Necrotising otitis externa and how to reduce length of hospital stay: a complete audit cycle of 77 hospital admissions.

BACKGROUND: Necrotising otitis externa is a serious condition that requires hospital admission. Longer hospital stays are associated with increased complications. METHOD: This was a closed audit cycle in a tertiary ENT centre of patients presenting with necrotising otitis externa to the ENT department between 2015 and 2019. The aim was to audit the length of hospital stay in comparison to national figures as well as the time needed for investigations. RESULTS: The number of patients with necrotising otitis externa is increasing in England. Length of stay, however, appears to be more stable. A total of 66 admissions occurred over the study period for 48 patients in total, and mean length of stay was 12.4 days. After implementation of a new protocol, length of stay was reduced to 7.1 days. CONCLUSION: Patients with necrotising otitis externa require prompt diagnosis and management in order to shorten length of stay in hospital and avoid serious complications. Multi-disciplinary protocol development and implementation could help in reducing length of stay of necrotising otitis externa patients.

Outcomes of examination under anaesthesia of post-nasal space and grommet insertion in adults presenting with otitis media with effusion.

BACKGROUND: In adults, otitis media with effusion causes considerable morbidity and has poorly established outcomes. A small number of nasopharyngeal carcinoma patients present with isolated ear-related symptoms. The investigation of choice for these patients is a point of debate. METHODS: A retrospective cohort study was conducted using a local database of adult patients who underwent examination under anaesthesia of the post-nasal space with grommet insertion for otitis media with effusion between January 2014 and January 2016. RESULTS: Ninety-eight patients met the inclusion criteria. Follow-up duration ranged from 39 to 63 months. Complications of grommets were present in 36 out of 98 patients (36.73 per cent). The findings of examination under anaesthesia of the post-nasal space were documented as abnormal in three patients. No patient was diagnosed with nasopharyngeal carcinoma. CONCLUSION: Grommets in adults with otitis media with effusion as the sole presenting feature carry a high complication rate, especially in those with previously inserted grommets. Examination under anaesthesia of the post-nasal space offers a low yield. A magnetic resonance imaging scan of the post-nasal space may be a more sensitive alternative.

Increased weight loss with reduced viral replication in interleukin-10 knock-out mice infected with murine cytomegalovirus.

The anti-inflammatory cytokine interleukin (IL)-10 plays an important role in the regulation of host-immune responses. Here we studied the role IL-10 plays in host responses to cytomegalovirus (CMV) infection. We demonstrate that manifestations of murine CMV (MCMV) disease are more severe in IL-10 knock-out mice, despite significantly reduced levels of viral replication. Cytokine analysis of serum revealed increased levels of interferon (IFN)-gamma, monocyte chemotactic protein 1 (MCP-1) and IL-6, all of which are potent stimulators of inflammatory responses. Depletion of IFN-gamma by monoclonal antibodies in IL-10 knock-out mice failed to improve the physical condition of the mice, while increasing viral replication. In contrast, serum levels of IL-6 in the knock-out animals were unaffected by IFN-gamma depletion and remained significantly elevated early in the course of infection. These data suggest that increased weight loss observed in IL-10 knock-out mice may be attributed to the uncontrolled production of proinflammatory cytokines, including IL-6.

A novel aco-type cytochrome-c oxidase from a facultative alkalophilic Bacillus: purification, and some molecular and enzymatic features.

A novel aco-type cytochrome-c oxidase was highly purified from the facultative alkalophilic bacterium, Bacillus YN-2000, grown at pH 10. The enzyme contained 9.0 nmol heme a/mg protein. It contained 1.23 mol of protoheme, 1.06 mol of heme c, 2.0 g atoms of copper, 2.5 g atoms of iron, and 1.8 g atoms of magnesium per mol of heme a. The enzyme molecule seemed to be composed of two subunits with Mrs of 52,000 and 41,600. On the basis of these results, the enzyme seemed to contain one molecule each of heme a, protoheme, and heme c per minimal structural unit (Mr, 93,600). Only protoheme among the three kinds of hemes in the enzyme reacted with CO and CN-. Heme a did not react with CO; cytochrome a3 did not seem to be present in the enzyme. The enzyme oxidized 314 mol of horse ferrocytochrome c per heme a per sec at pH 6.5 and the catalytic activity was 50% inhibited by 7.65 microM KCN. The enzymatic activity was found to be optimal at pH 6.0.

