Residual Population Research Articles

Mapping landscape friction to locate isolated tsetse populations that are candidates for elimination

Tsetse flies are the cyclical vectors of deadly human and animal trypanosomes in sub-Saharan Africa. Tsetse control is a key component for the integrated management of both plagues, but local eradication successes have been limited to less than 2% of the infested area. This is attributed to either resurgence of residual populations that were omitted from the eradication campaign or reinvasion from neighboring infested areas. Here we focused on Glossina palpalis gambiensis, a riverine tsetse species representing the main vector of trypanosomoses in West Africa. We mapped landscape resistance to tsetse genetic flow, hereafter referred to as friction, to identify natural barriers that isolate tsetse populations. For this purpose, we fitted a statistical model of the genetic distance between 37 tsetse populations sampled in the region, using a set of remotely sensed environmental data as predictors. The least-cost path between these populations was then estimated using the predicted friction map. The method enabled us to avoid the subjectivity inherent in the expert-based weighting of environmental parameters. Finally, we identified potentially isolated clusters of G. p. gambiensis habitat based on a species distribution model and ranked them according to their predicted genetic distance to the main tsetse population. The methodology presented here will inform the choice on the most appropriate intervention strategies to be implemented against tsetse flies in different parts of Africa. It can also be used to control other pests and to support conservation of endangered species.

Passenger Lymphocyte Syndrome After Simultaneous Pancreas-Kidney Transplantation: A Case Report of an Unusual Cause of Alloimmune Hemolytic Anemia

BackgroundPassenger lymphocyte syndrome (PLS) is a disease in which the donor's lymphocytes produce antibodies to the red blood cell antigens of the recipient, causing alloimmune hemolysis. Case ReportWe report the case of a 39-year-old woman with stage V chronic kidney disease on hemodialysis secondary to poorly controlled diabetes mellitus type 1. She received a simultaneous pancreas-kidney transplant from a cadaver donor. The donor was A− and the recipient was A+ without initial complications with normal renal and pancreatic function, and her hemoglobin (Hb) level was 10.2 g/dL at discharge. Four weeks later she was admitted with acute pyelonephritis of the renal graft, with a Hb level of 7.5 g/dL, creatinine level of 0.7 mg/dL, and glucose level of 80 mg/dL. The study of anemia showed direct polyspecific direct Coombs weakly positive (w/+), presenting 2 alloantibodies against the Rh system: anti-D, anti-E. We increased Prednisone dose to 1 mg/kg/d and then decreased it in a pattern. Eight days after discharge, without transfusion, her Hb level was 9.9 g/dL and then it normalized. ConclusionsPLS is a very rare condition and should be suspected in the first few weeks after transplantation. In our case anemia was probably due to a residual population of Rh-negative donor cells in the transplanted pancreas-kidney received. It is usually a sudden onset of hemolytic anemia in patients with a solid organ transplant and different Rh or ABO lower incompatibility.

Origin and genetic structure of a recovering bobcat (Lynxrufus) population

Genetic analyses can provide important insights into the demographic processes that underlie recovering populations of mammals of conservation concern such as felid species. To better understand the recent and rapid recovery of bobcats (Lynx rufus (Schreber, 1777)) in Ohio, we analyzed samples from four states in the lower Great Lakes Region using 12 microsatellite DNA loci and a portion of the mtDNA control region. Our results showed that a newly established population of bobcats in the eastern part of Ohio was genetically distinct from a multistate population distributed across Kentucky, southern Ohio, West Virginia, and western Pennsylvania. There was no direct genetic evidence of a bottleneck or inbreeding in this population. A lack of private alleles and only slightly lower levels of allelic richness and heterozygosity compared with its neighbors suggest that the eastern Ohio population likely originated from the migration of relatively large numbers of individuals from a source population rather than re-emerging from an undetected residual population. We recommend that a management plan should define the areas occupied by the two populations in Ohio as separate management units at least for the near future.

