Effects of recombinant human soluble thrombomodulin on neutrophil extracellular traps in the kidney of a mouse model of endotoxin shock.

Objectives: Sepsis is a life-threatening condition characterized by multi-organ dysfunction due to host immune system dysregulation in response to an infection. During sepsis, neutrophils release neutrophil extracellular traps (NETs) as part of the innate immune response. However, excessive NETs play a critical role in the development of organ failure during sepsis. Although recombinant human soluble thrombomodulin (rTM) can inhibit NET formation in the lungs and liver of a mouse model of endotoxin shock, its effects on the kidneys are unclear. Methods: The specific effects of NETs and rTM on the renal cortex and renal medulla were examined in a mouse model of endotoxin shock generated by intraperitoneal (i.p.) injection of lipopolysaccharide (LPS), followed by i.p. injection of rTM or an identical volume of saline 1 h later. Results: LPS injection increased serum creatinine, blood urea nitrogen, and histone H3 levels. However, rTM administration significantly decreased histone H3 and citrullinated histone H3 (citH3) levels. Immunohistochemical analysis revealed no significant changes in citH3 quantity in the renal cortex of any group. However, in the renal medulla, the increase in citH3 induced by LPS was abolished in the LPS+rTM group. Conclusions: Our findings demonstrate that rTM can suppress NETs in the renal medulla of mice with endotoxin-induced acute kidney injury.

Association of circulating histone H3 and high mobility group box 1 levels with postoperative prognostic indicators in intensive care unit patients: a single-center observational study.

Objectives: Damage associated molecular patterns (DAMPs) levels are associated with sepsis severity and prognosis. Histone and high mobility group box 1 (HMGB1) levels are also potential indicators of prognosis. We investigated the relationship between serum histone H3 and HMGB1 levels and the illness severity score and prognosis in postoperative patients. Methods: Postoperative serum histone H3 and HMGB1 levels in 39 intensive care unit (ICU) patients treated at our institution were measured. The correlation between peak histone H3 and HMGB1 levels in each patient and clinical data (age, sex, surgical time, length of ICU stay, and survival after ICU discharge), which also included the patients' illness severity score, was examined. Results: Histone H3 but not HMGB1 levels were positively correlated with surgical time, the Sequential Organ Failure Assessment score, the Japanese Association for Acute Medicine acute phase disseminated intravascular coagulation diagnosis score, and the length of ICU stay. Both histone H3 and HMGB1 levels were negatively correlated with age. However, survival post-ICU discharge was not correlated with histone H3 or HMGB1 levels. Conclusions: Histone H3 levels are correlated with severity scores and the length of ICU stay. Serum histone H3 and HMGB1 levels are elevated postoperatively. These DAMPs, however, are not prognostic indicators in postoperative ICU patients.

Effects of Thrombomodulin in Reducing Lethality and Suppressing Neutrophil Extracellular Trap Formation in the Lungs and Liver in a Lipopolysaccharide-Induced Murine Septic Shock Model.

Neutrophil extracellular trap (NET) formation, an innate immune system response, is associated with thrombogenesis and vascular endothelial injury. Circulatory disorders due to microvascular thrombogenesis are one of the principal causes of organ damage. NET formation in organs contributes to the exacerbation of sepsis, which is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. We have previously reported that recombinant human soluble thrombomodulin (rTM) reduces lipopolysaccharide (LPS)-induced NET formation in vitro. Here, we aimed to show that thrombomodulin (TM)-mediated suppression of NET formation protects against organ damage in sepsis. Mice were injected intraperitoneally (i.p.) with 10 mg/kg LPS. rTM (6 mg/kg/day) or saline was administered i.p. 1 h after LPS injection. In the LPS-induced murine septic shock model, extracellular histones, which are components of NETs, were observed in the liver and lungs. In addition, the serum cytokine (interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), macrophage chemotactic protein-1 (MCP-1), and interleukin-10 (IL-10)) levels were increased. The administration of rTM in this model prevented NET formation in the organs and suppressed the increase in the levels of all cytokines except IL-1ß. Furthermore, the survival rate improved. We provide a novel role of TM in treating inflammation and NETs in organs during sepsis.

Adsorption kinetics of high mobility group box 1 protein in a polyacrylonitrile hemofiltration membrane.

