Implementation of an extracorporeal resuscitation (ECPR) program for out-of-hospital cardiac arrest in Stockholm, Sweden: Feasibility, safety, and outcome.

Background: The aim of this study was to evaluate the implementation of a novel extra corporeal cardiopulmonary (ECPR) program in the greater Stockholm area with focus on feasibility, safety aspects and clinical outcomes. Methods: Prospective observational study of ECPR program including patients with OHCA from January 2020 to December 2022, fulfilling ECPR criteria: age 18-65 years, initial shockable rhythm or pulseless electrical activity, witnessed arrest, bystander cardiopulmonary resuscitation and refractory arrest after three cycles of advance cardiac life support. The predefined time threshold from collapse to extracorporeal membrane oxygenation (ECMO) initiation was set at 60 min. Results: We included 95 patients. Of these, 22/95 (23%) had return of spontaneous circulation before ECMO initiation, 39/95 (41%) were excluded for ECMO and 34/95 (36%) had ECMO initiated out of which 23 patients were admitted alive to the ICU. ECMO-initiation within 60 min was met in 9%. In 6 patients vascular access was complicated, 2 patients had severe bleeding at access site requiring intervention. Survival to discharge among all cases was 25% (24/95). Among patients admitted to ICU on ECMO 39% (9/23) survived to discharge, of these 78% had cerebral performance category scale score 1-2 within 12 months. 8 out of 9 survivors had time from OHCA to ECMO-initiation >60 min. Conclusion: The implementation of an ECPR protocol was feasible without any major, unexpected safety aspects but did not meet the intended target time intervals. Despite this, survival rates were similar to previous studies although most survivors had >60 min to ECMO-initiation.

Physician-staffed prehospital units: a retrospective follow-up from an urban area in Scandinavia.

BACKGROUND: The aim of this study was to determine when and how rapid response vehicles (RRVs) make a difference in prehospital care by investigating the number and kinds of RRV assignment dispatches and the prehospital characteristics and interventions involved. METHODS: This retrospective cohort study was based on data from a quality assurance system where all assignments are registered. RRV staff register every assignment directly at the site, using a smartphone, tablet, or computer. There is no mandatory information requirement or time limit for registration. The study includes data for all RRVs operating in Region Stockholm, three during daytime hours and one at night - from January 1, 2021 to December 31, 2021. RESULTS: In 2021, RRVs in Stockholm were dispatched on 11,283 occasions, of which 3,571 (31.6%) resulted in stand-downs. In general, stand-downs were less common for older patients. The most common dispatch category was blunt trauma (1,584 or 14.0%), which accounted for the highest frequency of stand-downs (676 or 6.0%). The second most common category was cardiac arrest (1,086 or 9.6%), followed by shortness of breath (691 or 6.1%), medical not specified (N/S) (596 or 5.3%), and seizures (572 or 5.1%). CONCLUSION: The study findings confirm that RRVs provide valuable assistance to the ambulance service in Stockholm, especially for cardiac arrest and trauma patients. In particular, RRV personnel have more advanced medical knowledge and can administer medications and perform interventions that the regular ambulance service cannot provide.

Glycolipid transfer protein knockout disrupts vesicle trafficking to the plasma membrane.

The glycolipid transfer protein (GLTP) has been linked to many cellular processes aside from its best-known in vitro function as a lipid transport protein. It has been proposed to act as a sensor and regulator of glycosphingolipid homeostasis in cells. Furthermore, through its previously determined interaction with the endoplasmic reticulum membrane protein VAP-A (vesicle-associated membrane protein-associated protein A), GLTP may also be involved in facilitating vesicular transport in cells. In this study, we characterized the phenotype of CRISPR/Cas9-mediated GLTP KO HeLa cells. We showed that motility, three-dimensional growth, and cellular metabolism were all altered by GLTP knockout. Expression of a GLTP mutant incapable of binding VAP disrupted cell spheroid formation, indicating that the GLTP-VAP interaction is linked to cellular adhesion, cohesion, and three-dimensional growth. Most notably, we found evidence that GLTP, through its interaction with VAP-A, affects vesicular trafficking, marking the first cellular process discovered to be directly impacted by a change in GLTP expression.

