Optimized flow cytometry assays to monitor neutrophil activation in human and mouse whole blood samples.

Polymorphonuclear neutrophils (PMNs) protect the host from invading microorganisms. However, excessively activated PMNs can also cause damage to host tissues under inflammatory conditions. Here we developed simple assays to determine the activation state of PMNs in human whole blood that contains soluble mediators known to influence PMN functions. Because mouse models are widely used to study the role of PMNs in infectious and inflammatory diseases, we adapted these assays for the rapid and reliable assessment of PMN functions in murine blood samples. Freshly collected whole blood samples were stimulated with agonists of the formyl peptide receptors (FPR) of PMNs and changes in reactive oxygen species (ROS) production and the expression of CD11b, CD62L (L-selectin), CD66b, and CD63 on the cell surface were analyzed with flow cytometry. We optimized these assays to minimize inadvertent interferences such as cell stress generated during sample handling and the loss of plasma mediators that regulate PMN functions. Human PMNs readily responded to the FPR agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP). The most sensitive responses of human PMNs to fMLP were CD11b, CD62L, and CD66b expression with half maximal effective concentrations (EC50) of 5, 8, and 6 nM fMLP, respectively. CD63 expression and ROS production required markedly higher fMLP concentrations with EC50 values of 19 and 50 nM fMLP, respectively. Mouse PMNs did not respond well to fMLP and required significantly higher concentrations of the FPR agonist WKYMVm (W-peptide) to achieve equivalent cell activation. The most sensitive response of mouse PMNs was ROS production with an EC50 of 38 nM W-peptide. Because mice do not express CD66b, we only assessed the expression of CD62L, CD11b, and CD63 with EC50 values of 54, 119, and 355 nM W-peptide, respectively. Validation of our optimized assays showed that they sensitively detect the responses of human PMNs to priming with endotoxin in vitro as well as the corresponding responses of murine PMNs to bacterial infection in a sepsis model. We conclude that these optimized assays could be useful tools for the monitoring of patients with infections, sepsis, and other inflammatory conditions as well as for the design and interpretation of preclinical studies of these diseases in mouse models.

Errate: Hypoprothrombinemia During Cefmetazole Treatment: A Case Report.

The authors contacted the journal with the following corrections to the published article: 1. Table 1. PT-INR at 6th hospital day. 2.46 is incorrect; Correct: 2.47. 2. Table 2. Line 5, at Purpose of use of CMZ. NA is incorrect; Correct: Infection secondary to Pneumothorax. 3. Table 2. Line 11, at PT-INR, 2.46 is incorrect; Correct: 2.47. 4. References No. 22 and No. 23 are reversed. The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated. Reference: Yuichiro Haba, Hikaru Akizuki, Naoyuki Hashiguchi, Toshio Naito. Hypoprothrombinemia During Cefmetazole Treatment: A Case Report. Am J Case Rep. 2022; 23: e936712. DOI: 10.12659/AJCR.936712.

Hypoprothrombinemia During Cefmetazole Treatment: A Case Report.

BACKGROUND Cefmetazole (CMZ), containing an N-methyl-tetrazole-thiol (NMTT) side chain, is a therapeutic option for diverticulitis in Japan. Cephems containing an NMTT, a methyl-thiadiazol, and a thiadiazolethiol side chain are known to induce coagulation disorders. CASE REPORT A 76-year-old woman developed hypoprothrombinemia after receiving oral levofloxacin (LVFX) 250 mg q24h for 2 days followed by intravenous CMZ 2 g q8h for sigmoid diverticulitis. On day 5 of CMZ administration (after 12 doses in total), black stool was observed. On the following day (after 14 doses), prothrombin time (PT) prolongation was noted; PT and international normalized ratio (INR) were 37.1 s and 2.47, respectively. We diagnosed the patient with hypoprothrombinemia because of vitamin K deficiency caused by markedly elevated protein levels induced by vitamin K absence or antagonist-II on day 6 of CMZ administration. Intravenous vitamin K administration and CMZ cessation rapidly restored PT and led to the disappearance of black stool. CONCLUSIONS The causes of vitamin K deficiency were considered to be an impaired vitamin K cycle due to CMZ and decreased vitamin K intake because of malnutrition. These findings are consistent with CMZ's reported adverse effects. Decreased vitamin K production due to alterations in the gut bacterial flora by LVFX and CMZ was also postulated as a cause. If a bleeding tendency is noted during diverticulitis treatment with NMTT-containing cephems, switching to intravenous quinolones or carbapenems is recommended. It remains unclear how this reaction can be avoided; however, prudent monitoring of bleeding signs and PT-INR is recommended.

