IL-2 delivery by engineered mesenchymal stem cells re-invigorates CD8+ T cells to overcome immunotherapy resistance in cancer.

Immune checkpoint blockade (ICB)-based immunotherapy depends on functional tumour-infiltrating lymphocytes (TILs), but essential cytokines are less understood. Here we uncover an essential role of endogenous IL-2 for ICB responsiveness and the correlation between insufficient IL-2 signalling and T-cell exhaustion as tumours progress. To determine if exogenous IL-2 in the tumour microenvironment can overcome ICB resistance, we engineered mesenchymal stem cells (MSCs) to successfully deliver IL-2 mutein dimer (SIL2-EMSC) to TILs. While MSCs have been used to suppress inflammation, SIL2-EMSCs elicit anti-tumour immunity and overcome ICB resistance without toxicity. Mechanistically, SIL2-EMSCs activate and expand pre-existing CD8+ TILs, sufficient for tumour control and induction of systemic anti-tumour effects. Furthermore, engineered MSCs create synergy of innate and adaptive immunity. The therapeutic benefits of SIL2-EMSCs were also observed in humanized mouse models. Overall, engineered MSCs rejuvenate CD8+ TILs and thus potentiate ICB and chemotherapy.

Three-dimensional versus two-dimensional laparoscopic surgery for rectal cancer: better promote postoperative sexual and urinary function of a propensity-matched study.

Laparoscopic total mesorectal excision (TME) with autonomic nerve preservation (ANP) is a common procedure for rectal cancer (RC), associated with a high prevalence of postoperative urogenital and anorectal dysfunctions. Compared to 2D laparoscopy, 3D laparoscopy provides better depth perception of the surgical field and hand-eye coordination to achieve better outcomes. We compared the performance of 2D and 3D laparoscopy on preserving urogenital and anorectal function in TME+ANP surgery for rectal cancer using propensity-score matching. Data were collected from consecutive male patients who underwent 3D or 2D laparoscopic TME+ANP for primary RC at our institution between March 2012 and December 2020. The primary outcome was sexual and urinary function 1 year after surgery. A total of 450 male patients were eligible. After 1:1 matching, 146 cases were included in each group for analysis. One year after surgery, the prevalence of sexual dysfunction (International Index of Erectile Function score <26) was 8.22% in the 3D laparoscopic group and 44.52% in the 2D laparoscopic group, respectively (P=0.000) and a significant difference in the incidence of urinary retention was observed (n=3 and 24, respectively (P=0.000)). Moreover, blood loss, operative time, duration of hospital stay, and the time to first flatus in the 3D laparoscopic group were significantly less than in the 2D laparoscopic group. In conclusion, 3D laparoscopic TME is associated with lower incidences of postoperative sexual and urinary dysfunction than 2D laparoscopic TME for rectal cancer in male patients.

Risk factors of postoperative nausea and vomiting following ambulatory surgery: A retrospective case-control study.

Objective: To explore potential risk factors of postoperative nausea and vomiting (PONV) following ambulatory surgery. Method: Clinical data of 1670 cases receiving ambulatory surgery in Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School from September 2017 to December 2019 were retrospectively analyzed. They were categorized to PONV group and non-PONV group, and perioperative data in both groups were analyzed for assessing risk factors of PONV following ambulatory laparoscopy. Results: There were 156/1,670 (9.3%) PONV cases, and the female and male incidence in recruited cases was 12.0% and 6.0%, respectively. Analyses on perioperative data of them identified that female gender [adjusted odds ratio (aOR) = 2.060, P < 0.001], operation time >1 h (aOR = 1.554, P = 0.011), postoperative pain at rest (aOR = 1.909, P = 0.013) and postoperative pain during activities (aOR = 3.512, P < 0.001) were independent risk factors of PONV following ambulatory surgery. Furthermore, postoperative pain at rest and during activities were linearly, positively correlated to the incidence of PONV. Conclusion: Female gender, operation time >1 h and postoperative pain are closely related with the incidence of PONV following ambulatory surgery. Alleviating postoperative pain properly is one of the methods to reduce risk factors of PONV following ambulatory surgery.

Next generation of tumor-activating type I IFN enhances anti-tumor immune responses to overcome therapy resistance.