Who moves the sphinx? An overview of intracellular sphingolipid transport.

Lipid bilayers function as boundaries that enclose their content from the surrounding media, and the composition of different membrane types is accurately and dynamically tailored so that they can perform their function. To achieve this balance, lipid biosynthetic machinery and lipid trafficking events are intertwined into an elegant network. In this review, we focus on the intracellular movement of sphingolipids mediated by sphingolipid transfer proteins. Additionally, we will focus on the best characterized and understood mammalian sphingolipid transfer proteins and provide an overview of how they are hypothesized to function. Some are already well understood, while others remain enigmatic. A few are actual lipid transfer proteins, moving lipids from membrane to membrane, while others may have more of a sensor role, possibly reacting to changes in the concentrations of their ligands. Considering the substrates available for cytosolic sphingolipid transfer proteins, one open question that is discussed is whether galactosylceramide is a target. Another question is the exact mechanics by which sphingolipid transfer proteins are targeted to different organelles, such as how four phosphate adapter protein-2, FAPP2 is targeted to the endoplasmic reticulum. The aim of this review is to discuss what is known within the field today and to provide a basic understanding of how these proteins may work.

LAPTM4B controls the sphingolipid and ether lipid signature of small extracellular vesicles.

Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is a four-membrane spanning ceramide interacting protein that regulates mTORC1 signaling. Here, we show that LAPTM4B is sorted into intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) and released in small extracellular vesicles (sEVs) into conditioned cell culture medium and human urine. Efficient sorting of LAPTM4B into ILV membranes depends on its third transmembrane domain containing a sphingolipid interaction motif (SLim). Unbiased lipidomic analysis reveals a strong enrichment of glycosphingolipids in sEVs secreted from LAPTM4B knockout cells and from cells expressing a SLim-deficient LAPTM4B mutant. The altered sphingolipid profile is accompanied by a distinct SLim-dependent co-modulation of ether lipid species. The changes in the lipid composition of sEVs derived from LAPTM4B knockout cells is reflected by an increased stability of membrane nanodomains of sEVs. These results identify LAPTM4B as a determinant of the glycosphingolipid profile and membrane properties of sEVs.

Caspase-1 inflammasome activity in patients with Staphylococcus aureus bacteremia.

The inflammasome is a multiprotein complex that mediates caspase-1 activation with subsequent maturation of the proinflammatory cytokines IL-1ß and IL-18. The NLRP3 inflammasome is known to be activated by Staphylococcus aureus, one of the leading causes of bacteremia worldwide. Inflammasome activation and regulation in response to bacterial infection have been found to be of importance for a balanced host immune response. However, inflammasome signaling in vivo in humans initiated by S. aureus is currently sparsely studied. This study therefore aimed to investigate NLRP3 inflammasome activity in 20 patients with S. aureus bacteremia (SAB), by repeated measurement during the first week of bacteremia, compared with controls. Caspase-1 activity was measured in monocytes and neutrophils by flow cytometry detecting FLICA (fluorescent-labeled inhibitor of caspase-1), while IL-1ß and IL-18 was measured by Luminex and ELISA, respectively. As a measure of inflammasome priming, messenger RNA (mRNA) expression of NLRP3, CASP1 (procaspase-1), and IL1B (pro-IL-1ß) was analyzed by quantitative PCR. We found induced caspase-1 activity in innate immune cells with subsequent release of IL-18 in patients during the acute phase of bacteremia, indicating activation of the inflammasome. There was substantial interindividual variation in caspase-1 activity between patients with SAB. We also found an altered inflammasome priming with low mRNA levels of NLRP3 accompanied by elevated mRNA levels of IL1B. This increased knowledge of the individual host immune response in SAB could provide support in the effort to optimize management and treatment of each individual patient.

