Transplantation of dental pulp-derived cell sheets cultured on human amniotic membrane induced to differentiate into bone.

OBJECTIVE: The usefulness of the amniotic membrane as a cell culture substrate has led to its use in the development of dental pulp-derived cell sheets. We induced osteoblastic differentiation of dental pulp-derived cell sheets and conducted histological and immunological examinations in addition to imaging assessments for regeneration of bone defects. METHODS: Dental pulp cells were obtained by primary culture of the dental pulp tissue harvested from extracted wisdom teeth. These cells were maintained for three to four passages. Subsequently, the dental pulp cells were seeded onto an amniotic membrane to produce dental pulp-derived cell sheets. Following the induction of osteoblastic differentiation, the sheets were grafted into the subcutaneous tissue of the lower back and maxillary bone defect of a nude mouse. Histological and immunological examinations of both grafts were performed. RESULTS: Dental pulp-derived cell sheets cultured on an osteoblast differentiation-inducing medium demonstrated resemblance to dental pulp tissue and produced calcified tissue. Mineralization was maintained following grafting of the sheets. Regeneration of the maxillary bone defect was observed. CONCLUSION: Induction of osteoblastic differentiation of the dental pulp-derived cell sheets may be indicated for the regeneration of periodontal tissue.

Biomechanical force induces the growth factor production in human periodontal ligament-derived cells.

Although many reports have been published on the functional roles of periodontal ligament (PDL) cells, the mechanisms involved in the maintenance and homeostasis of PDL have not been determined. We investigated the effects of biomechanical force on growth factor production, phosphorylation of MAPKs, and intracellular transduction pathways for growth factor production in human periodontal ligament (hPDL) cells using MAPK inhibitors. hPDL cells were exposed to mechanical force (6 MPa) using a hydrostatic pressure apparatus. The levels of growth factor mRNA and protein were examined by real-time RT-PCR and ELISA. The phosphorylation of MAPKs was measured using BD™ CBA Flex Set. In addition, MAPKs inhibitors were used to identify specific signal transduction pathways. Application of biomechanical force (equivalent to occlusal force) increased the synthesis of VEGF-A, FGF-2, and NGF. The application of biomechanical force increased the expression levels of phosphorylated ERK and p38, but not of JNK. Furthermore, the levels of VEGF-A and NGF expression were suppressed by ERK or p38 inhibitor. The growth factors induced by biomechanical force may play a role in the mechanisms of homeostasis of PDL.

Enhancement of alcohol drinking in mice depends on alterations in RNA editing of serotonin 2C receptors.

Serotonin 2C receptors (5-HT(2C)R) are G-protein-coupled receptors with various actions, including involvement in drug addiction. 5-HT2CR undergoes mRNA editing, converting genomically encoded adenosine residues to inosines via adenosine deaminases acting on RNA (ADARs). Here we show that enhanced alcohol drinking behaviour in mice is associated with the degree of 5-HT(2C)R mRNA editing in the nucleus accumbens and dorsal raphe nuceus, brain regions important for reward and addiction. Following chronic alcohol vapour exposure, voluntary alcohol intake increased in C57BL/6J mice, but remained unchanged in C3H/HeJ and DBA/2J mice. 5-HT(2C)R mRNA editing frequency in both regions increased significantly in C57BL/6J mice, as did expressions of 5-HT(2C)R, ADAR1 and ADAR2, but not in other strains. Moreover, mice that exclusively express the unedited isoform (INI) of 5-HT(2C)R mRNA on a C57BL/6J background did not exhibit increased alcohol intake compared with wild-type mice. Our results indicate that alterations in 5-HT(2C)R mRNA editing underlie alcohol preference in mice.

Combined administration of oseltamivir and hochu-ekki-to (TJ-41) dramatically decreases the viral load in lungs of senescence-accelerated mice during influenza virus infection.

To enhance the effect of anti-influenza-virus agent treatment, the effect of combined administration of oseltamivir phosphate and hochu-ekki-to (Japanese traditional herbal medicine, HET) on early viral clearance was examined. Senescence-accelerated mice were given HET in drinking water for 2 weeks, followed by intranasal infection with influenza A virus strain PR8. After 4 hours of infection, oseltamivir was administered orally for 5 days. The viral loads in the lungs of the group receiving combined treatment were dramatically lower when compared with the viral loads in the lungs of the group receiving oseltamivir alone. HET significantly increased the induction of IL-1ß and TNF-α in the lungs of PR8-infected mice and stimulated alveolar macrophage phagocytosis. From these results, we conclude that these functions may be responsible the increased effect on viral load reduction. Here, we show that the combined administration of oseltamivir and HET is very useful for influenza treatment in senescence-accelerated mice.