Type I interferon is promising in treating different kinds of tumors, but has been limited by its toxicity, lack of tumor targeting, and very short half-life. To target tumors, reduce systemic toxicity, and increase half-life, here we engineer a masked type I IFN-Fc (ProIFN) with its natural receptor connected by a cleavable linker that can be targeted by tumor-associated proteases. ProIFN has a prolonged serum half-life and shows an improved tumor-targeting effect. Interestingly, ProIFN-treated mice show enhanced DC cross-priming and significant increased CD8+ infiltration and effector function in the tumor microenvironment. ProIFN is able to improve checkpoint blockade efficacy in established tumors, as well as radiation efficacy for both primary and metastatic tumors. ProIFN exhibits superior long-term pharmacokinetics with minimal toxicity in monkeys. Therefore, this study demonstrates an effective tumor-activating IFN that can increase targeted immunity against primary tumor or metastasis and reduce periphery toxicity to the host.

Identification and characteristics of a novel gland-forming gene in cotton.

The cotton (Gossypium hirsutum) pigment gland is a distinctive structure that functions as the main deposit organ of gossypol and its derivatives. It is also an ideal system in which to study cell differentiation and organogenesis. However, only a few genes that determine the process of gland formation have been reported, including GoPGF, CGP1, and CGFs; the molecular mechanisms underlying gland initiation are still largely unclear. Here, we report the discovery of the novel stem pigment gland-forming gene GoSPGF by map-based cloning; annotated as a GRAS transcription factor, this gene is responsible for the glandless trait specifically on the stem. In the stem glandless mutant T582, a point mutation (C to A) was found to create a premature stop codon and truncate the protein. Similarly, virus-induced gene silencing of GoSPGF resulted in glandless stems and dramatically reduced gossypol content. Comparative transcriptomic data showed that loss of GoSPGF significantly suppressed expression of many genes involved in gossypol biosynthesis and altered expression of genes involved in gibberellic acid signaling/biosynthesis. Overall, these findings provide more insight into the networks regulating glandular structure differentiation and formation in cotton, which will be helpful for understanding other plants bearing special gland structures such as tobacco (Nicotiana benthamiana), artemisia annua, mint (Mentha spp.), and rubber (Hevea brasiliensis).

Tumor-conditional IL-15 pro-cytokine reactivates anti-tumor immunity with limited toxicity.

IL-15 is a promising cytokine to expand NK and CD8+ T cells for cancer immunotherapy, but its application is limited by dose-limiting, on-target off-tumor toxicity. Here, we have developed a next-generation IL-15 that is activated inside the tumor microenvironment (TME). This pro-IL-15 has the extracellular domain of IL-15Rß fused to the N-terminus of sIL-15-Fc through a tumor-enriched Matrix Metalloproteinase (MMP) cleavable peptide linker to block its activity. Unlike sIL-15-Fc, pro-IL-15 does not activate the peripheral expansion of NK cells and T cells, thus reducing systemic toxicity, but it still preserves efficient anti-tumor abilities. In various mouse tumors, the anti-tumor effect of pro-IL-15 depends on intratumoral CD8+ T cells and IFN-γ. Pro-IL-15 increases the stem-like TCF1+Tim-3-CD8+ T cells within tumor tissue and helps overcome immune checkpoint blockade (ICB) resistance. Moreover, pro-IL-15 synergizes with current tyrosine kinase inhibitor (TKI) targeted-therapy in a poorly inflamed TUBO tumor model, suggesting that pro-IL-15 helps overcome targeted-therapy resistance. Our results demonstrate a next-generation IL-15 cytokine that can stimulate potent anti-tumor activity without severe toxicity.

Efficacy and safety of once-weekly dulaglutide in adult Chinese patients with type 2 diabetes and lower baseline body mass index.

Highlights In Chinese patients with type 2 diabetes (T2D) and body mass index (BMI) <25 kg/m2 , dulaglutide demonstrated great improvements in glycemic control with mild body weight reduction and low hypoglycemia risk. The results indicate that dulaglutide is effective and safe in patients with T2D and lower BMI; therefore, BMI should not be a consideration when dulaglutide is prescribed to Chinese patients with T2D.

DNA Sensing in Mismatch Repair-Deficient Tumor Cells Is Essential for Anti-tumor Immunity.

