A peritonitis model with low mortality and persisting intra-abdominal abscesses

Intra-abdominal abscesses are a potential source of recurrent or residual infection after surgical intervention for secondary peritonitis. The development of therapies requires a model which combines low mortality with the formation of persisting abscesses and which is also suitable to study the local inflammatory response. Male Wistar rats were injected intraperitoneally with a mixture of sterile rat faeces, increasing doses of E. coli (10(4)-10(8) cfu/ml) and a fixed dose of B. Fragilis (10(4) cfu/ml). After one h a laparotomy was performed and the peritoneal cavity was debrided. Blood samples were taken under anaesthesia after 6 and 24 h. Abdominal fluid samples were collected (by laparotomy) after 24 and 72 h. The rats were killed after 5 days and the abdomen was inspected for abscesses. Mortality was 90% in the two groups with the highest doses of E. coli and 30% in those with the two lowest doses. In the latter groups all surviving rats but one showed intraabdominal abscesses and bacteremia was encountered frequently, especially after 24 h in the 10(5) cfu E. coli group. The groups receiving 10(4)-10(6) cfu E. coli showed similar plasma IL-6 concentrations after 6 h which were lowered significantly after 24 h. No circulating TNF-alpha was found. Considerable concentrations of TNF-alpha, IL-6, IL-1beta, and IL-10, were found in the peritoneal fluid after 24 h but no differences were observed between the contro groups and those receiving 10(4)-10(6) cfu E. coli. At 72 h cytokine levels were reduced significantly and remained the highest in the animals dosed with 10(6) cfu E. coli. The present model is suitable to study the mechanisms involved in, and prevention of, intra-abdominal abscess formation after surgical treatment of generalized peritonitis.     

Cardiovascular risk factors in women with inheritable thrombophilia a decade after single or recurrent hypertensive disorder of pregnancy

Objective: To described cardiovascular risk factors in women with inheritable thrombophilia 8–19 years after early-onset hypertensive disorders of pregnancy (HD) with or without recurrent HD. Methods: Women with recurrent HD were compared with women with single HD, for physical examination and cardiovascular parameters in serum. Results: Systolic blood pressure, diastolic blood pressure, and albumin: creatinine ratio were higher in women with recurrent HD compared with women with single HD (p = 0.046, p = 0.029, and p = 0.008, respectively). In both groups 72.7% had an increased cardiovascular risk. Conclusion: Women with inheritable thrombophilia after single or recurrent HD have a high cardiovascular risk.     

Transfusion of red blood cells in venoarterial extracorporeal membrane oxygenation: A multicenter retrospective observational cohort study

Background

Evidence-based recommendations for transfusion in patients with venoarterial extracorporeal membrane oxygenation (VA ECMO) are scarce. The current literature is limited to single-center studies with small sample sizes, therefore complicating generalizability. This study aims to create an overview of red blood cell (RBC) transfusion in VA ECMO patients.

Methods

This international mixed-method study combined a survey with a retrospective observational study in 16 centers. The survey inventoried local transfusion guidelines. Additionally, retrospective data of all adult patients with a VA ECMO run >24 h (January 2018 until July 2019) was collected of patient, ECMO, outcome, and daily transfusion parameters. All patients that received VA ECMO for primary cardiac support were included, including surgical (i.e., post-cardiotomy) and non-surgical (i.e., myocardial infarction) indications. The primary outcome was the number of RBC transfusions per day and in total. Univariable logistic regressions and a generalized linear mixed model (GLMM) were performed to assess factors associated with RBC transfusion.

Results

Out of 419 patients, 374 (89%) received one or more RBC transfusions. During a median ECMO run of 5 days (1st–3rd quartile 3–8), patients received a median total of eight RBC units (1st–3rd quartile 3–17). A lower hemoglobin (Hb) prior to ECMO, longer ECMO-run duration, and hemorrhage were associated with RBC transfusion. After correcting for duration and hemorrhage using a GLMM, a different transfusion trend was found among the regimens. No unadjusted differences were found in overall survival between either transfusion status or the different regimens, which remained after adjustment for potential confounders.

