scholarly journals Lactobacillus rhamnosus GG increases Toll-like receptor 3 gene expression in murine small intestine ex vivo and in vivo

2016 ◽  
Vol 7 (3) ◽  
pp. 421-429 ◽  
Author(s):  
A. Aoki-Yoshida ◽  
S. Saito ◽  
S. Fukiya ◽  
R. Aoki ◽  
Y. Takayama ◽  
...  
Keyword(s):  
Gene Expression ◽  
Small Intestine ◽  
Ex Vivo ◽  
Lactobacillus Rhamnosus ◽  
Poly I:C ◽  
Mrna Levels ◽  
Intestinal Organoids ◽  
Tlr3 Gene ◽  
Tlr3 Mrna

Administration of Lactobacillus rhamnosus GG (LGG) has been reported to be therapeutically effective against acute secretory diarrhoea resulting from the structural and functional intestinal mucosal lesions induced by rotavirus infection; however, the underlying mechanisms remain to be completely elucidated. Because Toll-like receptor 3 (TLR3) plays a key role in the innate immune responses following the recognition of rotavirus, the present study examined whether LGG influences TLR3 gene expression in murine small intestine ex vivo and in vivo. We employed cultured intestinal organoids derived from small intestinal crypts as an ex vivo tissue model. LGG supplementation increased TLR3 mRNA levels in the intestinal organoids, as estimated by quantitative real-time polymerase chain reaction. Likewise, single and 7-day consecutive daily administrations of LGG increased TLR3 mRNA levels in the small intestine of C57BL/6N mice. The mRNA levels of other TLRs were not substantially altered both ex vivo and in vivo. In addition, LGG supplementation increased the mRNA levels of an antiviral type 1 interferon, interferon-α (IFN-α), and a neutrophil chemokine, CXCL1, upon stimulation with a synthetic TLR3 ligand, poly(I:C) in the intestinal organoids. LGG administration did not alter IFN-α and CXCL1 mRNA levels in the small intestine in vivo. Supplementation of other bacterial strains, Bifidobacterium bifidum and Lactobacillus paracasei, failed to increase TLR3 and poly(I:C)-stimulated CXCL1 mRNA levels ex vivo. We propose that upregulation of TLR3 gene expression may play a pivotal role in the therapeutic efficacy of LGG against rotavirus-associated diarrhoea. In addition, we demonstrated that intestinal organoids may be a promising ex vivo tissue model for investigating host-pathogen interactions and the antiviral action of probiotics in the intestinal epithelium.

scholarly journals A119 AN EX VIVO MODEL TO STUDY THE GUT SEROTONERGIC SYSTEM RESPONSE TO LIVE AND HEAT-KILLED LACTOBACILLUS RHAMNOSUS STRAIN JB-1

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 138-140
Author(s):  
T Javkar ◽  
M Paul ◽  
A Stanisz ◽  
P Forsythe
Keyword(s):  
Gene Expression ◽  
Tryptophan Hydroxylase ◽  
Ex Vivo ◽  
Lactobacillus Rhamnosus ◽  
Monoamine Oxidase A ◽  
Serotonergic System ◽  
In Vivo Experiments ◽  
Mao A ◽  
Ec Cells