Differential effect of Cryptococcus neoformans on the production of IL-12p40 and IL-10 by murine macrophages stimulated with lipopolysaccharide and gamma interferon.

In the present study, we examined the in vitro effect of Cryptococcus neoformans on the production of interleukin-12 (IL-12) and IL-10 by murine macrophages. At a dose of 1 x 10(5), 1 x 10(6) or 1 x 10(7) ml-1, a highly virulent strain of C. neoformans (strain YC-11) suppressed the production of IL-12p40 by a murine macrophage cell line, J774.1 stimulated with lipopolysaccharide (LPS) and interferon (IFN)-gamma, while the production of IL-10 was not inhibited, but rather slightly augmented. The suppression of IL-12p40 production did not change by neutralizing anti-IL-10 mAb. A direct contact of C. neoformans with macrophages was largely involved in this inhibitory effect, since placement of a 0.45 micron pore membrane between the organism and macrophages prevented such effect. On the other hand, the culture supernatant of YC-11 did not inhibit macrophage IL-12p40 production when used at a lower dose, which contained an equivalent amount of capsular polysaccharide to that in the supernatant of YC-11 cultured at 1 x 10(5) or 1 x 10(6) ml-1, although it showed a small suppression at higher doses. Our results suggest that C. neoformans may suppress the induction of Th1 responses by inhibiting macrophage IL-12 production predominantly through a direct contact-dependent mechanism and to a lesser extent by a certain soluble factor(s) released from this microorganism.

Reduced host resistance and Th1 response to Cryptococcus neoformans in interleukin-18 deficient mice.

Using interleukin (IL)-18 deficient (IL-18(-/-)) mice, we examined the role of IL-18 in the host resistance and Th1 response against infection with Cryptococcus neoformans. Fungal clearance in the lung was reduced in IL-18(-/-) mice, although there was no significant change in the level of dissemination to the brain. The DTH response, as determined by footpad swelling, was also diminished in IL-18(-/-) mice compared to control wild-type (WT) mice. The levels of IL-12 and interferon (IFN)-gamma in the sera were significantly lower in IL-18(-/-) mice than in WT mice. Spleen cells from infected WT mice produced a high level of IFN-gamma upon stimulation with the microbe, while only a low level of IFN-gamma production was detected in spleen cells from infected IL-18(-/-) mice. Administration of IL-18 almost completely restored the reduced response in IL-18(-/-) mice, while IL-12 showed a marginal effect. These results demonstrated the important role of IL-18 in the resistance and Th1 response of mice to C. neoformans by potentiating the production of IFN-gamma.

Involvement of endogenously synthesized interleukin (IL)-18 in the protective effects of IL-12 against pulmonary infection with Cryptococcus neoformans in mice.

We previously demonstrated that interleukin (IL)-12 protected mice against fatal pulmonary infection with a highly virulent strain of Cryptococcus neoformans, which correlated well with the production of interferon (IFN)-gamma as well as IL-18 in the primary infected site. In the present study, we examined the role of endogenously synthesized IL-18 in IL-12-induced host resistance to this pathogen. There was little or no production of IFN-gamma and IL-18 both at mRNA and protein levels in lungs of mice infected with C. neoformans, while treatment with IL-12 induced a marked production of these cytokines. Caspase-1 mRNA was expressed in infected mice even without IL-12 treatment. Administration of neutralizing anti-IFN-gamma monoclonal antibody (mAb) clearly inhibited production of IFN-gamma and IL-18 induced by IL-12, while control IgG did not show such an effect. However, administration of IFN-gamma did not induce the production of both cytokines in infected mice, although tumor necrosis factor (TNF)-alpha and IFN-gamma-inducible protein (IP)-10 were synthesized by the same treatment. Finally, neutralizing anti-IL-18 antibody (Ab) significantly interfered with the production of IFN-gamma and elimination of the microorganism from the lung induced by IL-12 treatment. Furthermore, both IFN-gamma synthesis and host protection caused by IL-12 were profoundly diminished in IL-18 gene-disrupted mice. Considered collectively, our results indicated that host protection against C. neoformans induced by IL-12 involved endogenously synthesized IL-18 and that the production of IL-18 was mediated at least in part by endogenous IFN-gamma.

Chemokine responses and accumulation of inflammatory cells in the lungs of mice infected with highly virulent Cryptococcus neoformans: effects of interleukin-12.

