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1.
J Laryngol Otol ; 136(7): 668-673, 2022 Jul.
Article in English | MEDLINE | ID: mdl-34674778

ABSTRACT

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.


Subject(s)
Otitis Externa , England/epidemiology , Hospitals , Humans , Length of Stay , Otitis Externa/complications , Retrospective Studies
2.
J Laryngol Otol ; 135(7): 620-624, 2021 Jul.
Article in English | MEDLINE | ID: mdl-34078484

ABSTRACT

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.


Subject(s)
Middle Ear Ventilation , Nasopharyngeal Carcinoma/diagnosis , Nasopharyngeal Neoplasms/diagnosis , Otitis Media with Effusion/surgery , Postoperative Complications/epidemiology , Adolescent , Adult , Aged , Aged, 80 and over , Anesthesia, General , Cohort Studies , Female , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Nasopharynx , Physical Examination , Retrospective Studies , Young Adult
3.
Clin Exp Immunol ; 151(1): 155-64, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18005264

ABSTRACT

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.


Subject(s)
Herpesviridae Infections/immunology , Interleukin-10/physiology , Muromegalovirus/physiology , Weight Loss , Animals , CD4-Positive T-Lymphocytes/immunology , Chemokine CCL2/analysis , Female , Flow Cytometry , Herpesviridae Infections/virology , Interferon-gamma/analysis , Interleukin-10/genetics , Interleukin-6/analysis , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/analysis , Up-Regulation , Virus Replication
4.
J Biol Chem ; 276(49): 46422-8, 2001 Dec 07.
Article in English | MEDLINE | ID: mdl-11584006

ABSTRACT

The strong biotin-streptavidin interaction limits the application of streptavidin as a reversible affinity matrix for purification of biotinylated biomolecules. To address this concern, a series of single, double, and triple streptavidin muteins with different affinities to biotin were designed. The strategy involves mutating one to three strategically positioned residues (Ser-45, Thr-90, and Asp-128) that interact with biotin and other framework structure-maintaining residues of streptavidin. The muteins were produced in soluble forms via secretion from Bacillus subtilis. The impact of individual residues on the overall structure of streptavidin is reflected by the formation of monomeric streptavidin to different extents. Of the three targeted residues, Asp-128 has the most dramatic effect (Asp-128 > Thr-90 > Ser-45). Conversion of all three targeted residues to alanine results in a soluble biotin binding mutein that exists 100% in the monomeric state. Both wild-type and mutated (monomeric and tetrameric) streptavidin proteins were purified, and their kinetic parameters (on- and off-rates) were determined using a BIAcore biosensor with biotin-conjugated bovine serum albumin immobilized to the sensor chip. This series of muteins shows a wide spectrum of affinity toward biotin (K(d) from 10(-6) to 10(-11) m). Some of them have the potential to serve as reversible biotin binding agents.


Subject(s)
Biotin/metabolism , Streptavidin/metabolism , Amino Acid Sequence , Bacillus subtilis/metabolism , Base Sequence , Biopolymers , Biosensing Techniques , DNA , Models, Molecular , Molecular Sequence Data , Mutagenesis , Protein Binding , Solubility , Streptavidin/chemistry , Streptavidin/genetics , Streptavidin/isolation & purification
5.
Biosci Biotechnol Biochem ; 65(3): 690-3, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11330692

ABSTRACT

We have isolated the genes for quinol oxidase from a deep-sea piezophilic bacterium, Shewanella violacea. Analysis of the deduced amino acid sequences of the cyo subunits showed that this oxidase has high similarity to Escherichia coli bo-type quinol oxidase. Northern blot analysis showed that these genes are expressed at a high level when the bacterium is grown at elevated pressure. Upstream in the cyo-operon, a sigma54-binding motif and an octamer sequence unit were found, suggesting that these elements may play a role in regulation of expression of the cyo-operon in response to changes in pressure.


