Fibrinogen γ-Chain Peptide-Coated Adenosine 5' Diphosphate-Encapsulated Liposomes Rescue Mice From Lethal Blast Lung Injury via Adenosine Signaling.

OBJECTIVES: Fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes can accumulate via dodecapeptide HHLGGAKQAGDV interactions at bleeding sites where they release adenosine 5'-diphosphate that is rapidly metabolized to adenosine, which has tissue-protective effects. We investigated the efficacy of fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes to treat blast lung injury, with a focus on adenosine signaling. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Adult male C57BL/6 mice. INTERVENTIONS: Mice were pretreated with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes, dodecapeptide HHLGGAKQAGDV-(phosphate-buffered saline)-liposomes, adenosine 5' diphosphateliposomes, or phosphate-buffered saline-liposomes. Five minutes after treatment the mice received a single laser-induced shock wave (1.8 J/cm) that caused lethal blast lung injury, and their survival times and lung injuries were then assessed. We also evaluated the therapeutic effect of posttreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes or H12-(phosphate-buffered saline)-liposomes 1 minute after laser-induced shock wave exposure. To examine the effect of adenosine signaling, adenosine A2A receptor (ZM241385) or adenosine A2B receptor (PSB 1115) antagonists were administered to the mice 1 hour before the pretreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes that was followed by laser-induced shock wave exposure. MEASUREMENTS AND MAIN RESULTS: Pre- and posttreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes significantly increased mouse survival [fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes: 58% survival vs H12-(phosphate-buffered saline)-liposomes: 8%; p < 0.05 (posttreatment)] and mitigated pulmonary tissue damage/hemorrhage and neutrophil accumulation after laser-induced shock wave exposure. fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes accumulated at pulmonary vessel injury sites after laser-induced shock wave exposure with both pre- and posttreatment. Furthermore, pretreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes reduced albumin and macrophage inflammatory protein-2 levels in bronchoalveolar lavage fluid. Although fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes pretreatment did not affect blood coagulation activity in the injured mice, its beneficial effect on blast lung injury was significantly abrogated by A2A or A2B adenosine receptor antagonists (A2A antagonist: 17% survival; A2B antagonist: 33% vs dimethyl sulfoxide control: 80%; p < 0.05, respectively). CONCLUSIONS: Fibrinogen γ-chain (dodecapeptide HHLGGAKQA GDV)-coated adenosine 5'-diphosphate-encapsulated liposomes may be effective against blast lung injury by promoting tissue-protective adenosine signaling and could represent a novel controlled-release drug delivery system.

Activation of CD11b+ Kupffer cells/macrophages as a common cause for exacerbation of TNF/Fas-ligand-dependent hepatitis in hypercholesterolemic mice.

We have reported that the mouse hepatic injury induced by either α-galactosylceramide (α-GalCer) or bacterial DNA motifs (CpG-ODN) is mediated by the TNF/NKT cell/Fas-ligand (FasL) pathway. In addition, F4/80(+) Kupffer cells can be subclassified into CD68(+) subset with a phagocytosing capacity and CD11b(+) subset with a TNF-producing capacity. CD11b(+) subset increase if mice are fed high-fat and cholesterol diet (HFCD). The present study examined how a HFCD affects the function of NKT cells and F4/80(+) CD11b(+) subset and these hepatitis models. After the C57BL/6 mice received a HFCD, high-cholesterol diet (HCD), high-fat diet (HFD) and control diet (CD) for four weeks, the HFCD mice increased surface CD1d and intracellular TLR-9 expression by the CD11b(+) population compared to CD mice. Hepatic injury induced either by α-GalCer or CpG-ODN was more severe in HCD and HFCD mice compared to CD mice, which was in proportion to the serum TNF levels. In addition, liver cholesterol levels but not serum cholesterol levels nor liver triglyceride levels were involved in the aggravation of hepatitis. The FasL expression of NKT cells induced by both reagents was upregulated in HFCD mice. Furthermore, the liver mononuclear cells and purified F4/80(+) CD11b(+) subset from HFCD mice stimulated with either reagent in vitro produced a larger amount of TNF than did those from CD mice. Intracellular TNF production in F4/80(+) CD11b(+) cells was confirmed. The increased number of F4/80(+) CD11b(+) Kupffer cells/macrophages by HFCD and their enhanced TNF production thus play a pivotal role in TNF/NKT cell/FasL dependent hepatic injury.