Administration of ß-lactam antibiotics and delivery method correlate with intestinal abundances of Bifidobacteria and Bacteroides in early infancy, in Japan.

The intestinal microbiome changes dynamically in early infancy. Colonisation by Bifidobacterium and Bacteroides and development of intestinal immunity is interconnected. We performed a prospective observational cohort study to determine the influence of antibiotics taken by the mother immediately before delivery on the intestinal microbiome of 130 healthy Japanese infants. Faecal samples (383) were collected at 1, 3, and 6 months and analysed using next-generation sequencing. Cefazolin was administered before caesarean sections, whereas ampicillin was administered in cases with premature rupture of the membranes and in Group B Streptococcus-positive cases. Bifidobacterium and Bacteroides were dominant (60-70% mean combined occupancy) at all ages. A low abundance of Bifidobacterium was observed in infants exposed to antibiotics at delivery and at 1 and 3 months, with no difference between delivery methods. A lower abundance of Bacteroides was observed after caesarean section than vaginal delivery, irrespective of antibiotic exposure. Additionally, occupancy by Bifidobacterium at 1 and 3 months and by Bacteroides at 3 months differed between infants with and without siblings. All these differences disappeared at 6 months. Infants exposed to intrapartum antibiotics displayed altered Bifidobacterium abundance, whereas abundance of Bacteroides was largely associated with the delivery method. Existence of siblings also significantly influenced the microbiota composition of infants.

Boerhaave syndrome due to excessive alcohol consumption: two case reports.

BACKGROUND: Spontaneous esophageal rupture, or Boerhaave syndrome, is a fatal disorder caused by an elevated esophageal pressure owing to forceful vomiting. Patients with Boerhaave syndrome often present with chest pain, dyspnea, and shock. We report on two patients of Boerhaave syndrome with different severities that was triggered by excessive alcohol consumption and was diagnosed immediately in the emergency room. CASE PRESENTATION: The patient in case 1 complained of severe chest pain and nausea and vomited on arrival at the hospital. He was subsequently diagnosed with Boerhaave syndrome coupled with mediastinitis using computed tomography (CT) and esophagogram. An emergency operation was successfully performed, in which a 3-cm tear was found on the left posterior wall of the distal esophagus. The patient subsequently had anastomotic leakage but was discharged 41 days later. The patient in case 2 complained of severe chest pain, nausea, vomiting, and hematemesis on arrival. He was suggested of having Boerhaave syndrome without mediastinitis on CT. The symptoms gradually disappeared after conservative treatment. Upper gastrointestinal endoscopy performed on the ninth day revealed a scar on the left wall of the distal esophagus. The patient was discharged 11 days later. In addition to the varying severity between the cases, the patient in case 2 was initially considered to have Mallory-Weiss syndrome. CONCLUSION: Owing to similar histories and symptoms, Boerhaave syndrome and Mallory-Weiss syndrome must be accurately distinguished by emergency clinicians. CT can be a useful modality to detect any severity of Boerhaave syndrome and also offers the possibility to distinguish Boerhaave syndrome from Mallory-Weiss syndrome.

Heart rate variability in patients presenting with neurally mediated syncope in an emergency department.