The high mobility group box 1 protein (HMGB1) is recognized as a prototypical endogenous danger cytokine in sepsis. We previously reported that a polyacrylonitrile (AN69ST) membrane rapidly adsorbed HMGB1. Herein, an in vitro hemofiltration system was designed to assess the HMGB1 adsorption capacity, adsorption sites, and adsorption mechanism of the AN69ST membrane. HMGB1 was repeatedly added seven times during hemofiltration. A rapid decrease in circulating HMGB1 was observed after every addition with no sign of saturation. Presence of HMGB1 on the filter membrane was observed on both membrane surfaces and within the bulk layer using a high concentration of HMGB1 by immunoelectron microscopy. We hypothesized that the addition of heparin to the membrane surface or filtration rate would contribute to the adsorption mechanism. We could not measure the influence of heparin and filtration. Although the membrane was too large to saturate under the µg/mL HMGB1 conditions, our results show that the AN69ST membrane has a robust absorption capacity that could be used to treat sepsis.

Impact of Blood Type O on Mortality of Sepsis Patients: A Multicenter Retrospective Observational Study.

ABO blood groups have been implicated as potential risk factors for various diseases. However, no study has investigated the association between sepsis mortality and ABO blood types. We aimed to evaluate the impact of these blood types on mortality in patients with sepsis and septic shock. This retrospective observational study was conducted at two general hospitals in Japan. Patients diagnosed with sepsis or septic shock were included and divided into four groups based on blood type (O, A, B, and AB). The association between type O vs. other types and 28- and 90-day mortalities was evaluated using multivariate logistic regression analysis adjusted for age, sex, and Sequential (Sepsis-related) Organ Failure Assessment score. This study included 415 patients, of whom 131 (31.6%), 171 (41.2%), 81 (19.5%), and 32 (7.7%) had type O, A, B, and AB, respectively. Blood type O was not associated with 28-day (odds ratio: 1.7 p = 0.08) or 90-day mortality (odds ratio: 1.53, p = 0.091). However, type O was significantly associated with higher 90-day mortality (odds ratio: 3.26, p = 0.009) in patients with septic shock. The role of ABO blood type in risk stratification for septic shock and the mechanisms that potentially affect the prognosis of sepsis patients need further investigation.

Nafamostat mesilate inhibits linezolid metabolism via its antioxidant effects.

Patients who undergo renal replacement therapy often exhibit a high plasma linezolid concentration. Linezolid is metabolized via oxidation. Nafamostat mesilate has antioxidant effects and is frequently used as an anticoagulant during renal replacement therapy. We aimed to investigate the effect of nafamostat mesilate on plasma linezolid concentration. We examined whether the co-administration of linezolid and nafamostat had any effect on plasma linezolid concentration. Mice were randomly allocated to two groups (n = 18/group): linezolid (100 mg kg-1 , subcutaneous injection) + nafamostat (30 mg kg-1 , intraperitoneal injection) and linezolid + saline. At 5 hours, the linezolid concentration was significantly higher in the linezolid + nafamostat co-administration group than that in the linezolid + saline group (20.6 ± 9.8 vs 3.6 ± 1.2 µg/mL, respectively P < .001). The antioxidant effects of nafamostat may inhibit linezolid metabolism, resulting in the adverse event of high linezolid concentration if both are administered concurrently during renal replacement therapy.

Involvement of ionic interactions in cytokine adsorption of polyethyleneimine-coated polyacrylonitrile and polymethyl methacrylate membranes in vitro.

Polyethyleneimine-coated polyacrylonitrile (AN69ST) and polymethyl methacrylate (PMMA) membranes are effective cytokine-adsorbing hemofilters; however, the cytokine-adsorption mechanism remains elusive. This study investigated the involvement of ionic interactions in cytokine adsorption to a negatively charged AN69ST membrane and neutral-charged PMMA membrane. Experimental hemofiltration was performed for 30 min in a closed-loop circulation system using AN69ST and PMMA hemofilters. Tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 concentrations in the test solutions were measured at baseline and at 10 min and 30 min into hemofiltration. To investigate the involvement of ionic interactions in cytokine adsorption, cytokine clearance (CL) was calculated at 10 min into hemofiltration and with three types of solutions at various pH levels (7.6, 7.2, and 6.8). During AN69ST hemofiltration, the CLs of TNF-α, IL-6, and IL-8 were 38 ± 6 mL/min, 23 ± 7 mL/min, and 78 ± 3 mL/min, respectively, demonstrating a relationship with their respective isoelectric points. During PMMA hemofiltration, the CL of IL-6 peaked at 31 ± 76 mL/min, with no relationship observed between the CL and isoelectric point. When the pH of the test solution shifted from 7.6 to 6.8, the CLs of TNF-α, IL6, and IL-8 increased in the AN69ST hemofilter; whereas, no such trend was observed in the PMMA hemofilter. These results indicated that Ionic interactions play a role in cytokine adsorption by the AN69ST membrane but not the PMMA membrane and highlight the clinical relevance of this finding, as well as the potential practical applications for further hemofilter design.