Scavenging rate constants of hydrophilic antioxidants against multiple reactive oxygen species.

Scavenging rate constants of eight hydrophilic antioxidants, including caffeic acid, chlorogenic acid, genistein, glutathione, N-acetylcysteine, rutin, trolox, and uric acid against multiple ROS, namely superoxide anion, hydroxyl radical, singlet oxygen, and alkoxyl radical were determined with the electron spin resonance method. Direct flash photolysis measurement of the second-order rate constant in the reaction of alkoxyl radical plus the spin trap 5,5-dimethyl-pyrroline N-oxide made it possible to evaluate scavenging rate constants in antioxidants. The magnitudes of scavenging rate constants were notably dependent on the character of each ROS and the overall rate constants were highest in hydroxyl radical scavenging and the lowest in superoxide anion. The highest scavenging rate constant against superoxide anion was obtained by chlorogenic acid (2.9 × 10(5) M(-1) s(-1)) and the lowest was by N-acetylcysteine (5.0 × 10(2) M(-1) s(-1)). For singlet oxygen, the highest was by glutathione (9.4 × 10(8) M(-1) s(-1)) and the lowest was by uric acid (2.3 × 10(6) M(-1) s(-1)). All other numbers are listed and illustrated. Redox potential measurements of the antioxidants indicated that the antioxidants are likely to react with superoxide anion and singlet oxygen through electron transfer processes.

Reduced small-intestinal injury induced by indomethacin in interleukin-17A-deficient mice.

BACKGROUND AND AIMS: The pathogenesis of enteropathy induced by non-steroidal anti-inflammatory drugs (NSAIDs) is still unclear, and there are no established treatments. Interleukin-17A (IL-17A) is a pro-inflammatory cytokine that has been associated with the development of chronic inflammatory diseases, including autoimmune diseases. To define the role of IL-17A in small intestinal injury and inflammation, we studied the effects of indomethacin administration in mice with targeted deletions of the IL-17A gene. METHODS: Male C57BL/6 (wild-type) and homozygous IL-17A(-/-) C57BL/6 mice were subjected to this study. Indomethacin (10 mg/kg) was subcutaneously administered to induce small-intestinal damage. Indomethacin-induced lesions in the small intestine were evaluated by measuring the injured area and by histopathology. Also assessed were myeloperoxidase (MPO) activity, as an index of neutrophil accumulation, and intestinal mRNA expression for inflammatory cytokines. RESULTS: The area of macroscopic ulcerative lesions, the MPO activity and the mRNA expression of inflammatory-associated chemokines, such as keratinocyte chemoattractant (KC), monocyte chemotactic protein-1 (MCP-1), and granulocyte-colony stimulating factor (G-CSF), were significantly increased in indomethacin-treated groups compared with the sham groups. The development of intestinal lesions by indomethacin was inhibited in IL-17A(-/-) mice compared with wild-type mice, together with significant suppression of the increased levels of MPO activities and KC, MCP-1, and G-CSF levels. CONCLUSION: These findings demonstrate that IL-17A contributes to the development of indomethacin-induced small intestinal injury through upregulation of G-CSF, KC, and MCP-1. IL-17A might be a promising new therapeutic target to treat NSAID-induced enteritis.

Nitric oxide (NO) scavenging capacity of natural antioxidants.

Nitric oxide (NO)-scavenging capacities of several hydrophilic antioxidants were determined by using the PTIO method, a competitive NO-scavenging method with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Relative NO-scavenging rates of antioxidants were measured with respect to PTIO and the scavenging rate constants were calculated based on PTIO's rate constant. Results indicated that NO-scavenging rate constants of the antioxidants were: uric acid (2.5)>caffeic acid (1.2)>trolox (1.0)>genistein (0.19)>glutathione (0) ≈N-acetylcysteine (0), where the numbers are expressed in trolox equivalent unit. The oxidation potentials of these antioxidants were measured and the order in the magnitude of oxidation potential was in good accordance with NO-scavenging capacity. Based on the results, we have suggested that the primary chemical process of the antioxidant reaction with NO can be characterised with the electron transfer from NO to the antioxidant.