Increased neoantigens in hypermutated cancers with DNA mismatch repair deficiency (dMMR) are proposed as the major contributor to the high objective response rate in anti-PD-1 therapy. However, the mechanism of drug resistance is not fully understood. Using tumor models defective in the MMR gene Mlh1 (dMLH1), we show that dMLH1 tumor cells accumulate cytosolic DNA and produce IFN-ß in a cGAS-STING-dependent manner, which renders dMLH1 tumors slowly progressive and highly sensitive to checkpoint blockade. In neoantigen-fixed models, dMLH1 tumors potently induce T cell priming and lose resistance to checkpoint therapy independent of tumor mutational burden. Accordingly, loss of STING or cGAS in tumor cells decreases tumor infiltration of T cells and endows resistance to checkpoint blockade. Clinically, downregulation of cGAS/STING in human dMMR cancers correlates with poor prognosis. We conclude that DNA sensing within tumor cells is essential for dMMR-triggered anti-tumor immunity. This study provides new mechanisms and biomarkers for anti-dMMR-cancer immunotherapy.

Magnetic resonance neuroimaging promotes the preservation of pelvic autonomic nerves in laparoscopic total mesorectal excision: a comparative study.

BACKGROUND: How to preserve pelvic autonomic nerves system (PANS) in total mesorectal excision (TME) is still a technical challenge for gastrointestinal surgeons, and nerve preservation according to preoperative magnetic resonance imaging (MRI) is a hot topic in pelvic surgery. The purpose of this study was to assess the postoperative urogenital function of patients with rectal cancer (RC) who underwent preoperative and postoperative neuroimaging of PANS vs. patients who did not. METHODS: Patients meeting the inclusion criteria were prospectively enrolled in a magnetic resonance neuroimaging (MRN) group from June 2018, while primary RC patients from January 2016 to May 2018 who met the inclusion criteria were enrolled in a non-MRN group. Patients in the MRN group underwent MRN examination before operation and 6 months after operation, while those in the non-MRN group were collected and analyzed retrospectively. RESULTS: Based on International Prostate Symptom Score (IPSS) and International Index of Erectile Function 5 (IIEF5) scores at 6 months, the postoperative urinary and sexual function of male patients in the MRN group were significantly better than that in the non-MRN group (P<0.05). In addition, based on International Consultation on Incontinence modular Questionnaire on Female Lower Urinary Tract Symptoms (ICIQ-FLUTS) and Female Sexual Function Index (FSFI) scores at 6 months, the postoperative sexual function of female patients in the MRN group was significantly better than that in the non-MRN group (P<0.05). CONCLUSIONS: In the present study, we constructed a three-dimensional (3D) presentation of PANS based on preoperative MRN which showed in vivo pelvic autonomous innervation. This may promote the preservation of PANS during TME and reduce the postoperative urogenital dysfunction rate.

Telomere Stress Potentiates STING-Dependent Anti-tumor Immunity.

Telomerase is an attractive target for anti-tumor therapy as it is almost universally expressed in cancer cells. Here, we show that treatment with a telomere-targeting drug, 6-thio-2'-deoxyguanosine (6-thio-dG), leads to tumor regression through innate and adaptive immune-dependent responses in syngeneic and humanized mouse models of telomerase-expressing cancers. 6-thio-dG treatment causes telomere-associated DNA damages that are sensed by dendritic cells (DCs) and activates the host cytosolic DNA sensing STING/interferon I pathway, resulting in enhanced cross-priming capacity of DCs and tumor-specific CD8+ T cell activation. Moreover, 6-thio-dG overcomes resistance to checkpoint blockade in advanced cancer models. Our results unveil how telomere stress increases innate sensing and adaptive anti-tumor immunity and provide strong rationales for combining telomere-targeting therapy with immunotherapy.

Curcumin Attenuates Both Acute and Chronic Immune Nephritis.

Curcumin is known to have immunomodulatory potential in addition to anti-oxidant, anti-inflammatory and anti-carcinogenic effects. The aim of the present study is to investigate the therapeutic effects of curcumin on immune-mediated renal disease in an anti-glomerular basement membrane (GBM) model (representing acute kidney Injury, AKI) and murine lupus model (representing chronic kidney disease, CKD). In the AKI model, female anti-GBM 129/svj mice were administered with curcumin right before disease induction. In the CKD model, female MRL.lpr mice at the age of 8-10 weeks old were treated with curcumin or placebo via oral gavage daily for two months. After treatment, serum autoantibody levels, splenomegaly and spleen cellularity were reduced in murine lupus. Collectively, curcumin ameliorated kidney disease in the two mouse models with either acute or chronic nephritis, as marked by reduced proteinuria, blood urea nitrogen, glomerulonephritis, crescent formation, tubule-interstitial disease, and renal infiltration by lymphocytes. In addition, curcumin treatment reduced activation of the NFkB, MAPK, AKT and pBAD pathways either systemically, or within the inflamed kidneys. These findings suggest that natural food supplements could become an alternative approach to ameliorating immune-mediated kidney diseases.