Abstract Background Enterochromaffin (EC) cells are one of the most abundant enteroendocrine cells in the intestinal epithelium, responsible for producing and storing the largest pool of serotonin or 5-hydroxytryptamine (5-HT) in the body. 5-HT has been shown to be important for modulating a large number of gastrointestinal reflexes in health and disease. 5-HT can stimulate extrinsic (vagal and spinal afferents) or intrinsic primary afferent neurons (IPANs) which are involved in motility, secretion and vasodilation within the intestines. Where EC cell localized enzyme tryptophan hydroxylase (TpH) isoform 1 is responsible for 5-HT synthesis, serotonin reuptake transporter (Sert) and monoamine oxidase A (Mao A) are responsible for termination by uptake and metabolism of 5-HT respectively. Our previous research has demonstrated the effects Lactobacillus rhamnosus (JB-1) on the firing frequency of spinal nerve fibres and motility. Increasing interest is being focused on potential health benefits of heat-inactivated microbes and purified bacterial components. However, the effect of these heat-killed bacteria on the intestinal epithelium cells, particularly on EC cells, is unknown. Aims Small intestinal organoids are shown to recapitulate in vivo characteristics of the small intestine epithelium. The present study aims to assess the suitability of intestinal organoids to study bacterial effects on the serotonergic system in the gut. Here we determined changes in the gene expression of key mediators in the serotonergic system [serotonin reuptake transporter (Sert), tryptophan hydroxylase-1 (Tph-1) and monoamine oxidase A (Mao A)] in response to live and heat-killed JB-1. Methods Male C57bl/6 mice aged 6–8 weeks were used for both ex vivo and in vivo experiments. Jejunal organoids were grown from whole crypts isolated using DTT-EDTA solution. Live and heat-killed JB-1 bacteria were used as treatments. Gene expression analysis was performed on jejunal organoids and jejunum tissue using qRT-PCR. Results JB-1 induced a significant increase in gene expression of Sert, Mao A and Tph-1. No significant difference was observed between the effects of live and heat-killed bacteria. In contrast the JB-1 increased expression of the peptide hormone CCK. Effects of JB-1 on gene expression in organoid culture were reflective of changes observed in in vivo experiments involving feeding of the bacteria. Conclusions Ex vivo organoid culture could be a useful tool in studying mechanisms underlying bacterial effects on serotonergic signalling. The observation that heat-killed bacteria produced comparable effects to the live organism suggests the possibility of isolating active 5-HT modulating components from these strains. Future research will focus on identifying such bacterial components and how their effects on gene expression influence serotonin availability Funding Agencies None

scholarly journals Regulation of GLUT5 gene expression in rat intestinal mucosa: regional distribution, circadian rhythm, perinatal development and effect of diabetes

1995 ◽  
Vol 309 (1) ◽  
pp. 271-277 ◽  
Author(s):  
A Castelló ◽  
A Gumá ◽  
L Sevilla ◽  
M Furriols ◽  
X Testar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Small Intestine ◽  
Circadian Rhythm ◽  
Intestinal Absorption ◽  
Light Period ◽  
Proximal Jejunum ◽  
Mrna Levels ◽  
Dark Cycle ◽  
Perinatal Development ◽  
Streptozotocin Induced Diabetes

1. GLUT5 gene expression was studied in small intestine under a variety of conditions characterized by altered intestinal absorption of monosaccharides. 2. RNA-blotting studies showed that GLUT5 mRNA was abundantly expressed in rat and rabbit intestine and kidney, but it was not detected in heart or brown adipose tissue. GLUT5 mRNA levels were higher in the upper segments of the small intestine (duodenum and proximal jejunum) than in the lower segments (distal jejunum and ileum). 3. The intestinal expression of GLUT5 mRNA in rat proximal jejunum showed circadian rhythm. A 12-fold increase in GLUT5 mRNA levels was detected at the end of the light cycle and at the beginning of the dark cycle when compared with the early light period. In keeping with this, GLUT5 protein content in brush-border membranes was also increased at the beginning of the dark cycle compared with that in the light period. 4. In streptozotocin-induced diabetes an 80% increase in GLUT5 mRNA levels in mucosa from the proximal jejunum was detected under conditions in which enhanced intestinal absorption of monosaccharides has been reported. 5. The intestinal expression of GLUT5 mRNA showed regulation during perinatal development. Levels of GLUT5 mRNA were low during fetal life, increased progressively during the postnatal period and reached levels comparable with the adult state after weaning. Weaning on to a high-fat diet partially prevented the induction of GLUT5 gene expression. 6. Our results indicate that GLUT5 gene expression is tightly regulated in small intestine. Regulation involves maximal expression in the upper part of the small intestine, circadian rhythm, developmental regulation dependent on the fat and carbohydrate content in the diet at weaning and enhanced expression in streptozotocin-induced diabetes. Furthermore, changes observed in intestinal GLUT5 expression correlate with reported alterations in intestinal absorption of fructose. This suggests a regulatory role for GLUT5 in fructose uptake by absorptive enterocytes.

scholarly journals Healthy versus inflamed lung environments differentially effect MSCs

2021 ◽  
pp. 2004149
Author(s):  
Sara Rolandsson Enes ◽  
Thomas H. Hampton ◽  
Jayita Barua ◽  
David H. McKenna ◽  
Claudia C. dos Santos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Healthy Volunteers ◽  
Cell Injury ◽  
Ex Vivo ◽  
Lavage Fluid ◽  
Cell Therapies ◽  
Inflammatory Microenvironment ◽  
Self Recognition ◽  
Clinical Investigations