Conclusion

RBC transfusion in patients on VA ECMO is very common. The sum of RBC transfusions increases rapidly after ECMO initiation, and is dependent on the Hb threshold applied. This study supports the rationale for prospective studies focusing on indications and thresholds for RBC transfusion.     

Contractile effects and intracellular Ca2+ signalling induced by motilin and erythromycin in the circular smooth muscle of human colon

Motilin has excitatory effects on the colon of the rabbit and the dog, but little is known of its effect on the human colon. The aim of this study was to investigate the effects induced by motilin and erythromycin A (EMA) on muscle strips and on single cells from primary cultures from human colon. Isotonic contraction was recorded in circular muscle strips from macroscopically normal resection specimens of patients operated on for colonic neoplasm. Agonist-induced intracellular Ca2+ ([Ca2+]i) signalling was studied in primary cultures of colonic smooth-muscle cells using the ratiometric Ca2+ indicator Indo 1, on a laser-scanning confocal epifluorescence microscope. In circular muscle strips, norleucine13-porcine motilin ([Nle13]-pm)and EMA induced tonic contractions with an EC50 of 92 +/- 21 nmol L(-1) and 31 +/- 16 micromol L(-1), respectively. The maximal contraction was 21 +/- 4% (motilin) and 33 +/- 12% (EMA) of the response to 10(-4) mol L(-1) acetylcholine (ACh). The motilin antagonist OHM-11526 (10(-5.5) mol L(-1)) abolished the effects of both [Nle13]-pm and EMA. Neither tetrodotoxin (10(-5.5) mol L(-1)), L-nitro-D-arginine methyl ester (L-NAME) (10(-3.5) mol L(-1)) nor guanethidine (10(-5) mol L(-1)) interfered with the effects of [Nle13]-pm or EMA. [Nle13]-pm (10(-11)-10(-6) mol L(-1)) induced rises of [Ca2+]i in cultured colonic myocytes. At 10(-6) mol L-1, 94% of the cells responded, and half of the cells responded at 1.4 nmol L(-1) [Nle13]-pm. 81% (35/43) and 95% (75/79) responded to EMA (10(-6) mol L(-1)) and acetylcholine (ACh, 10(-4) mol L(-1)), respectively. The motilin antagonist GM-109 inhibited motilin- and EMA-induced [Ca2+]i rises. In the absence of extracellular Ca2+, only 13% (7/52) of the cells responded to [Nle13]-pm (10(-6) mol L(-1)) vs. 90% (47/52) to ACh (10(-4) mol L(-1)). Motilin and EMA have direct excitatory effects on circular smooth muscle from the human colon and these effects are mediated via a smooth-muscle motilin receptor. These findings suggest that motilin may regulate colonic motility and that motilides may have therapeutic potential for the treatment of colonic hypomotility.     

Immunity to the Bacteriocin Sublancin 168 Is Determined by the SunI (YolF) Protein of Bacillus subtilis