The effect of synthetic C-reactive protein on the in vitro immune response of human PBMCs stimulated with bacterial reagents.

Synthetic C-reactive protein (CRP) rescues mice from lethal endotoxin shock or bacterial infection by suppressing tumor necrosis factor (TNF-α), but in turn, enhances Kupffer cell phagocytic activity. We herein assessed the influence of CRP in human peripheral blood mononuclear cells (PBMCs). When human PBMCs were stimulated in vitro with penicillin-treated Streptococcus pyogenes, bacterial DNA motifs and lipopolysaccharide with or without synthetic CRP, CRP suppressed the production of TNF-α and IL-12, but not that of IFN-γ. This was also the case for the in vitro Shwartzman reaction induced in PBMCs. CRP also decreased high-mobility group box 1 production from macrophages, which is crucial in the later phase of endotoxin/septic shock. However, CRP upregulated the perforin expression by CD56(+) NK cells and increased their antitumor cytotoxicity. CRP may thus be a potent immunomodulatory factor in the human immune system, suggesting its therapeutic potential for use against human septic shock.

Effective control of massive venous bleeding by "multioverlapping therapy" using polysaccharide nanosheets in a rabbit inferior vena cava injury model.

OBJECTIVE: To investigate the efficacy of multioverlapping therapy using a polysaccharide nanosheet having 75-nm thickness for sealing and stopping massive venous hemorrhage. METHODS: The hydrostatic durability of the polysaccharide nanosheet was evaluated in vitro when secured to an incised silicon tube. For in vivo studies, the inferior vena cava (IVC) of rabbits was cut longitudinally, and multiple polysaccharide nanosheets were overlapped onto the injured IVC. RESULTS: The mechanical hydrostatic durability of the nanosheets was gradually augmented by an increasing number of multilayered nanosheets in vitro. This durability was saturated at 80 ± 6 mm Hg by four layers of nanosheets, which was robust enough to seal injured vessel walls of the large IVC. Multioverlapping therapy using nanosheets effectively sealed and stopped bleeding from the injured IVC in vivo. One month later, no inflammatory tissue response was observed around the nanosheet attachment sites of the IVC, while conventional suturing repair in control rabbits showed a severe inflammatory response around the sutured area. CONCLUSIONS: The multioverlapping therapy using the polysaccharide nanosheets will effectively stop massive venous bleeding without adverse effects in the immediate or chronic postoperative setting.

Liposome-encapsulated hemoglobin attenuates cardiac dysfunction and sympathetic activity during hypohemoglobinemic shock.

The use of liposome-encapsulated hemoglobin (LHb), which is a cellular Hb, has been demonstrated to be beneficial in the treatment of hypohemoglobinemic shock. As a molecule of appropriate size (220 nm) that can carry oxygen, LHb may ameliorate cardiac dysfunction during lethal hemodilation. The purpose of this study was to determine the efficacy of LHb transfusion in relieving cardiovascular dysfunction in a rat model of lethal progressive hemodilution. Over the course of 150 min, rats were subjected to blood withdrawal (0.2 mL/min) and simultaneously transfused with LHb, washed rat red blood cells, or 5% albumin. Temporal changes in cardiac function, heart-type fatty acid-binding protein levels, plasma levels of catecholamines, heart rate variability, and hypoxia-inducible factor 1α expression were measured during lethal progressive hemodilution. More than 80% of the rats transfused with either LHb or washed rat red blood cells survived for 8 days. Liposome-encapsulated hemoglobin transfusion suppressed hypoxia-inducible factor 1α expression in the heart, maintained low levels of heart-type fatty acid-binding protein, and attenuated sympathetic nerve activity as reflected by changes in heart rate variability and plasma levels of epinephrine and norepinephrine. The results indicate that LHb attenuates cardiac dysfunction and sympathetic overactivity during lethal hemorrhage.

Pivotal advance: characterization of mouse liver phagocytic B cells in innate immunity.

