[show abstract][hide abstract] ABSTRACT: During antimicrobial treatment of classic Whipple's disease (CWD), the chronic systemic infection with Tropheryma whipplei, immune reconstitution inflammatory syndrome (IRIS), is a serious complication. The aim of our study was to characterize the immunological processes underlying IRIS in CWD. Following the definition of IRIS, we describe histological features of IRIS and immunological parameters of 24 CWD IRIS patients, 189 CWD patients without IRIS, and 89 healthy individuals. T cell reconstitution, Th1 reactivity, and the phenotype of T cells were described in the peripheral blood, and infiltration of CD4(+) T cells and regulatory T cells in the duodenal mucosa was determined. During IRIS, tissues were heavily infiltrated by CD3(+), predominantly CD45RO(+)CD4(+) T cells. In the periphery, initial reduction of CD4(+) cell counts and their reconstitution on treatment was more pronounced in CWD patients with IRIS than in those without IRIS. The ratio of activated and regulatory CD4(+) T cells, nonspecific Th1 reactivity, and the proportion of naive among CD4(+) T cells was high, whereas serum IL-10 was low during IRIS. T. whipplei-specific Th1 reactivity remained suppressed before and after emergence of IRIS. The findings that IRIS in CWD mainly are mediated by nonspecific activation of CD4(+) T cells and that it is not sufficiently counterbalanced by regulatory T cells indicate that flare-up of pathogen-specific immunoreactivity is not instrumental in the pathogenesis of IRIS in CWD.
The Journal of Immunology 01/2013; · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: OBJECTIVE: Effects of immune cells on the beta 2 (β2)-defensin (HBD2) expression and its antibacterial activity in the intestinal mucosa of patients with inflammatory bowel diseases remains unclear. The small size of these proteins presents a major challenge in localizing antibacterial activities in human intestinal tissue. In this study, we evaluated the detection limits at mRNA and protein level by approaching HBD2 from small tissue samples. METHODS: HT-29 colonic epithelial cells were incubated with proinflammatory cytokines before HBD2 mRNA was investigated by quantitative polymerase chain reaction. The HBD2 protein was assessed by Western blot analysis using HBD2 fused with enhanced green fluorescent protein (HBD2-EGFP). Purified HBD2 fused with the glutathione-S-transferase (GST-HBD2) was used to detect antibacterial activity in a densitometric assay. RESULTS: Interleukin (IL)-1β induced HBD2 mRNA in HT-29 cells; however, tumor necrosis factor-α, IL-6 and IL-17 did not. The Western blot had a sensitivity of 1.5 pmol to detect recombinant HBD2, but did not detect HBD2 in either human intestinal or IL-1β-treated HT-29 cells. HBD2-EGFP was detected by HBD2-specific Western blot within cell lysates and culture supernants of transfected HT-29 and primary cells. In nanomolar ranges, GST-HBD2 reduced bacterial growth. The HBD2 bioactivity depended on solution conditions, but not on the size of the fusion partner. CONCLUSION: The established fusion proteins provide excellent tools to evaluate expression patterns and antibacterial effects of HBD2 in human intestinal tissue samples.
[show abstract][hide abstract] ABSTRACT: Obesity and its associated health risks still demand for effective therapeutic strategies. Drugs and compositions derived from Oriental medicine such as green tea polyphenols attract growing attention. Previously, an extract from the Japanese spice bush Lindera obtusiloba (L. obtusiloba) traditionally used for treatment of inflammation and prevention of liver damage was shown to inhibit adipogenesis. Aiming for the active principle of this extract (+)-episesamin was identified, isolated and applied in adipogenic research using 3T3-L1 (pre)adipocytes, an established cell line for studying adipogenesis. With an IC(50) of 10μM (+)-episesamin effectively reduced the growth of 3T3-L1 preadipocytes and decreased hormone-induced 3T3-L1 differentiation as shown by reduced accumulation of intracellular lipid droplets and diminished protein expression of GLUT-4 and vascular endothelial growth factor. Mechanistically, the presence of (+)-episesamin during hormone-induced differentiation provoked a reduced phosphorylation of ERK1/2 and β-catenin along with a reduced protein expression of peroxisome proliferator-activated receptor γ and a strongly increased protein expression of iNOS. Treatment of mature adipocytes with (+)-episesamin resulted in a reduction of intracellular stored lipid droplets and induced the proapoptotic enzymes caspases-3/-7. Besides interfering with adipogenesis, (+)-episesamin showed anti-inflammatory activity by counteracting the lipopolysaccharide- and tumor necrosis factor α-induced secretion of interleukin 6 by 3T3-L1 preadipocytes. In conclusion, (+)-episesamin seems to be the active drug in the L. obtusiloba extract being responsible for the inhibition of adipogenesis and, thus, should be evaluated as a novel potential complementary treatment for obesity.