Subject(s)
Operon , Oxidoreductases/genetics , Shewanella/enzymology , Base Sequence , DNA, Bacterial , Molecular Sequence Data , Shewanella/genetics
6.
J Immunol ; 166(9): 5704-11, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11313412

ABSTRACT

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.


Subject(s)
Aging/immunology , Animals, Newborn/immunology , Lung/immunology , Lung/microbiology , Pneumonia, Pneumocystis/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/microbiology , Animals , Animals, Newborn/growth & development , Antibodies, Monoclonal/pharmacology , Bronchi , CD3 Complex/immunology , Cell Movement/immunology , Cells, Cultured , Concanavalin A/pharmacology , Cytokines/biosynthesis , Cytokines/genetics , Female , Lung/cytology , Lung/metabolism , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymphocyte Transfusion , Mice , Mice, Inbred BALB C , Mice, SCID , Pneumocystis/growth & development , Pneumocystis/immunology , Pneumonia, Pneumocystis/pathology , Pneumonia, Pneumocystis/prevention & control , RNA, Messenger/biosynthesis , Spleen/cytology , Spleen/transplantation , T-Lymphocyte Subsets/metabolism , Trachea
7.
J Immunol ; 165(11): 6480-6, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-11086088

ABSTRACT

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.


Subject(s)
Animals, Newborn/immunology , Animals, Newborn/microbiology , Lung/immunology , Lung/pathology , Pneumonia, Pneumocystis/immunology , Pneumonia, Pneumocystis/pathology , Adoptive Transfer , Age Factors , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/pathology , Cell Movement/immunology , Chemokines/biosynthesis , Chemokines/genetics , Cytokines/biosynthesis , Cytokines/genetics , Female , Inflammation/immunology , Inflammation/microbiology , Lung/metabolism , Lung/microbiology , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymphocyte Subsets/immunology , Lymphocyte Subsets/pathology , Mice , Mice, Inbred BALB C , Mice, SCID , Pneumocystis/immunology , Pneumonia, Pneumocystis/etiology , RNA, Messenger/biosynthesis , Spleen/cytology , Spleen/transplantation
8.
J Immunol ; 165(2): 941-7, 2000 Jul 15.
Article in English | MEDLINE | ID: mdl-10878369

ABSTRACT

The aim of this study was to examine the contribution of IL-18 in host defense against infection caused by Cryptococcus neoformans in mice with defective IL-12 production. Experiments were conducted in mice with a targeted disruption of the gene for IL-12p40 subunit (IL-12p40-/- mice). In these mice, host resistance was impaired, as shown by increased number of organisms in both lungs and brains, compared with control mice. Serum IFN-gamma was still detected in these mice at a considerable level (20-30% of that in control mice). The host resistance was moderately impaired in IL-12p40-/- mice compared with IFN-gamma-/- mice. Neutralizing anti-IFN-gamma mAb further increased the lung burdens of organisms. In addition, treatment with neutralizing anti-IL-18 Ab almost completely abrogated the production of IFN-gamma and also impaired the host resistance. Host resistance in IL-12p40-/- IL-18-/- mice was more profoundly impaired than in IL-12p40-/- mice. Administration of IL-12 as well as IL-18 increased the serum levels of IFN-gamma and significantly restored the reduced host resistance. Spleen cells obtained from infected IL-12p40-/- mice did not produce any IFN-gamma upon restimulation with the same organisms, while those from infected and IL-12-treated mice produced IFN-gamma. In contrast, IL-18 did not show such effect. Finally, depletion of NK cells by anti-asialo GM1 Ab mostly abrogated the residual production of IFN-gamma in IL-12p40-/- mice. Our results indicate that IL-18 contributes to host resistance to cryptococcal infection through the induction of IFN-gamma production by NK cells, but not through the development of Th1 cells, under the condition in which IL-12 synthesis is deficient.


