of a systemic inflammatory -response was accompanied by a moderately severe thymic involution, apoptosis of thymocytes, an increase in the absolute number of NK, and rise of testosterone and corticosterone contents. LPS injection into low-resistant rats, if compared to females highly resistant to hypoxia, led to more severe manifestations of systemic inflammation, i.e., a pronounced inflammatory reaction in the lungs and a more extensive liver necrotic area accompanied by increased absolute numbers of regulatory T lymphocytes and T helper cells, and more pronounced thymic accidental involution with apoptotic death of thymocytes. Systemic manifestations of inflammation were less pronounced in hypoxia-resistant female rats, which was apparently associated with activation of lymphocyte migration from the thymus and blood to the inflammation focus, and development of more effective immune response.Conclusion: immune reactions in the systemic inflammatory response induced by LPS in female Wistar rats depend on individual resistance to hypoxia. These data should be used to develop approaches to personalized therapy of infectious and inflammatory diseases in women.

Assessment of the genetic diversity and population structure of rice genotypes using SSR markers

ABSTRACTGenetic diversity is a pre-requisite for rice (Oryza sativa L.) breeding and population development. Hence, the objective of this study was to assess the genetic diversity and population structure of 54 rice accessions using 14 polymorphic simple sequence repeats (SSR) markers to select unique parents for breeding. Data analysis was based on marker and population genetic parameters. The mean polymorphic information content (PIC) was 0.61 suggesting high polymorphisms for the selected SSR markers among the rice accessions. The population structure revealed a narrow genetic base with only two major sub-populations. Analysis of molecular variance revealed that only 30% of the variation was attributed to population differences while 47% and 23% were due to variation among individuals within populations and within individual variation, respectively. The genetic distance and identity among genotypes varied from 0.083 to 1.834 and 0.159 to 0.921, respectively. Dendrogram grouped the genotypes into three clusters with wide variation among the accessions. The study established the existence of considerable genetic diversity among the tested 54 accessions. The selected genetic resources will be useful resources for rice breeding in Tanzania or other African countries.

A Minor Subpopulation of Mycobacteria Inherently Produces High Levels of Reactive Oxygen Species That Generate Antibiotic Resisters at High Frequency From Itself and Enhance Resister Generation From Its Major Kin Subpopulation.

Antibiotic-exposed bacteria produce elevated levels of reactive oxygen species (ROS), to which either they succumb or get mutated genome-wide to generate antibiotic resisters. We recently showed that mycobacterial cultures contained two subpopulations, short-sized cells (SCs; ∼10%) and normal/long-sized cells (NCs; ∼90%). The SCs were significantly more antibiotic-susceptible than the NCs. It implied that the SCs might naturally be predisposed to generate significantly higher levels of ROS than the NCs. This in turn could make the SCs more susceptible to antibiotics or generate more resisters as compared to the NCs. Investigation into this possibility showed that the SCs in the actively growing mid-log phase culture naturally generated significantly high levels of superoxide, as compared to the equivalent NCs, due to the naturally high expression of a specific NADH oxidase in the SCs. This caused labile Fe2+ leaching from 4Fe-4S proteins and elevated H2O2 formation through superoxide dismutation. Thus, the SCs of both Mycobacterium smegmatis and Mycobacterium tuberculosis inherently contained significantly higher levels of H2O2 and labile Fe2+ than the NCs. This in turn produced significantly higher levels of hydroxyl radical through Fenton reaction, promoting enhanced antibiotic resister generation from the SCs than from the NCs. The SCs, when mixed back with the NCs, at their natural proportion in the actively growing mid-log phase culture, enhanced antibiotic resister generation from the NCs, to a level equivalent to that from the unfractionated whole culture. The enhanced antibiotic resister generation from the NCs in the reconstituted SCs-NCs natural mixture was found to be due to the high levels of H2O2 secreted by the SCs. Thus, the present work unveils and documents the metabolic designs of two mycobacterial subpopulations where one subpopulation produces high ROS levels, despite higher susceptibility, to generate significantly higher number of antibiotic resisters from itself and to enhance resister generation from its kin subpopulation. These findings show the existence of an inherent natural mechanism in both the non-pathogenic and pathogenic mycobacteria to generate antibiotic resisters. The presence of the SCs and the NCs in the pulmonary tuberculosis patients’ sputum, reported by us earlier, alludes to the clinical significance of the study.

