Inactivated Serum Research Articles

Thromboelastography and the Functional Confirmation of Heparin-Induced Thrombocytopenia in Donor Whole Blood

Thromboelastography (TEG) presents a novel testing environment to detect functional antibodies associated with heparin-induced thrombocytopenia (HIT). TEG is considered a qualitative and qualitative method for assess global hemostasis in a test environment that includes shear stresses. In this study we explore the sensitivity of TEG to detect a HIT-associated reaction when whole blood or platelet rich plasma (PRP) is mixed with heat inactivated positive HIT sera in the presence of low dose (0.2U/mL) or high dose (100U/mL) sodium heparin. TEG is designed to record the kinetic changes in whole blood or plasma-based samples during clot initiation, propagation and fibrinolysis. Here we show that HIT positive sera can cause a reaction in both whole blood and PRP when exposed to reactive normal donor whole blood or PRP. Curiously, whole blood was shown to be more sensitive to the presence of HIT positive sera than PRP. The ration of heat-inactivated sera to donor whole blood or PRP was 1 to 2 parts. HIT positive sera was obtained from a patient that was clinical diagnosed with HIT type II and had a clinically significant positive ELISA with an optical density of 1.735 ± 0.032. HIT positive sera with ELISA optical densities above 1.000 or associated with an enhanced likelihood of developing thrombosis. Additionally, the HIT positive sera was functionally positive with 13 qualified, reactive HIT normal platelet donors with a washed-platelet heparin-induced platelet activation (wp-HIPA) average result of 66 ± 11%. The wp-HIPA is an established reference method or GOLD STANDARD assay for the diagnosis of HIT. The positive TEG detection of this functional HIT antibody may reveal another, more rapid test approach for the hospital and reference laboratory diagnosis of HIT. Additional studies are warranted to further characterize the comparative sensitivities of TEG and wp-HIPA and other reference methods for the diagnosis of HIT. DisclosuresNo relevant conflicts of interest to declare.

자몽종자추출물, 구연산, 사과산 그리고 염화벤잘코늄을 주성분으로 하는 스프레이형 소독제의 조류인플루엔자바이러스에 대한 살바이러스 효과

Objectives: This study evaluated the virucidal efficacy against avian influenza virus (AIV) of a disinfectant spray containing 0.25% grapefruit seed extract, 0.2% citric acid, 0.0625% malic acid and 0.0125% benzalkonium chloride. Methods: The disinfectant spray was diluted several times with hard water (HW) and organic matter (OM). Two point five mL of each diluent was added into each test tube, and 2.5 mL of AIV suspension was inserted into each test tube. After 30 minutes of virus-disinfectant contact reaction at 4℃, 2.5 mL of 10% inactivated fetal bovine serum was added into each test tube to neutralize the sanitizer efficacy. The neutralized solutions were serial 10-fold dilutions with phosphate buffer solution, and 0.2 mL of the diluents was injected into the allantoic cavity of five ten-day-old-chickens per dilution time. After incubation of the embryos for five days, the viability of the AIV was examined by hemagglutination titer. The valid dilution of the disinfectant spray was estimated according to the dilution time that the virus titer was inactivated more than 104 50% egg-infective dose (EID50)/mL compared with pathogen control. Results: In HW and OM conditions, the valid dilutions of the disinfectant spray against AIV were seven- and three-fold dilutions, respectively. The AIV titer of the pathogen control was more than 6.1 log10EID50/mL, and there was no embryonic toxicity.

OR6 The positive virtual crossmatch. Correlation between HLA DSA and flow cytometry crossmatch results