We examined the mechanisms involved in the development of lung lesions after infection with Cryptococcus neoformans by comparing the histopathological findings and chemokine responses in the lungs of mice infected with C. neoformans and assessed the effect of interleukin (IL) 12 which protects mice from lethal infection. In mice infected intratracheally with a highly virulent strain of C. neoformans, the yeast cells multiplied quickly in the alveolar spaces but only a poor cellular inflammatory response was observed throughout the course of infection. Very little or no production of chemokines, including MCP-1, RANTES, MIP-1alpha, MIP-1beta and IP-10, was detected at the mRNA level using RT-PCR as well as at a protein level in MCP-1, RANTES and MIP-1alpha. In contrast, intraperitoneal administration of IL-12 induced the synthesis of these chemokines and a marked cellular inflammatory response involving histiocytes and lymphocytes in infected mice. Our findings were confirmed by flow cytometry of intraparenchymal leukocytes obtained from lung homogenates which showed IL-12-induced accumulation of inflammatory cells consisting mostly of macrophages and CD4+ alphabeta T cells. On the other hand, C-X-C chemokines including MIP-2 and KC, which attract neutrophils, were produced in infected and PBS-treated mice but treatment with IL-12 showed a marginal effect on their level, and neutrophil accumulation was similar in PBS- and IL-12-treated mice infected with C. neoforman. Our results demonstrate a close correlation between chemokine levels and development of lung lesions, and suggest that the induction of chemokine synthesis may be one of the mechanisms of IL-12-induced protection against cryptococcal infection.

Sex differential in life expectancy in Japan and Scotland: age and causes of death.

The contribution of age groups and causes of death to the sex difference in life expectancy (SDLE) at birth in Japan and Scotland was estimated for the period 1965-1990. The purpose was to determine the particular age groups and causes of death responsible for the opposite trend of SDLE in the two countries. SDLE has been widening and narrowing in Japan and Scotland, respectively. The availability of complete and reliable data for these two developed countries facilitated the study. A method of decomposing the total SDLE into age and cause of death components was employed. About 40-60% contribution to SDLE was observed for ages after 65 years. Marked increase in the contribution of the 75+ age group and marked decrease in the contribution of ages 45-64 for Japan and Scotland, respectively, had a major effect on the widening and narrowing of SDLE in the two countries, respectively. The contribution of diseases of the circulatory system was the maximum until 1980 in Japan (< or = 1.8 years or 33.6%; cerebrovascular disease alone < or = 23.4%) and until 1990 in Scotland (< or = 3.1 years or 47.0%; ischemic heart disease alone < or = 42.0%). In Japan, the contribution of malignancy had a marked increased from 0.7 year (12.3%) to 2.0 years (32.6%), particularly for the trachea, bronchus and lung, while there was only a small increase in Scotland from 1.0 year (16.6%) to 1.2 years (19.8%) with an increase in the negative contribution of female breast malignancy. In Japan, the contribution of diseases of the respiratory system increased considerably from 0.5 year (8.5%) to 1.1 years (18.1%) while it decreased in Scotland from 1.0 year (16.5%) to 0.6 year (10.7%). About 60-75% of SDLE is due to the above three groups of causes of death. Malignancy and diseases of the respiratory system had a persistently increased contribution in Japan with resultant widening of SDLE by 0.9 year. Diseases of the circulatory system have always had a high contribution. On the contrary, in Scotland the contribution of diseases of the circulatory system and malignancy was practically unchanged and diseases of the respiratory system had a decrease with a consequent narrowing of SDLE by 0.4 year. Further epidemiological study is necessary to detect and analyze in detail the internal gradients (environmental and genetic-biological) of major contributor diseases to SDLE in Japan and Scotland.

Demographic processes of urbanisation in Delhi.

"Metropolitanisation has been observed by experts as one of the main characteristics of Indian urbanisation. Rural to urban migration has been a major player in urban growth of metropolitan cities, particularly Delhi. Not only Delhi but its hinterland [are] urbanising fast as compared to the hinterlands of other major cities of India. There are several processes of urbanization viz. historical, physical, demographic and economic. In this paper the role of demographic processes in urban expansion of Delhi [is examined]."

Pressure response in deep-sea piezophilic bacteria.

