Endotoxin Penetration Research Articles

Role of Akkermansia muciniphila in insulin resistance.

Insulin resistance (IR) is a pathogenic factor in numerous metabolic diseases. The gut microbiota plays a crucial role in maintaining the function of the intestinal barrier and overall human health, thereby influencing IR. Dysbiosis of the gut microbiota can contribute to the development of IR. Therefore, it is essential to maintain a balanced and diverse gut microbiota for optimal health. Akkermansia muciniphila, a widely present microorganism in the human intestine, has been shown to regulate gastrointestinal mucosal barrier integrity, reduce endotoxin penetration, decrease systemic inflammation levels, and improve insulin sensitivity. Reduced abundance of A.muciniphila is associated with an increased risk of IR and other metabolic diseases, highlighting its correlation with IR. Understanding the role and regulatory mechanism of A.muciniphila is crucial for comprehending IR pathogenesis and developing novel strategies for preventing and treating related metabolic disorders. Individual variations may exist in both the gut microbiota composition and its impact on IR among different individuals. Further investigation into individual differences between A.muciniphila and IR will facilitate advancements in personalized medicine by promoting tailored interventions based on the gut microbiota composition, which is a potential future direction that would optimize insulin sensitivity while preventing metabolic disease occurrence. In this review, we describe the physiological characteristics of A.muciniphila, emphasize its roles in underlying mechanisms contributing to IR pathology, and summarize how alterations in its abundance affect IR development, thereby providing valuable insights for further research on A.muciniphila, as well as new drug development targeting diabetes.

Endotoxins Induced ECM-Receptor Interaction Pathway Signal Effect on the Function of MUC2 in Caco2/HT29 Co-Culture Cells.

Endotoxins are toxic substances that widely exist in the environment and can enter the intestine with food and other substances. Intestinal epithelial cells are protected by a mucus layer that contains MUC2 as its main structural component. However, a detailed understanding of the mechanisms involved in the function of the mucus barrier in endotoxin penetration is lacking. Here, we established the most suitable proportion of Caco-2/HT-29 co-culture cells as a powerful tool to evaluate the intestinal mucus layer. Our findings significantly advance current knowledge as focal adhesion and ECM-receptor interaction were identified as the two most significantly implicated pathways in MUC2 small interfering RNA (siRNA)-transfected Caco-2/HT-29 co-culture cells after 24 h of LPS stimulation. When the mucus layer was not intact, LPS was found to damage the tight junctions of Caco-2/HT29 co-cultured cells. Furthermore, LPS was demonstrated to inhibit the integrin-mediated focal adhesion structure and damage the matrix network structure of the extracellular and actin microfilament skeletons. Ultimately, LPS inhibited the interactive communication between the extracellular matrix and the cytoskeleton for 24 h in the siMUC2 group compared with the LPS(+) and LPS(-) groups. Overall, we recognized the potential of MUC2 as a tool for barrier function in several intestinal bacterial diseases.

Validation of Two Kinetic Assays for the Quantification of Endotoxin in Human Serum.

Background: There is an emerging evidence of the role of the microbiome in neurological diseases. Endotoxin is a component of gram-negative bacteria and thought to be one of the possible signals between the gut microbiota and the immune system. Previous studies explored the blood levels of endotoxin using an endpoint chromogenic assay.Methods: We validated and compared the analytical performance of two kinetic assays for the quantification of endotoxin in serum: (1) the Limulus Amebocyte Lysate (LAL) Kinetic-QCL assay and (2) the turbidimetric LAL Pyrogent-5000 assay. We used the best-performing validated assay to measure the endotoxin level in 20 patients with multiple sclerosis (MS) and eight healthy controls.Results: The Pyrogent-5000 and QCL assay achieved similar performance in regard to spike recovery and linear dilution; however, the Pyrogent-5000 had a better signal to noise in the calibrator curve. By using the Pyrogent-5000 assay, we found that serum samples from MS patients and healthy controls have a similar level of endotoxin; hence, we did not find evidence to support a penetration of endotoxin in the blood of MS patients. Our findings do not exclude a role of endotoxin in mediating signals from the gut microbiota in MS patients directly at the gut–blood barrier where numerous antigen-presenting cells are actively sensing metabolites and bacterial products.