Different photodynamic effects of blue light with and without riboflavin on methicillin-resistant Staphylococcus aureus (MRSA) and human keratinocytes in vitro.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of infections in humans. Photodynamic therapy using blue light (450 nm) could possibly be used to reduce MRSA on different human tissue surfaces without killing the human cells. It could be less harmful than 300-400 nm light or common disinfectants. We applied blue light ± riboflavin (RF) to MRSA and keratinocytes, in an in vitro liquid layer model, and compared the effect to elimination using common disinfection fluids. MRSA dilutions (8 × 105/mL) in wells were exposed to blue light (450 nm) ± RF at four separate doses (15, 30, 56, and 84 J/cm2). Treated samples were cultivated on blood agar plates and the colony forming units (CFU) determined. Adherent human cells were cultivated (1 × 104/mL) and treated in the same way. The cell activity was then measured by Cell Titer Blue assay after 24- and 48-h growth. The tested disinfectants were chlorhexidine and hydrogen peroxide. Blue light alone (84 J/cm2) eliminated 70% of MRSA. This dose and riboflavin eradicated 99-100% of MRSA. Keratinocytes were not affected by blue light alone at any dose. A dose of 30 J/cm2 in riboflavin solution inactivated keratinocytes completely. Disinfectants inactivated all cells. Blue light alone at 450 nm can eliminate MRSA without inactivation of human keratinocytes. Hence, a high dose of blue light could perhaps be used to treat bacterial infections without loss of human skin cells. Photodynamic therapy using riboflavin and blue light should be explored further as it may perhaps be possible to exploit in treatment of skin diseases associated with keratinocyte hyperproliferation.

Indirect Lipid Transfer Protein Activity Measurements Using Quantification of Glycosphingolipid Production.

Here we summarize how glycosphingolipid production can be followed using metabolic labeling with radiolabeled lipid precursors. No assays are available yet that directly would address the lipid transfer protein activity in vivo. Therefore, these approaches can serve as tools to indirectly study the lipid transfer protein activity in cells, by monitoring their impact on the glycosphingolipid homeostasis.

Glucosylceramide acyl chain length is sensed by the glycolipid transfer protein.

The glycolipid transfer protein, GLTP, can be found in the cytoplasm, and it has a FFAT-like motif (two phenylalanines in an acidic tract) that targets it to the endoplasmic reticulum (ER). We have previously shown that GLTP can bind to a transmembrane ER protein, vesicle-associated membrane protein-associated protein A (VAP-A), which is involved in a wide range of ER functions. We have addressed the mechanisms that might regulate the association between GLTP and the VAP proteins by studying the capacity of GLTP to recognize different N-linked acyl chain species of glucosylceramide. We used surface plasmon resonance and a lipid transfer competition assay to show that GLTP prefers shorter N-linked fully saturated acyl chain glucosylceramides, such as C8, C12, and C16, whereas long C18, C20, and C24-glucosylceramides are all bound more weakly and transported more slowly than their shorter counterparts. Changes in the intrinsic GLTP tryptophan fluorescence blueshifts, also indicate a break-point between C16- and C18-glucosylceramide in the GLTP sensing ability. It has long been postulated that GLTP would be a sensor in the sphingolipid synthesis machinery, but how this mechanistically occurs has not been addressed before. It is unclear what proteins the GLTP VAP association would influence. Here we found that if GLTP has a bound GlcCer the association with VAP-A is weaker. We have also used a formula for identifying putative FFAT-domains, and we identified several potential VAP-interactors within the ceramide and sphingolipid synthesis pathways that could be candidates for regulation by GLTP.

Preoperative preparation of eye with chlorhexidine solution significantly reduces bacterial load prior to 23-gauge vitrectomy in Swedish health care.