In vitro Evaluation of Linezolid and Doripenem Clearance with Different Hemofilters.

INTRODUCTION: Renal replacement therapy (RRT) is widely used in the treatment of septic acute kidney injury. However, little is known about how the adsorption properties of hemofilters used in RRT affect antibiotic concentration. Because a cytokine-adsorption membrane is frequently used in RRT, it is important to determine the antibiotic adsorption capacity of this membrane. OBJECTIVE: The present study aimed to investigate the antibiotic adsorption capacity of different hemofilter membranes by in vitro experiments using 2 antibacterial agents (linezolid and doripenem). METHODS: We performed experimental hemofiltration in vitro using polyacrylonitrile (AN69ST), polymethylmethacrylate (PMMA), and polysulfone (PS) hemofilters for 1,440 min. The test solution was a 1,000-mL substitution fluid containing 30 µg/mL linezolid and 120 µg/mL doripenem. We measured drug concentrations at the inlet, outlet, and filtrate ports of the hemofilters for 1,440 min and calculated the sieving coefficient (SC) and adsorption rate (Ra) of the drugs onto the hemofilters. RESULTS: The amount of linezolid adsorbed onto AN69ST, PMMA, and PS membranes was decreased relative to that in the control group at 15 min (p < 0.05). However, no SC for linezolid was obtained thereafter. The Ra of linezolid onto AN69ST, PMMA, and PS membranes was higher than that in the control group (p < 0.05). In contrast, no significant differences were observed in the concentrations and Ra values of doripenem adsorbed onto AN69ST, PMMA, and PS membranes compared with those in the control group. CONCLUSIONS: Doripenem was not adsorbed onto PMMA, PS, and AN69ST membranes. Linezolid was adsorbed onto PMMA, PS, and AN69ST membranes, but only temporarily, and this did not affect drug bioavailability.

Suppressive effects of the neutrophil elastase inhibitor sivelestat sodium hydrate on interleukin-1ß production in lipopolysaccharide-stimulated porcine whole blood.

OBJECTIVE: Sivelestat sodium hydrate (Siv) is expected to be an effective therapy for acute respiratory distress syndrome, although its mechanism of action is not understood. In this study, we investigated which myeloid cells-derived cytokines were suppressed by Siv. METHODS: Continuous hemofiltration was performed by circulating fresh porcine blood through a semi-closed circuit. To ensure that leukocytes survived for 360 min, 5% glucose, heparin, and air were continuously injected. The control group received continuous administration of lipopolysaccharide (LPS) only, whereas the Siv group received LPS and Siv. Complete blood count, levels of various cytokines, and other variables were compared between the groups. RESULTS: Interleukin (IL)-1ß level was significantly suppressed in the Siv group compared with that in the control group (p<0.05). CONCLUSIONS: The results suggested that Siv suppressed the production of IL-1ß and possibly other cytokines by myeloid cells. Whether this suppression of cytokine production is caused directly by Siv or mediated via suppression of granulocyte elastase should be evaluated in the future.

Differential effect of lactate in predicting mortality in septic patients with or without disseminated intravascular coagulation: a multicenter, retrospective, observational study.

BACKGROUND: We examined whether high lactate level in septic patients was associated with 90-day mortality based on the patients' disseminated intravascular coagulation (DIC) status. METHODS: We conducted a multicenter, retrospective, observational study of patients admitted to the intensive care unit (ICU) with a suspicion of severe infection and diagnosed with sepsis. Regression analyses were performed to estimate the interaction effect between DIC status and the lactate level. Then, the association between the lactate level and 90-day mortality was assessed in the DIC and non-DIC subgroups. RESULTS: The data of 415 patients were analyzed. We found a significant interaction between DIC status and the lactate level for predicting 90-day mortality (p interaction = 0.04). Therefore, we performed a subgroup analysis and found that high lactate concentration was significantly associated with 90-day mortality in the DIC group (odds ratio = 2.31, p = 0.039) but not in the non-DIC group. CONCLUSIONS: In patients with DIC, a high lactate level significantly predicted 90-day mortality; no such association was found in the non-DIC group. Thus, DIC status may serve as a possible effect modifier of lactate level in predicting mortality in patients with sepsis.