Formulation and in vivo evaluation of probiotics-encapsulated pellets with hydroxypropyl methylcellulose acetate succinate (HPMCAS)

The aim of this study was to formulate probiotics-encapsulated pellets with hydroxypropyl methylcellulose acetate succinate (HPMCAS) using a dry powder coating technique to improve the storage stability, acid resistance, and intestinal adherence of viable bacteria (Lactobacillus acidophilus and Bifidobacteria animalis ssp. Lactis). Dry coated pellet (DCP) loaded with probiotics was optimized with respect to the quantity of the HPMCAS, an enteric coating polymer (108mg), and the kinds and amounts of plasticizer (triethyl citrate, 15.7mg; acetylated monoglyceride, 6.8mg), by evaluating the survival rate of the bacteria during preparation process and in an acidic medium. Dry coating process allows the whole survivals of living bacteria during preparation process. The DCP formulation exhibited markedly higher acid tolerability and storage stability compared to uncoated viable bacteria. In an in vivo mucosal adherence study in rats, a profound colonization of viable bacteria in the small and large intestine was observed in rats receiving DCP system (p<0.05) compared to rats receiving uncoated probiotics. Moreover, we found that the repeated DCP administration noticeably inhibited intestinal penetration of endotoxin, a potent inflammatory stimulant, from intestinal mucus. The novel DCP system may be an alternative approach for improving bacterial viability in the preparation process and in an acidic medium, and to promote mucosal colonization of probiotic bacteria in the human gut.

The Role of Chlorhexidine on Endotoxin Penetration to the Implant‐Abutment Interface (IAI)

The aim of this study is to assess the risk of endotoxin penetration to the implant-abutment interface (IAI) of implants with Morse-taper connection and the effect of chlorhexidine in the prevention of such penetration. Thirty implants with Morse-taper connection were divided into three groups (n = 10/group) based on type of inoculation of the internal aspect of the implant. Implants in Group 1 were inoculated with 1 μl Escherichia coli for 24 hours; supernatant was removed and 0.5 μl of sterile saline was added. Implants in Group 2 were inoculated with 1 μl E. coli for 24 hours; supernatant was removed and 0.5 μl 0.2% chlorhexidine solution was added. Implants in Group 3 were inoculated with 0.5 μl of sterile saline and served as controls. Following inoculation procedures, implants were connected to standard abutments, immersed in sterile culture media, and loaded with 200,000 cycles of 160 N in a wear simulator. Samples were collected from the supernatant solution of each implant for endotoxin identification at the beginning of the loading cycle (T0) and following 9 hours (T9), 18 hours (T18), 27 hours (T27), 36 hours (T36), 45 hours (T45), and 54 hours (T54). For Group 1 and Group 2, there were statistically significant differences between the endotoxin concentration at T0 and the endotoxin concentration at the subsequent sampling points (p < .05 Kruskal-Wallis with Bonferoni corrections for intragroup comparisons). There were no statistically significant differences between Group 1 and Group 2 at all sampling points. This study indicates that bacterial endotoxin can penetrate the IAI of implants with Morse-taper connection, and 0.2% chlorhexidine solution had no significant effect on that penetration.

Zinc and Hepatocyte Nuclear Factor-4α in Alcohol-Induced Intestinal Barrier Dysfunction

Zinc is an essential micronutrient and plays an important role in maintaining intestinal epithelial integrity. Increasing evidence support that zinc homeostasis has significant impact on the intestinal barrier via regulation of epithelial tight junction proteins. Intestinal barrier plays a critical role in the prevention of endotoxin penetration from the intestinal lumen to the blood. Disruption of the intestinal barrier leads to elevation of blood endotoxin level, namely endotoxemia. Endotoxemia may lead to proinflammatory cytokine production and inflammation, thereby being an etiological factor in the pathogenesis of alcoholic liver disease (ALD). Recent studies demonstrated that oxidative stress and zinc deficiency correlate well with alcohol-induced gut leakiness. Alcohol exposure induces oxidative stress which, in turn, releases zinc from proteins. Hepatocyte nuclear factor-4 (HNF-4 ) is a zinc finger transcription factor which abundantly distributes in the intestine, particularly the distal intestine. Inactivation of HNF-4 correlates well with alcohol-induced down- regulation of tight junction proteins. This review discusses mechanisms involved in alcohol-induced intestinal epithelial barrier disruption with emphasis on the relationship among oxidative stress, zinc deficiency, and HNF-4 inactivation.