BACKGROUND: Bacteria in the conjunctiva present a potential risk of vitreous cavity infection during 23-gauge pars plana vitrectomy (PPV). Current preoperative procedures used in Sweden include irrigation with chlorhexidine solution (CHX) 0.05% only and no iodine solutions. We evaluated the bacterial diversity and load before and after this single antibacterial measure. METHODS: In a prospective, consecutive cohort we investigated bacterial growth in samples from 40 eyes in 39 consecutive individuals subjected to vitrectomy. A conjunctival specimen was collected from each preoperative patient before and after irrigating of eye with CHX, 0.05% solution. Iodine was not used during any part of the surgery. One drop of chloramphenicol was administered prior to surgery. Samples from vitreous cavity were collected at the beginning and end of vitrectomy. All conjunctival specimens were cultured for different species and quantified using colony forming units (CFU). RESULTS: There was a significant 82% reduction in the total number of CFUs for all bacteria in all eyes (P < 0.0001), and 90% reduction for coagulase negative staphylococci (CoNS) alone (P = 0.0002). The number of eyes with positive bacterial growth in conjunctival samples decreased from 33 to 18 after irrigation with CHX (P = 0.0023). The most common bacteria prior to surgery were CoNS (70%), Propionibacterium acnes (55%) and Corynebacterium species (36%). No case of post-vitrectomy endophthalmitis was reported during mean follow-up time, which was 4.6 ± 2.3 (range; 1.5 to 9) months. CONCLUSIONS: Patients undergoing PPV harbored bacteria in conjunctiva capable of causing post-vitrectomy endophthalmitis. Preoperative preparation with CHX significantly reduced the bacterial load in the conjunctival samples subsequently leading to very low inoculation rates in recovered vitreous samples. Thus, CHX used as a single disinfectant agent might be an effective preoperative procedure for eye surgery in Sweden. This is a relatively small study but the results could be a reference for other intraocular surgeries.

Drones for Provision of Flotation Support in Simulated Drowning.

OBJECTIVE: The feasibility and potential of using drones for providing flotation devices in cases of drowning have not yet been assessed. We hypothesize that a drone carrying an inflatable life buoy is a faster way to provide flotation compared with traditional methods. The purpose of this study is to explore the feasibility and efficiency of using a drone for delivering and providing flotation support to conscious simulated drowning victims. METHODS: A simulation study was performed with a simulated drowning victim 100 m from the shore. A drone (DJI Phantom 4; dji, Shenzhen, China) equipped with an inflatable life buoy of 60 N was compared with traditional surf rescue swimming for providing flotation. The primary outcome was delay (minutes:seconds). RESULTS: A total number of 30 rescues were performed with a median time to delivery of the floating device of 30 seconds (interquartile range [IQR] = 24-32 seconds) for the drone compared with 65 seconds (IQR = 60-77 seconds) with traditional rescue swimming (P < .001). The drone had an accuracy of 100% in dropping the inflatable life buoy < 5 m from the victim, with a median of 1 m (IQR = 1-2 m). CONCLUSION: Using drones to deliver inflatable life buoys is safe and may be a faster method to provide early flotation devices to conscious drowning victims compared with rescue swimming.

Expression of HLA-DRA and CD74 mRNA in whole blood during the course of complicated and uncomplicated Staphylococcus aureus bacteremia.

To improve management of Staphylococcus aureus bacteremia (SAB), better understanding of host-pathogen interactions is needed. In vitro studies have shown that S. aureus bacteria induce dose-dependent immunosuppression that is evidenced by reduced expression of major histocompatibility complex (MHC) class II on antigen presenting cells. Thus, the aim of this study was to determine whether expression of the MHC class II-related genes HLA-DRA and CD74 is more greatly reduced in complicated SAB, with its probable higher loads of S. aureus, than in uncomplicated SAB. Adult patients with SAB were prospectively included and blood samples taken on the day of confirmation of SAB (Day 1) and on Days 2, 3, 5 and 7. HLA-DRA and CD74 mRNA expression was determined by quantitative reverse transcription PCR. Sepsis was defined according to the Sepsis-3 classification and SAB was categorized as complicated in patients with deep-seated infection and/or hematogenous seeding. Twenty patients with SAB were enrolled and samples obtained on all assessment days. HLA-DRA and CD74 expression did not differ significantly between patients with SAB and sepsis (n = 13) and those without sepsis (n = 7) on any assessment day. However, patients with complicated SAB (n = 14) had significantly weaker HLA-DRA expression on all five assessment days than patients with uncomplicated SAB (n = 6). Additionally, they tended to have weaker CD74 expressions. Neutrophil, monocyte and leukocyte counts did not differ significantly between complicated and uncomplicated SAB. In conclusion, patients with complicated SAB show weaker HLA-DRA expression than those with uncomplicated SAB during the first week of bacteremia.