BACKGROUND: Neurally mediated syncope (NMS) is a disorder of autonomic nervous system (ANS) regulation. Orthostatic stress is one of the most common causative factors seen in clinical practice. Analysis of heart rate variability (HRV) is a non-invasive method that is used to assess ANS regulation. In this study, we investigated the pathophysiology of NMS using HRV in our emergency department. METHODS: The subjects were 19 patients (age 25.8 ±â€¯6.2 years old) who presented with NMS and 20 healthy individuals (age 26.6 ±â€¯2.7 years old) who served as controls. HRV was measured in supine, sitting and standing positions. Heart rate (HR), low frequency (LF 0.04-0.15 Hz), high frequency (HF > 0.15 Hz), and coefficient of variation of the R-R interval (CVRR) were determined. RESULTS: LF and HF in the supine position were significantly lower in the patients with NMS (p < 0.05). HR was higher in all positions in patients with NMS than in healthy individuals (p < 0.05). CVRR in the supine position was lower in the patients with NMS (p < 0.001), and it was significantly lower in patients who were positive in an orthostatic test (p = 0.0017). Area under the curve was calculated to be 0.824, and at the cutoff value of 4.997 of CVRR in supine, the sensitivity and the specificity were 78.9% and 85.0%. CONCLUSION: The sympathetic and parasympathetic nervous systems were both suppressed in patients with NMS. In post-syncope, parasympathetic withdrawal, rather than sympathetic reactivation, was responsible for the increased HR after syncope. CVRR may serve as a new clinical biomarker in the emergency department.

Acute Myocardial Infarction Following Administration of Polyethylene Glycol Electrolyte Solution with Ascorbic Acid (MoviPrep®) at Home: A Case Report.

A male individual aged 82 years with hypertension who had a smoking history, but no history of cardiovascular events, developed acute myocardial infarction immediately after he took oral polyethylene glycol electrolyte solution with ascorbic acid as a pretreatment for a colonoscopy to examine anemia. He took polyethylene glycol electrolyte solution with ascorbic acid at twice (2 L/h) the rate recommended in the package insert and by the physician. The patient showed impaired consciousness 2 h after taking polyethylene glycol electrolyte solution with ascorbic acid and his family called the emergency medical service. Upon arrival of the emergency medical service, his systolic blood pressure was 60 mmHg and heart rate was 50 bpm. Systolic blood pressure and impaired consciousness were slightly improved, but compensatory shock remained, at arrival at the emergency outpatient service at our hospital. No dyspnea or rash was apparent. The patient had no subjective chest pain; however, ST-segment elevation was detected in the electrocardiogram at II, III, aVF, V3R, and V4R. He was diagnosed with ST-segment elevation myocardial infarction and underwent a coronary catheter intervention for total occlusion of the right coronary artery. His shock state was abolished by this intervention. The patient was pretreated with polyethylene glycol electrolyte solution with ascorbic acid under close watch in the coronary care unit 4 days later, with no relapse of symptoms. Advanced cancer was found in the ileocecum by colonoscopy; consequently, the patient underwent a colectomy and was discharged from our hospital and transferred to another hospital for rehabilitation on hospital day 74. A Naranjo assessment score of 4 was obtained, indicating a possible relationship of acute myocardial infarction with misuse of the suspect drug, polyethylene glycol electrolyte solution with ascorbic acid.

Maternal antimicrobial use at delivery has a stronger impact than mode of delivery on bifidobacterial colonization in infants: a pilot study.

OBJECTIVE: To investigate factors related to bifidobacterial colonization in early infancy, with a focus on maternal antimicrobial use at delivery. STUDY DESIGN: A cross-sectional pilot study was performed. Feces samples of 33 Japanese healthy infants were collected over 10 months and analyzed by next-generation sequencing to examine the diversity and abundance of the gut microbiota. RESULTS: The beta diversity index of the gut microbiota differed significantly based on maternal antimicrobial use at delivery (P < 0.05). The most dominant genus was bifidobacteria, and the relative abundance of bifidobacteria in infants exposed to maternal antibiotics was significantly lower than in those who were not exposed (P < 0.05). In contrast, the delivery mode showed no significant relationship with gut microbiota diversity. CONCLUSIONS: Maternal antimicrobial use at delivery has a stronger effect than delivery mode on the gut microbiota, especially for colonization of bifidobacteria.

Heparins attenuated histone-mediated cytotoxicity in vitro and improved the survival in a rat model of histone-induced organ dysfunction.