Subject(s)
Cryptococcosis/immunology , Interferon-gamma/biosynthesis , Interleukin-12/biosynthesis , Interleukin-12/deficiency , Interleukin-18/physiology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/physiology , Animals , Cryptococcosis/genetics , Cryptococcosis/prevention & control , Cryptococcus neoformans/immunology , Humans , Immunity, Innate/genetics , Injections, Intraperitoneal , Interferon-gamma/physiology , Interleukin-12/genetics , Interleukin-18/administration & dosage , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Th1 Cells/immunology
9.
FEMS Microbiol Lett ; 186(1): 121-6, 2000 May 01.
Article in English | MEDLINE | ID: mdl-10779723

ABSTRACT

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.


Subject(s)
Cryptococcosis/immunology , Cryptococcus neoformans/immunology , Interleukin-18/immunology , Th1 Cells/immunology , Animals , Brain/immunology , Brain/microbiology , Colony Count, Microbial , Crosses, Genetic , Cryptococcosis/microbiology , Cryptococcus neoformans/isolation & purification , Cytokines/blood , Hypersensitivity, Delayed/physiopathology , Interferon-gamma/blood , Interleukin-12/blood , Interleukin-18/blood , Lung/immunology , Lung/microbiology , Mice , Mice, Inbred C57BL , Spleen/cytology , Spleen/immunology
10.
FEMS Immunol Med Microbiol ; 27(3): 191-200, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10683463

ABSTRACT

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.


Subject(s)
Cryptococcosis/immunology , Interleukin-12/immunology , Interleukin-18/biosynthesis , Interleukin-18/immunology , Lung Diseases, Fungal/immunology , Animals , Bronchoalveolar Lavage Fluid/immunology , Caspase 1/biosynthesis , Caspase 1/genetics , Cryptococcosis/microbiology , Cryptococcus neoformans/immunology , Cryptococcus neoformans/isolation & purification , Female , Humans , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-12/pharmacology , Interleukin-18/genetics , Lung/microbiology , Lung Diseases, Fungal/microbiology , Mice , Mice, Inbred C57BL , RNA, Fungal/analysis , Rabbits , Reverse Transcriptase Polymerase Chain Reaction
11.
Microbiol Immunol ; 44(12): 1033-41, 2000.
Article in English | MEDLINE | ID: mdl-11220677

ABSTRACT

In the present study, we examined the effect of soluble CD4 (sCD4) on host resistance and delayed-type hypersensitivity (DTH) response to Cryptococcus neoformans using a novel mutant mouse that exhibits a defect in the expression of membrane-bound CD4 but secretes high levels of sCD4 in the serum. In these mice, host resistance to this pathogen was impaired as indicated by an increased number of live pathogens in the lung. To elucidate the mechanism of immunodeficiency, three different sets of experiments were conducted. First, administration of anti-CD4 mAb restored the attenuated host defense. Second, in CD4 gene-disrupted (CD4KO) mice, host resistance was not attenuated compared to control mice. Third, implantation of sCD4 gene-transfected myeloma cells rendered the CD4KO mice susceptible to this infection, while similar treatment with mock-transfected cells did not show such an effect. These results indicated that immunodeficiency in the mutant mice was attributed to the circulating sCD4 rather than to the lack of CD4+ T cells. In addition, DTH response to C. neoformans evaluated by footpad swelling was reduced in the mutant mice compared to that in the control, and the reduced response was restored by the administration of anti-CD4 mAb. Finally, serum levels of IFN-gamma, IL-12 and IL-18 in the mutant mice were significantly reduced, while there was no difference in Th2 cytokines, such as IL-4 and IL-10. Considered collectively, our results demonstrated that sCD4 could directly prevent host resistance and DTH response to C. neoformans through interference with the production of Th1-type cytokines.