Comparison of wiping and rinsing techniques after oral care procedures in critically ill patients during endotracheal intubation and after extubation: A prospective cross-over trial.

AIM: Endotracheal intubation of critically ill patients increases the risk of aspiration pneumonia, which can be reduced by regular oral care. However, the rinsing of the residual oral contaminants after mechanical cleaning carries the risk of aspirating the residue during the intubation period. Removing the contaminants by wiping with mouth wipes could be an alternative to rinsing with water because of no additional fluid. This study tested: (i) the amount of oral bacteria during endotracheal intubation and after extubation; and (ii) the changes in the bacterial count during oral care procedures. METHODS: Thirty-five mechanically ventilated patients in the intensive care unit were enrolled. The amount of bacteria on the dorsal tongue surface was counted before and following oral care and then after the elimination of contaminants either by rinsing with water and suctioning or by wiping with mouth wipes. The oral bacterial amount was compared statistically between the intubation and extubation status and among set time points during the oral care procedure. RESULTS: The oral bacterial count was significantly decreased after extubation. During the oral care procedure, the oral bacterial amount was significantly lower after eliminating the contaminants either by rinsing or wiping, with no remarkable difference between the elimination techniques. CONCLUSIONS: The findings suggest that the oral bacterial amount is elevated during endotracheal intubation, which could increase the risk of aspiration pneumonia. The significant reduction in the bacterial count by wiping indicates that it might be a suitable alternative to rinsing for mechanically ventilated patients.

In Vitro Study of Endotoxin Adsorption by a Polymyxin B-Immobilized Fiber Column.

BACKGROUND: Polymyxin B-immobilized fiber (PMX-F) columns are used as therapeutic interventions for septic shock. The clinical efficacy has been reported for 2-h applications, but their ability to adsorb endotoxin over longer treatments has not been fully elucidated. We hypothesized that PMX-F columns are capable of endotoxin removal for more than 2 h. METHOD: We designed closed circuits incorporating either a PMX-F column with an 8.5-mL priming volume (PMX-01R) or a sham-control column, and used inactivated fetal bovine serum as the circulating perfusate. Endotoxin was continuously injected at a fixed rate for 24 h, and perfusate endotoxin concentrations were measured at fixed time points. PMX-01R endotoxin adsorption was calculated from the difference in the endotoxin concentrations. RESULTS: PMX-01R endotoxin adsorption increased continuously in a virtually linear manner. CONCLUSIONS: The PMX-01R column showed sustained endotoxin adsorption for at least 24 h. This indicated that PMX-F columns would be capable of clinical endotoxin removal for 24 h.

Influence of Blood Purification and Differential Injection Sites of Cold Saline on Transpulmonary Thermodilution Values.

We aimed to investigate the effects of blood purification and cold saline injection sites on the transpulmonary thermodilution values. We measured the cardiac output of eight pigs in every combination of cold saline injection (left jugular and femoral veins) and blood purification sites (right jugular and femoral veins), with or without blood purification. We examined the influence of the difference between the presence and absence of blood purification, vascular sites for blood purification, and sites for cold saline injection on the transpulmonary thermodilution values. Cardiac output measured during blood purification using transpulmonary thermodilution was underestimated; however, there was no difference between vascular sites. Cardiac output measured via injection of cold saline into the femoral vein was higher than that obtained through injection of cold saline into the jugular vein, with or without blood purification.

A devised strategy for tracheal extubation for predicted difficult airway in a child with unilateral vocal cord paralysis: a case report.