Group 3 Innate Lymphoid Cells Protect Steatohepatitis From High-Fat Diet Induced Toxicity.

Background and Aims: Emerging evidence has revealed that innate lymphoid cells (ILCs) play a key role in regulating metabolic disorders. Here, we investigated the role of group 3 ILCs (ILC3s) in the modulation of Non-alcoholic fatty liver disease (NAFLD). Methods: RORγ gfp/gfp (RORgt KI/KI) and Rag2-/- mice with the administration of A213, RORgt antagonist, fed with a high-fat-diet (HFD) for 12 weeks, were used. We performed flow cytometry, real time PCR, and lipidomics analysis of serum and liver, and used RAW264.7 cells and murine primary hepatocytes in vitro. Results: HFD increased ILC3s and M1 macrophages in the liver, and RORgt KI/KI mice deficient in ILC3 showed significant fatty liver, liver fibrosis and significantly increased palmitic acid levels in serum and liver. In addition, administration of A213 to Rag2-/- mice caused significant fatty liver, liver fibrosis, and a significant increase in serum and liver palmitate concentrations, as in RORgt KI/KI mice. Addition of palmitc acid stimulated IL-23 production in cell experiments using RAW264.7. IL-22 produced by ILC3s inhibited the palmitate-induced apoptosis of primary hepatocytes. Conclusions: HFD stimulates IL-23 production by M1 macrophages, thus promoting ILC3 proliferation, whereas IL-22 secreted by ILC3s contributes to the upregulation of hepatic lipid metabolism and has anti-apoptosis activity.

Prevalence of Helicobacter pylori infection in long-term hemodialysis patients.

Patients on hemodialysis often have gastrointestinal complications; however, it is unclear if Helicobacter pylori infection is present in these patients. Here we determined the prevalence of H. pylori infection in 539 Japanese hemodialysis patients by measuring serum anti-H. pylori IgG antibodies. Endoscopy was performed on 299 of these patients and the results were compared to 400 patients with normal renal function who had also undergone endoscopy and sero-testing. A second cohort of 478 dialysis patients, within the original group, was checked serologically for H. pylori infection three times over a four-year observation period. The prevalence of infection in these patients was significantly lower than in those patients with normal renal function, irrespective of the clinical outcomes. The prevalence of H. pylori infection significantly decreased as the duration of dialysis increased, particularly within the first four years following initiation of dialysis. About one-third of patients on dialysis for less than four years became serologically negative for H. pylori infection within this observation period. Our study suggests that although long-term dialysis patients have low prevalence of H. pylori, they still have significant gastroduodenal diseases, such as peptic ulcers, that require endoscopic follow-up.

Involvement of IL-17A in the pathogenesis of DSS-induced colitis in mice.

To investigate the etiological implication of IL-17A in inflammatory bowel disease (IBD), dextran sodium sulfate (DSS) was administered to the mice deficient for the IL-17A gene. They showed only faint manifestations of colitis, as revealed by body weight loss, shrinkage in the colon length, serum haptoglobin concentration, and disease activity index. Although the mortality rate of WT mice reached approximately 60%, more than 90% of the IL-17A KO mice survived the DSS treatment. Histological change was also marginal in the IL-17A KO intestine, in which epithelial damage and inflammatory infiltrates were not obvious and the myeloperoxidase activity elevated only slightly. G-CSF and MCP-1 were abundantly produced in WT mouse intestine, whereas the production of these chemokines was drastically hampered in IL-17A-null intestine. The present results show that IL-17A plays a pivotal role in the pathogenesis of DSS-induced colitis, while MCP-1 and G-CSF may be crucially involved in the IL-17A-induced inflammation.

IL-17 is involved in Helicobacter pylori-induced gastric inflammatory responses in a mouse model.