Although B cells in vertebrates have been thought to lack phagocytic activity, there has been a recent report of such ability by the B cells of early vertebrates such as fish and frogs. Here, we show for the first time that mouse liver IgM(+) B cells actively phagocytose microsphere beads and Escherichia coli and that they effectively kill bacterial cells. Such phagocytic activity is not observed in other liver MNCs, except for F4/80(+) Kupffer cells. In the presence of fresh mouse serum (but not heat-inactivated serum), the heat-killed E. coli phagocytic activity of liver B cells increased significantly but was inhibited significantly by anticomplement component C3 antibody, suggesting E. coli opsonization by serum factors, including complement components. Upon i.v. injection of FITC-labeled E. coli into mice, a substantial proportion of liver B cells phagocytosed the bacteria, as compared with spleen B cells. Functional phagolysosome formation in liver B cells was supported by several reagents showing an acidic change and lysosomes in the phagocytosed vacuoles. Indeed, mouse liver B cells killed viable E. coli more efficiently than did spleen B cells in vitro. Further, E. coli-phagocytic liver B cells produced a substantial amount of IL-12. These results indicate that liver B cells have phagocytic and bactericidal activities similar to those of dedicated phagocytes and may contribute to bacterial clearance.

The immunologic outcome of enhanced function of mouse liver lymphocytes and Kupffer cells by high-fat and high-cholesterol diet.

Dietary lipids/cholesterol may modulate liver immune function. We have recently found that mouse F4/80 Kupffer cells are classified into phagocytic CD68 Kupffer cells and cytokine-producing CD11b Kupffer cells. We here investigate how a high-fat and/or high-cholesterol diet affects innate immune liver mononuclear cells. For 4 weeks, C57BL/6 mice were fed a high-fat and high-cholesterol diet (HFCD), a high-cholesterol diet (HCD), a high-fat diet (HFD), or a control diet (CD). High-fat and high-cholesterol diet and HCD increased liver cholesterol levels; serum cholesterol levels increased in HFCD and HFD mice but not in HCD mice. The increased proportion of natural killer (NK) cells, downregulated NK1.1 expression of natural killer T cells, and enhanced CD69 and IL-12 receptor ß mRNA expression of liver lymphocytes indicate the activation of them by HFCD. IL-12 production from Kupffer cells and interferon γ production from NK/natural killer T cells activated by LPS and/or IL-12 both increased. IL-12 pretreatment more effectively improved the survival of HFCD mice relative to the survival of CD mice upon injections of liver metastatic EL-4 cells. In contrast, HFCD mouse survival decreased after LPS injection and generalized Shwartzman reaction. Consistently in HFCD mice, Toll-like receptor 4 mRNA expression of whole Kupffer cells was upregulated, and CD11b Kupffer cells proportionally increased. Although the proportion of CD68 Kupffer cells decreased in HFCD mice, phagocytic activity of them was enhanced. Mice fed with HCD rather than those fed with HFD showed features closer to HFCD mice. Thus, enhanced function of mouse liver mononuclear cells is likely dependent on the liver cholesterol level, rather than the liver triglyceride level.

Intraosseous transfusion with liposome-encapsulated hemoglobin improves mouse survival after hypohemoglobinemic shock without scavenging nitric oxide.

Recently, we developed liposome-encapsulated hemoglobin (LEH), a novel cellular hemoglobin-based oxygen carrier. We hypothesized that the LEH effectively suppresses scavenging of nitrogen oxides by sequestering hemoglobin, thereby being useful for resuscitation from hemorrhagic shock, especially in prehospital settings where blood transfusion is not available. However, putting a catheter into the peripheral vessels is sometimes difficult in prehospital resuscitation, because these vessels collapse in patients with hemorrhagic shock. The intraosseous route does not collapse under such conditions. We here studied the resuscitation of severe hypohemoglobinemia following massive hemorrhage using intraosseous (intrafemur) transfusion with LEH in mice. First, we examined the effect of intravenous transfusion with LEH on the resuscitation of mice with fatal hypohemoglobinemia that was made with progressive hemodilution by blood exchanges. Despite a success in initial resuscitation without scavenging of NO2 or NO3, LEH transfusion did not significantly improve mouse survival 72 h later as compared with red blood cell (RBC) transfusion. In other experiments, hypohemoglobinemic mice were also made with blood withdrawal and intraosseous infusion with 5% albumin. Thereafter, the mice were rescued with intraosseous transfusion of LEH or RBCs. Unlike intravenous transfusion, intraosseous transfusion with LEH (but not such transfusion with RBCs) significantly increased mouse survival without scavenging of NO2 or NO3, presumably because LEH vesicles were much smaller than RBCs, thereby effectively flowing into the circulation from the femur. Thus, intraosseous transfusion with LEH may be a candidate strategy for efficient prehospital resuscitation from hemorrhagic shock.