Aim Virtual crossmatch (VXM) is a good tool to expedite and streamline pre-transplant recipient selection and facilitate national organ sharing. However, VXM can be a barrier to transplantation, based on donor specific antibodies (DSA) detected by single antigen bead (SAB) testing, especially when low MFI cutoffs are used to assign unacceptable antigens. Recent studies report good clinical outcomes in patients transplanted across DSA with a negative FCXM, emphasizing that the choice of VXM cutoffs should be based on careful correlation with FCXM. The purpose of this study was to assess the correlation between MFI of DSA and FCXM results. Methods The results of 2843 deceased donor FCXM performed at our center between 03/2015 and 03/2016 were analyzed and correlated with cumulative MFI of locus specific DSA. Donors were typed (HLA-A, B, C, DR, DQ) by SSP method. FCXM were performed as per Halifax protocol (Liwski et al., ASHI Q 2015) with spleen or lymph node derived donor cells. SAB testing (LABScreen) was performed with heat inactivated sera to prevent prozone. DSA were assigned based on 1000 (1K) MFI cutoff. All sera with DP antibodies were excluded. Results Of 1859 VXM −ve samples (no DSA), 56 were FCXM +ve (3% false positive rate). Of 318 VXM +ve (DSA; >1K MFI) samples, only 214 (67.3%) were FCXM +ve. The degree of concordance, VXM vs FCXM, was locus and MFI dependent (Fig. 1). HLA-A and B DSA predicted a +ve FCXM (T and/or B) in 80%, 93% and 100% of cases using 1K, 2K and 5K cutoffs respectively. HLA-DR DSA predicted positive B cell FCXM in 54%, 89% and 100% of cases with same MFI cutoffs. HLA-C and DQ DSA predicted positive FCXM reliably only when 10K cutoff was used (100% and 95% respectively; Fig. 1). Conclusions Concordance between VXM and FCXM depends on MFI cutoff and DSA locus. 1K cutoff is not useful for FCXM prediction regardless of DSA specificity. For HLA-A, B and DR DSA, 2–5K cutoff can be used to reliably predict FCXM, while for HLA-C and DQ only 10K cutoff is predictive. Download : Download high-res image (138KB) Download : Download full-size image

The role of membrane complement regulatory protein in transplantation of bone marrow mesenchymal stem cell

Objective To explore the effect of complement activation on bone marrow mesenchymal stem cells (BMSCs) and evaluate the effect after transfection of complement regulatory proteins. Methods Bone marrow aspirate was harvested from 10 cases of patients suffered from fractures. Mesenchymal stem cells were isolated, indentified cultured and then experimented in vitro. The complement cytotoxicity on the mesenchymal stem cells in autologous serum was measured by Europium cytotoxicity assay. The samples were divided into BMSCs group, BMSCs+ autologous human serum (AHS) group and BMSCs+ inactivated autologous human serum (iAHS) group. The complement membrane attack complex (MAC) deposited on the membranes was detected by flow cytometry. Finally, the cytotoxicity on BMSCs was measured after transfected with membrane complement regulatory proteins (mCRPs). All samples were divided into BMSCs with mCRPs untransfected group and BMSCs with mCRPs transfected group. Results More than 95% of cells derived from bone marrow were identified to be mesenchymal stem cells through detection of cell surface markers by flow cytometry. The cytotoxicity of untreated cells was 0.41%±1.48%. BMSCs harvested from the 10 patients all had cytotoxicity after incubated with autologous serum, and the cytotoxicity was 32.59%±2.73%, while cytotoxicity after incubated with complement inactivated autologous serum was 2.59%±3.08%, which was similar to control group. Complement attack complex (MAC) could be detected on the BMSCs incubated with autologous serum, which implied the complement activation. After transfection of mCRPs, the cytotoxicity of autologous serum on transfected cells was decreased. The cytotoxicity of untransfected cells (41.70%±4.47%) had significant difference compared to the cells transfected with CD55 (21.87%±2.19%), the cells transfected with CD59 (18.67%±1.42%), and the cells transfected with CD46+CD55+CD59 (28.43%±2.14%). CD55, CD59 and CD46+ CD55+CD59 transfected groups could impair effectively the cytotoxicity from complement. However, the cytotoxicity impairment was less effective in CD46 transfected cells (39.30%±3.96%), which had no significant difference compared to untransfected cells. Conclusion Membrane complement regulatory proteins could effectively protect bone marrow mesenchymal stem cells from attacks by complement. Key words: Bone marrow; Mesenchymal stem cells; Complement membrane attack complex

Candida albicans Shaving to Profile Human Serum Proteins on Hyphal Surface.