Subject(s)
CD4 Antigens/immunology , Cryptococcus neoformans/immunology , Hypersensitivity, Delayed/immunology , Animals , CD4 Antigens/genetics , Cryptococcosis/blood , Cryptococcosis/immunology , Female , Humans , Immunity, Innate/immunology , Interferon-gamma/blood , Interleukin-12/blood , Interleukin-18/blood , Mice , Mice, Inbred C57BL , Mice, Knockout , Solubility
12.
Microbiol Immunol ; 44(12): 1043-50, 2000.
Article in English | MEDLINE | ID: mdl-11220678

ABSTRACT

In the present study, we examined whether natural killer (NK) cells have direct fungicidal activity against Cryptococcus neoformans. Splenic NK cells were obtained from SCID mice and stimulated with a combination of interleukin (IL)-12 and IL-18 in flat culture plates or round tubes. They were then or at the same time cultured with the yeast cells and the number of viable yeast cells was examined. We could not detect direct fungicidal activity by NK cells under any culture condition, although they produced a large amount of IFN-gamma and exerted marked cytotoxic activity against YAC-1 cells. On the other hand, NK cells significantly potentiated the nitric oxide-mediated cryptococcocidal activity of thioglycolate-elicited peritoneal macrophages obtained from SCID mice upon stimulation with IL-12 and IL-18. The culture supernatants of NK cells stimulated with IL-12 and IL-18 provided similar results when used in place of NK cells. The induction of macrophage anticryptococcal activity by NK cells and NK cell culture supernatants were both mediated by IFN-gamma because the specific mAb almost completely abrogated such effect. Considered collectively, our results suggested that NK cells may play a regulatory role in potentiating macrophage-mediated fungicidal mechanisms in host resistance to infection with C. neoformans rather than exerting a direct killing activity against the fungal pathogen.


Subject(s)
Cryptococcus neoformans/immunology , Interleukin-12/immunology , Interleukin-8/immunology , Killer Cells, Natural/immunology , Macrophage Activation/immunology , Macrophages/immunology , Animals , Cells, Cultured , Female , Interferon-gamma/immunology , Interleukin-12/pharmacology , Interleukin-8/pharmacology , Killer Cells, Natural/drug effects , Killer Cells, Natural/microbiology , Mice , Mice, SCID , Spleen/cytology
13.
FEMS Immunol Med Microbiol ; 25(4): 391-402, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10497871

ABSTRACT

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.


Subject(s)
Cryptococcosis/immunology , Cryptococcosis/pathology , Interleukin-12/pharmacology , Lung/pathology , Animals , Chemokine CCL2/genetics , Chemokine CCL2/immunology , Chemokine CCL3 , Chemokine CCL4 , Chemokine CCL5/genetics , Chemokine CCL5/immunology , Chemokine CXCL10 , Chemokines, CXC/genetics , Chemokines, CXC/immunology , Cryptococcus neoformans/immunology , Cryptococcus neoformans/pathogenicity , Female , Flow Cytometry , Leukocytes/immunology , Lung/cytology , Lung/immunology , Macrophage Inflammatory Proteins/genetics , Macrophage Inflammatory Proteins/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Virulence
14.
Clin Exp Immunol ; 117(1): 113-22, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10403924

ABSTRACT

We have recently demonstrated that IL-12 induced cellular inflammatory responses consisting mainly of accumulation of mononuclear leucocytes in the lungs of mice infected with Cryptococcus neoformans and protected mice against fulminant infection. We examined the involvement of endogenously synthesized IFN-gamma in such a response by investigating the effects of a neutralizing monoclonal antibody against this cytokine. The latter treatment completely abrogated the positive effects of IL-12 on survival of infected mice and prevented IL-12-induced elimination of microbials from the lungs. Histopathological examination showed that accumulation of mononuclear leucocytes in the infected lungs caused by IL-12 was clearly inhibited by anti-IFN-gamma MoAb. We also examined the local production of mononuclear cell-attracting chemokines such as monocyte chemotactic protein-1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta and IFN-gamma-inducible protein 10 (IP-10) in the lungs using a reverse transcriptase-polymerase chain reaction (RT-PCR) method. We found that these chemokines were not synthesized in the infected lungs, while IL-12 treatment markedly induced their production. Interestingly, neutralizing anti-IFN-gamma MoAb strongly suppressed IL-12-induced production of these chemokines. Similar results were obtained with MCP-1 and MIP-1alpha when their synthesis was measured at the protein level using respective ELISA kits. Our results indicate that IFN-gamma plays a central role in the protective effects of IL-12 by inducing mononuclear leucocyte-attracting chemokines and cellular inflammatory responses.