Implication of the Autologous Immune System in BCR–ABL Transcript Variations in Chronic Myelogenous Leukemia Patients Treated with Imatinib

Imatinib and other tyrosine kinase inhibitors (TKI) have improved treatment of chronic myelogenous leukemia (CML); however, most patients are not cured. Deeper mechanistic understanding may improve TKI combination therapies to better control the residual leukemic cell population. In analyzing our patients' data, we found that many patients who otherwise responded well to imatinib therapy still showed variations in their BCR-ABL transcripts. To investigate this phenomenon, we applied a mathematical model that integrates CML and an autologous immune response to the patients' data. We define an immune window or a range of leukemic loads for which the autologous immune system induces an improved response. Our modeling results suggest that, at diagnosis, a patient's leukemic load is able to partially or fully suppress the autologous immune response developed in a majority of patients, toward the CML clone(s). Imatinib therapy drives the leukemic population into the "immune window," allowing the patient's autologous immune cells to expand and eventually mount an efficient recognition of the residual leukemic burden. This response drives the leukemic load below this immune window, allowing the leukemic population to partially recover until another weaker immune response is initiated. Thus, the autologous immune response may explain the oscillations in BCR-ABL transcripts regularly observed in patients on imatinib.

Cattle mange in Great Britain

Ectoparasites are almost ubiquitous on British cattle, reflecting the success of these parasites at retaining a residual population in the national herd. Chorioptes is the most common cause of mange in cattle and given its surface feeding habits can be difficult to eradicate with current treatments. Psoroptic mange has re-emerged in British cattle in recent years and while the prevalence of infestation is low this parasite poses a significant challenge for treatment especially in dairy cattle. Diagnosis of such ectoparasites is usually made by interpretation of signs of skin disease; definitive diagnosis requires microscopic examination of the ectoparasite, which can more accurately inform the implementation of control measures.

Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations

The recent introduction of Droplet Digital PCR (ddPCR) has provided researchers with a tool that permits direct quantification of nucleic acids from a wide range of samples with increased precision and sensitivity versus RT-qPCR. The sample interdependence of RT-qPCR stemming from the measurement of Cq and ΔCq values is eliminated with ddPCR which provides an independent measure of the absolute nucleic acid concentration for each sample without standard curves thereby reducing inter-well and inter-plate variability. Well-characterized RNA purified from H275-wild type (WT) and H275Y-point mutated (MUT) neuraminidase of influenza A (H1N1) pandemic 2009 virus was used to demonstrate a ddPCR optimization workflow to assure robust data for downstream analysis. The ddPCR reaction mix was also tested with RT-qPCR and gave excellent reaction efficiency (between 90% and 100%) with the optimized MUT/WT duplexed assay thus enabling the direct comparison of the two platforms from the same reaction mix and thermal cycling protocol. ddPCR gave a marked improvement in sensitivity (>30-fold) for mutation abundance using a mixture of purified MUT and WT RNA and increased precision (>10 fold, p<0.05 for both inter- and intra-assay variability) versus RT-qPCR from patient samples to accurately identify residual mutant viral population during recovery.

Amorphous intergranular films act as ultra-efficient point defect sinks during collision cascades

Atomistic simulations are used to explore the effect of interfacial structure on residual radiation damage. Specifically, an ordered grain boundary is compared to a disordered amorphous intergranular film, to investigate how interface thickness and free volume impacts point defect recombination. Collision cascades are simulated and residual point defect populations are analyzed as a function of boundary type and primary knock on atom energy. While ordered grain boundaries easily absorb interstitials, these interfaces are inefficient vacancy sinks. Alternatively, amorphous intergranular films act as ultra-efficient, unbiased defect sinks, providing a path for the creation of radiation-tolerant materials.

Abstract 4378: Targeting the embryonic morphogen Nodal reduces viability of doxorubicin-treated breast cancer cells in vitro