Purification and Validation of Lipid Transfer Proteins.

Understanding the holistic picture of lipid homeostasis not only involves the analysis of synthesis and breakdown of lipids but also requires a thorough understanding of their transport. The transport of lipid monomers in an aqueous environment is facilitated by different lipid transfer proteins. Their universal feature is the shielding or encapsulation of the hydrophobic part of the lipid, consequently overcoming the poor solubility of lipids in water. Here we describe a method to purify lipid transfer proteins using bacterial expression. We also present three methods to validate their transfer activity.

Photochemical eradication of methicillin-resistant Staphylococcus aureus by blue light activation of riboflavin.

PURPOSE: To compare elimination of methicillin-resistant Staphylococcus aureus (MRSA) by exposure of blue light alone and with riboflavin. METHODS: A reference strain of MRSA was cultured and diluted in PBS with and without riboflavin (0.01%). Fifteen microlitre was added on a microscope slide, creating a fluid layer with a thickness of around 400 microns. Both of the bacterial suspensions were exposed to blue light, and the effect between exposure with and without riboflavin was compared. Evaluation involved two different wavelengths (412 and 450 nm) of blue light with a lower (5.4 J/cm2 ) and higher dose (approximately 28.5 J/cm2 ). The effect of 412 nm light was also evaluated for a thicker fluid layer (1.17 mm). After exposure, colony-forming units (CFUs) were determined for each solution. All measurements were repeated eight times. RESULTS: The reductions in bacteria were similar for both wavelengths. With riboflavin, a statistically significant elimination was observed for both 412 and 450 nm (p < 0.001). At both dosages, the mean reduction was more pronounced with the presence of riboflavin than without it. Using the higher dose, CFU reduction was 99% and 98%, respectively, for 412 and 450 nm light. The bactericidal efficacy was high also in the deeper fluid layer (93%, higher dose). CONCLUSION: Riboflavin enhanced the antibacterial effect on the exposed MRSA strain of blue light for both 412 and 450 nm blue light. This indicates that blue light could be considered for possible implementation in deep corneal infections.

Quantitative Real-Time Polymerase Chain Reaction Measurement of HLA-DRA Gene Expression in Whole Blood Is Highly Reproducible and Shows Changes That Reflect Dynamic Shifts in Monocyte Surface HLA-DR Expression during the Course of Sepsis.