BACKGROUND: Helicobacter pylori (H. pylori) is the major cause of chronic active gastritis and peptic ulcer disease. Recent studies have shown that H. pylori produces various cytokines that are related to neutrophil or mononuclear cell accumulation. Interleukin-17 (IL-17) is the founding member of an emerging family of inflammatory cytokines whose biological activities remain incompletely defined. In this study, the contributions of IL-17 to the induction of gastric inflammation and to the protection from H. pylori infection were investigated using IL-17 gene-knockout (IL-17(-/-)) mice. MATERIALS AND METHODS: IL-17(-/-)and wild-type C57BL/6 mice were challenged with H. pylori CPY2052 (2 x 10(8) CFU/mL) and the histological and microbiological evaluation were carried out at specified times. IL-17 and myeloperoxidase (MPO) protein levels in tissues were assayed in duplicate using ELISA kits. RESULTS: In wild-type mice, IL-17 was undetected at baseline; however, the protein expression of IL-17 was induced after infection with H. pylori. A severe infiltration of neutrophils appeared in the submucosa and the lamina propria in wild-type mice. In contrast, the degree of neutrophil infiltration in IL-17(-/-) mice was significantly lower than that in wild-type mice. Although wild-type mice infected with H. pylori showed drastically higher MPO activity compared with uninfected wild-type mice, any significant increase in the enzyme activity was not revealed in infected IL-17(-/-) mice. The number of H. pylori colonized in the stomach of IL-17(-/-) mice was significantly lower than that of wild-type mice from 1 to 6 months after infection. CONCLUSIONS: These results suggest that IL-17 may play an important role in the inflammatory response to the H. pylori infection and ultimately influence the outcome of the H. pylori-associated disease.

Serological evidence for hepatitis e virus infection in laboratory monkeys and pigs in animal facilities in Japan.

In laboratory animal facilities, monkeys and pigs are used for animal experiments, but the details of hepatitis E virus (HEV) infection in these animals are unknown. The risk of infection from laboratory animals to humans has become a concern; therefore, much attention should be paid to the handling of these animals during their care and use, including surgical procedures performed on infected animals. In this connection, serum samples collected from 916 monkeys and 77 pigs kept in 23 animal facilities belonging to the Japanese Association of Laboratory Animal Facilities of National University Corporations (JALAN) and the Japanese Association of Laboratory Animal Facilities of Public and Private Universities (JALAP) in Japan were examined for the purpose of detecting antibodies to HEV and HEV RNA by using ELISA and RT-PCR, respectively. One hundred and seven serum samples of 916 (11.7%) monkeys were positive for anti-HEV IgG, and 7 and 17 serum samples of 916 (0.8% and 5.3%) monkeys were positive for anti-HEV IgM and IgA, respectively. Thirty-six samples from 62 (58.1%) farm pigs were positive for anti-HEV IgG, whereas all samples tested from miniature pigs were negative (0/15, 0%). Seven samples from 62 (9.1%) farm pigs and 7 samples from 916 (0.8%) monkeys were positive for IgM antibody, but these HEV-IgM antibody positive serum samples were HEV-RNA negative by RT-PCR. The IgM antibody positive rate (9.1%) of farm pigs was much higher than that of monkeys (0.8%). These results suggest the relative levels of risk of HEV infection from these animals to animal handlers and researchers who work with them in laboratory animal facilities.

Effects of far infrared radiation by isotropic high-density carbon on the human oral mucosa.

OBJECTIVES: Wound healing of the oral mucosal epithelium through the application of far infrared radiation emitted by isotropic high-density carbon was investigated in order to clarify the preventive and therapeutic effects of isotropic high-density carbon on oral mucosal injury. MATERIALS AND METHODS: A carbon massager with an isotropic high-density carbon tip was used. Far infrared radiation was applied to the human buccal mucosal squamous cell carcinoma cell line, HO-1-N-1 using a carbon massager, and cell growth factors and heat shock protein levels were measured using real-time RT-PCR and ELISA. Far infrared radiation was applied to oral mucosal injury in SD rats over time using the carbon massager, and its effects were examined by HE staining and immunostaining. The immunostaining positive rate was measured and analyzed using image analysis software. RESULTS: Far infrared radiation induced stronger mRNA expression and higher HSP27 and HSP70 protein levels on real-time RT-PCR and ELISA than in the control group. The far infrared radiation of oral mucosal injury in rats induced strong positive reactions, and positive rates for Ki67, HSP27, and HSP70 were higher than those in the control group. CONCLUSIONS: The treatment of oral mucosal injury with far infrared radiation emitted by isotropic high-density carbon appears to have promoted heat shock protein production and induced regenerative reactions more strongly than in the control group.

[Role of IFN-gamma in nonviral infection].