Several piezophilic bacteria have been isolated from deep-sea environments under high hydrostatic pressure. Taxonomic studies of the isolates showed that the piezophilic bacteria are not widely distributed in terms of taxonomic positions, and all were assigned to particular branches of the Proteobacteria gamma-subgroup. A pressure-regulated operon from piezophilic bacteria of the genus Shewanella, S. benthica and S. violacea, was cloned and sequenced, and downstream of this operon another pressure regulated operon, cydD-C, was found. The cydD gene was found to be essential for the bacterial growth under high-pressure conditions, and the product of this gene was found to play a role in their respiratory system. Results obtained later indicated that the respiratory system in piezophilic bacteria may be important for survival in a high-pressure environment, and more studies focusing on other components of the respiratory chain have been conducted. These studies suggested that piezophilic bacteria are capable of changing their respiratory system in response to pressure conditions, and a proposed respiratory chain model has been suggested in this regard.

Neonatal T cells in an adult lung environment are competent to resolve Pneumocystis carinii pneumonia.

Initiation of the pulmonary inflammatory response to Pneumocystis carinii is delayed by 3 wk in mice infected as neonates compared with adults. There was no difference in the proliferative response of draining lymph node T cells from mice infected as neonates compared with adults when stimulated in vitro with either Con A or anti-CD3 mAB: However, TNF-alpha and IFN-gamma mRNA expression in the lungs of P. carinii-infected neonates was significantly lower than in adults indicating a lack of appropriate activation signaling in the local environment. This may have been due to active suppression because TGF-beta mRNA expression was significantly elevated in neonatal lungs compared with adults. To determine whether T cells from 10-day-old mice would effect resolution of P. carinii if harbored in an adult lung environment, cells were adoptively transferred to SCID mice with established P. carinii infections. There was no difference in the kinetics of T cell migration into the lungs or of clearance of P. carinii organisms when SCID mice were reconstituted with splenocytes from young mice as compared with adult mice. Furthermore, splenocytes from young mice stimulated both TNF-alpha and IFN-gamma mRNA expression to levels that were similar to that in the lungs of SCID mice reconstituted with adult cells. These data indicate that neonatal lymphocytes are competent to resolve P. carinii infection when harbored in an adult lung environment, suggesting that the neonatal lung environment, and not the T cells, is ineffective at responding to P. carinii infection.

Delayed inflammatory response to Pneumocystis carinii infection in neonatal mice is due to an inadequate lung environment.

Challenge of neonatal mice with an intranasal inoculation of Pneumocystis carinii results in a subclinical infection that takes 6 wk to resolve, whereas adult mice resolve a comparable challenge within 3 wk. This delayed clearance is due to a delayed inflammatory response in neonatal mice; however, the reason for this delay has been unknown. To determine whether the neonatal lung environment is sufficient to attract immunocompetent lymphocytes into the lungs, an adoptive transfer strategy was employed in which splenocytes from adult BALB/c mice were transferred into P. carinii-infected neonatal or adult SCID mice. All adults, but no pups, resolved their infections by day 37 postreconstitution. Despite reconstitution with adult splenocytes, pups had a negligible lung inflammatory response until day 24, whereas adult mice had activated CD4(+) and CD8(+) cells in the lung by day 13. The delay in neonates corresponded to delayed kinetics of expression of lung cytokines TNF-alpha and IFN-gamma mRNA and chemokines lymphotactin, RANTES, and macrophage inflammatory protein-1ss mRNA. Phagocytic cells from neonatal mice were significantly less efficient than adult cells at migrating to the draining lymph nodes after phagocytosing fluorescent beads. There were fewer dendritic cells and Ia(+) myeloid cells in the lungs of P. carinii-infected neonatal mice compared with adults. These data indicate that the lung environment of neonatal mice is insufficient for migration of T cells, due at least in part to inefficient phagocytosis and migration of APCs to the lymph nodes as well as delayed chemokine and TNF-alpha mRNA expression.

Cryptococcus neoformans inhibits nitric oxide production by murine peritoneal macrophages stimulated with interferon-gamma and lipopolysaccharide.