The Journal of nutritional biochemistry 07/2012; · 4.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: In Crohn's disease bacteria could be detected in the adjacent mesenteric fat characterized by hypertrophy of unknown function. This study aimed to define effector responses of this compartment induced by bacterial translocation during intestinal inflammation. Dextran sulfate sodium-induced colitis served as a model of intestinal inflammation. Translocation of peptides and bacteria into mesenteric fat was evaluated. Innate functions of mesenteric fat and epithelium were characterized at whole tissue, cellular, and effector molecule levels. Orally applied peptides translocated in healthy wild-type (WT) mice. Bacterial translocation was not detected in healthy and acute but increased in chronic colitis. Mesenteric fat from colitic mice released elevated levels of cytokines and was infiltrated by immune cells. In MyD88(-/-) mice bacterial translocation occurred in health and increased in colitis. The exaggerated cytokine production in mesenteric fat accompanying colonic inflammation in WT mice was less distinct in MyD88(-/-) mice. In vitro studies revealed that fat not only increases cytokine production following contact with bacterial products, but also that preadipocytes are potent phagocytes. Colonic inflammation is accompanied by massive cytokine production and immune cell infiltration in adjacent adipose tissue. These effects can be considered as protective mechanisms of the mesenteric fat in the defense of bacterial translocation.
[show abstract][hide abstract] ABSTRACT: OBJECTIVE: The creeping fat in Crohn's disease (CD) is infiltrated by macrophages; local adipokine levels are increased. This study aimed to link these observations to define a role for macrophages in the pathology of human CD. METHODS: Human peripheral blood CD14 cells were polarised in vitro into M1 and M2 macrophages. The effects on adipokine receptors, phenotypic surface markers, cytokines and chemokines were assessed after treatment with leptin and adiponectin. Immunohistochemistry visualised macrophage subtypes in samples of mesenteric fat tissue from patients with CD. The chemotactic potential of secreted macrophage products was determined by T cell migration and chemokine production in vitro. RESULTS: Although both adipokines altered the phenotype and function of M1 and M2 macrophages, M2 macrophages were more susceptible. M1 responded to leptin by increased cytokine production, but the stronger effect was seen in M2 macrophages with high expression of interleukin (IL)-10, IL-6 and tumour necrosis factor α. Adiponectin exerted similar effects and led to upregulated mannose receptor expression by M2 macrophages. Large macrophage numbers within the mesenteric fat tissue of patients with CD comprise a unique infiltration predominantly of M2 macrophages, leading to an IL-10-rich environment. While leptin increased the potency of both subtypes to attract CD3 T cells, adiponectin only affected M2 macrophages. CONCLUSION: The adipocyte-dependent microenvironment within the creeping fat of patients with CD modulates the local macrophage compartment to a preference for the M2 subtype. The findings in this study with human cells suggest a protective role for the mesenteric fat in CD in terms of an enveloping barrier with the potential to limit intestinal inflammation.