Bacillus subtilis strain 168 produces the extremely stable lantibiotic sublancin 168, which has a broad spectrum of bactericidal activity. Both sublancin 168 production and producer immunity are determined by the SPβ prophage. While the sunA and sunT genes for sublancin 168 production have been known for several years, the genetic basis for sublancin 168 producer immunity has remained elusive. Therefore, the present studies were aimed at identifying an SPβ gene(s) for sublancin 168 immunity. By systematic deletion analysis, we were able to pinpoint one gene, named yolF, as the sublancin 168 producer immunity gene. Growth inhibition assays performed using plates and liquid cultures revealed that YolF is both required and sufficient for sublancin 168 immunity even when heterologously produced in the sublancin-sensitive bacterium Staphylococcus aureus. Accordingly, we propose to rename yolF to sunI (for sublancin immunity). Subcellular localization studies indicate that the SunI protein is anchored to the membrane with a single N-terminal membrane-spanning domain that has an N_out-C_in topology. Thus, the bulk of the protein faces the cytoplasm of B. subtilis. This topology has not yet been reported for known bacteriocin producer immunity proteins, which implies that SunI belongs to a novel class of bacteriocin antagonists.Lantibiotics are small posttranslationally modified peptides with antimicrobial activity and are produced by gram-positive bacteria (7, 31, 44). In general, this class of bacteriocins is characterized by the presence of the unusual dehydrated amino acids 2,3-didehydroalanine (Dha) and/or 2,3-didehydrobutyrine (Dhb). With neighboring cysteine residues, Dha and Dhb can form thioether-linked lanthionine and 3-methyllanthionine bridges, respectively (15, 35).Two major types of lantibiotics have been previously identified (21). Type A lantibiotics such as nisin (26, 47), epidermin (40), and Pep5 (34) are flexible, elongated, amphipathic molecules with a positive charge. They usually act by forming pores in the cytoplasmic membrane of a sensitive target organism in processes that may involve other molecules such as the cell wall precursor lipid II (3, 55). In contrast, type B lantibiotics such as cinnamycin (14) and mersacidin (8) are globular, conformationally defined peptides that inhibit enzyme functions. Type A lantibiotics are further subdivided into type AI and AII lantibiotics on the basis of their structures; type AI lantibiotics are linear whereas type AII lantibiotics are globular at the C-terminal region. Type A lantibiotics are usually synthesized with an N-terminal leader peptide. Subsequently, they are translocated across the membrane by an ABC transporter. During membrane translocation, the leader peptide is cleaved either by a protease domain of the ABC transporter or by a separate protease (15). The leader sequences are thought to prevent lantibiotic activation prior to membrane translocation (6, 53).The sequenced Bacillus subtilis 168 strain is known to produce an extremely stable lantibiotic, named sublancin 168, which exhibits bactericidal activity against other gram-positive bacteria, including important pathogens such as Bacillus cereus, Streptococcus pyogenes, and Staphylococcus aureus (38, 49). Sublancin 168 has been classified as a type AII lantibiotic, although it displayed the, for lantibiotics, extraordinary characteristic of having two disulfide bonds in addition to a β-methyllanthionine bridge (38). The gene encoding sublancin 168, named sunA, was identified by sequencing the SPβ prophage region of the B. subtilis 168 chromosome (29). SunA is transcribed into a monocistronic mRNA (46). An operon of four successive genes (sunT, bdbA, yolJ, and bdbB) was found to be located downstream of sunA (46). The sunT gene, immediately downstream of sunA, encodes a bifunctional ABC transporter with an ATP-binding cassette domain and a proteolytic domain (31). SunT is indispensable for sublancin 168 production. This ABC transporter is therefore thought to be required for sublancin 168 export from the cytoplasm and concomitant removal of the leader peptide (10). The bdbA and bdbB genes encode thiol-disulfide oxidoreductases. Whereas BdbA is dispensable for sublancin 168 production, BdbB is of major importance for this process (2, 10, 24). A possible role of the yolJ gene in sublancin 168 production has not yet been documented.Any bacterium producing a bacteriocin must be immune to its bactericidal activity. To date, two general mechanisms for bacteriocin producer immunity have been reported. Firstly, dedicated ABC transporters of the LanFEG type can actively pump bacteriocins out of the membrane, thereby preventing their accumulation to toxic levels (32, 40, 43). Secondly, the bacterial cell can employ dedicated small producer immunity proteins of the LanI type that are usually weakly associated with the extracytoplasmic membrane surface. Such immunity proteins bind specific lantibiotics to intercept them before they can cause cell damage (20, 50, 51). An alternative type of producer immunity protein, NukH, was more recently described (36, 37). Although the function of NukH resembles that of LanI, its topology is very different, since NukH is a membrane protein with three transmembrane domains. In addition to these active immunity mechanisms, cells can also achieve resistance to lantibiotics by modifying the charge of the cell wall or cytoplasmic membrane. For example, the d-alanylation of teichoic acids or the lysinylation of phospholipids will make the cell wall or membrane, respectively, more positively charged (39, 41). As a consequence, bacterial cells with such modifications will be more resistant to cationic bacteriocins than cells lacking these modifications.Recent studies by Butcher and Helmann have shown that the yqeZ and yqfAB genes of the σ^W regulon confer resistance to sublancin 168 (5). However, full producer immunity to sublancin 168 is known to require gene functions of the SPβ prophage (18), while none of the σ^W regulon genes implicated in sublancin 168 resistance are located on this prophage. Thus, it has remained unclear which SPβ gene or genes are required for sublancin 168 producer immunity. Notably, our previous studies have shown that the ABC transporter SunT, the thiol-disulfide oxidoreductases BdbA and BdbB, and the YolJ protein of unknown function are fully dispensable for sublancin 168 producer immunity (10). Moreover, none of the 187 SPβ genes show homology to known bacteriocin producer immunity genes (29).In the present studies, we have addressed the issue of which SPβ gene or genes are required for sublancin 168 producer immunity. Our results show that only 1 of the 187 genes of the SPβ prophage, yolF, is both required and sufficient for immunity of B. subtilis to sublancin 168. We therefore propose changing the name of this gene to sunI. Interestingly, SunI (YolF) seems to belong to a new class of bacteriocin producer immunity proteins.     