Characterization of two F4/80-positive Kupffer cell subsets by their function and phenotype in mice.

BACKGROUND & AIMS: Liver Kupffer cells have been suggested to be heterogeneous macrophage lineage cells. We explored this possibility by classifying the mouse Kupffer cells into subpopulations and characterizing them by their phenotype and function. METHODS: Liver mononuclear cells (MNCs) from C57BL/6 mice were isolated and their phenotypes and functions were analyzed. The effects of clodronate liposomes and gadolinium chloride (GdCl(3)) on Kupffer cells were also investigated. RESULTS: Approximately 25% of liver MNCs were F4/80(+) Kupffer cells. Of these, 46% were CD11b(-)CD68(+), 22% were CD11b(+)CD68(-), and 6% were CD11b(+)CD68(+). CD68(+) cells showed potent phagocytic activity and reactive oxygen species (ROS) production capacity after lipopolysaccharide (LPS) stimulation, whereas CD11b(+) cells did not. CD11b(+) cells showed a strong capacity for the production of cytokines (TNF and IL-12), which was much less prominent in CD68(+) cells. At 24h after LPS or Escherichia coli injection into mice, the proportions of CD11b(+)CD68(-) and CD11b(+)CD68(+) cells increased but that of CD11b(-)CD68(+) cells decreased. The increase in CD11b(+)CD68(+) cells appeared to be derived from the CD11b(+)CD68(-) subset. Although the CD11b(+) cells augmented phagocytic activity after LPS injection, they did not increase ROS production, suggesting their weak lytic activity. Injection of clodronate or GdCl(3) into mice depleted the CD68(+) cells but increased CD11b(+) cells proportionally because CD68(+) cells may phagocytose these toxic reagents and undergo apoptosis. GdCl(3)-treated mice also consistently increased serum TNF after LPS challenge. CONCLUSIONS: Two F4/80(+) Kupffer cell subsets may exist, a CD68(+) subset with phagocytic activity and a CD11b(+) subset with cytokine-producing capacity.

The effect of ketamine anesthesia on the immune function of mice with postoperative septicemia.

BACKGROUND: It is unknown how ketamine anesthesia immunologically affects the outcome of patients with postoperative septicemia. We investigated the effects of ketamine anesthesia on mice with an Escherichia coli or lipopolysaccharide (LPS) challenge after laparotomy, focusing on phagocytosis by liver macrophages (Kupffer cells) and cytokine production. METHODS: C57BL/6 mice received ketamine or sevoflurane anesthesia during laparotomy, which was followed by an E. coli or LPS challenge; thereafter, mouse survival rates and cytokine secretions were examined. The effects of a ß-adrenoceptor antagonist, nadolol, on ketamine anesthesia were also assessed to clarify the mechanisms of ketamine-induced immunosuppressive effects. RESULTS: Ketamine anesthesia increased the mouse survival rate after LPS challenge after laparotomy compared with sevoflurane anesthesia, whereas such an effect of ketamine was not observed after E. coli challenge. Ketamine suppressed tumor necrosis factor (TNF) and interferon (IFN)-γ secretion after LPS and E. coli challenge. When bacterial growth was inhibited using an antibiotic, ketamine anesthesia effectively improved mouse survival after E. coli challenge compared with sevoflurane anesthesia. Neutralization of TNF also improved survival and decreased IFN-γ secretion after bacterial challenge in antibiotic-treated mice with sevoflurane anesthesia, suggesting that ketamine's suppression of TNF may improve survival. Ketamine also suppressed in vivo phagocytosis of microspheres by Kupffer cells in LPS-challenged mice. Concomitant use of nadolol with an anesthetic dose of ketamine did not restore TNF suppression in LPS-challenged mice, suggesting a mechanism independent of the ß-adrenergic pathway. However, it restored TNF secretion under low-dose ketamine (10% anesthetic dose). In contrast, nadolol restored the decrease in phagocytosis by Kupffer cells, which was induced by the anesthetic dose of ketamine via the ß-adrenergic pathway, suggesting distinct mechanisms. CONCLUSION: Ketamine suppresses TNF production and phagocytosis by Kupffer cells/macrophages. Therefore, unless bacterial growth is well controlled (by an antibiotic), postoperative infection might not improve despite reduction of the inflammatory response.