IFN-gamma is induced by receptor-mediated stimulation or in response to early produced cytokines, including interleukin-12 (IL-12), IL-18, and IFN-alpha/beta, or by stimulation through T cell receptors (TCRs) or natural killer (NK) cell receptors. Cellular effects of IFN-gamma are described, including up-regulation of pathogen recognition, antigen processing and presentation, the antiviral state, inhibition of cellular proliferation and effects on apoptosis, activation of microbicidal effector functions, immunomodulation, and leukocyte trafficking. Innate production of IFN-gamma is a critical step in immunological defense against certain pathogen, such as intracellular bacteria, viruses and fungi. This review focuses on recent progress of the role of IFN-gamma in nonviral infections.

In vivo evaluation of a porous hydroxyapatite/poly-DL-lactide composite for use as a bone substitute.

We investigated the biocompatibility, osteoconductivity, and biodegradability of a porous composite of hydroxyapatite (HA) and poly-DL-lactide (PDLLA) implanted into rabbit femoral condyles and compared it with porous HA. Six weeks after implantation, the HA/PDLLA was covered with bone and contacted the bone directly. The amount of newly formed bone in the pores was similar in both materials during the examined period. The newly formed bone in the HA/PDLLA tended to increase over 26 weeks, but that in the HA did not show a significant increase after 12 weeks. By 26 weeks, remodeling of the newly formed bone in the pores was seen and bone marrow tissue was found in the pores of the HA/PDLLA. The porous HA/PDLLA was resorbed much faster than the porous HA. Porous HA/PDLLA was resorbed continuously through bone formation and remodeling. Conversely, porous HA was scarcely resorbed throughout the period. HA/PDLLA is thought to be degraded almost completely after about 1 year, and in this study, porous HA/PDLLA showed excellent osteoconductivity and faster resorption than HA. Therefore, HA/PDLLA might be a desirable material for bone substitutes.

Antitumor activity of gefitinib in malignant rhabdoid tumor cells in vitro and in vivo.

PURPOSE: Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. Current treatments have had only limited success. Epidermal growth factor receptor (EGFR) was found recently to be expressed on MRT cell lines. Gefitinib (trade name Iressa) is an oral and selective EGFR-tyrosine kinase inhibitor and has been demonstrated to be effective in inhibiting the proliferation of cancer cells in vivo as well as in clinical trials. This encouraged us to examine the antitumor effects of gefitinib on MRT cells in vitro and in vivo. EXPERIMENTAL DESIGN: The expression of EGFR in two MRT tumors and two MRT cell lines (MP-MRT-AN and KP-MRT-NS), established from these two tumor tissues, was examined by immunohistochemistry, immunofluorescence, and immunoblot. The effect of gefitinib on EGFR phosphorylation was examined by immunoblot. The effects of gefitinib on cell growth and apoptosis were examined by cell growth assay and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. The in vivo effect of gefitinib was assessed in athymic mice that had been xenografted with MRT cells. RESULTS: The expression of EGFR was detected in both tumor tissues and cell lines. Gefitinib inhibited EGFR-phosphorylation (IC(50) < 0.1 micromol/L) and in vitro cell growth (IC(50) = approximately 10-12 micromol/L), and a high concentration of gefitinib (20 micromol/L) induced apoptosis in vitro (MP-MRT-AN, 42.9% and KP-MRT-NS, 47.2%). Furthermore, gefitinib at 150 mg/kg had a cytostatic effect on established MRT xenografts (MP-MRT-AN, P = 0.039 and 0.0014; and KP-MRT-NS, P = 0.048 and 0.0086). CONCLUSIONS: Our results demonstrate that gefitinib has antitumor effects in MRT cells in vitro and in vivo and, thus, has promise as a novel and therapeutic strategy for MRT.

Quantitative analysis of chloride in brackish water: an application to the hyperchromic effect of copper(II) ion with chloride ion.

A new modified method was applied to the determination of chloride anion in brackish water by using a common copper(II) sulfate solution and a spectrophotometer. The result of the quantitative analysis demonstrated that chloride concentrations were 1.61% w/v and 1.74% w/v at ebb tide and flood tide, respectively, of the Muya River. On the other hand, chloride concentrations were 1.17% w/v at ebb tide and 1.65% w/v at flood tide of the Yoshino River. The new method proved that chloride concentration corresponded well with results from Mohr and conductive methods. Therefore, the newly developed method can be exploited as common means to measure chloride concentration in aqueous solutions. Moreover, it is also valuable for application to other types of environmental samples.