BACKGROUND: The beneficial effects of heparin in the treatment of severe sepsis, septic shock, and sepsis-associated disseminated intravascular coagulation (DIC) have recently been reported. However, the mechanisms underlying the therapeutic benefits of heparin in these conditions have not yet been clearly elucidated. The purpose of this study was to confirm the effect of heparin of neutralizing histone toxicity. METHODS: Rat models of histone H3-induced organ dysfunction were administered in a low or high dose of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), or argatroban, and the therapeutic effects of each anticoagulant were examined. In another series, the survival of the histone H3-administered animals was evaluated. Furthermore, the effect of each of the aforementioned anticoagulants on cell death induced by histone H3 was examined in cultured vascular endothelial cells and leukocytes. RESULTS: Although UFH, LMWH, and argatroban significantly suppressed the histone-induced decrease of the WBC and platelet counts in the animal models of organ dysfunction, only UFH and LMWH attenuated hepatic and renal dysfunction. In addition, the mortality was significantly reduced only by high-dose UFH and LMWH. The in vitro study revealed that both vascular endothelial cell death and leukocyte cell death were significantly attenuated by UFH and LMWH but not by argatroban. CONCLUSIONS: The histone-neutralizing effect of heparin may contribute to the beneficial effects of heparins observed in the animal study. The results of the in vitro study further confirmed the above contention and suggested that heparin binds to histones to attenuate the cytotoxic actions of the latter. Since heparin has been demonstrated to protect animals from the organ damage induced by histones and consequently reduce the mortality, administration of heparin could become a treatment of choice for patients suffering from severe sepsis.

Is the neutrophil a 'prima donna' in the procoagulant process during sepsis?

Activation of the coagulation system is a fundamental host defense mechanism. Microorganisms that have invaded the body are trapped and disposed of in clots. Monocytes/macrophages are widely accepted as the main players in the procoagulant process; however, recent evidence suggests that neutrophils also play important roles. Tissue factor, which initiates the extrinsic coagulation cascade, is reportedly expressed on the surface of neutrophils, as well as on microparticles derived from neutrophils. Neutrophil extracellular traps (NETs) are another source of tissue factor. The components of NETs, such as DNA, histones, and granule proteins, also provide procoagulant activities. For instance, DNA initiates the intrinsic pathway, histones are a strong generator of thrombin, and granule proteins such as neutrophil elastase, cathepsin G and myeloperoxidase contribute to the suppression of the anticoagulation systems. Although understanding of the mechanisms that are involved in coagulation/fibrinolysis in sepsis has gradually progressed, the impact of neutrophils on thrombogenicity during sepsis remains to be addressed. Since the importance of the connection between coagulation and inflammation is advocated nowadays, further research on neutrophils is required.

Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels in endotoxemic rats.

INTRODUCTION: The activation of coagulation is recognized as a universal event in severe sepsis. Both antithrombin and thrombomodulin play pivotal roles as suppressors of coagulation. Since the levels of both anticoagulants decrease significantly, we hypothesized that a combination therapy would be beneficial. METHODS: A sepsis model was established using the intravenous infusion of lipopolysaccharide (LPS). Either 125 IU/kg of antithrombin, 0.25mg/kg of recombinant thrombomodulin, or a combination of both agents was injected immediately after LPS infusion (n=7 each), while only a physiological saline was injected in the control group (n=7). Blood samples were obtained at eight hours after LPS infusion, and organ damage markers and the plasma levels of damage-associated molecular patterns (DAMPs), such as histone H3 and cell-free DNA (cf-DNA), were measured. In another series, the leukocytes harvested from normal rats were cultured in plasma obtained from each group (n=7). Eight hours later, the leukocytes were stained with green fluorescent protein, Annexin V and 7-AAD, and the proportion of alive+apoptic/necrotic cells was calculated. RESULTS: Organ damage markers such as ALT and BUN were maintained best in the combination group (P<0.05). The circulating levels of histone H3 and cf-DNA were both significantly lower in the combination therapy group (P<0.01, 0.05, respectively). The proportion of alive+apoptic/necrotic cells was significantly higher in the combination therapy group (P<0.05). CONCLUSION: The coadministration of antithrombin and recombinant thrombomodulin can modulate cell death and decrease the circulating levels of histone H3 and cf-DNA, leading to protection against organ damage in a rat model of sepsis.

New therapeutic options for patients with sepsis and disseminated intravascular coagulation.