Abstract Triple negative breast cancer represents an aggressive malignancy for which few targeted therapies exist. The embryonic morphogen Nodal is re-expressed in highly aggressive breast cancers and reduction of Nodal signaling decreases tumor cell metastatic characteristics. This represents a potential avenue for the development of therapeutics aimed at inhibiting the Nodal pathway. To explore this further, we evaluated Nodal expression in breast cancer cells following chemotherapy. In vitro treatment of MDA-MB-231 and MDA-MB-468 human breast cancer cell lines with Doxorubicin, a chemotherapy commonly used for the treatment of malignant breast cancer, resulted in the generation of surviving subpopulations that retained Nodal expression (&amp;gt;80%) and remained viable. To validate these findings in vivo, we analyzed Doxorubicin treated metastatic patient derived breast cancer xenografts (PDX) which displayed a residual population of Nodal positive cells when analyzed by immunohistochemistry. Based on these findings, we hypothesize that targeting Nodal in a combinatorial approach with chemotherapy will lead to a reduction in remaining viable populations of cancer cells. When we utilized an anti-Nodal neutralizing antibody to inhibit Nodal signaling, antibody treatment reduced cell proliferation, attachment and increased rates of apoptosis in Doxorubicin treated breast cancer cell lines. Decreases in phosphorylation of Histone 3, a marker of cellular proliferation, and increases in the cleavage of PARP, correlating to induction of apoptosis, were noted in response to antibody treatment. These data suggest that Nodal signaling plays a critical role in the growth of metastatic breast cancer cells and targeting Nodal may improve therapeutic outcome in malignancies such as triple negative breast cancer. The initial results highlight translational potential for the development of humanized antibodies capable of inhibiting Nodal function. Citation Format: Thomas M. Bodenstine, Grace S. Chandler, Naira V. Margaryan, Luigi Strizzi, Alina Gilgur, Elisabeth A. Seftor, Richard E B Seftor, Zhila Khalkhali-Ellis, Andrey Ugolkov, Andrew P. Mazar, Mary J C Hendrix. Targeting the embryonic morphogen Nodal reduces viability of doxorubicin-treated breast cancer cells in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4378. doi:10.1158/1538-7445.AM2015-4378

Thermal and Residual Excited-State Population in a 3D Transmon Qubit.

Remarkable advancements in coherence and control fidelity have been achieved in recent years with cryogenic solid-state qubits. Nonetheless, thermalizing such devices to their milliKelvin environments has remained a long-standing fundamental and technical challenge. In this context, we present a systematic study of the first-excited-state population in a 3D transmon superconducting qubit mounted in a dilution refrigerator with a variable temperature. Using a modified version of the protocol developed by Geerlings et al., we observe the excited-state population to be consistent with a Maxwell-Boltzmann distribution, i.e., a qubit in thermal equilibrium with the refrigerator, over the temperature range 35-150 mK. Below 35 mK, the excited-state population saturates at approximately 0.1%. We verified this result using a flux qubit with ten times stronger coupling to its readout resonator. We conclude that these qubits have effective temperature T(eff)=35 mK. Assuming T(eff) is due solely to hot quasiparticles, the inferred qubit lifetime is 108 μs and in plausible agreement with the measured 80 μs.

Expansion of Activated Peripheral Blood Memory B Cells in Rheumatoid Arthritis, Impact of B Cell Depletion Therapy, and Biomarkers of Response

Although B cell depletion therapy (BCDT) is effective in a subset of rheumatoid arthritis (RA) patients, both mechanisms and biomarkers of response are poorly defined. Here we characterized abnormalities in B cell populations in RA and the impact of BCDT in order to elucidate B cell roles in the disease and response biomarkers. In active RA patients both CD27+IgD- switched memory (SM) and CD27-IgD- double negative memory (DN) peripheral blood B cells contained significantly higher fractions of CD95+ and CD21- activated cells compared to healthy controls. After BCD the predominant B cell populations were memory, and residual memory B cells displayed a high fraction of CD21- and CD95+ compared to pre-depletion indicating some resistance of these activated populations to anti-CD20. The residual memory populations also expressed more Ki-67 compared to pre-treatment, suggesting homeostatic proliferation in the B cell depleted state. Biomarkers of clinical response included lower CD95+ activated memory B cells at depletion time points and a higher ratio of transitional B cells to memory at reconstitution. B cell function in terms of cytokine secretion was dependent on B cell subset and changed with BCD. Thus, SM B cells produced pro-inflammatory (TNF) over regulatory (IL10) cytokines as compared to naïve/transitional. Notably, B cell TNF production decreased after BCDT and reconstitution compared to untreated RA. Our results support the hypothesis that the clinical and immunological outcome of BCDT depends on the relative balance of protective and pathogenic B cell subsets established after B cell depletion and repopulation.