Breast MRI increases the number of mastectomies for ductal cancers,but decreases them for lobular cancers

Purpose

Mammographic density is a measurable and modifiable biomarker that is strongly and independently associated with breast cancer risk. Paradoxically, although Asian women have lower risk of breast cancer, studies of minority Asian women in predominantly Caucasian populations have found that Asian women have higher percent density. In this cross-sectional study, we compared the distribution of mammographic density for a matched cohort of Asian women from Malaysia and Caucasian women from Sweden, and determined if variations in mammographic density could be attributed to population differences in breast cancer risk factors.

Methods

Volumetric mammographic density was compared for 1501 Malaysian and 4501 Swedish healthy women, matched on age and body mass index. We used multivariable log-linear regression to determine the risk factors associated with mammographic density and mediation analysis to identify factors that account for differences in mammographic density between the two cohorts.

Results

Compared to Caucasian women, percent density was 2.0% higher among Asian women (p < 0.001), and dense volume was 5.7 cm³ higher among pre-menopausal Asian women (p < 0.001). Dense volume was 3.0 cm³ lower among post-menopausal Asian women (p = 0.009) compared to post-menopausal Caucasian women, and this difference was attributed to population differences in height, weight, and parity (p < 0.001).

Conclusions

Our analysis suggests that among post-menopausal women, population differences in mammographic density and risk to breast cancer may be accounted for by height, weight, and parity. Given that pre-menopausal Asian and Caucasian women have similar population risk to breast cancer but different dense volume, development of more appropriate biomarkers of risk in pre-menopausal women is required.

     

Minimally invasive pancreatoduodenectomy

Background

Minimally invasive pancreatoduodenectomy (MIPD) is increasingly performed with several institutional series and comparative studies reported. The aim was to conduct an assessment of the best-evidence and expert opinion on the current status and future challenges of MIPD.

Prevention of lethal endotoxemia in actinomycin D-sensitized mice by incubation of Salmonella minnesota R595 lipopolysaccharide with monoclonal antibodies to R595

Murine monoclonal antibodies reacting with lipopolysaccharide (LPS) of Salmonella minnesota strain R595 (Re chemotype) were prepared, and tested for their ability to protect actinomycin D-sensitized mice against lethal endotoxemia. Protection was found with some antibodies up to a 90-fold increase in LD50, whereas others exhibited no protection. The various protective antibodies did not all bind to the same epitope. The same applied for non-protective clones. Protective and non-protective clones could not be discriminated by ELISA. One protective monoclonal antibody (clone 20) was specific for ketodeoxyoctonate, a structural element common to various LPS. These findings show that the involvement of lipid A in the binding site of monoclonal antibodies is no prerequisite for protection.