Molecular leakage at implant-abutment connection—in vitro investigation of tightness of internal conical implant-abutment connections against endotoxin penetration

Microleakage has been discussed as a major contributing factor for inflammatory reactions at the implant-abutment connection. In previous studies, the tightness against corpuscular bodies (viable bacteria) has been successfully investigated under static and dynamic conditions. The aim of this study was to investigate the tightness against endotoxins of two implant systems (AstraTech and Ankylos) with conical internal connections under static conditions. The inner parts of eight implants of each system were inoculated with endotoxin. Implants were screwed together with the respective abutments and stored under isostatic conditions in a supernatant of pyrogen-free water for 168 h. Supernatant samples were taken after 5 min, 24 h, 72 h, and 168 h, and endotoxin contamination was determined by the amebocyte-lysate test. Only one implant in the AstraTech group showed no sign of endotoxin contamination after 168 h, while the other implants showed contamination after varying storage times, respectively. The implants in the Ankylos group showed endotoxin contamination after only 5 min of storage in the supernatant solution. The tested internal conical implant-abutment connections appear to be unable to prevent endotoxin leakage. In average, Astra implants showed a higher tightness than Ankylos implants.

Effect of Root Canal Filling/Sealer Systems on Apical Endotoxin Penetration: A Coronal Leakage Evaluation

Endotoxin, elaborated by gram-negative organisms, is an important factor in apical periodontitis. The objective of this study was to evaluate the magnitude of endotoxin penetration through root canal treated teeth using a dual chamber model system. Forty-four maxillary anterior teeth were prepared endodontically and canals filled either by lateral condensation or a warm thermoplasticized technique in combination with either Roth's 801 or AH 26 sealer. Teeth were suspended in the model system with a mixed anaerobic bacterial suspension in the upper chamber and HBSS in the lower chamber. The QCL-1000 LAL assay was used to measure endotoxin at 0, 1, 7, 14, and 21 days. Response feature analysis using trapezoidal area under the curve was performed; the four treatment groups were compared using nonparametric methods. Groups differed (p = 0.028), with thermoplasticized root canal filling/Roth's 801 sealer permitting the least apical endotoxin penetration. Results suggest that Roth's 801 sealer may have a role in inhibiting endotoxin penetration.

New strategies for prevention and treatment of graft-versus-host disease and for induction of graft-versus-leukemia effects.

Graft-versus-host disease (GVHD) continues to be a problem in allogeneic hemopoietic stem cell transplantation; however, our understanding of the basic pathophysiology of GVHD has improved. Although not all data obtained from murine or other animal models can be extrapolated to the clinic, there are leads that deserve to be pursued. The skin, intestinal tract, and liver are the 3 major target organs of GVHD and share the feature of presenting a barrier to the "environment" of the host. There is evidence that the damage inflicted to these organs, the epithelial and endothelial cells in particular, by the conditioning regimen causes a release of various cytokines and a penetration of endotoxin into the systemic circulation. According to these observations, the nonimmunologic aspects of GVHD have been likened to an inflammatory process. If this characterization is valid, blocking these nonspecific inflammatory changes would ameliorate GVHD without interfering with the graft-versus-leukemia (GVL) reaction. In fact, one study has shown a substantial amelioration of GVHD with a molecule that directly blocks endotoxin. Clinical data also suggest that patients with organ dysfunction early after transplantation that is presumed to be treatment related may benefit from preemptive interventions aimed at controlling GVHD. Furthermore, there is growing evidence that the mechanisms involved in GVHD may differ from organ to organ (for example, Fas/Fas-ligand interactions in the liver versus tumor necrosis factor alpha/receptor interactions in the intestinal tract), and from a therapeutic point of view, the time of onset of clinical GVHD may be important in choosing the appropriate therapy. Thus, combinations of interventions chosen and timed appropriately may be more effective in preventing and managing GVHD than are the standard across-the-board approaches that have been used so far. Such a strategy may also be successful in maintaining a GVL effect and possibly in incorporating direct antileukemic therapy, such as the use of cytotoxic T-cells directed at minor histocompatibility antigens, without increasing the risk of GVHD. The development of nonmyeloablative conditioning regimens and the observations on GVHD kinetics and the progression or eradication of leukemia with that strategy are likely to add new insights into how one can optimally combine various modalities to achieve engraftment, prevent GVHD, and at the same time maintain a GVL effect.