[show abstract][hide abstract] ABSTRACT: In experimental models of and humans with intestinal inflammation, increased levels of the matrix-degrading gelatinases MMP-2 and -9 in inflamed tissues can be detected. The synthetic collagen analogue (Gly-Pro-Hyp) 10 , (GPO) 10 , has been identified as a relevant binding structure for proMMP-2/-9 and promotes enzymatic activity of proMMP-2. Since targeted MMP strategies might offer promising anti-inflammatory treatment options, we for the first time studied in vivo actions exerted by (GPO) 10 applying an acute dextrane sulfate sodium (DSS) induced colitis model. Seven-day intraperitoneal (GPO) 10 treatment ameliorated clinical symptoms and histopathological colonic changes as compared to placebo controls with severe colitis. (GPO) 10 -treated mice displayed a diminished influx of neutrophils, and T- and B-lymphocytes into their colonic mucosa whereas numbers of regulatory T-cells and regenerative cells were higher as compared to placebo controls. Furthermore, IL-6 secretion was down-regulated in ex vivo colonic biopsies derived from (GPO) 10 -treated mice whereas higher concentrations of the anti-inflammatory cytokine IL-10 in extra-intestinal compartments such as MLN and spleen could be detected. Strikingly, influx of inflammatory cells into lungs was abolished following (GPO) 10 application. We therefore propose (GPO) 10 as a promising effective and safe treatment option of intestinal and extra-intestinal inflammatory conditions in humans.
European Journal of Microbiology and Immunology. 01/2012; 2(3):192-200.
[show abstract][hide abstract] ABSTRACT: Well-established differences in Coxsackievirus B3 (CVB3) elimination in resistant C57BL/6 and permissive A.SW/SnJ mice provide suitable models for studying the significance of the link between mitochondrial respiratory chain (RC), antioxidative stress components and mitochondrion-related apoptosis in the context of myocardial virus elimination. Distinct myocardial CVB3 titer in C57BL/6 (2.5 ± 1.4 × 10(4) plaque-forming units (p.f.u.)/g tissue) and A.SW/SnJ mice (1.4 ± 0.8 × 10(7) p.f.u./g) were associated with differences in the cardiac mitochondrial function 8 days post infection (p.i.). Infected C57BL/6 mouse hearts disclosed increased complex I (CI) and CIII activity, but restricted CII and normal CIV activity of RC. Reduced expression of the antioxidative catalase was accompanied by elevated lipid peroxidation (LPO), indicating oxidative stress. Intrinsic apoptosis was activated demonstrated by elevated levels of Bax, Bcl-2, caspase 3 and DNA degradation. In contrast, all myocardial RC complex activities were restricted in CVB3-infected A.SW/SnJ mice. The antioxidative system provided sufficient protection against oxidative stress shown by an elevated catalase expression and unaltered LPO. Bax and Bcl-2 levels were unchanged in CVB3-infected A.SW/SnJ mice, while caspase 3 was moderately increased but no DNA degradation was detectable. Correlation analyses including data from the two mouse strains revealed that reduced CVB3 titer correlated with increased CI and CIII activity, oxidative stress as well as active apoptosis during acute myocarditis (MC). C57BL/6 mice completely eliminated CVB3 and inflammation and normalized all intracellular parameters, while A.SW/SnJ mice showed permanently restricted CI activity in chronic MC 90 days p.i., at which time the replicating virus was no longer detectable but immunological processes were still active. Consequently, the regulation of energy metabolism appears crucial for an effective virus elimination and may be of prognostic and therapeutic significance for patients with virus-induced MC.
[show abstract][hide abstract] ABSTRACT: Diseases such as liver fibrosis and intestinal inflammation are characterized by accumulated components of the extracellular matrix (ECM). Given that fibrillar collagen structures were shown to serve as storage site for inactive proforms of matrixmetalloproteinases (MMPs), modulating this MMP-collagen interaction might offer a rational interventional (therapeutic) approach to enhance degradation of accumulated ECM. The synthetic triple helical collagen analogue (Gly-Pro-Hyp) 10 — (GPO) 10 — was shown to trigger release and enzymatic activation of collagen sequestered proMMP-2. In the presented study, we, for the first time, investigated how MMP-(GPO) 10 interaction impacts cellular responses in vitro . We found that recombinant proMMP-2 induced proliferation of hepatic stellate cells (HSC), which was enhanced after addition of (GPO) 10 reaching comparable levels following incubation with fully activated MMP-2. In addition, (GPO) 10 induced HSC migration similar to the platelet-derived growth factor subunit-B. Further, the MMP-2-dependent invasion of HT1080 fibrosarcoma cells through an ECM membrane was enhanced after addition of (GPO) 10 . Since cellular proliferation and migration concomitant with matrix degradation is stimulated, we conclude that the MMP-(GPO) 10 interaction also functions in a physiological environment. Thus, a potential therapeutic effect of (GPO) 10 should be further tested in animal models for MMP-associated diseases such as colitis or fibrosis.