Co-expression of C/EBPγ and ATF5 in mouse vomeronasal sensory neurons during early postnatal development.

The differentiation of sensory neurons involves gene expression changes induced by specific transcription factors. Vomeronasal sensory neurons (VSNs) in the mouse vomeronasal organ (VNO) consist of two major subpopulations of neurons expressing vomeronasal 1 receptor (V1r)/Gαi2 or vomeronasal 2 receptor (V2r)/Gαo, which differentiate from a common neural progenitor. We previously demonstrated that the differentiation and survival of VSNs were inhibited in ATF5 transcription factor-deficient mice (Nakano et al. Cell Tissue Res 363:621-633, 2016). These defects were more prominent in V2r/Gαo-type than in V1r/Gαi2-type VSNs; however, the molecular mechanisms responsible for the differentiation of V2r/Gαo-type VSNs by ATF5 remain unclear. To identify a cofactor involved in ATF5-regulated VSN differentiation, we investigated the expression and function of CCAAT/enhancer-binding protein gamma (C/EBPγ, Cebpg), which is a major C/EBP family member expressed in the mouse VNO and dimerizes with ATF5. The results obtained showed that C/EBPγ mRNAs and proteins were broadly expressed in the postmitotic VSNs of the neonatal VNO, and their expression decreased by the second postnatal week. The C/EBPγ protein was expressed in the nuclei of approximately 70% of VSNs in the neonatal VNO, and 20% of the total VSNs co-expressed C/EBPγ and ATF5 proteins. We examined the trans-acting effects of C/EBPγ and ATF5 on V2r transcription and found that the co-expression of C/EBPγ and ATF5, but not C/EBPγ or ATF5 alone, increased Vmn2r66 promoter reporter activity via the C/EBP:ATF response element (CARE) in Neuro2a cells. These results suggest the role of C/EBPγ on ATF5-regulated VSN differentiation in early postnatal development.

Abstract 3768: Single-cell characterization and lineage tracking of recurrent pediatric rhabdomyosarcoma

Abstract A critical challenge for the effective treatment of pediatric solid tumors is tumor recurrence. In pediatric rhabdomyosarcoma (RMS), 70% of patients with metastatic disease will experience relapse despite treatment with chemotherapy, radiation and/or surgery. Those patients with relapse have particularly dismal outcomes with less than 20% survival over 5 years. These poor outcomes underscore the need for a better understanding of RMS, particularly the biologic processes responsible for recurrence and progression. Currently, a major obstacle towards improving RMS treatment has been that we, as a field, do not know which cells within RMS tumors evade treatment toxicity, and how these cells are selected for in the presence of chemotherapy. We have previously developed a panel of orthotopic patient-derived xenografts (O-PDXs) that encompass the clinical and molecular diversity of RMS. In this study, we have leveraged single-cell RNA-sequencing (scRNA-seq) of our O-PDX system to characterize the intratumoral heterogeneity within this panel. Analysis of single-cell transcriptomic data demonstrate that fusion-negative RMS O-PDXs bear two distinct subpopulations: (1) a major subpopulation encompassing approximately 99% of the tumor cells, and (2) a minor population that are non-cycling and express genes from a variety of mesenchymal lineages. This two-population intratumoral structure is stable within O-PDXs during passaging of xenografts. To extend these findings, we have developed a workflow using lentiviral transduction of O-PDX cells with a high complexity barcode library. This library integrates a unique 18-mer oligonucleotide barcode into each transduced cell within the 3’-untranslated region of blue fluorescent protein. Thus, all cells within an O-PDX with the same barcode are progeny of the same ancestral cell. We have validated longitudinal tracking of barcode diversity using genomic DNA and within the libraries generated during scRNA-seq. Mice bearing barcoded O-PDXs have undergone chemotherapy dosed to pharmacokinetically mimic pediatric therapy regimens; O-PDXs regress during treatment and ultimately recur following completion of therapy, akin to the clinical experience with RMS. Recurrent O-PDXs bear divergent barcodes, indicating that chemotherapy selects for tumor cells independent of the originating lineage of those cells. Further studies to evaluate the changes that occur in recurrent O-PDXs cells are underway. Citation Format: Anand G. Patel, Elizabeth Stewart, Michael Clay, Xiang Chen, Michael Dyer. Single-cell characterization and lineage tracking of recurrent pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3768.