Candida albicans is a human opportunistic fungus and it is responsible for a wide variety of infections, either superficial or systemic. C. albicans is a polymorphic fungus and its ability to switch between yeast and hyphae is essential for its virulence. Once C. albicans obtains access to the human body, the host serum constitutes a complex environment of interaction with C. albicans cell surface in bloodstream. To draw a comprehensive picture of this relevant step in host-pathogen interaction during invasive candidiasis, we have optimized a gel-free shaving proteomic strategy to identify both, human serum proteins coating C. albicans cells and fungi surface proteins simultaneously. This approach was carried out with normal serum (NS) and heat inactivated serum (HIS). We identified 214 human and 372 C. albicans unique proteins. Proteins identified in C. albicans included 147 which were described as located at the cell surface and 52 that were described as immunogenic. Interestingly, among these C. albicans proteins, we identified 23 GPI-anchored proteins, Gpd2 and Pra1, which are involved in complement system evasion and 7 other proteins that are able to attach plasminogen to C. albicans surface (Adh1, Eno1, Fba1, Pgk1, Tdh3, Tef1, and Tsa1). Furthermore, 12 proteins identified at the C. albicans hyphae surface induced with 10% human serum were not detected in other hypha-induced conditions. The most abundant human proteins identified are involved in complement and coagulation pathways. Remarkably, with this strategy, all main proteins belonging to complement cascades were identified on the C. albicans surface. Moreover, we identified immunoglobulins, cytoskeletal proteins, metabolic proteins such as apolipoproteins and others. Additionally, we identified more inhibitors of complement and coagulation pathways, some of them serpin proteins (serine protease inhibitors), in HIS vs. NS. On the other hand, we detected a higher amount of C3 at the C. albicans surface in NS than in HIS, as validated by immunofluorescence.

The effect of foetal bovine serum supplementation upon the lactate dehydrogenase cytotoxicity assay: Important considerations for in vitro toxicity analysis

The lactate dehydrogenase (LDH) assay is a commonly-used tool for assessing toxicity in vitro. However, anecdotal reports suggest that foetal bovine serum (FBS) may contain LDH at concentrations significant enough to interfere with the assay and thus reduce its sensitivity. A series of experiments were performed to determine whether addition of FBS to culture medium significantly elevated culture media LDH content, and whether replacement of FBS with heat inactivated foetal bovine serum (HI-FBS) reduced LDH content and interfered with cell response to cytotoxic challenge. The addition of FBS at 5, 10 and 15% final concentrations increased culture medium LDH content in a dose-dependent manner. The substitution of HI-FBS for FBS reduced culture medium LDH content and increased the dynamic range of the assay. Cell viability of the SH-SY5Y human neuroblastoma and N27 rat mesencephalic neurone cell lines were significantly reduced as measured using the MTT reduction assay, whilst HI-FBS only affected toxicity response in a cell- and toxin-specific manner, although these effects were small. Hence, for cell lines with a high FBS requirement, the use of HI-FBS or alternative toxicity assays can be considered, or the use of alternative formulations, such as chemically-defined serum-free media, be adopted.

Case report: Post-transplantation acute humoral rejection apparently due to igm donor specific antibodies and/or anti-angiotensin type 1 receptor antibodies

Patient 46 yo (gravida 6) BF, IgA nephropathy. Pre-transplant LABScreen SA beads (SAB): anti-B57 (MFI = 11,125), B58 (9163), DP1 (1871). Class I and Class II DSA not detected. See Table 1. Virtual-XM predicted negative; deceased donor pre-transplant flow- and CDC-XM were negative. Renal function was poor following transplant. By day 3 post-transplant clinical findings were consistent with AMR. Day 4 renal biopsy revealed patchy C4d+ in peritubular capillaries consistent with AMR. Nephrectomy was performed on post-transplant day 7. Additional assays were performed on pre- and 3 wk post-transplant sera to evaluate for reasons for AMR, including: One Lambda LABScreen SAB assay for C1q; and modified for C1q + AHG, detection of IgM, and use of non-heat inactivated serum for assays; and One Lambda MICA, anti-AT1R, and anti-ETAR antibody assays. See Tables. Results Pre-transplant IgG DSA were negligible. IgM DSA were elevated. Anti-AT1R antibody was elevated. Interpretation: immediate post-transplant humoral rejection correlates with presence of IgM DSA and anti-AT1R (IgG) antibodies. Discussion IgM anti-HLA antibodies are not considered to be relevant for AMR. Anti-AT1R is reported as a cause of acute AMR, but not typically in the immediate post-transplant period. We believe this case argues that IgM DSA appear be relevant, but cannot exclude that either anti-AT1R IgG antibodies alone, or in combination with IgM DSA, may result in AMR. Conclusions IgM DSA and/or anti-AT1R may result in post-transplant humoral rejection, and therefore should be considered in evaluation for episodes of AMR, especially when IgG DSA are negligible. Download : Download full-size image Download : Download full-size image T. Harville: Scientific/Medical Advisor; Company/Organization; Baxter Healthcare, CSL Behring. 7. Other (Identify); Company/Organization; Medical Advisory Board for Arkansas Regional Organ Recovery Agency.