BACKGROUND: Extubation is a more challenging medical practice than intubation, and countermeasures against it are similar to those described in the Difficult Intubation Guidelines, but problems cannot be overcome by completely the same methods. We predicted difficult extubation in a pediatric patient with left recurrent laryngeal nerve paralysis and devised an extubation method. CASE PRESENTATION: The patient was a 2-year-and-8-month-old boy scheduled for cleft palate repair. Concomitant cardiac anomaly and first and second branchial arch syndrome-associated facial malformations, such as mandibular micrognathia and auricular malformation, were observed. He had a past medical history of difficult intubation and respiratory arrest on a catheter test under intravenous sedation at 4 months old. Left recurrent laryngeal nerve paralysis was discovered on preoperative examination of the cleft palate, based on which difficulty in postoperative extubation was predicted. A catheter for tracheal tube exchange proposed by the extubation guidelines of the Difficult Airway Society (DAS) was placed, endoscopic examination was performed while inducing spontaneous breathing and swallowing reflex by an otolaryngologist, and the tube was removed while movement of the tissue around the glottis was visually evaluated. The patient was managed in an ICU after extubation, and both the systemic and respiratory conditions were favorable. CONCLUSIONS: Extubation and airway management could be safely performed by devising extubation while conforming to the DAS guidelines.

Recombinant human thrombomodulin inhibits neutrophil extracellular trap formation in vitro.

The aim of this study was to investigate the effects of recombinant human-soluble thrombomodulin (rTM) on lipopolysaccharide (LPS)-induced, platelet-dependent neutrophil extracellular trap (NET) formation (NETosis). Human peripheral blood neutrophils and platelets were co-incubated with or without LPS (0.2 µg/ml) in the presence and absence of rTM (2 µg/ml). NETosis was confirmed by immunostaining and confocal microscopy. In the absence of platelets, LPS did not induce NETosis in the neutrophils. NETosis, however, was induced by LPS when neutrophils were co-cultured with platelets (64 % of neutrophils). Notably, rTM was able to fully inhibit NETosis in neutrophils cultured with platelets and in the presence of LPS. rTM did not induce NETosis in this co-culture system (p < 0.01 versus LPS in the absence of rTM). These results show that rTM can suppress LPS-induced platelet-dependent NETosis in vitro.

Novel Blood Purification System for Regulating Excessive Immune Reactions in Severe Sepsis and Septic Shock: An Ex Vivo Pilot Study.

Promising results have been reported with blood purification as adjuvant treatment; however, the immunological mechanisms remain unclear. We have been developing a new blood purification system for regulating excessive immune reactions in severe sepsis and septic shock using a granulocyte adsorbing column (Adacolumn [Ada]), and a cytokine-adsorbing hemofilter (AN69ST hemofilter [AN69]). Fresh porcine blood was circulated for 6 h in five experimental groups including Ada and AN69 to assess the effects of leukocyte adsorption, phagocytic activity and adhesiveness of granulocytes. In the present study, we found that Ada mainly adsorbed granulocytes and monocytes, but not lymphocytes. The phagocytic activity level of granulocytes decreased, and adhesiveness increased, but the number of CD11b-positive cells markedly decreased in the current system. Elevated cytokine levels (IL-1ß, IL-8 and IL-10) at the outlet of Ada were significantly lower than at the outlet of AN69 due to cytokine adsorption. Further studies are needed to better understand cellular interactions.

In vitro evaluation of high mobility group box 1 protein removal with various membranes for continuous hemofiltration.

The high mobility group box 1 protein (HMGB1) is an alarmin that plays an important role in sepsis and has been recognized as a promising target with a wide therapeutic window; however, no drugs and devices are currently in practical use. We hypothesized that hemofilters composed of porous membranes or cytokine-adsorbing membranes could remove HMGB1 from the blood. We performed experimental hemofiltration in vitro using four types of hemofilters composed of different membranes specifically designed for continuous hemofiltration. The test solution was a 1000-mL substitution fluid containing 100 µg of HMGB1 and 35 g of bovine serum albumin. Experimental hemofiltration was conducted for 360 min in a closed loop circulation system. Among the four membranes, surface-treated polyacrylonitrile (AN69ST) showed the highest capacity to adsorb HMGB1; it adsorbed nearly 100 µg of HMGB1 in the initial 60 min and showed a markedly high clearance rate (60.8 ± 5.0 mL/min) at 15 min. The polymethylmethacrylate membrane had half of the adsorption capacity of the AN69ST membrane. Although the highest sieving coefficient for HMGB1 was obtained with the high cut-off polyarylethersulfone membrane, which correlated with a constant filtrate clearance rate, albumin loss was observed. However, no such removal of both HMGB1 and albumin was observed with the polysulfone membrane and tubing. We conclude that continuous hemofiltration using the AN69ST membrane is a promising approach for HMGB1-related sepsis.