INTRODUCTION: A decrease in the expression of monocyte surface protein HLA-DR (mHLA-DR), measured by flow cytometry (FCM), has been suggested as a marker of immunosuppression and negative outcome in severe sepsis. However, FCM is not always available due to sample preparation that limits its use to laboratory operational hours. In this prospective study we evaluated dynamic changes in mHLA-DR expression during sepsis in relation to changes in HLA-DRA gene expression and Class II transactivator (CIITA), measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). AIMS: The aims of this study were: 1. to validate the robustness of qRT-PCR measurement of HLA-DRA- and CIITA-mRNA expression, in terms of reproducibility; and 2. to see if changes in expression of these genes reflect changes in mHLA-DR expression during the course of severe and non-severe bacteraemic sepsis. METHODS AND FINDINGS: Blood samples were collected from 60 patients with bacteraemic sepsis on up to five occasions during Days 1-28 after hospital admission. We found the reproducibility of the qRT-PCR method to be high by demonstrating low threshold variations (<0.11 standard deviation (SD)) of the qRT-PCR system, low intra-assay variation of Ct-values within triplicates (≤0.15 SD) and low inter-assay variations (12%) of the calculated target gene ratios. Our results also revealed dynamic HLA-DRA expression patterns during the course of sepsis that reflected those of mHLA-DR measured by FCM. Furthermore, HLA-DRA and mHLA-DR recovery slopes in patients with non-severe sepsis differed from those in patients with severe sepsis, shown by mixed model for repeated measurements (p<0.05). However, during the first seven days of sepsis, PCR-measurements showed a higher magnitude of difference between the two sepsis groups. Mean differences (95% CI) between severe sepsis (n = 20) and non-severe sepsis (n = 40) were; on day 1-2, HLA-DRA 0.40 (0.28-0.59) p<0.001, CIITA 0.48 (0.32-0.72) p = 0.005, mHLA-DR 0.63 (0.45-1.00) p = 0.04, day 7 HLA-DRA 0.59 (0.46-0.77) p<0.001, CIITA 0.56 (0.41-0.76) p<0.001, mHLA-DR 0.81 (0.66-1.00) p = 0.28. CONCLUSION: We conclude that qRT-PCR measurement of HLA-DRA expression is robust, and that this method appears to be preferable to FCM in identifying patients with severe sepsis that may benefit from immunostimulation.

Photodynamic UVA-riboflavin bacterial elimination in antibiotic-resistant bacteria.

BACKGROUND: To evaluate the bactericidal effect of clinical ultraviolet A (UVA) settings used in photoactivated chromophore for infectious keratitis (PACK)-collagen cross-linking (CXL) in antibiotic-resistant and non-resistant bacterial strains. METHODS: Well-characterized bacterial strains from clinical isolates, without and with antibiotic resistance, were studied in a pairwise comparison. The evaluated pathogens were Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis. Bacteria were dispersed in PBS and diluted to a concentration of approximately 4 × 105 /ml. Riboflavin was added to a concentration of 0.01%. By spreading the solution on a microscope slide, a fluid film layer, with a thickness of around 400 mm, was formed and UVA exposure followed. Eight separate exposures were made for each strain (n = 8). The degree of elimination in resistant and non-resistant pathogens was compared. RESULTS: The bactericidal efficacy of exposure differed between the tested microorganisms, and the mean elimination ranged between 60 and 92%, being most extensive in both of the evaluated Pseudomonas strains and least in the E. faecalis strains. Similar reductions were seen in antibiotic-resistant and non-resistant strains, with the exception of S. aureus, in which the resistant strain metchicillin-resistant Staphylococcus aureus (MRSA) was eradicated in a greater extent than the non-resistant strain (P = 0.030). CONCLUSION: UVA-riboflavin settings used in PACK-CXL are effective in reducing both antibiotic-resistant and non-resistant bacteria. Antibiotic resistance does not appear to be protective against the photooxidative exposure.

The efficiency of cross-linking methods in eradication of bacteria is influenced by the riboflavin concentration and the irradiation time of ultraviolet light.

PURPOSE: To determine bacterial eradication using numerous riboflavin concentrations and different ultraviolet light A (UVA) radiant and exposure time in an experimental model. METHODS: Dilutions of Staphylococcus epidermidis were mixed with riboflavin at varying concentrations (0.007­0.09%). Effects on bacterial growth were evaluated after 0, 3, 6, 30 and 60 min of UVA exposure (irradiance 30 and 3 mW/cm2). Standard settings of UVA were compared with high-power UVA approach. Different fluid thicknesses of the exposed dilutions were also examined to improve the model. RESULTS: Bacterial eradication (%) was increased after 60 compared with 30 min of UVA exposure for concentrations of 0.03­0.07% but not for 0.09% riboflavin.There was a significant difference between the efficacy between 0.03 and 0.09% and eradication dropped from 80%to 50%(p = 0.01).A correlation could be calculated for the amount of riboflavin at 60 min of UVA and the ability to kill bacteria(p = 0.01). The antibacterial effect was more pronounced when the tested bacterial suspension thickness was reduced. High-power UVA method was less potent in microbial elimination, eradicating only 60%of bacteria after 6 min versus 97­99%after 60 min in the low-power setting, compared with respective controls (p = 0.02). CONCLUSIONS: In these in vitro experiments, a longer UVA exposure time in combination with lower riboflavin levels were found to be favourable in killing bacteria as compared to the standard cross-linking settings. Further studies are needed to evaluate the clinical relevance of these findings.