The severity of sepsis increases along with the degree of coagulation disorder, and a fulminant coagulation abnormality is recognized as disseminated intravascular coagulation (DIC). The mortality in sepsis­associated DIC remains as high as 40%, which is comparable to that in septic shock. Even though intensive research is ongoing, there is currently no established therapy for this life­threatening complication. Heparins are the oldest, most popular, and least­expensive anticoagulants available; however, their usefulness for the treatment of septic DIC has never been proved. Expectations for antithrombin concentrate were once high, but high­dose antithrombin failed to demonstrate a survival benefit, and global sepsis guidelines no longer recommend its use. Recombinant activated protein C was the only recommended anticoagulant for the treatment of severe sepsis until 2011, but it was subsequently withdrawn from the world market after the failure of the latest clinical trial. Recombinant thrombomodulin is newly developed and has been utilized in Japan since 2008; however, its efficacy has not yet been proved. As shown above, progress has not been as fast as expected, but some new agents are upcoming. The efficacy of anticoagulant therapy for septic DIC has long been discussed and aggressively studied, and we have finally realized that correcting the coagulation disorder is not sufficient to conquer this deadly complication. Since many natural anticoagulants have pleiotropic functions, we need to examine these effects and apply them to the right target at the right timing.

Combination of antithrombin and recombinant thrombomodulin attenuates leukocyte-endothelial interaction and suppresses the increase of intrinsic damage-associated molecular patterns in endotoxemic rats.

INTRODUCTION: Both antithrombin (AT) and thrombomodulin are key players in physiological anticoagulant systems. Because the levels of both factors are known to decrease significantly during severe sepsis, we hypothesized that a combination therapy would be effective. METHODS: A sepsis model was established using the intravenous infusion of lipopolysaccharide (LPS). A dose of 125 IU/kg of AT, 0.25 mg/kg of recombinant thrombomodulin, or a combination of both agents was injected immediately after LPS infusion (n = 7, each). Intravital observation of the mesenteric microcirculation was performed, and leukocyte adhesion and blood flow were calculated at 3 h after LPS infusion. Immediately after the observation, blood samples were obtained and coagulation markers, organ damage markers, the circulating levels of nucleosome and high-mobility group box 1 were measured. RESULTS: Microscopic findings revealed the suppression of leukocyte adhesion and thrombus formation in the combination group. The number of adhesive leukocytes on the endothelium was significantly suppressed (P < 0.01), and the blood flow in venules was better maintained in the combination group compared with the placebo control (P < 0.01). The blood samples showed the suppressed activation in coagulation, no significant changes were observed in the organ damage markers in the treatment groups. The circulating levels of nucleosome and high-mobility group box 1 were both decreased significantly in the combination group compared with the placebo control (P < 0.01). CONCLUSIONS: The coadministration of AT and recombinant thrombomodulin is effective for the suppression of leukocyte activation and cell death during sepsis.

Neutrophil cell death in response to infection and its relation to coagulation.

Neutrophil is a major player in the pathophysiology of severe sepsis. Recent studies have revealed that the cell death mechanism of neutrophils directly relates to the development of organ dysfunction during sepsis. Here we discuss about the different types of neutrophil cell death such as necrosis, apoptosis, autophagy, and the unique cell death style dubbed NETosis. NETosis cells release neutrophil extracellular traps (NETs), which are composed of chromatin bound to granular and nucleic proteins. The primary purpose of NET release is thought to be the control of microbial infections; however, it acts as a danger signal for the host as well. The harmful substances such as DNA, histones, and high-mobility group box 1 (HMGB1) and many other danger-associated molecular patterns (DAMPs) released along with NETosis or from necrotic neutrophils also contribute to the pathogenesis of sepsis. At the same time, the coagulation system, which is closely tied to these neutrophil cell death mechanisms, is often over-activated. It is well known that individual bacterial pathogens express virulence factors that modulate cell death pathways and influence the coagulation disorder during sepsis. Moreover, extensive cross talk exists between these two phenomena, whereby inflammation leads to activation of coagulation and coagulation considerably affects inflammatory activity. A greater knowledge of cell death pathways in sepsis informs the potential for future therapies designed to ameliorate excessive immune responses during sepsis.

Hypertonic saline increases gammadeltaT cell-mediated killing of activated neutrophils.