BackgroundDespite increased interest in MSC-based cell therapies for the acute respiratory distress syndrome (ARDS), clinical investigations have not yet been successful and understanding of the potential in vivo mechanisms of MSC actions in ARDS remain limited. ARDS is driven by an acute severe innate immune dysregulation, often characterised by inflammation, coagulation, and cell injury. How this inflammatory microenvironment influences MSC functions remains to be determined.AimTo comparatively assess how the inflammatory environment present in ARDS lungs versus the lung environment present in healthy volunteers alters MSC behaviors.MethodsClinical grade human bone marrow-derived MSCs (hMSCs) were exposed to bronchoalveolar lavage fluid (BALF) samples obtained from ARDS patients or from healthy volunteers. Following exposure, hMSCs and their conditioned media were evaluated for a broad panel of relevant properties including viability, levels of expression of inflammatory cytokines, gene expression, cell surface HLA expression, and activation of coagulation and complement pathways.ResultsPro-inflammatory, pro-coagulant, and major histocompatibility complex (self recognition) related gene expression was markedly up-regulated in hMSCs exposed ex vivo to BALF obtained from healthy volunteers. In contrast, these changes were less apparent and often opposite in hMSCs exposed to ARDS BALF samples.ConclusionThese data provide new insights into how hMSCs behave in healthy versus inflamed lung environments strongly suggesting that the inflamed environment in ARDS induces hMSC responses potentially benefical for cell survival and actions. This further highlights the need to understand how different disease environments affect hMSC functions.

scholarly journals Effect of the proinflammatory cytokine TNF-α on intestinal riboflavin uptake: inhibition mediated via transcriptional mechanism(s)

2018 ◽  
Vol 315 (5) ◽  
pp. C653-C663 ◽  
Author(s):  
Kasin Yadunandam Anandam ◽  
Omar A. Alwan ◽  
Veedamali S. Subramanian ◽  
Padmanabhan Srinivasan ◽  
Rubina Kapadia ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Significant Inhibition ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Uptake Inhibition ◽  
Tnf Α ◽  
Vitro Model ◽  
Factor Α

Riboflavin (RF), is essential for normal cellular metabolism/function. Intestinal RF absorption occurs via a specific carrier-mediated process that involves the apical transporter RFVT-3 ( SLC52A3) and the basolateral RFVT-1 (SLC52A1). Previously, we characterized different cellular/molecular aspects of the intestinal RF uptake process, but nothing is known about the effect of proinflammatory cytokines on the uptake event. We addressed this issue using in vitro, ex vivo, and in vivo models. First, we determined the level of mRNA expression of the human (h)RFVT-3 and hRFVT-1 in intestinal tissue of patients with inflammatory bowel disease (IBD) and observed a markedly lower level compared with controls. In the in vitro model, exposing Caco-2 cells to tumor necrosis factor-α (TNF-α) led to a significant inhibition in RF uptake, an effect that was abrogated upon knocking down TNF receptor 1 (TNFR1). The inhibition in RF uptake was associated with a significant reduction in the expression of hRFVT-3 and -1 protein and mRNA levels, as well as in the activity of the SLC52A3 and SLC52A1 promoters. The latter effects appear to involve Sp1 and NF-κB sites in these promoters. Similarly, exposure of mouse small intestinal enteroids and wild-type mice to TNF-α led to a significant inhibition in physiological and molecular parameters of intestinal RF uptake. Collectively, these findings demonstrate that exposure of intestinal epithelial cells to TNF-α leads to inhibition in RF uptake and that this effect is mediated, at least in part, via transcriptional mechanism(s). These findings may explain the significantly low RF levels observed in patients with IBD.

Abstract 249: Delayed Gene Transfer Increases Transgene Expression in Vein Grafts

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Liang Du ◽  
Jingwan Zhang ◽  
Alexander Clowes ◽  
David Dichek
Keyword(s):  
Gene Expression ◽  
Blood Flow ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Arterial Blood Flow ◽  
Vein Grafts ◽  
Arterial Blood ◽  
En Face