Prevention of murine lupus nephritis by targeting multiple signaling axes and oxidative stress using a synthetic triterpenoid.

OBJECTIVE: Current treatment options for lupus are far from optimal. Previously, we reported that phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin, MEK-1/ERK-1,2, p38, STAT-3, STAT-5, NF-κB, multiple Bcl-2 family members, and various cell cycle molecules were overexpressed in splenic B cells in an age-dependent and gene dose-dependent manner in mouse strains with spontaneous lupus. Since the synthetic triterpenoid methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) has been shown to inhibit AKT, MEK-1/2, and NF-κB, and to induce caspase-mediated apoptosis, we tested the therapeutic potential of this agent in murine lupus nephritis. METHODS: The synthetic triterpenoid CDDO-Me or placebo was administered to 2-month-old B6.Sle1.Sle3 mice or MRL/lpr mice, which develop spontaneous lupus. All mice were phenotyped for disease. RESULTS: CDDO-Me-treated mice exhibited significantly reduced splenic cellularity, with decreased numbers of both CD4+ T cells and activated CD69+/CD4+ T cells compared to the placebo-treated mice. These mice also exhibited a significant reduction in serum autoantibody levels, including anti-double-stranded DNA (anti-dsDNA) and antiglomerular antibodies. Finally, CDDO-Me treatment attenuated renal disease in mice, as indicated by reduced 24-hour proteinuria, blood urea nitrogen, and glomerulonephritis. At the mechanistic level, CDDO-Me treatment dampened MEK-1/2, ERK, and STAT-3 signaling within lymphocytes and oxidative stress. Importantly, the NF-E2-related factor 2 pathway was activated after CDDO-Me treatment, indicating that CDDO-Me may modulate renal damage in lupus via the inhibition of oxidative stress. CONCLUSION: These findings underscore the importance of AKT/MEK-1/2/NF-κB signaling in engendering murine lupus. Our findings indicate that the blockade of multiple signaling nodes and oxidative stress may effectively prevent and reverse the hematologic, autoimmune, and pathologic manifestations of lupus.

Peritoneal catheter implantation elicits IL-10-producing immune-suppressor macrophages through a MyD88-dependent pathway.

Catheters are implanted into the peritoneal cavity during the process of peritoneal dialysis. Though these catheters may be effective and beneficial, the impact of catheters on the immune system is poorly understood. Catheters and other devices implanted in the peritoneal cavity elicit a foreign body reaction. However, the immunological consequences of this remain uncharacterized. To model this, catheters were implanted into the peritoneal cavity of healthy mice. Catheter implantation induced rapid cellular changes within the peritoneal cavity. Whereas B-cells and T-cells were reduced, catheter implantation was associated with the rapid expansion of F4/80-low-positive, CD11b-positive macrophages that elaborated IL-10, and suppressed T-cell division and Th1 skewing in co-culture assays. Peritoneal catheter elicited macrophages had increased Jmjd3 but reduced NF-κB activation, and their emergence was MyD88-dependent. Collectively, these studies indicate that foreign body implantation into the peritoneal cavity is associated with the expansion of suppressor macrophages. Whether peritoneal cavity catheter implantation may have systemic immunoregulatory roles remains to be explored.

Adverse effects of simulated hyper- and hypo-phosphatemia on endothelial cell function and viability.

BACKGROUND: Dysregulation of phosphate homeostasis as occurs in chronic kidney disease is associated with cardiovascular complications. It has been suggested that both hyperphosphatemia and hypophosphatemia can cause cardiovascular disease. The molecular mechanisms by which high or low serum phosphate levels adversely affect cardiovascular function are poorly understood. The purpose of this study was to explore the mechanisms of endothelial dysfunction in the presence of non-physiologic phosphate levels. METHODOLOGY/PRINCIPAL FINDINGS: We studied the effects of simulated hyper- and hypophosphatemia in human umbilical vein endothelial cells in vitro. We found both simulated hyperphosphatemia and hypophosphatemia decrease eNOS expression and NO production. This was associated with reduced intracellular calcium, increased protein kinase C ß2 (PKCß2), reduced cell viability, and increased apoptosis. While simulated hyperphosphatemia was associated with decreased Akt/p-Akt, Bcl-xl/Bax ratios, NFkB-p65 and p-Erk abundance, simulated hypophosphatemia was associated with increased Akt/p-Akt and Bcl-xl/Bax ratios and p-Mek, p38, and p-p38 abundance. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration of endothelial dysfunction with hypophosphatemia. Our data suggests that both hyperphosphatemia and hypophosphatemia decrease eNOS activity via reduced intracellular calcium and increased PKCß2. Hyperphosphatemia also appears to reduce eNOS transcription via reduced signaling through PI3K/Akt/NF-kB and MAPK/NF-kB pathways. On the other hand, hypophosphatemia appears to activate these pathways. Our data provides the basis for further studies to elucidate the relationship between altered phosphate homeostasis and cardiovascular disease. As a corollary, our data suggests that the level of phosphate in the culture media, if not in the physiologic range, may inadvertently affect experimental results.