Coronal leakage: Endotoxin penetration from mixed bacterial communities through obturated, post-prepared root canals

Coronal leakage has been considered as one of the major causes of failure in root canal treatment. Bacteria have been shown to penetrate the root canal obturating materials and influence the periapical tissues. Endotoxin, a component of Gram-negative bacteria, is a potent inflammatory agent and may be able to penetrate obturating materials faster than bacteria. This in vitro study examined the possible penetration of post-prepared canals by endotoxin and bacterial cells derived from mixed bacterial communities. Results showed that both bacteria and endotoxin were able to penetrate the obturating materials in post-prepared canals; however, endotoxin penetration was faster than bacterial. If one speculates that a similar sequence of events occurs clinically, then such a rapid penetration of endotoxin could lead to an early periapical reaction, with subsequent treatment failure and need for retreatment or periapical surgery. The need for an immediate and proper coronal restoration after root canal treatment is therefore reinforced.

PR 21 Endotoxin penetration from mixed culture through obturated, post-prepared canals: J. Alves*, K. Barrieshi, R. Walton, L. Wilcox, P. Wertz, D. Drake. University of Iowa, Iowa City

PR 21 Endotoxin penetration from mixed culture through obturated, post-prepared canals: J. Alves*, K. Barrieshi, R. Walton, L. Wilcox, P. Wertz, D. Drake. University of Iowa, Iowa City

In vitro endotoxin penetration of coronally unsealed endodontically treated teeth

The purpose of the study was to evaluate the penetration of bacterial endotoxin (L.P.S.) through obturated root canals. Twenty-four single-rooted teeth were instrumented in a uniform manner. The root canals of 16 teeth were obturated with guttapercha and Roth's sealer and 8 teeth were similarly obturated but without sealer. The teeth were then divided into three groups. Group 1-Positive control (4 teeth). Teeth obturated without sealer were used. Sticky wax was softened in an open flame and painted over the external root surface except the apical 2 mm and coronal canal orifice which were left free of the sticky wax. Group 2-Negative control (4 teeth). Again teeth without sealer comprised the teeth in this group. However, in this group the sticky wax covered the apical area of the root and the canal orifice coronally, in addition to the rest of the root. Group 3-Experimental (16 teeth). The teeth obturated in conjunction with sealer were used. Otherwise the teeth were treated as in group 1. The coronal root half was then hermetically sealed from the apical root half so that LPS placed coronally could move only through the obturated canal space to the apical segment. One ml of water containing 100 micrograms/ml endotoxin was placed in the upper chambers and pyrogen-free water in the lower chambers. The upper and lower chambers were then tested for endotoxin at different time periods upto 21 days. The upper chambers tested positive for endotoxin at all test times for all teeth. All 4 positive control teeth showed endotoxin in the lower chambers by 24 hrs.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract #8 — In vitro endotoxin penetration of coronally unsealed endodontically treated teeth

Abstract #8 — In vitro endotoxin penetration of coronally unsealed endodontically treated teeth

Effect of Milking Without Pulsation on Teat Duct Colonization with Streptococcus agalactiae and Penetrability to Endotoxin

Forty quarters of 10 cows were milked for a 9-d period with one of four treatments: 1) no liner pulsation, 2) conventional milking, 3) no pulsation for 4 d followed by conventional milking for 5 d, and 4) conventional milking for 4 d followed by milking without pulsation for 5 d. All teat orifices were inoculated with approximately .5million cfu of Streptococcus agalactiae and Streptococcus dysgalactiae on d 1 and 5. Recoveries of Strep. agalactiae from the teat end were increased for teats milked without pulsation. Recoveries of Strep. dysgalactiae were lower than those of Strep. agalactiae and not increased by milking without pulsation.In a second experiment, teats of 20 cows were milked for a 15-d period with or without liner pulsation. For 10 successive milkings, all teats were inoculated with 1.0µg of Escherichia coli endotoxin either immediately or 2.5h after each milking. The frequency of endotoxin penetration, measured by the Wisconsin Mastitis Test, in pulsated quarters and in nonpulsated quarters was similar. For quarters milked without pulsation but not for pulsated quarters, inoculation of endotoxin immediately after milking led to greater incidence of teat duct penetration than for inoculation 2.5h after milking.

Localization of rate-limiting barrier to penetration of endotoxin through nonkeratinized oral mucosa in vitro.

The penetration of tritiated bacterial endotoxin through nonkeratinized oral mucosal epithelium was studied using an in vitro model system and radioautographic tracer techniques. The basement membrane region of the epithelium was the rate-limiting barrier to penetration, and this barrier effect was independent of the direction and duration of penetration as well as any clearance effects of the vasculature.