608. Evaluation of Pre-Existing Neutralizing Antibodies Against Tyrosine-Mutant AAV2 in Leber Hereditary Optic Neuropathy (LHON) Patients

PURPOSE: Pre-existing neutralizing antibodies against adeno-associated virus (AAV), due to natural AAV infections could interfere with the recombinant AAV vector-mediated gene transfer, thus decreasing the efficiency and preventing effective vector readministration. Here, we measured neutralizing antibody (NAb) titers against tyrosine-mutant AAV serotype 2 in Leber Hereditary Optic Neuropathy (LHON) patients.METHODS: Serum samples from LHON patients (n=53, age =33 + 1.9 yrs, average + se) were screened for the existence of NAbs to AAV2 using an invitro assay. For this, a self-complimentary AAV2(Y444,500,703F)-smCBA-mCherry vector was pre-incubated with heat inactivated LHON patient serum samples at various dilutions, beginning with a 1:5 dilution and ending at 1:20,480. The serum-vector mixtures were used to infect RGC5 cells at multiplicity of infection of 5000 vector genomes/per cell. Two days post-infection, cell viability was confirmed by microscopy and transduction scored by flow cytometry. Expression of mCherry was quantified by multiplying the percentage of cells positive for mCherry by the mean fluorescence intensity. The neutralizing antibody (NAb) titer was reported as the highest serum dilution that inhibited self-complementary AAV2 (Y444,500,703F)-smCBA-mCherry transduction >50% compared to no serum control.RESULTS: We found that the prevalence of NAbs to AAV2 (Y444,500,703F) among the LHON subjects was variable with majority, 49% (26/53) showing low NAb titers ( 0.05).CONCLUSIONS: Previous AAV2 exposure among LHON patients is low to high and these findings as they relate to the ongoing clinical human gene therapy trial (NCT02161380) will be discussed.

Hypertrophy Inducing Factor In Pediatric Idiopathic Dilated Cardiomyopathy Serum

IntroductionHeart failure (HF) is a devastating and costly disease that results in about 50% mortality within 5 years of diagnosis. Despite improvements in adult HF outcomes, children with HF have had no substantial improvement in survival over the past 3 decades. Our work has shown that the myocardium of children with idiopathic dilated cardiomyopathy (IDC) demonstrates a gene expression pattern consistent with pathologic remodeling. The objective of this study was to investigate whether serum from pediatric IDC patients would recapitulate this pathologic gene expression pattern in neonatal rat ventricular myocytes (NRVMs).MethodsNRVMs were treated with: 1) whole serum from IDC and non‐failing (NF) control patients; 2) heat inactivated serum; or 3) exosomes isolated from serum. Serum protein identification was performed by mass spectrometry.ResultsTreatment of NRVMs with whole serum from pediatric IDC patients induced a pathologic hypertrophic response that was characterized by changes in gene expression and increase in cell size. These changes were prevented by heat inactivation, indicating a protein factor. Exosome treatment suggests that the observed effect is also mediated by RNAs/miRNAs.ConclusionWe show that circulating factors (RNA and protein) present in the serum of pediatric IDC patients but not in NF controls, induce a pathologic response in NRVMs. Identification of these circulating factors could represent a novel approach to age‐specific HF drug discovery.Source of Research Support: NIH‐NHLBI R01 HL107715 (BLS), APS Undergraduate Summer Research Fellowship (JSC)

Membrane‐Spanning DNA Nanopores with Cytotoxic Effect

Membrane‐Spanning DNA Nanopores with Cytotoxic Effect

Anti-leishmanial Activity of Gossypium hirsutum L., Ferula assa-foetida L. and Artemisia aucheri Boiss. Extracts by Colorimetric Assay