Alternation in the glycolipid transfer protein expression causes changes in the cellular lipidome.

The glycolipid transfer protein (GLTP) catalyzes the binding and transport of glycolipids, but not phospholipids or neutral lipids. With its all-alpha helical fold, it is the founding member for a new superfamily, however its biological role still remains unclear. We have analyzed changes in the HeLa cell lipidome in response to down- and up-regulation of GLTP expression. We used metabolic labeling and thin layer chromatography analysis, complemented with a lipidomics mass spectroscopic approach. HeLa cells were treated with GLTP siRNA or were transiently overexpressing the GLTP gene. We identified eight different lipid classes that changed as a result of the GLTP down- or up-regulation treatments; glucosylceramide, lactosylceramide, globotriaosylceramide, ceramide, sphingomyelin, cholesterol-esters, diacylglycerol and phosphatidylserine. We discovered that the amount of globotriaosylceramide (Gb3) was extensively lowered after down-regulation of GLTP. Further, an up-regulation of GLTP caused a substantial increase in both the Gb3 and glucosylceramide levels compared to the controls. Total galactosylceramide levels remained unchanged. Both lactosylceramide and ceramide showed small changes, an increase with increasing GLTP and a decrease in the HeLa cell GLTP knockdowns. The cholesterol-esters and diacylglycerol masses increased in cells that had upregulated GLTP protein levels, wheras down-regulation did not affect their amounts. For the glycerophospholipids, phosphatidylserine was the only species that was lower in GLTP overexpressing cells. Phosphatidylethanolamine, phosphatidylglyerol and phosphatidylinositol remained unaltered. A total of 142 lipid species were profiled and quantified using shotgun lipidomics analyses. This work provides for the first time insights into how alternations in the levels of a protein that binds and transfers glycolipids affects the cellular lipid metabolism. We discuss the observed changes in the lipidome and how these relate to GLTP. We suggest, that GLTP not only could be a significant player in cellular sphingolipid metabolism, but also could have a much broader role in the overall lipid metabolism.

Evaluation of a PCR method to determine the clinical significance of blood cultures with Staphylococcus epidermidis in patients with hematological malignancies.

The aim was to investigate whether the detection and quantification of Staphylococcus epidermidis DNA in blood could distinguish S. epidermidis blood stream infections (BSIs) from blood culture contaminations in patients with hematological malignancies. The hld gene was chosen to identify S. epidermidis DNA and DNA in blood samples was detected by real-time PCR. Blood samples were obtained simultaneously with blood cultures positive for S. epidermidis (n = 30), during blood culture-negative episodes (n = 10) and episodes of bacteremia with other bacteria than S. epidermidis (n = 4) and from healthy blood donors (n = 10). In addition, DNA from S. epidermidis and a selection of other bacterial species were analyzed. Three different sets of criteria were used to classify episodes with positive blood cultures with S. epidermidis as BSIs or contaminations. All DNA preparations from S. epidermidis (n = 48) were hld-positive, but other bacterial species (n = 13) were negative. Sixteen (53%) of 30 blood samples from patients with blood cultures positive for S. epidermidis were hld-positive, but none of the controls. There was no clear association between a positive hld PCR and episodes interpreted as BSIs. In conclusion, hld PCR failed to distinguish S. epidermidis BSIs from blood culture contaminations in patients with hematological malignancies.