Sealing effect of a polysaccharide nanosheet for murine cecal puncture.

BACKGROUND: Recent developments in nanobiotechnology have led us to develop a method of producing a free-standing polymer nanosheet composed of polysaccharides (ie, polysaccharide nanosheet) with a thickness of tens of nanometers. Owing to its enormous aspect ratio, the polysaccharide nanosheet is semi-absorbent and has a physical adhesive strength 7.5-fold greater than that of conventional films of >1 microm thickness. Herein, we have investigated the therapeutic sealing effect of this polysaccharide nanosheet on murine cecal puncture as a wound dressing material. METHODS: Murine cecum was punctured and then overlapped with the polysaccharide nanosheet. Thereafter, we evaluated its sealing effect on bacterial peritonitis as well as the protection offered by the polysaccharide nanosheet against bacterial permeability using an in vitro transmembrane assay. RESULTS: The 39-nm-thick polysaccharide nanosheet overlapped tightly the perforated cecum. No adhering agents were required because of the ability of the polysaccharide nanosheet to adhere to the tissue surface by physical adsorption (eg, van der Waals interaction). Sealing the perforated cecum with the polysaccharide nanosheet increased survival rate without postoperative adhesion by comparison with untreated mice (90 vs 30%; P < .01). These data were supported by the improvement in peritonitis related to bacterial counts, white blood cell counts, and the serum tumor necrosis factor level. Moreover, using an in vitro transmembrane assay, we showed that the polysaccharide nanosheet inhibited effectively bacterial penetration. CONCLUSION: We have demonstrated the potential clinical benefits of the nanosheet-type biomaterial that can be used for repairing a cecal colotomy without chemical bonding agents.

Immunosuppression in the livers of mice with obstructive jaundice participates in their susceptibility to bacterial infection and tumor metastasis.

Although patients with obstructive jaundice are susceptible to bacterial infections and cancers, the mechanisms remain to be elucidated. In the present study, liver mononuclear cells (MNCs) of bile duct-ligated (BDL) mice were immunologically assessed. Liver natural killer T cells were greatly decreased within 24 h after BDL. Upon injection of Escherichia coli (E. coli; 10 colony-forming units) at 7 days after the procedure, all BDL mice had died, but no sham mice died. Consistently, an overgrowth of E. coli was seen in the livers of BDL mice. Although the serum IL-12 and IL-18 levels after E. coli challenge in BDL mice were higher than those in sham mice, the IFN-gamma level was greatly suppressed. However, exogenous IFN-gamma injection significantly increased BDL mouse survival after E. coli challenge. Furthermore, liver MNC of BDL mice exhibited a lower cytotoxic activity against tumors, and BDL mice intravenously injected with liver metastatic EL-4 cells showed markedly increased EL-4 metastases. The total bile acids, as well as the bile acid fractions, increased in the sera and liver. IFN-gamma production by liver MNC from normal mice stimulated with LPS in vitro was inhibited by the addition of bile acids, whereas, conversely, the production of IL-12 and IL-18 increased. In conclusion, liver natural killer T cells were diminished in BDL mice, and the function of liver MNC (IFN-gamma production) was also impaired presumably due to increased bile acids. This may partly explain the increased susceptibility of BDL mice to bacterial infections and tumor metastasis.

Superoxide produced by Kupffer cells is an essential effector in concanavalin A-induced hepatitis in mice.