Electron Beam Irradiation of Wine Grapes: Effect on Microbial Populations, Phenol Extraction and Wine Quality

Vitis vinifera L. (variety Tempranillo) grapes were subjected to electron beam irradiation at nominal doses of 0.5, 1 and 10 kGy, and the effect on microbial populations, phenol extraction and wine quality was examined. No external modifications of fruit shape or colour were observed with any of the doses tested. The 1 kGy dose reduced initial must bacterial and yeast counts by 1 log cycle, while the 10 kGy dose left only a residual population of <10 colony-forming units (cfu)/mL. Irradiation was associated with a dose-dependent increase in phenolic compounds in the must. However, the wines produced from grapes irradiated at different doses showed no significant differences in their total phenolic compound contents. All the wines had a good sensory profile; those irradiated at 10 kGy had an increased fruity odour.

Ash regeneration capacity after emerald ash borer (EAB) outbreaks: Some early results

The emerald ash borer (EAB), a wood-boring beetle native of Asia, has killed millions of ash trees in North America since its detection in 2002. The rapid spread of the infestation and the widespread distribution and importance of ash in North America, coupled with the genus’ high vulnerability raise the crucial question of ash regeneration capacity following EAB infestation and its potentially broad ecological implications. We report on ash regeneration and infestation at the epicentre of the initial EAB invasion in Canada (near Windsor, Ontario). Up to 12 years after detection of the outbreak, we found abundant ash regeneration in the impacted area. However, the likelihood of these stems reaching maturity appears low. Dissection of a subsample of saplings allowed us to confirm the presence of an active residual EAB population, with infestation in 19% of regenerating stems (including stems as small as 2 cm in basal diameter). A vigorous regenerating ash cohort comprising a large portion of stump resprout...

Tamaño, composición y patrones diarios de actividad de grupos de pecarí de labios blancos (Tayassu pecari) en el Parque Nacional Mirador-Río Azul, Guatemala

Introduction: The white-lipped peccary is a highly social species that forms large, cohesive groups of 10 to over 300 individuals. They are vulnerable to human presence and habitat fragmentation because they require large extensions of undisturbed forest with sufficient availability of food to maintain their biological requirements. This species has disappeared in 84% of its historic range in Guatemala. In Guatemala, the only place containing a viable population of white-lipped peccary is the Maya Biosphere Reserve although it is possible that residual populations exist in Punta de Manabique Wildlife Refuge, Laguna Lachua National Park and Maya Mountains Biosphere Reserve. The specific objectives of this study were to describe the size and composition of white-lipped peccary groups in Mirador-Rio Azul National Park, a protected area within the Maya Biosphere Reserve, and describe their daily activity patterns at waterholes using camera traps and direct observations. Methods: For analysis with camera traps we used photo-captures of white-lipped peccary obtained in a Rapid Ecological Assessment conducted in Mirador-Rio Azul National Park in 2004. In addition, as part of this study, in 2006 we studied 12 waterholes, from April 22 to May 25. In 2007 direct observations were also made on waterholes from 8:00 at 17:00 during the months of February to May 2007 (dry season), and opportunistic encounters occurred on roads during June to January (rainy season). When a group was observed we recorded the date, time, number of newborns, juveniles and adults. Results: In 2007 we recorded 11 groups of white-lipped peccary, five in waterholes and six in roads totaling 344 individuals. The estimated average of groups during the dry season in the Mirador-Rio Azul National Park, was larger compared with Calakmul Biosphere Reserve and Corcovado National Park although these differences were not statistically significant (U = 9, P = 0.08, U = 31.5, P = 0.63). Group composition included 94.2 % adults, 3.8 % juveniles, and 2 % newborns. The groups visited waterholes more frequently between 8:00 and 12:00 . Discussion and conclutions: The average group size of white-lipped peccary reported during the dry season in Mirador-Rio Azul National Park is larger than the estimates reported in studies at the Calakmul Biosphere Reserve in Mexico and the Corcovado National Park in Costa Rica. This may be due to the moderate level of conservation within the Mirador-Rio Azul National Park, high connectivity to protected areas of Mexico and Belize, and high availability of water for the presence of the Rio Azul, which provides surface water availability, even during the dry season. White-lipped peccaries visited waterholes mainly during the day, registering two peaks of visits between 8:00 and 12:00 . It is necessary to increase the protection in the Mirador-Rio Azul National Park, especially at the borders with Mexico and Belize, and coordinate actions should be implemented in Mexico, Belize and Guatemala to preserve the natural heritage of these protected areas.