Toll-like receptor signaling pathways--therapeutic opportunities.

Toll-like receptors (TLRs) are transmembrane proteins acting mainly as sensors of microbial components. Triggering TLRs results in increased expression of multiple inflammatory genes, which then play a protective role against infection. However, aberrant activation of TLR signaling has a significant impact on the onset of cancer, allergy, sepsis and autoimmunity. Various adaptor proteins, including MyD88, IRAKs, TIRAP, TRIF, and TRAM, are involved in specific TLR signaling pathways. This article reviews the role of these molecules in TLR signaling, and discusses the impact of this pathway on various disease scenarios. Given their important role in infectious and non-infectious disease settings, TLRs and their signaling pathways emerge as attractive targets for therapeutics.

PI3K/AKT/mTOR hypersignaling in autoimmune lymphoproliferative disease engendered by the epistatic interplay of Sle1b and FASlpr.

Previous studies have demonstrated that the NZM2410/NZW 'z' allele of Sle1 on telomeric murine chromosome 1 led to lymphoproliferative autoimmunity, when acting in concert with the FAS(lpr) defect on the C57BL/6 background. The present report shows that the Sle1b sub-locus, harboring the NZM2410/NZW 'z' allele of SLAM, in epistasis with FAS(lpr), may be sufficient to induce lymphoproliferative autoimmunity. Disease in this simplified genetic model is accompanied by significant activation of the AKT signaling axis in both B- and T cells, as evidenced by increased phosphorylation of AKT, mTOR, 4EBP-1 and p70S6K, resulting from increased PI3K and reduced PTEN activity. In addition, blocking this axis using RAD001, an mTOR inhibitor, ameliorated lymphoproliferation and modulated serum IgG anti-nuclear auto-antibodies. Finally, mTOR inhibition also dampened signaling via parallel axes, including the MAPK and NFkB pathways. Hence, hypersignaling via the PI3K/AKT/mTOR axis appears to be an important mechanism underlying autoimmune lymphoproliferative disease, presenting itself as a potential target for therapeutic intervention.

Innate stimuli accentuate end-organ damage by nephrotoxic antibodies via Fc receptor and TLR stimulation and IL-1/TNF-alpha production.

Innate stimuli are well recognized as adjuvants of the systemic immune response. However, their role in driving end-organ disease is less well understood. Whereas the passive transfer of glomerular-targeting Abs alone elicited minimal renal disease, the concomitant delivery of innate stimuli triggered severe nephritis, characterized by proliferative glomerulonephritis with crescent formation, and tubulointerstitial disease. Specifically, stimulating TLR2, TLR3, TLR4, and TLR5 by using peptidoglycan, poly(I:C), LPS, and flagellin, respectively, all could facilitate anti-glomerular Ab-elicited nephritis. In this model, innate and immune triggers synergistically activated several cytokines and chemokines, including IL-1, IL-6, TNF-alpha, and MCP-1, some of which were demonstrated to be absolutely essential for the development of renal disease. Genetic studies revealed that, whereas the innate trigger is dependent on TLR/IL-1R-associated kinase-mediated signaling, the immune component was contingent on FcR-mediated signals. Importantly, infiltrating leukocytes as well as intrinsic glomerular cells may both serve to integrate these diverse signals. Extrapolating to spontaneous immune-mediated nephritis, although the adaptive immune system may be important in generating end-organ targeting Abs, the extent of damage inflicted by these Abs may be heavily dependent on cues from the innate immune system.

Enhanced expression of stem cell antigen-1 (Ly-6A/E) in lymphocytes from lupus prone mice correlates with disease severity.