Aim: Plant extracts or plant-derived compounds are likely to provide a valuable source of new medicinal agents. In the present work anti-leishmanial activity of methanolic extracts of Gossypium hirsutum, Ferula assa-foetida and Artemisia aucheri, was studied on Leishmania major promastigotes by colorimetric assay in comparison to a trivalent antimony compound (tartar emetic). Methods: L. major promastigotes were cultured at 24±1°C to stationary phase in RPMI 1640 medium, supplemented with 10% heat inactivated fetal calf serum (FCS) and antibiotics. The biological activity of three plant extracts was studied against L. major promastigotes in comparison to tartar emetic. The optical density (OD) due to cleavage of the tetrazolium salt MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] into a colored product formazan by the parasite was measured using an ELISA reader, to determine IC50 values (50% inhibitory concentrations). All experiments were repeated in triplicate. Results: All three plant extracts and tartar emetic inhibited the growth of promastigote stage of L.major after 72 hours of incubation. Tartar emetic as positive control gave a 50% inhibitory concentration (IC50) of 4.7µg/ml, while the IC50 values of G. hirsutum, F. assa-foetida and A. aucheri, were determined as 3.6, 5.9 and 7.5µg/ml, respectively. Although G. hirsutum was more active than the others, but all extracts had profound effects on promastigotes of L. major. Conclusion: This study provides valuable data on potent activity of three plant extracts. Further works are required to evaluate the leishmanicidal effect of these plant extracts on animal models or human volunteers. Keywords: Artemisia aucheri, Ferula assa-foetida, Gossypium hirsutum, Leishmania major, MTT assay.

BMPs Enhance in Vitro Tissue Regeneration by Human Degenerated Nucleus Pulposus Cells

Introduction Bone morphogenetic proteins (BMPs) are currently being investigated for differentiation of stem cells or enhancement of tissue production for various cell types. It has been shown that heterodimers of BMPs can be more effective than the combined homodimers.1 In this study, we investigate whether this is also true for tissue regeneration by human degenerated NP cells. Materials and Methods Nucleus pulposus (NP) cells from a human donor (Thompson grade III) were cultured for 14 days in high density (1 × 10^6 cells/cm2) on collagen II coated filters. Several rhBMPs (BMP2, BMP4, BMP7, BMP2+7, BMP4+7 and the heterodimers of BMP2,7 and BMP4,7) were added to the culture medium (DMEM + 10% heat inactivated fetal bovine serum + 2% ascorbic acid-2-phosphate) in two different concentrations (total concentration 10 or 100 ng/mL medium). Control medium contained 10 ng/mL TGF-β2. Samples were stained with Safranin O for glycosaminoglycans (GAGs) and with immunohistochemistry for collagen II. Matrix content as reflected by GAG production was measured with a dimethylmethylene blue (DMMB) assay and DNA content with a PicoGreen assay. Statistical analyses included one-way ANOVA and post hoc Bonferroni. Results GAG per DNA was highest for BMP4 (see Fig. 1 for p values). At 10 ng/mL, the heterodimer of BMP2,7 resulted in more GAG/DNA than the homodimers combined ( p = 0.003) or BMP7 alone ( p = 0.003), but not for 100 ng/mL. GAG per DNA was higher at 100 ng/mL BMP versus 10 ng/mL for all BMPs ( p < 0.001). Conclusion In conclusion, BMP4 seems to be the most potent factor for improving matrix production by NP cells in vitro. The heterodimers of BMP2,7 and BMP4,7 do not seem to be clearly more effective for tissue production than the homodimers combined or alone, especially for higher concentrations. Disclosure of Interest None declared Reference Israel DI, Nove J, Kerns KM, et al. Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo. Growth Factors 1996;13(3-4):291–300

Effectiveness of a peracetic acid-based disinfectant against spores of Bacillus cereus under different environmental conditions