Sustained high-efficiency daily diafiltration using a mediator-adsorbing membrane (SHEDD-fA) in the treatment of patients with severe sepsis.

Sustained high-efficiency daily diafiltration using a mediator-adsorbing membrane (SHEDD-fA) is an effective, intensive modality for sepsis treatment. Here we describe the effectiveness of SHEDD-fA, which makes the best use of three principles: dialysis, filtration and adsorption, for mediator removal in the treatment of severe sepsis. SHEDD-fA was initiated after adequate fluid resuscitation and catecholamine support had been provided. A large (2.1 m(2)) polymethylmethacrylate membrane dialyzer was placed in the blood circuit. Operation conditions were as follows: blood flow rate 150 ml/min, filtration rate 1,500 ml/h (post-dilution), and dialysate flow rate 300-500 ml/min over 8-12 h daily. 55 consecutive patients with severe sepsis were studied. The following results were obtained: pressure catecholamine index significantly decreased at 3 h after initiation of septic shock, PaO(2)/F(IO2) significantly increased at 1 h after initiation of septic acute respiratory distress syndrome, a significant decrease in interleukin (IL)-6 level for 3 days was observed, and IL-6 was effectively adsorbed in one pass through the filter. The average sequential organ failure assessment score of patients was 10.1 and the mortality at 28 days was 16.4% (46 survived, 9 died). Because SHEDD-fA is an intensive and high-efficiency modality, removal of useful drugs or nutrients may be observed. Despite the fact that removal of useful substances cannot be ignored, we believe that an appropriate stage or timing can be identified so that we can avoid a vicious cycle and use blood purification with effective diffusion, filtration and adsorption. We demonstrate that SHEDD-fA may be an effective, intensive modality for the treatment of patients with severe sepsis and is a possible modality for cytokine modulation therapy.

A unique B2 B cell subset in the intestine.

Over 80% of the body's activated B cells are located in mucosal sites, including the intestine. The intestine contains IgM(+) B cells, but these cells have not been characterized phenotypically or in terms of their developmental origins. We describe a previously unidentified and unique subset of immunoglobulin M(+) B cells that present with an AA4.1(-)CD21(-)CD23(-) major histocompatibility complex class II(bright) surface phenotype and are characterized by a low frequency of somatic hypermutation and the potential ability to produce interleukin-12p70. This B cell subset resides within the normal mucosa of the large intestine and expands in response to inflammation. Some of these intestinal B cells originate from the AA4.1(+) immature B2 cell pool in the steady state and are also recruited from the recirculating naive B cell pool in the context of intestinal inflammation. They develop in an antigen-independent and BAFF-dependent manner in the absence of T cell help. Expansion of these cells can be induced in the absence of the spleen and gut-associated lymphoid tissues. These results describe the existence of an alternative pathway of B cell maturation in the periphery that gives rise to a tissue-specific B cell subset.

Regulatory role of B-1 B cells in chronic colitis.

According to the 'hygiene hypothesis', enhanced microbial exposure due to early childhood infections leads to a reduction of T(h)2-mediated allergic diseases and inflammatory bowel disease. To begin to elucidate the mechanisms underlying this hypothesis, we studied development of T(h)2-mediated colitis of the TCRalpha knockout (KO) mouse in both a specific pathogen-free (SPF) facility and a conventional (CV) facility. After more than five generations in each facility, TCRalpha KO mice kept in the CV facility developed dramatically less colitis than mice that were kept in the SPF facility. Surprisingly, the suppression of colitis in the CV facility correlated with a significant increase in natural IgM production by B-1 B cells. In contrast, B cell-deficient TCRalpha double-knockout (alphamu DKO) mice maintained in the CV facility continued to develop severe colitis, strongly suggesting that B-1 B cells contributed to the suppression of colitis. Indeed, the adoptive transfer of B-1 B cells isolated from the peritoneal cavity of TCRalpha KO mice (SPF) into alphamu DKO mice (CV) suppressed the development of colitis in the recipient mice. We conclude that B-1 cells play a regulatory role in T(h)2-mediated colitis under non-hygienic conditions, possibly by generating natural antibodies in response to microbial flora.