European Journal of Microbiology and Immunology. 01/2012; 2(3):186-191.
[show abstract][hide abstract] ABSTRACT: Gut-activated T cells migrating into the liver can cause extraintestinal manifestations of inflammatory bowel disease. T cells acquire a gut-homing phenotype dependent on retinoic acid (RA) provided by intestinal dendritic cells (DC). We investigated whether liver antigen-presenting cells can induce gut tropism supporting an enterohepatic lymphocyte circulation. Priming of CD4(+) T cells by liver sinusoidal endothelial cells (LSEC) supported migration into gut and gut-associated lymphoid tissue. As observed for T cells primed by intestinal DCs, this gut tropism depended on α(4) β(7) integrin and CC chemokine receptor 9 (CCR9) expression by LSEC-primed CD4(+) T cells. The induction of gut-homing molecules was mediated by RA, a derivate of vitamin A that is stored in large amounts within the liver. LSECs expressed functional retinal dehydrogenases and could convert vitamin A to RA. Conversely, the lack of signaling via the RA receptor prevented the expression of α(4) β(7) integrin and CCR9 on LSEC-primed CD4(+) T cells, consequently reducing their in vivo migration to the intestine. Other liver antigen-presenting cells failed to support high expression of α(4) β(7) integrin on CD4(+) T cells, thus, the potential to induce gut homing is restricted to LSECs. CONCLUSION: The capacity to promote gut tropism via vitamin A use is not unique for intestinal DCs but is also a feature of LSECs. Our data support the assumption that CD4(+) T cells can migrate from the liver to the gut as one branch of a postulated enterohepatic lymphocyte circulation.
[show abstract][hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Treatment options, especially in advanced tumor stages, are still limited. Inhibition of signaling cascades involved in the pathogenesis of HCC - such as NF-ĸB - offer a promising therapeutic approach. Aim of this study was to examine anti-neoplastic effects of (+)-episesamin which has been isolated from an anti-fibrotic extract of Lindera obtusiloba on human HCC cells with particular interest in activation of NF-κB. The human HCC cell lines HepG2, Huh-7 and SK-Hep1 were treated with (+)-episesamin. Beside measurement of proliferation, invasion and apoptosis, effects of (+)-episesamin on NF-κB-activity, VEGF secretion and enzymatic MMP-9 activity were determined. Anti-inflammatory effects were assessed by IL-6 ELISA using HCC cells and RAW264.7 macrophages. 10 μM (+)-episesamin reduced the proliferation of HCC cells by ~50%, suppressed invasion and induced apoptosis. DNA-binding ELISA experiments revealed that (+)-episesamin treated HCC cells showed a suppressed basal and TNFα-induced activation of NF-κB and a subsequent suppression of TNFα- and LPS-induced IL-6 production. Further, (+)-episesamin exhibited inhibitory effects on the enzymatic activity of recombinant MMP-9 and the secretion of MMP-9 and VEGF by HCC cells into their supernatants. Our findings show that anti-neoplastic effects of (+)-episesamin are mediated via suppressed activation of NF-κB which entails a decreased release of pro-inflammatory IL-6. In addition, (+)-episesamin inhibits MMP-9, which is strongly expressed in invasive HCC, and the production of proangiogenic VEGF. We conclude that (+)-episesamin has the potential to be further explored as a complementary treatment for HCC.