Background Autogenous vein grafts are effective therapies for obstructive arterial disease. However, their long-term utility is limited by stenosis and occlusion. Genetic engineering of veins that prevents intimal hyperplasia and atherosclerosis could significantly improve the clinical utility of vein grafts. We recently reported that a helper-dependent adenoviral vector (HDAd) reduces atherosclerosis 4 wks after gene transfer in fat-fed rabbits and can express a therapeutic transgene (apo AI) in normal rabbit carotids for at least 48 wks. Use of HDAd for vein graft gene therapy will depend on achievement of similarly high and persistent transgene expression in grafted veins. Hypothesis We tested the hypothesis that Ad-mediated transgene expression in grafted veins (at an early time point) can be increased by varying the timing of gene transfer. Methods Rabbit external jugular veins were transduced by exposure to a beta galactosidase (b-gal)-expressing Ad: in situ either without (a) or with (b) immediate arterial grafting; c) ex vivo with grafting after overnight incubation with Ad; d) in vivo immediately after grafting and e) in vivo 4 wks after grafting (n = 6 - 19 veins/group). Transgene expression was measured in veins removed 3 d after Ad exposure by PCR quantitation of b-gal mRNA and by en-face planimetry of blue-stained area. Results B-gal transgene expression was higher in ungrafted veins than in veins grafted immediately after gene transfer (84 ± 17 vs 9.4 ± 2.0 arbitrary units (AU); P < 0.0001). Overnight incubation of veins with Ad increased gene expression ex vivo by 10-fold but neither this nor performing vector infusion immediately after grafting improved gene expression (11 ± 4.7 and 9.1 ± 1.8 AU; P > 0.9 for both vs immediately grafted veins). Delaying gene transfer until 4 wks after grafting significantly increased gene expression, to a level equivalent to transgene expression in ungrafted veins (61 ± 11 AU; P = 0.3 vs ungrafted veins). En face planimetry yielded similar results. Conclusions Exposure of a transduced vein to arterial blood flow is associated with significant loss of transgene expression. Transgene expression in grafted veins is significantly higher when gene transfer is performed 4 wks after exposure of the vein to arterial blood flow.

scholarly journals Polyinosinic: Polycytidylic Acid and Murine Cytomegalovirus Modulate Expression of Murine IL-10 and IL-21 in White Adipose Tissue

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 569
Author(s):  
Pablo Garcia-Valtanen ◽  
Ruth Marian Guzman-Genuino ◽  
John D. Hayball ◽  
Kerrilyn R. Diener
Keyword(s):  
Adipose Tissue ◽  
B Cells ◽  
White Adipose Tissue ◽  
Ex Vivo ◽  
Interleukin 10 ◽  
Poly I:C ◽  
Murine Cytomegalovirus ◽  
Polycytidylic Acid ◽  
Polyinosinic Polycytidylic Acid

White adipose tissue (WAT) produces interleukin-10 and other immune suppressors in response to pathogen-associated molecular patterns (PAMPs). It also homes a subset of B-cells specialized in the production of IL-10, referred to as regulatory B-cells. We investigated whether viral stimuli, polyinosinic: polycytidylic acid (poly(I:C)) or whole replicative murine cytomegalovirus (MCMV), could stimulate the expression of IL-10 in murine WAT using in vivo and ex vivo approaches. Our results showed that in vivo responses to systemic administration of poly(I:C) resulted in high levels of endogenously-produced IL-10 and IL-21 in WAT. In ex vivo WAT explants, a subset of B-cells increased their endogenous IL-10 expression in response to poly(I:C). Finally, MCMV replication in WAT explants resulted in decreased IL-10 levels, opposite to the effect seen with poly(I:C). Moreover, downregulation of IL-10 correlated with relatively lower number of Bregs. To our knowledge, this is the first report of IL-10 expression by WAT and WAT-associated B-cells in response to viral stimuli.

scholarly journals Osmanthus fragrans Flower Aqueous Extract and its Enriched Acteoside inhibit Melanogenesis and Ultraviolet-induced Pigmentation

2018 ◽  
Vol 13 (5) ◽  
pp. 1934578X1801300
Author(s):  
Shuo Liu ◽  
Zhen Zhao ◽  
Zhijun Huo ◽  
Zhiru Xu ◽  
Yan Zhong ◽  
...  
Keyword(s):  
Aqueous Extract ◽  
Ex Vivo ◽  
Melanoma Cells ◽  
B16 Melanoma ◽  
Tyrosinase Activity ◽  
In Vivo Model ◽  
Mrna Levels ◽  
Osmanthus Fragrans ◽  
B16 Melanoma Cells