We examined the effect of Cryptococcus neoformans on nitric oxide (NO) production by activated cultured macrophages. C. neoformans suppressed NO production by murine peritoneal macrophages stimulated with bacterial lipopolysaccharide (LPS) and interferon (IFN)-gamma, while it did not influence the production of IL-1 beta. This effect was observed when 1 x 10(6) or 10(7) of C. neoformans was added to macrophage cultures. A direct contact of C. neoformans with macrophages was essential for this inhibitory effect, since placement of a 0.45-micron-pore membrane between the organism and macrophages prevented such effect. In addition, C. neoformans killed by heat or paraformaldehyde did not show this inhibitory activity. Capsular polysaccharide did not mediate the inhibitory effect, since two nonencapsulated mutant strains of C. neoformans showed an inhibitory activity similar to that of encapsulated wild strains, and culture supernatants of C. neoformans, rich in polysaccharide antigens, did not inhibit macrophage NO production compared with control culture medium. The inhibitory effect was also not mediated by interleukin (IL)-10 and transforming growth factor (TGF)-beta since neutralizing specific antibodies to these cytokines did not influence C. neoformans-induced reductions in macrophage NO production. Our results suggest that C. neoformans may cause a direct suppression of NO-mediated fungicidal activity of macrophages, and the effect is independent of the capsular polysaccharide and production of IL-10 and TGF-beta.

Candida albicans suppresses nitric oxide (NO) production by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages.

We examined the in vitro effect of Candida albicans on NO production by macrophages. Candida albicans suppressed not only NO production but also expression of inducible NO synthase (iNOS) mRNA by murine IFN-gamma and bacterial LPS-stimulated peritoneal macrophages. The suppression was not associated with inhibition but rather stimulation of IL-1 beta production. This effect was observed when more than 1 x 10(3)/ml of Candida albicans were added to macrophage cultures (1 x 10(6) cells/ml) and reached a maximal level at 1 x 10(6)/ml. The NO inhibitory effect of Candida albicans was mediated predominantly by as yet unidentified soluble factor(s) and to a lesser extent by direct contact. In addition, heat- or paraformaldehyde-killed Candida albicans did not show this inhibitory activity. Culture supernatant of Candida albicans also inhibited NO production by activated macrophages in a dose-dependent manner, and increased IL-1 beta production. Finally, the inhibitory effect was not mediated by IL-10 and transforming growth factor-beta (TGF-beta), since neutralizing antibodies to these cytokines did not influence Candida albicans-induced reduction in macrophage NO production. Our results suggest that Candida albicans may evade host defence mechanism(s) through a soluble factor-mediated suppression of NO production by stimulated macrophages, and that the effect is independent of production of immunosuppressive cytokines such as IL-10 and TGF-beta.

Purification of a ccb-type quinol oxidase specifically induced in a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F.

We investigated for the first time the respiratory chain system of a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F. A membrane-bound ccb-type quinol oxidase, from cells grown at 60 MPa pressure, was purified to an electrophoretically homogeneous state. The purified enzyme complex consisted of four kinds of subunits with molecular masses of 98, 66, 18.5, and 15kDa, and it contained 0.96 mol of protoheme and 1.95mol of covalently bound heme c per mol of enzyme. Only protoheme in the enzyme reacted with CO and CN-, and the catalytic activity of the enzyme was 50% inhibited by 4 microM CN-. The isoelectric point of the native enzyme complex was determined to be 5.0. This enzyme was specifically induced only under conditions of elevated hydrostatic pressure, and high levels were expressed in cells grown at 60 MPa. The membranes isolated from cells grown at atmospheric pressure (0.1 MPa) exhibited high levels of both cytochrome c oxidase and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPDH2)-oxidase activity. These results suggest the presence of two kinds of respiratory chains regulated in response to pressure in the deep-sea bacterium DB-172F.

Succinate:quinone oxidoreductase (complex II) containing a single heme b in facultative alkaliphilic Bacillus sp. strain YN-2000.

Succinate:quinone oxidoreductase (EC 1.3.5.1) was first purified from the facultative alkaliphilic Bacillus sp. strain YN-2000 in the presence of Triton X-100. The isolated enzyme showed high succinate-ubiquinone oxidoreductase activity at pH 8.5. The Km for ubiquinone 1 and the Vmax of the enzyme were determined to be about 5 microM and 48 micromol of ubiquinone 1 per min per mg, respectively. The catalytic activity of the enzyme was 50% inhibited by 9 microM 2-thenoyltrifluoroacetone or 0.8 microM 2-n-heptyl-4-hydroxyquinoline- N-oxide. The enzyme consisted of three kinds of subunits with molecular masses of 66, 26, and 15 kDa, respectively, and contained 1.28 mol of covalently bound flavin adenine dinucleotide, 0.9 mol of heme b, 1.35 mol of menaquinone, 8.3 mol of nonheme iron, and 7.5 mol of inorganic sulfide per mol of enzyme. The enzyme showed symmetrical alpha absorption peaks at 556.5 and 554 nm in the reduced state at room temperature and 77 K, respectively. The potentiometric analysis of the enzyme yielded an Em,7 of heme b of about -64 mV (n = 1). Furthermore, the content of the enzyme was increased up to fivefold when the bacterium was grown at pH 10 compared with pH 7. These results indicate that the succinate:quinone oxidoreductase with a single heme b is involved in the respiratory chain of the alkaliphile at a very alkaline pH.