Investigational New Drugs 11/2011; · 3.50 Impact Factor
[show abstract][hide abstract] ABSTRACT: Classical Whipple's disease (CWD) is caused by chronic infection with Tropheryma whipplei that seems to be associated with an underlying immune defect. The pathognomonic hallmark of CWD is a massive infiltration of the duodenal mucosa with T. whipplei-infected macrophages that disperse systemically to many other organ systems. An alleviated inflammatory reaction and the absence of T. whipplei-specific Th1 reactivity support persistence and systemic spread of the pathogen. In this article, we hypothesized that regulatory T cells (T(reg)) are involved in immunomodulation in CWD, and we asked for the distribution, activation, and regulatory capacity of T(reg) in CWD patients. Whereas in the lamina propria of CWD patients before treatment numbers of T(reg) were increased, percentages in the peripheral blood were similar in CWD patients and healthy controls. However, peripheral T(reg) of CWD patients were more activated than those of controls. Elevated secretion of IL-10 and TGF-β in the duodenal mucosa of CWD patients indicated locally enhanced T(reg) activity. Enhanced CD95 expression on peripheral memory CD4(+) T cells combined with reduced expression of IFN-γ and IL-17A upon polyclonal stimulation by CD4(+) cells from untreated CWD patients further hinted to T(reg) activity-related exhaustion of effector CD4(+) T cells. In conclusion, increased numbers of T(reg) can be detected within the duodenal mucosa in untreated CWD, where huge numbers of T. whipplei-infected macrophages are present. Thus, T(reg) might contribute to the chronic infection and systemic spread of T. whipplei in CWD but in contrast prevent mucosal barrier defect by reducing local inflammation.
The Journal of Immunology 09/2011; 187(8):4061-7. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: In traditional Chinese and Korean medicine, an aqueous extract derived from wood and bark of the Japanese spice bush Lindera obtusiloba (L.obtusiloba) is applied to treat inflammations and chronic liver diseases including hepatocellular carcinoma. We previously demonstrated anti-fibrotic effects of L.obtusiloba extract in hepatic stellate cells. Thus, we here consequently examine anti-neoplastic effects of L.obtusiloba extract on human hepatocellular carcinoma (HCC) cell lines and the signaling pathways involved.
Four human HCC cell lines representing diverse stages of differentiation were treated with L.obtusiloba extract, standardized according to its known suppressive effects on proliferation and TGF-β-expression. Beside measurement of proliferation, invasion and apoptosis, effects on signal transduction and NF-κB-activity were determined.
L.obtusiloba extract inhibited proliferation and induced apoptosis in all HCC cell lines and provoked a reduced basal and IGF-1-induced activation of the IGF-1R signaling cascade and a reduced transcriptional NF-κB-activity, particularly in the poorly differentiated SK-Hep1 cells. Pointing to anti-angiogenic effects, L.obtusiloba extract attenuated the basal and IGF-1-induced expression of hypoxia inducible factor-1α, vascular endothelial growth factor, peroxisome proliferator-activated receptor-γ, cyclooxygenase-2 and inducible nitric oxide synthase.
The traditional application of the extract is confirmed by our experimental data. Due to its potential to inhibit critical receptor tyrosine kinases involved in HCC progression via the IGF-1 signaling pathway and NF-κB, the standardized L.obtusiloba extract should be further analysed for its active compounds and explored as (complementary) treatment option for HCC.
BMC Complementary and Alternative Medicine 01/2011; 11:39. · 2.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fibrolytic and profibrotic activities of the matrix metalloproteinases (MMPs)-2 and -9 play a central role in liver fibrosis. Since binding to the extracellular matrix influences the activity of both gelatinases, here the role of fibrillar collagens as the most abundant matrix components in fibrotic tissue was investigated.
In situ zymography and immunohistology showed association of enzymatically inactive prodomain-containing proMMP-2 and proMMP-9 but not of their activated forms to fibrillar collagen structures, which are not substrates of these gelatinases. In solid-phase binding studies with human collagens and collagen fragments, up to 45% of [125I]-labeled proMMP-2 and proMMP-9 but not of active (act)MMP-2 and actMMP-9 were retained by natural collagenous molecules and by synthetic analogs containing repeated Gly-Pro-Hyp triplets (GPO). Surface plasmon resonance yielded binding constants for the interaction of collagen type I (CI) with proMMP-2 and proMMP-9 in a nanomolar range. Values for actMMP-2 and actMMP-9 were 30-40 times higher. Tenfold molar excesses of (GPO)10 reduced the interaction of CI with pro- and actMMP-2 by 22- or 380-fold and resulted in prodomain release accompanied by high enzymatic activation and activity. Pointing to gelatine substrate displacement, higher (GPO)10 concentrations blocked the enzymatic activity. The MMP-2 prodomain-derived collagen-binding domain peptide (P33-42) binds to the collagen-binding domain of MMP-2, thereby preserving enzymatic inactivity. Synthetic P33-42 peptide competed with proMMP-2 binding to CI and prevented (GPO)10-mediated proMMP-2 activation. In contrast to (GPO)10, P33-42 did not activate proMMP-2, making triple helical and hydroxyproline-containing (GPO)10 unique in modulating gelatinase availability and activity.