The Osmanthus fragrans flower (OFF) is commonly used as an additive for tea in China and as a traditional medicine to treat dysentery, asthma and hepatitis. In the current study, we have acquired the aqueous extract of the dried OFF (OFFE) and determined its enriched acteoside contents. However, whether OFFE and acteoside can modulate melanogenesis and pigmentation has yet to be determined. We here provide novel data revealing that OFFE and acteoside inhibit melanogenesis induced by α-MSH in B16 melanoma cells via the MITF-tyrosinase signaling pathway. Treatment with α-MSH (1μM) enhanced melanin levels and tyrosinase activity, up-regulated the mRNA levels of MITF and tyrosinase and increased the dendritic number in B16 melanoma cells, effects all being intervened by OFFE and acteoside. Of interest, OFFE and acteoside showed no direct inhibition of tyrosinase activity as revealed by our ex vivo tyrosinase activity assay. In addition, OFFE produced a depigmenting action on UVB-induced hyperpigmentation in guinea pigs, as shown by the improved skin brightness and the decreased melanin staining. Our data have demonstrated that OFFE can alter melanogenesis via modulating the MITF-tyrosinase signaling thereby leading to its depigmenting action in the in vivo model. OFFE could be a substitute for acteoside as a promising skin-whitening agent.

scholarly journals Perlecan regulates Oct-1 gene expression in vascular smooth muscle cells.

1997 ◽  
Vol 8 (6) ◽  
pp. 999-1011 ◽  
Author(s):  
M C Weiser ◽  
N A Grieshaber ◽  
P E Schwartz ◽  
R A Majack
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Type Iv Collagen ◽  
Muscle Cells ◽  
Mrna Levels ◽  
Type Iv

Vascular smooth muscle cells (SMCs) are very quiescent in the mature vessel and exhibit a remarkable phenotype-dependent diversity in gene expression that may reflect the growth responsiveness of these cells under a variety of normal and pathological conditions. In this report, we describe the expression pattern of Oct-1, a member of a family of transcription factors involved in cell growth processes, in cultured and in in vivo SMCs. Oct-1 mRNA was undetectable in the contractile-state in vivo SMCs; was induced upon disruption of in vivo SMC-extracellular matrix interactions; and was constitutively expressed by cultured SMCs. Oct-1 transcripts were repressed when cultured SMCs were plated on Engelbreth-Holm-Swarm tumor-derived basement membranes (EHS-BM) but were rapidly induced after disruption of SMC-EHS-BM contacts; reexpression was regulated at the transcriptional level. To identify the EHS-BM component involved in the active repression of Oct-1 mRNA expression, SMCs were plated on laminin, type IV collagen, fibronectin, or perlecan matrices. Oct-1 mRNA levels were readily detectable when SMCs were cultured on matrices composed of laminin, type IV collagen, or fibronectin but were repressed when SMCs were cultured on perlecan matrices. Finally, the Oct-1-suppressing activity of EHS-BM was sensitive to heparinase digestion but not to chondroitinase ABC or hyaluronidase digestion, suggesting that the heparan sulfate side chains of perlecan play a biologically important role in negatively regulating the expression of Oct-1 transcripts.

Acute hypoxia stimulates renin secretion and renin gene expression in vivo but not in vitro

1997 ◽  
Vol 272 (4) ◽  
pp. R1105-R1111 ◽  
Author(s):  
T. Ritthaler ◽  
K. Schricker ◽  
F. Kees ◽  
B. Kramer ◽  
A. Kurtz
Keyword(s):  
Gene Expression ◽  
Acute Hypoxia ◽  
Primary Cultures ◽  
Renin Secretion ◽  
Plasma Norepinephrine ◽  
Mrna Levels ◽  
Sprague Dawley ◽  
Renin Gene ◽  
Renin Mrna

This study aimed at examining the influence of acute hypoxia on renin secretion and renin gene expression in the kidney. To this end, male Sprague-Dawley rats were exposed to severe hypoxic stress (8% O2) or to carbon monoxide (0.1% CO) for 6 h, and plasma renin activity (PRA) and renal renin mRNA levels were determined. PRA values increased from 3 to 13 and 10 ng angiotensin I x h(-1) x ml(-1), and renin mRNA levels increased by 120 and 100% during hypoxia and CO, respectively. Lowering the PO2 from 150 to 20 or 7 mmHg in the gas atmosphere of primary cultures of renal juxtaglomerular cells had no influence on renin secretion and renin gene expression after 6 and 20 h. Our findings thus suggest that both arterial and venous hypoxia can be powerful stimulators of renin secretion and renin gene expression in vivo. Because renal denervation did not prevent stimulation of the renin system by hypoxia, the effect could be indirectly mediated via the baroreceptor-macula densa mechanism. Another potential mediator of the effect could be circulating catecholamines, since we found that plasma norepinephrine increased from 0.7 to 1.5 and 2.4 ng/ml and plasma epinephrine increased from 0.3 to 1.4 and 2.7 ng/ml during hypoxia and CO inhalation, respectively.