A different view on immunity: spatial mapping of human T cells over a lifetime.

A different view on immunity: spatial mapping of human T cells over a lifetime.

A Convenient Human Hepatocyte Model for Molecular Mechanisms in Caffeine Preconditioning and Protection from Acetaminophen (APAP)‐Induced Hepatotoxicity

Long‐term low‐dose caffeine use may be tissue protective with superior outcomes in liver cirrhosis or other chronic diseases. However, molecular mechanisms underlying these effects of caffeine are unknown and suitable cell‐based models will be helpful for this purpose. Caffeine may affect cells through multiple pathways including interference with DNA damage/repair through ATM, ATR or DNA‐PK activity. Here, we focused on regulation by low‐dose caffeine of ATM‐related mechanisms. To induce hepatotoxicity, we cultured HuH‐7 cells with 10 mM APAP (IC50) for 16‐20h. The effects of no caffeine preconditioning or preconditioning over 5 d with 1‐10 mM caffeine were studied. Cell viability assays with MTT dye reduction indicated low‐dose caffeine preconditioning decreased but short‐term caffeine exposure increased APAP toxicity, p&lt;0.05. Flow cytometry showed low‐dose caffeine preconditioning depleted cells in S and G2/M and enriched cells in G0/G1, indicating replicative stress. This residual cell population contained several‐fold greater numbers of Side Population cells with putative stem cell properties, p&lt;0.05. Use of an ATM promoter construct indicated that caffeine interfered with ATM activity in HuH‐7 cells. Therefore, low‐dose caffeine preconditioning in HuH‐7 cells involved ATM‐related mechanisms, and offers convenient models for studies. Cytoprotection against APAP toxicity in caffeine‐preconditioned cells will allow insights into ATM signaling‐specific pathways, cell cycle controls, and stem‐like cells capable of withstanding injury.

Susceptibility characterization of residual Brazilian populations of Triatoma infestans Klug, 1834 (Hemiptera: Reduviidae) to deltamethrin pyrethroid.

Despite years of efforts towards the elimination of Triatoma infestans in Brazil, residual foci still persist in some areas of the States of Bahia and Rio Grande do Sul. The persistence of these T. infestans populations in the country has two different origins of equal concern: operational failures or insecticide resistance. Thus, the objective of this study was to characterize the susceptibility profile of the residual Brazilian populations of T. infestans to deltamethrin. The susceptibility reference lineage was derived from Cipein/Argentina. The populations studied were manually collected using a dislodging agent in peridomiciles in the States of Bahia (Novo Horizonte) and of Rio Grande do Sul (Santa Rosa and Doutor Maurício Cardoso). Serial dilutions of deltamethrin were prepared and applied at the dorsal abdomen of first instar nymphs. The control group received only pure acetone. Mortality was evaluated after 72h. Qualitative tests assessed the mortality of a diagnostic dose of 1xLD99 (2.76ng a.i./nymph) determined for the susceptibility reference lineage. The susceptibility profile characterization of the T. infestans populations revealed an RR50 ranging from 1.73 to 3.26. The mortality percentage in response to a diagnostic dose was 100%. The results obtained in the quantitative and qualitative assays corresponded for all populations. The results of this study indicate that the persistence of residual foci of T. infestans in Bahia and Rio Grande do Sul is not related to insecticide resistance but may be associated with operational failures. In Rio Grande do Sul, we must consider the possibility of continuous reinfestation by Argentinian individuals, which justifies active and efficient epidemiological surveillance.

Microbial population, chemical composition and silage fermentation of cassava residues.