These findings suggest novel strategies using collagen analogs for the resolution of liver fibrosis via fibrotic matrix-sequestered gelatinases.
[show abstract][hide abstract] ABSTRACT: Obesity, the related metabolic syndrome and associated liver diseases represent an epidemic problem and demand for effective therapeutic strategies. In this regard, natural compounds derived from Oriental medicine such as green tea polyphenols influencing adipogenesis attract growing attention. In Korea, an aqueous extract from the Japanese spice bush Lindera obtusiloba is traditionally used for treatment of inflammation and prevention of liver damage. We here investigated effects of the L. obtusiloba extract on cell growth, apoptosis, Wnt signaling and differentiation of (im)mature adipocytes using 3T3-L1, an established cell line for studying adipogenesis. L. obtusiloba extract reduced the de novo DNA synthesis of 3T3-L1 preadipocytes in a concentration dependent manner with an IC(50) of ∼135 μg/ml paralleled by induction of caspase 3/7 mediated apoptosis. Hormone-induced 3T3 L1 differentiation in the presence of L. obtusiloba extract resulted in a reduced accumulation of intracellular lipid droplets by 70%, in down-regulated expression of the adipogenesis-associated proteins glucose transporter-4 and vascular endothelial growth factor, in reduced secretion of the proadipogenic matrix metalloproteinase-2, and in dampened phosphorylation of the Wnt pathway effector protein β-catenin with subsequent diminished expression of the peroxisome proliferator-activated receptor-γ. Treatment of mature adipocytes with L. obtusiloba extract also significantly reduced intracellular lipid droplets. In addition to this strong interference of L. obtusiloba extract with adipogenesis, L. obtusiloba extract exerted anti-inflammatory effects. L. obtusiloba extract significantly attenuated lipopolysaccharide- and tumor necrosis factor α-induced secretion of IL-6 by preadipocytes, thus influencing insulin resistance and inflammatory state characterizing obesity. In conclusion, extracts of L. obtusiloba should be evaluated as a potential complementary treatment option for obesity associated with the metabolic syndrome.
The Journal of nutritional biochemistry 12/2010; 21(12):1170-7. · 4.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: A barrier defect of the intestinal mucosa is thought to affect the progression of human immunodeficiency virus (HIV) infection. It is not clear whether the mucosal barrier impairment already is present in acute infection and what mechanisms cause this defect. We analyzed T-cell subsets, epithelial apoptosis, and barrier function of the duodenal mucosa in patients with acute HIV infection.
Mucosal T-cell subsets, epithelial apoptosis, and barrier function were assessed by immunohistochemistry, immunofluorescence, flow cytometry, and impedance spectroscopy in duodenal samples from 8 patients with early acute infection, 8 patients with chronic infection, and 9 HIV-negative individuals (controls). One patient was analyzed serially, before and during acute infection.
Compared with controls, densities of mucosal CD8+ and, surprisingly, of mucosal CD4+ T cells too, increased in patients with acute infection. Most mucosal CD4+ T cells had an activated effector memory phenotype (CD45RA-CD45RO+CD62L-CD40L+CD38+) and did not proliferate. Perforin-expressing mucosal CD8+ T cells also were increased in acutely infected patients; their frequency correlated with epithelial apoptosis. The epithelial barrier was impaired significantly in patients with acute HIV infection. The patient analyzed serially developed increased densities of mucosal CD4+ and CD8+ T cells, increased apoptosis of epithelial cells, and mucosal barrier impairment during acute infection.
Before depleting CD4+ T cells, acute HIV infection induces infiltration of the mucosa with activated effector memory CD4+ and CD8+ T cells. The HIV-induced barrier defect of the intestinal mucosa is evident already in acute infection; it might arise from increased epithelial apoptosis, induced by perforin-positive mucosal cytotoxic T cells.
[show abstract][hide abstract] ABSTRACT: The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect.
PLoS ONE 01/2010; 5(3):e9488. · 3.73 Impact Factor