The Outgrowth of CLL Nurselike Cells in Vitro, and Its Relation to Clinical and Hematological Parameters.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2360-2360
Author(s):  
Agata A Filip ◽  
Dorota Koczkodaj ◽  
Tomasz Kubiatowski ◽  
Ewa Wasik-Szczepanek ◽  
Anna Dmoszynska
Keyword(s):  
Gene Expression ◽  
Serum Level ◽  
Ex Vivo ◽  
Hematological Parameters ◽  
Tp53 Gene ◽  
Leukemic Cells ◽  
Rt Pcr ◽  
Gene Status

Abstract Abstract 2360 Poster Board II-337 Introduction: Despite their longevity in vivo, CLL lymphocytes die rapidly when put to in vitro cultures, what proves that the resistance to apoptosis is not an intrinsic feature of leukemic cells, but depends on environmental signals. Recently it was shown that mononuclear cells from peripheral blood of CLL patients differentiate in vitro into large, adherent cells that grow in close contact with CLL lymphocytes. They were termed “nurselike cells” (NLCs), because they support leukemic lymphocyte survival in culture. The presence of the cells morphologically and phenotypically similar to NLCs was demonstrated in peripheral lymphatic organs of CLL patients. It may suggest their role in CLL lymphocytes protection in vivo and, as a consequence, point the new target in CLL treatment. Patients and Methods: The study included the group of 65 previously untreated CLL patients, 24 women and 41 men, aged from 36 to 86 yrs. 12 patients (18%) were diagnosed with stage 0 according to Rai, 15 patients (23%) with stage I, 30 patients (46%) with stage II, 5 patients (8%) with stage III and 3 patients (5%) with stage IV. Peripheral blood lymphocytes ex vivo were examined for CD14, CD38, BCL2 and ZAP70 expression by flow cytometry and for BCL2, SURVIVIN and ZAP70 gene expression by RT-PCR. TP53 gene status was assessed by FISH. Lymphocytes of 20 patients were assayed for apoptosis-related gene expression by means of cDNA macroarrays (Clontech). To generate NLCs, PB leukemic cells were cultured in vitro for 14 days on standard medium (RPMI 1640 with L-glutamine, 15% FCS, antibiotics/antimycotics; cell density 3 × 106/ml) and the outgrowth and number of NLCs was assessed in relation to clinical and hematological parameters. NLCs were identified morphologically and by CD31/VIMENTIN protein expression. Results: In 58 cases (89%) the outgrowth of NLCs was observed, while their number differed in cultures of the cells of different patients: in 49 cultures (84.5%) there were over 20 NLCs/mm2 (up to 52 NLCs/mm2), and in 9 cases (15.5%) less than 20 NLCs/mm2. Positive correlation was shown between NLC number and B2M serum level (p=0.044) and absolute monocyte count (p=0.019). Significantly higher NLC number was observed in case of patients with higher CD14+ cell number (p<0.0001) and higher SURVIVIN gene expression assessed by RT-PCR (p<0.0001) and macroarrays (p=0.013). We found no statistically significant relation of NLCs number and: the Rai stage of the disease, WBC, lymphocyte count, LDH serum level, BCL2, CD38 and ZAP70 expression and TP53 gene status. During the follow-up period of 6 years we observed the tendency for longer overall survival in patients that produce less than 20 NLCs/mm2 (fig. 1), but it was not statistically significant. Conclusions: The number of NLC cells obtained in vitro from PBL of CLL patients correlates with B2M serum level and SURVIVIN gene expression in CLL cells ex vivo. High B2M level is a marker of poor prognosis. SURVIVIN represents a family of IAP (Inhibitor of APoptosis) proteins. While rare in PBL of CLL patients, its expression is typical for proliferating leukemic cells pool in pseudofollicle microenvironment. SURVIVIN inhibits apoptosis by blocking caspase-3 and -7. Considering the protective role of NLC cells towards CLL lymphocytes in vitro, these results altogether with observed tendency to shorter survival of patients generating high NLCs number may prove the presence of supportive mechanisms exerted by NLCs in vivo. Disclosures: No relevant conflicts of interest to declare.

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