UNLABELLED: Although concanavalin A (Con-A)-induced experimental hepatitis is thought to be induced by activated T cells, natural killer T (NKT) cells, and cytokines, precise mechanisms are still unknown. In the current study, we investigated the roles of Kupffer cells, NKT cells, FasL, tumor necrosis factor (TNF), and superoxide in Con-A hepatitis in C57BL/6 mice. Removal of Kupffer cells using gadolinium chloride (GdCl(3)) from the liver completely inhibited Con-A hepatitis, whereas increased serum TNF and IFN-gamma levels were not inhibited at all. Unexpectedly, anti-FasL antibody pretreatment did not inhibit Con-A hepatitis, whereas it inhibited hepatic injury induced by a synthetic ligand of NKT cells, alpha-galactosylceramide. Furthermore, GdCl(3) pretreatment changed neither the activation-induced down-regulation of NK1.1 antigens as well as T cell receptors of NKT cells nor the increased expression of the CD69 activation antigen of hepatic T cells. CD68(+) Kupffer cells greatly increased in proportion in the early phase after Con-A injection; this increase was abrogated by GdCl(3) pretreatment. Anti-TNF antibody (Ab) pretreatment did not inhibit the increase of Kupffer cells, but it effectively suppressed superoxide/reactive oxygen production from Kupffer cells and the resulting hepatic injury. Conversely, depletion of NKT cells in mice by NK1.1 Ab pretreatment did suppress both the increase of CD68(+) Kupffer cells and Con-A hepatitis. Consistently, the diminution of oxygen radicals produced by Kupffer cells by use of free radical scavengers greatly inhibited Con-A hepatitis without suppressing cytokine production. However, adoptive transfer experiments also indicate that a close interaction/cooperation of Kupffer cells with NKT cells is essential for Con-A hepatitis. CONCLUSION: Superoxide produced by Kupffer cells may be the essential effector in Con-A hepatitis, and TNF and NKT cells support their activation and superoxide production.

Effects and cost of glycyrrhizin in the treatment of upper respiratory tract infections in members of the Japanese maritime self-defense force: Preliminary report of a prospective, randomized, double-blind, controlled, parallel-group, alternate-day treatment assignment clinical trial.

BACKGROUND: Upper respiratory tract infections (URTIs) account for at least half of all acute illnesses. Specific antiviral therapy has not been developed against most respiratory viruses thought to cause URTIs. The pharmacologic action of glycyrrhizin has been shown to produce anti-inflammatory activity, modulation of the immune system, inhibition of virus growth, and inactivation of viruses. OBJECTIVE: The aim of this study was to assess the tolerability, efficacy, and cost of glycyrrhizin in improving the severity and duration of signs and symptoms of URTIs. The primary end point was tolerability, and the secondary and points included improvement in signs and symptoms of URTI and cost. METHODS: Members of the Japanese Maritime Self-Defense Force (SDF) treated for URTIs from January 2002 to May 2002 in the SDF Etajima Hospital (Hiroshima, Japan) were eligible for this prospective, randomized, double-blind, controlled, parallel-group, alternate-day treatment assignment study. All patients in this study fulfilled the following enrollment criteria: admitted to the hospital on the first arrival day as an outpatient; fever (body temperature <38.0°C) with signs and symptoms of URTI (headache, sore throat, rhinorrhea, pharyngitis); and had not received antibiotics or oseltamivir phosphate for 4 weeks before the study. Patients who were admitted on an even day received an IV drip infusion of 40 mL of glycyrrhizin (0.2%) and 500 mL of lactated Ringer's solution daily during hospitalization (glycyrrhizin group). Patients who were admitted on an odd day received an IV drip infusion of 500 mL/d of lactated Ringer's solution only (control group). Adverse effects were assessed by the physicians during hospitalization, using patient interview and laboratory analysis. RESULTS: Forty-one consecutive patients entered the study; 15 patients (15 men, 0 women; mean [SD] age, 25.2 [1.5] years) were assigned to the glycyrrhizin group and 269 patients (24 men, 2 women; mean [SD] age, 22.6 [0.9] years) were assigned to the control group. The 2 groups were similar in terms of baseline characteristics. The mean duration of hospitalization was shorter (P = 0.01), the mean maximum body temperature 24 to 48 hours after admission was less (P = 0.05), and the cost of therapy (P = 0.03) was less in the glycyrrhizin group than the control group. No AEs were reported. CONCLUSIONS: In this study of hospitalized patients with URTIs, glycyrrhizin therapy was associated with a shorter hospitalization, lower-grade fever, and lower cost of therapy compared with controls, showing that it may be beneficial to patients with URTIs without acute bacterial infections.