In order to effectively use the cassava (Manihot esculenta Crantz) residues, including cassava leaves, peel and pulp for livestock diets, the chemical and microbiological composition, silage preparation and the effects of lactic acid bacteria (LAB) inoculants on silage fermentation of cassava residues were studied. These residues contained 10(4) to 10(5) LAB and yeasts, 10(3) to 10(4) coliform bacteria and 10(4) aerobic bacteria in colony forming units (cfu) on a fresh matter (FM) basis. The molds were consistently at or below the detectable level (10(2) cfu of FM) in three kinds of cassava residues. Dry matter (DM), crude protein (CP) and neutral detergent fiber (NDF) content of cassava residues were 17.50-30.95%, 1.30-16.41% and 25.40-52.90% on a DM basis, respectively. The silage treatments were designed as control silage without additive (CO) or with LAB inoculants Chikuso-1 (CH, Lactobacillus plantarum) and Snow Lacto (SN, Lactobacillus rhamnosus) at a rate of 5 mg/kg of FM basis. All silages were well preserved with a low pH (below 4.0) value and when cassava residues silage treated with inoculants CH and SN improved fermentation quality with a lower pH, butyric acid and higher lactic acid than control silage.

Whole-genome and multisector exome sequencing of primary and post-treatment glioblastoma reveals patterns of tumor evolution.

Glioblastoma (GBM) is a prototypical heterogeneous brain tumor refractory to conventional therapy. A small residual population of cells escapes surgery and chemoradiation, resulting in a typically fatal tumor recurrence ∼7 mo after diagnosis. Understanding the molecular architecture of this residual population is critical for the development of successful therapies. We used whole-genome sequencing and whole-exome sequencing of multiple sectors from primary and paired recurrent GBM tumors to reconstruct the genomic profile of residual, therapy resistant tumor initiating cells. We found that genetic alteration of the p53 pathway is a primary molecular event predictive of a high number of subclonal mutations in glioblastoma. The genomic road leading to recurrence is highly idiosyncratic but can be broadly classified into linear recurrences that share extensive genetic similarity with the primary tumor and can be directly traced to one of its specific sectors, and divergent recurrences that share few genetic alterations with the primary tumor and originate from cells that branched off early during tumorigenesis. Our study provides mechanistic insights into how genetic alterations in primary tumors impact the ensuing evolution of tumor cells and the emergence of subclonal heterogeneity.

Markers of squamocolumnar junction cells in normal tonsils and oropharyngeal cancer with and without HPV infection.

HPV infection has been identified recently as the causative agent of a subset of squamous cell carcinomas arising in oropharyngeal tonsils. Factors influencing the susceptibility of tonsillar epithelium to HPV-induced oncogenesis are far from being elucidated. A 5-protein signature including cytokeratin (CK)7, anterior gradient (AGR)2, cluster differentiation (CD)63, matrix metalloproteinase (MMP)7, and guanine deaminase (GDA) has recently been found to identify a residual embryonic cell population in the squamocolumnar (SC) junction of the cervix, susceptible to HPV infection, and cancers originating from these cells. The expression of SC junction markers was investigated with immunohistochemistry in normal tonsils and in oropharyngeal carcinomas (OPC) fully characterised for HPV. All markers were constantly expressed in the reticulated epithelial cells of the tonsillar crypts, with variable diffusion and intensity; in OPC, positivity was observed in 36,5%, 29,2%, 39%, 17%, and 25% of cases with respectively AGR2, CK7, GDA, CD63, and MMP7 antibodies. No OPC was positive for all markers; 6 were completely negative. AGR2 and CK7 showed significant association with tumor- and HPV-related parameters. AGR2 expression was associated with tumor origin in the tongue base (p=0.013); CK7 was associated with non-keratinising morphology (p=0.013). p16 tumor cell expression was associated with AGR2 (p=0.021); transcriptionally active HPV infection was associated with AGR2 and CK7 (p=0.024 and 0.043). Expression of SC junction markers in tonsillar crypt cells might be related to the embryological development of tonsillar structures; their partial association with HPV oncogenic infection could help to identify HPV-susceptible cells and related OPC.