A peracetic acid based disinfectant was tested for its efficacy against spores of different Bacillus cereus -strains (DSM 318, 4312, 4313 and 4384). To determine the influence of different factors like exposure-time, temperature and presence of protein quantitative and qualitative suspension tests were performed. Spore suspensions of B. cereus were treated with various concentrations of a representative peracetic acid based disinfectant at three temperatures (10, 15 and 20 °C), with protein load and with different exposure times (5, 30 and 60 min). Temperature, level of concentration and exposure-time had a significant influence on reduction of spores of B. cereus (p < 0.05). The susceptibility of spores of different strains greatly differed. A treatment of spores of DSM 4384 with 2.0% for 30 min even at 10 °C inactivated all present spores (initial number 6.18–6.71 log CFU/ml). Spores of B. cereus strain DSM 4313 had only reductions of 0.16–0.97 log CFU/ml at same treatment conditions. The presence of inactivated bovine serum as interfering substance had no significant influence on reduction (p > 0.05).

A new insect cell line from the pupal ovary of the Asian corn borer moth Ostrinia furnacalis

Ovaries were removed from Ostrinia furnacalis (Guenée) pupae and were placed in a flask containing TNM-FH medium with 10% inactivated fetal bovine serum. Cell migration occurred after about 1 wk of the initiation in June 2011. The migrated cells were distributed over most of the flask and were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a chemical carcinogen, after about 1 mo of initiation for 26 d. Cells were first subcultured successfully 12 d after the MNNG was removed, followed by subculturing for 30 passages. The established cell line, designated IOZCAS-Osfu-1, were analyzed by DNA fingerprinting-PCR (DAF-PCR) to confirm that it originated from O. furnacalis.

Phenotypic alterations in human saphenous vein culture induced by tumor necrosis factor-alpha and lipoproteins: a preliminary development of an initial atherosclerotic plaque model

BackgroundAtherosclerosis is a chronic progressive inflammatory disease of blood vessels particularly the arteries. The development of atherosclerotic plaques or atherogenesis is a complex process that is influenced by cardiovascular risk factors such as vascular inflammation and dyslipidemia. This study demonstrates the ability of tumor necrosis factor-alpha (TNF-α) and low density lipoproteins (LDL) to induce atherosclerotic plaque in human saphenous vein (HSV) organ culture.MethodsNormal HSV segments, from male patients who had coronary bypass graft, were cultured in DMEM containing 5% heat inactivated fetal bovine serum. TNF-α (5 ng/ml) was applied in combination with native LDL (nLDL) or oxidized LDL (oxLDL) at the dose of 50 μg/ml for 14 days. The phenotypic changes of the organ cultures characteristic of initial atherosclerotic plaques were evaluated. The effect of anti-atherogenic agent, 17-β estradiol (E2), was also determined.ResultsHistologic, histomorphometric, and immunohistochemical examinations revealed that HSV rings stimulated with TNF-α + nLDL or TNF-α + oxLDL can exhibit the essential morphological features of atherogenesis, including fibrous cap formation, cholesterol clefts, evident thickening of the intimal layer, increased proliferation of smooth muscle cells (SMC) and migration to the subendothelial layer, significant SMC foam cell formation, and increased expression of adhesion molecules in the vascular wall. Addition of E2 (50 nM) to the culture significantly modulated the critical changes. Consistently, mRNA profiling of the HSV model revealed that 50 of 84 genes of atherosclerosis were up-regulated.ConclusionsPhenotypic changes characteristic of the initial development of atherosclerotic plaques can be induced in HSV organ culture.

Ultralarge von Willebrand Factor Fibers Mediate Luminal Staphylococcus aureus Adhesion to an Intact Endothelial Cell Layer Under Shear Stress

During pathogenesis of infective endocarditis, Staphylococcus aureus adherence often occurs without identifiable preexisting heart disease. However, molecular mechanisms mediating initial bacterial adhesion to morphologically intact endocardium are largely unknown. Perfusion of activated human endothelial cells with fluorescent bacteria under high-shear-rate conditions revealed 95% attachment of the S aureus by ultralarge von Willebrand factor (ULVWF). Flow experiments with VWF deletion mutants and heparin indicate a contribution of the A-type domains of VWF to bacterial binding. In this context, analyses of different bacterial deletion mutants suggest the involvement of wall teichoic acid but not of staphylococcal protein A. The presence of inactivated platelets and serum increased significantly ULVWF-mediated bacterial adherence. ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13) caused a dose-dependent reduction of bacterial binding and a reduced length of ULVWF, but single cocci were still tethered by ULVWF at physiological levels of ADAMTS13. To further prove the role of VWF in vivo, we compared wild-type mice with VWF knockout mice. Binding of fluorescent bacteria was followed in tumor necrosis factor-α-stimulated tissue by intravital microscopy applying the dorsal skinfold chamber model. Compared with wild-type mice (n=6), we found less bacteria in postcapillary (60±6 versus 32±5 bacteria) and collecting venules (48±5 versus 18±4 bacteria; P<0.05) of VWF knockout mice (n=5). Our data provide the first evidence that ULVWF contributes to the initial pathogenic step of S aureus-induced endocarditis in patients with an apparently intact endothelium. An intervention reducing the ULVWF formation with heparin or ADAMTS13 suggests novel therapeutic options to prevent infective endocarditis.