Evaluation of a dental pulp-derived cell sheet cultured on amniotic membrane substrate.

OBJECTIVE: Mesenchymal stem cells (MSC) are transplanted for periodontal tissue regeneration, and the periodontal ligament (PDL) is regenerated using a cultured cell sheet. This cultured cell sheet is prepared using PDL-derived cells, growth factors, and amniotic membrane (AM). Dental pulp (DP)-derived cells can be easily obtained from extracted wisdom teeth, proliferate rapidly, and are less susceptible to bacterial infection than PDL-derived cells. Thus, to prepare a novel cell sheet, DP-derived cells were cultured on AM as a culture substrate for immunohistochemical examination. METHODS: Wisdom teeth extracted from three adults were cut along the cement-enamel border. DP tissue was collected, minced, and primarily cultured. After three or four passage cultures, DP-derived cells were cultured on AM, followed by hematoxylin-eosin (H-E) and immunofluorescence staining. RESULTS: DP-derived cells cultured on AM formed a layered structure. Cells positive for vimentin, Ki-67, ZO-1, desmoplakin, CD29, 44, 105 or 146, STRO-1, collagen IV or VII or laminin 5 or α5 chain were localized. CONCLUSIONS: DP-derived cells proliferated on AM, while retaining the properties of DP, which allowed the cultured cell sheet to be prepared. In addition, the cultured cell sheet contained MSC, which suggests its potential application in periodontal tissue regeneration.

Panax ginseng induces production of proinflammatory cytokines via toll-like receptor.

Ginseng radix, the dried root of Panax ginseng C. A. Meyer, has been shown to enhance the ability to resist microbial infections. Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and interferon-gamma (IFN-gamma) are produced by macrophages treated with ginseng radix extract (GRE) in vitro as well as in vivo. However, the molecular mechanisms of the production are still not clear. In the present study, we demonstrated that production of TNF-alpha and IFN-gamma was induced by GRE in spleen cells and peritoneal macrophages from C3H/HeN mice but was impaired in C3H/HeJ mice carrying a defective toll-like receptor-4 (TLR-4) gene. In addition to these cytokines, the expression of IFN-beta and inducible nitric oxide synthase (iNOS) mRNAs was also increased in GRE-treated C3H/HeN spleen cells. We investigated the possibility that GRE contains a lipopolysaccharide (LPS)-like component. However, GRE induced production of TNF-alpha and IFN-gamma in the presence of polymyxin B, an LPS inhibitor. Furthermore, a Limulus amebocyte lysate assay showed that the endotoxin content of GRE was below the threshold level of 1 ng/ml LPS. These results suggest that GRE contains a non-LPS agent that enhances innate immunity through production of proinflammatory cytokines via TLR-4.

Role of IFN-gamma and tumor necrosis factor-alpha in herpes simplex virus type 1 infection.

One of the characteristics of herpes simplex virus type 1 (HSV-1) is that recurrent diseases often develop from latent infection established after acute infection. Cytokines have been proposed to play an important role in each stage of HSV-1 infection, but the exact role of cytokines remains unclear. In the present study, we investigated the role of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in acute infection and reactivation using IFN-gamma gene knockout (IFN-gamma(-/-)) mice and TNF-alpha gene knockout (TNF-alpha(-/-)) mice. We first examined the survival rate after corneal infection with HSV-1. The survival rates of wild-type C57BL/6 (B6) mice, IFN-gamma(-/-) mice, and TNF-alpha(-/-) mice were 97% (73 of 75), 57% (24 of 42), and 83% (60 of 72), respectively. These results suggest that TNF-alpha and IFN-gamma play a protective role in acute infection with HSV-1. We also examined the rate of reactivation induced by ultraviolet (UV) light in latently infected mice over 60 days postinoculation. The reactivation was confirmed by detecting viral DNA extracted from eyeballs by the polymerase chain reaction (PCR) method at day 2 after the UV light stimulation. The rates of reactivation in IFN-gamma(-/-) mice and TNF-alpha(-/-) mice were significantly higher than that in B6 mice; 16% (4 of 25) showed reactivation in B6 mice, 47% (9 of 19) in IFN-gamma(-/-) mice, and 48% (10 of 21) in TNF-alpha(-/-) mice. These results suggest that IFN-gamma and TNF-alpha play an important role in acute infection and reactivation from latency.