Structure of a Micrococcus lysodeikticus cell wall fragment containing phosphorylated sugars.

A polysaccharide-peptidoglycan complex containing different phosphorylated sugars from Micrococcus lysodeikticus cell wall has been isolated and purified. The peptidoglycan contained muramic acid 6-phosphate and N-acetylglucosamine 6-phosphate as phosphorylated sugars in addition to other sugar residues. Mild acid hydrolysis of the peptidoglycan and subsequent reduction of the released polysaccharide showed therein the presence of glucose and N-acetyl-glucosamine in the linkage of the external polysaccharide residues to the peptidoglycan through phosphodiester linkage. These data suggest the presence of polysaccharide chains linked to a peptidoglycan core through two phosphorylated sugars via two different terminal carbohydrate residues of the external polysaccharide chains in a same polymer.

Contribution of tumour necrosis factor-alpha (TNF-alpha) in host defence mechanism against Cryptococcus neoformans.

We investigated the role of TNF-alpha in the host defence mechanism against infection with a virulent strain of Cryptococcus neoformans. Administration of exogenous recombinant human TNF-alpha significantly prolonged the survival time of mice infected by intratracheal instillation of the organism. Surprisingly, neutralizing MoAb to murine TNF-alpha did not shorten their survival time, a finding inconsistent with previous results. To investigate the cause of this inconsistency, we examined the production of TNF-alpha in the lungs of infected mice. During the course of cryptococcosis, there was little or no generation of TNF-alpha mRNA in the lung. This might be partly due to a direct inhibitory action of the fungal microorganism of TNF-alpha production by macrophages. In vitro production of TNF-alpha by murine interferon-gamma (IFN-gamma)- and lipopolysaccharide (LPS)-stimulated macrophages was strongly inhibited by co-culturing with the whole yeast cells. In contrast, administration of recombinant murine IL-12 markedly induced TNF-alpha production and the neutralizing anti-TNF-alpha MoAb strongly blocked IL-12-induced protection of mice against cryptococcal infection. These results indicate that endogenously synthesized TNF-alpha has the potential to contribute to the elimination of C. neoformans and partly mediates the protective effect of IL-12.

Interleukin-12 (IL-12) and IL-18 synergistically induce the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through production of gamma interferon by natural killer cells.

We examined the ability of interleukin-12 (IL-12) and IL-18 to induce the production of gamma interferon (IFN-gamma) and nitric oxide (NO) by murine peritoneal exudate cells (PEC) and to stimulate the growth-inhibitory activity of these cells against Cryptococcus neoformans. PEC produced IFN-gamma and NO when stimulated with a combination of IL-12 and IL-18 but little or no IFN-gamma or NO when either cytokine was used alone. PEC anticryptococcal activity was mediated by IFN-gamma and NO production, since it was completely inhibited by a neutralizing anti-IFN-gamma monoclonal antibody (MAb) and N(G)-monomethyl-L-arginine, a competitive inhibitor of NO synthesis, respectively. To identify the IFN-gamma-producing cells among PEC stimulated with IL-12 and IL-18, we depleted NK cells, gammadelta T cells, or CD4+ T cells by treating PEC with specific Abs and complement. NK cell depletion strongly suppressed IFN-gamma production and almost completely inhibited NO production and anticryptococcal activity, while depletion of other cells had no such influence. Alternatively, purified NK cells by two cycles of glass adherence and magnetic separation with anti-CD3, -CD4, -CD8, and -B220 MAbs produced a greater amount of IFN-gamma by stimulation with IL-12 and IL-18 than unseparated non-glass-adherent PEC. Our results demonstrated that IL-12 and IL-18 synergistically induced NO-dependent anticryptococcal activity of PEC by stimulating NK cells to produce IFN-gamma.