Protection of Bovine Chondrocyte Phenotype by Heat Inactivation of Allogeneic Serum in Monolayer Expansion Cultures

Cartilage defects can be addressed with replacement strategies such as autologous chondrocyte implantation (ACI). Expansion of autologous chondrocytes in vitro is an essential step to obtain the necessary cell numbers required for ACI. A major problem with this approach is dedifferentiation of chondrocytes during expansion, resulting in cells with fibroblast-like features. These cells generate cartilage tissue with fibrotic instead of hyaline characteristics. The use of serum is a common feature in most expansion protocols and a potential factor contributing to the dedifferentiation process. The aim of this study was to assess if heat inactivation of serum used in the expansion medium might be a valid approach to generate cells with an improved phenotype and in relevant numbers. We used bovine chondrocyte expansion cultures incubated with heat inactivated allogeneic serum (HIFBS) as a model system. We here show that heat inactivation protects the differentiated phenotype of chondrocytes compared to cultures with regular serum. This is not only true for primary cultures but holds up after two passages. Moreover, using relatively low cell seeding densities, clinically relevant cell numbers can already be reached after the first passage in cultures with HIFBS. In short we here introduce a simple way to improve cell quality while generating relevant amounts of cells during monolayer expansion of bovine chondrocytes in a relative short time period. Our results could have wider implications when translated to the expansion of human chondrocytes.

Genome-wide Gene Deletions in &lt;em&gt;Streptococcus sanguinis&lt;/em&gt; by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in &lt;em&gt;Streptococcus sanguinis&lt;/em&gt; by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria (1,2). Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Complement Inhibitors from Scabies Mites Promote Streptococcal Growth – A Novel Mechanism in Infected Epidermis?

BackgroundScabies is highly prevalent in socially disadvantaged communities such as indigenous populations and in developing countries. Generalized itching causes discomfort to the patient; however, serious complications can occur as a result of secondary bacterial pyoderma, commonly caused by Streptococcus pyogenes (GAS) or Staphylococcus aureus. In the tropics, skin damage due to scabies mite infestations has been postulated to be an important link in the pathogenesis of disease associated with acute rheumatic fever and heart disease, poststreptococcal glomerulonephritis and systemic sepsis. Treatment of scabies decreases the prevalence of infections by bacteria. This study aims to identify the molecular mechanisms underlying the link between scabies and GAS infections.Methodology/Principal FindingsGAS bacteria were pre-incubated with blood containing active complement, phagocytes and antibodies against the bacteria, and subsequently tested for viability by plate counts. Initial experiments were done with serum from an individual previously exposed to GAS with naturally acquired anti-GAS antibodies. The protocol was optimized for large-scale testing of low-opsonic whole blood from non-exposed human donors by supplementing with a standard dose of heat inactivated human sera previously exposed to GAS. This allowed an extension of the dataset to two additional donors and four proteins tested at a range of concentrations. Shown first is the effect of scabies mite complement inhibitors on human complement using ELISA-based complement activation assays. Six purified recombinant mite proteins tested at a concentration of 50 µg/ml blocked all three complement activation pathways. Further we demonstrate in human whole blood assays that each of four scabies mite complement inhibitors tested increased GAS survival rates by 2–15 fold.Conclusions/SignificanceWe propose that local complement inhibition plays an important role in the development of pyoderma in scabies infested skin. This molecular link between scabies and bacterial infections may provide new avenues to develop alternative treatment options against this neglected disease.