Emergence of Long-Lived Autoreactive Plasma Cells in the Spleen of Primary Warm Auto-Immune Hemolytic Anemia Patients Treated with Rituximab

Introduction: We recently proposed that B-cell depletion in immune thrombocytopenia (ITP) promotes the generation of long-lived plasma cells in the spleen, some of them being auto-reactive; but it remained possible that this observation was related to ITP itself rather than to B-cell depletion. Primary warm autoimmune hemolytic anemia (wAIHA) is a rare disease characterized by IgG auto-antibodies directed against antigens at the surface of red blood cells (RBCs) antigens, leading to their accelerated destruction. The use of B-cell depletion in wAIHA leads to 60 % to 70% of overall response at one-year and beyond. Nevertheless, 30-40 % of patients may resist to rituximab and then require a splenectomy. Nothing is known about antibody secreting cells (ASC) in the spleen of wAIHA patients, who have previously been treated or not with rituximab. In this study, we analyzed at the single cell level the splenic ASC from patients with chronic and active wAIHA, previously treated or not with rituximab (RTX), and we compared them with splenic ASC from ITP patients, and with splenic and bone marrow plasma cells from healthy donors (HD).Methods: We took advantage of the different therapeutic outcomes to analyze the splenic B-cell compartment of wAIHA patients, not previously treated with RTX (n=6), or after failure RTX treatment (n=3).Splenic tissues from organ donors and bone marrow from cardiovascular thoracotomy were used as controls. Blood samples from wAIHA (n=19), and (HD) (n=8) enrolled in this study were obtained after giving written informed consent in accordance with the Declaration of Helsinki.Results: We observed by flow cytometry and microscopy that the spleen from wAIHA patients who received less than 3 months of steroid therapy was the site of a B-cell response characterized by the presence of Bcl6+ germinal-center (GC) B-cells. Furthemore, splenic ASC secreted anti-red blood cell IgG in vitro. In line with this observation, we observed in the peripheral blood from patients with a newly diagnosed wAIHA (n=11), that short-lived IgG plasmablasts were increased compared with HD (n=8) (Mean 4.2 ± 0.84 % vs 0.99 ± 0.19% of CD19+ cells, p< 0.01). Moreover, for patients receiving long term steroid therapy (> 6 months) the plasmablast response was suppressed in the peripheral blood (Mean: 0.68 ± 0.2 % of CD19+ cells, n=8) and the splenic GC B-cell reaction was impaired (n=3). We conclude that short-lived IgG ASC result from an over-activity of GC reactions in wAIHA. We then analyzed the spleen of 3 patients who failed to respond to RTX, and observed a residual population of CD19+B-cells (median: 0.9% of lymphoid cells), including non-proliferative memory B-cells and plasma cells (PC). A fraction of these residual PC secreted anti-red blood cells IgG in vitro, thus accounting for the faillure of the B-cell depletion therapy. By using a single cell multiplex quantitative RT-PCR (Fluidigm dynamic arrays), we showed that such RTX-resistant plasma cells display a long-lived transcriptional program, which differs from PC from untreated wAIHA patients or HD, as well as from plasmablasts. Interestingly, the gene expression profile of wAIHA long-lived plasma cells segregated with long-lived PC previously observed in the spleen of ITP patients treated with rituximab. By a principal component analysis, we observed a gradient of maturation from plasmablasts to bone marrow plasma cells in which PC from RTX-treated spleens segregated close to bone marrow PC. We also observed that the cytokine BAFF was increased in the supernatants of spleen cell cultures from wAIHA patients treated with rituximab compared with controls (p< 0.05), suggesting, in keeping with our previous report in ITP, a role for BAFF in the differentiation of short-lived plasma cells into long-lived plasma cells.Conclusion: The presence of splenic long-lived autoreactive PC in wAIHA may explain why some patients cannot achieve a response after RTX. Our results show that, the B-cell depletion induced by rituximab itself, as opposed to the underlying auto-immune condition, promotes a suitable environment for the maturation of auto-immune long-lived plasma cells in the spleen. Targeting specifically some factors such as BAFF right after rituximab injection could be an interesting therapeutic option in the future. DisclosuresNo relevant conflicts of interest to declare.