B6.Sle1 mice, congenic for the NZM2410-derived lupus susceptibility locus, Sle1 on chromosome 1 exhibit many of the features seen in human lupus including activated lymphocytes and high titers of antinuclear autoantibodies. Among the different surface molecules that were aberrantly expressed on the B6.Sle1 lymphocytes was Ly-6A/E. Splenic B- and T-lymphocytes but not myeloid cells from B6.Sle1 mice exhibited enhanced levels of Ly-6A/E compared to B6 controls. In particular, MZ B cells, GC B cells and B-cell blasts expressed the highest levels of Ly-6A/E in both strains, with the levels being even higher on B6.Sle1 derived cells. Following stimulation with LPS or anti-IgM, there was a profound up-regulation in Ly-6A/E, particularly on MZ B cells and B-cell blasts. CD4 and CD8 T cells also up-regulated Ly-6A/E after stimulation with anti-CD3 and anti-CD28. These studies were extended to additional autoimmune strains including B6.Sle3, B6.Sle1.lpr and BXSB. Importantly, Ly-6A/E levels on lymphocytes were commensurate with the degree of disease exhibited by these lupus strains. Finally, it appears that increased interferon levels, in addition to antigen receptor stimulation, may also be a factor accounting for elevated Ly-6A/E in lupus. Given these observations it is important to elucidate the functional role of Ly-6A/E in lupus in future studies.

T cell hyperactivity in lupus as a consequence of hyperstimulatory antigen-presenting cells.

Sle3 is an NZM2410-derived lupus susceptibility locus on murine chromosome 7. Congenic recombination has resulted in a novel mouse strain, B6.Sle3, associated with serum antinuclear autoantibodies (ANAs), T cell hyperactivity, and elevated CD4/CD8 ratios. An OVA-specific TCR transgene was used as a tool to demonstrate that Sle3 facilitated heightened T cell expansion in vitro, and in vivo, following antigen challenge. Indeed, continued T cell expansion was noted even in response to a tolerogenic signal. However, these phenotypes did not appear to be T cell intrinsic but were dictated by hyperstimulatory B6.Sle3 APCs. Importantly, B6.Sle3-derived DCs and macrophages appeared to be significantly more mature/activated, less apoptotic, and more proinflammatory and were better at costimulating T cells in vitro, compared with the B6 counterparts. Finally, the adoptive transfer of B6.Sle3-derived DCs into healthy B6 recipients elicited increased CD4/CD8 ratios and serum ANAs, 2 cardinal Sle3-associated phenotypes. We posit that their heightened expression of various costimulatory molecules, including CD80, CD106, I-A, and CD40, and their elevated production of various cytokines, including IL-12 and IL-1beta, may explain why Sle3-bearing DCs may be superior at breaching self tolerance. These studies provide mechanistic evidence indicating that intrinsic abnormalities in DCs and possibly other myeloid cells may dictate several of the phenotypes associated with systemic lupus, including ANA formation and T cell hyperactivity.

Recombinant human Flt3 ligand exerts both direct and indirect effects on hematopoiesis.

OBJECTIVE: To investigate the direct effects of the Flt3 ligand (FL) on hematopoiesis, such as the stimulation of the formation of hematopoietic colonies and the proliferation of dendritic cells, as well as the indirect stimulation of hematopoiesis, especially via the proliferation of endothelial cells. METHODS: Mononuclear cells from human cord blood were plated in methylcellulose medium containing different cytokines to induce hematopoietic colony formation. Dendritic cells (DCs) were induced from the mononuclear cells with a cytokine cocktail with or without recombinant human soluble FL (rhFL; 100 ng/ml). The Flt3 receptors on the surface of a human microvascular endothelial cell line (ECV) were analyzed by flow cytometry. The proliferation of ECV stimulated by rhFL was measured with the microculture tetrazolium assay. The levels of FL, IL-6, IL-8, G-CSF and GM-CSF in the supernatant of ECV cultures were measured by enzyme linked immunoabsorbent assay (ELISA). RESULTS: rhFL stimulates colony formation from cord blood when used as a sole stimulant. FL in combination with other cytokines increased colony formation significantly. The number of DCs was approximately 2.5 times higher when rhFL was used. rhFL stimulates the proliferation of ECV on which Flt3 receptors are expressed. Furthermore, ECV secretes FL, IL-6, IL-8, G-CSF and GM-CSF, which were augmented by tumor necrosis factor-alpha and rhFL. CONCLUSIONS: rhFL enhances hematopoietic colony formation and DC proliferation from human cord blood cells. FL not only stimulates the proliferation of ECV, but is also secreted by ECV. FL may exert direct and indirect effects on hematopoiesis.