OBJECTIVE: Hypertonic saline fluids used to resuscitate trauma patients can prevent neutrophil-mediated lung tissue damage, making them attractive alternatives to conventional resuscitation fluids. We have previously shown that gammadeltaT cells, a small T lymphocyte subset, reduce acute inflammatory lung damage by eliminating activated neutrophils that express heat shock protein 72 on the cell surface. Here, we studied whether these protective effects of hypertonic saline are related to improved gammadeltaT cell-mediated neutrophil killing. DESIGN: Laboratory investigation. SETTING: University research laboratory. SUBJECTS: Human peripheral blood from healthy subjects--isolated gammadeltaT lymphocytes and neutrophils. INTERVENTIONS: Isolated blood cells were treated with different concentrations of hypertonic saline and endotoxin of Escherichia coli O111:B4 (lipopolysaccharide). In some experiments, gammadeltaT cells were activated by CD3 cross-linking or by phorbol-myristate acetate and ionomycin, or by phytohemagglutinin. MEASUREMENTS AND MAIN RESULTS: Clinically relevant concentrations of hypertonic saline (20 mM) significantly augmented CD69 expression of gammadeltaT cells that were stimulated with 100 ng/mL lipopolysaccharide. Additionally, lipopolysaccharide induced a three- to five-fold increase in tumor necrosis factor-alpha and interleukin-10 expression by gammadeltaT cells. This response was completely abrogated by hypertonic saline. These data indicate that hypertonic saline can modulate gammadeltaT cell functions. Stimulation of neutrophils with 1-1,000 ng/mL lipopolysaccharide caused a greater than 3-fold increase in heat shock protein-72 expression on the cell surface, which was significantly augmented by hypertonic saline. In cocultures of gammadeltaT cells with autologous neutrophils, 15.6 +/- 3.4% of all neutrophils were killed within 120 min. In the presence of lipopolysaccharide (1 microg/mL), this percentage increased to 23.7 +/- 2.1%, and it was further increased to 31.8 +/- 3.1% when 20 mM hypertonic saline was added with lipopolysaccharide. CONCLUSIONS: Our findings suggest that hypertonic saline enhances the elimination of inflammatory neutrophils by gammadeltaT cells by augmenting heat shock protein-72 expression on the cell surface of neutrophils. Hypertonic saline resuscitation may therefore protect host tissues by enhancing neutrophil clearance from the lungs.

Hypertonic saline resuscitation: efficacy may require early treatment in severely injured patients.

BACKGROUND: Activation of polymorphonuclear neutrophils (PMN) is a critical event leading to host tissue injury and organ damage after trauma. Hypertonic saline (HS) resuscitation prevents PMN activation in vitro and in animal models. Here, we studied how clinical parameters and timing requirements influence the efficacy of HS in suppressing PMN activation. MATERIALS AND METHODS: Twenty-six injured patients and 16 healthy volunteers were included as study subjects. To study how clinical parameters affect the efficacy of HS, whole blood samples from patients were collected 24 hours after admission, treated with HS and N-formyl-methionyl-leucyl-phenylalanine (fMLP), and PMN oxidative burst and degranulation were measured using flow cytometry. We studied the effect of timing on the ability of HS to inhibit PMN function by exposing blood of healthy volunteers to plasma samples from trauma patients before or after the addition of fMLP and HS. RESULTS: Age and gender did not significantly influence the effect of HS on PMN function. The suppressive effect of clinically relevant HS concentrations (20 mmol/L) on PMN oxidative burst correlated weakly with Sepsis Severity Score (SSS) and Acute Physiology and Chronic Health Evaluation II (APACHE II) score but not with the Injury Severity Score (ISS) or Multiple Organ Failure score (MOF). There was no correlation between any of these clinical scores and degranulation. HS was significantly less effective in suppressing oxidative burst of PMN from patients with ISS >10, APACHE II >5, MOF >0, or SSS >1 compared with patients with ISS < or =10, APACHE II < or =5, MOF = 0, or SSS < or =1. HS more effectively suppressed PMN activation when PMN were pretreatment with HS, whereas it was less effective on PMN previously primed in vivo or in vitro by adding trauma plasma. HS was ineffective on PMN previously stimulated in vitro with fMLP. CONCLUSIONS: Our data suggest that HS resuscitation may prevent PMN activation most effectively when patients are treated with HS early in the field.