Subject(s)
Chemotactic Factors/biosynthesis , Cryptococcosis/immunology , Interferon-gamma/physiology , Interleukin-12/pharmacology , Lung Diseases, Fungal/immunology , Lung/drug effects , Animals , Antibodies, Monoclonal/immunology , Chemokine CCL2/metabolism , Chemokine CCL3 , Chemokine CCL4 , Chemokine CCL5/metabolism , Chemokine CXCL10 , Chemokines, CXC/metabolism , Chemotaxis, Leukocyte , Cryptococcosis/prevention & control , Cryptococcus neoformans/isolation & purification , Female , Immunity, Cellular , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/immunology , Interleukin-12/antagonists & inhibitors , Interleukin-12/therapeutic use , Lung/immunology , Lung/metabolism , Lung/parasitology , Lung Diseases, Fungal/prevention & control , Macrophage Inflammatory Proteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Reverse Transcriptase Polymerase Chain Reaction
15.
FEMS Microbiol Lett ; 175(1): 87-94, 1999 Jun 01.
Article in English | MEDLINE | ID: mdl-10361712

ABSTRACT

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.


Subject(s)
Cryptococcus neoformans/immunology , Interleukin-10/biosynthesis , Interleukin-12/biosynthesis , Macrophages/immunology , Macrophages/microbiology , Animals , Cell Line , Cryptococcosis/microbiology , Cryptococcus neoformans/growth & development , Cryptococcus neoformans/isolation & purification , Humans , Interferon-gamma/pharmacology , Lipopolysaccharides/pharmacology , Lung Diseases, Fungal/microbiology , Macrophage Activation , Macrophages/drug effects , Mice
16.
Cell Immunol ; 193(1): 9-16, 1999 Apr 10.
Article in English | MEDLINE | ID: mdl-10202108

ABSTRACT

We have recently demonstrated that two IFN-gamma-inducing cytokines, interleukin (IL)-12 and IL-18, synergistically induced the fungicidal activity of mouse peritoneal exudate cells (PEC) against Cryptococcus neoformans through NK cell production of interferon (IFN)-gamma and nitric oxide (NO) synthesis. In the present study, we further dissected these effects by examining the involvement of tumor necrosis factor (TNF)-alpha in the induction of IL-12/IL-18-stimulated PEC fungicidal activity. The addition of neutralizing anti-TNF-alpha mAb significantly suppressed IL-12/IL-18-stimulated PEC anticryptococcal activity. This effect was ascribed to the inhibition of macrophage NO synthesis, but not of IFN-gamma production by NK cells, because the same treatment inhibited the former response, but not the latter one. On the other hand, combined treatment with IL-12 and IL-18 synergistically induced the production of TNF-alpha by PEC and this effect was almost completely abrogated by neutralizing anti-IFN-gamma mAb. The cell type producing TNF-alpha among PEC was mostly macrophage. TNF-alpha significantly promoted macrophage NO production and anticryptococcal activity induced by IFN-gamma, and furthermore anti-TNF-alpha mAb partially inhibited these responses. Considered together, our results indicated that TNF-alpha contributed to the potentiation of IL-12/IL-18-induced PEC fungicidal activity against C. neoformans through enhancement of IFN-gamma-induced production of NO by macrophages, but not through increased production of IFN-gamma by NK cells.


Subject(s)
Cryptococcus neoformans/immunology , Interleukin-12/pharmacology , Interleukin-18/pharmacology , Lymphocytes/immunology , Macrophages/immunology , Peritoneum/cytology , Tumor Necrosis Factor-alpha/physiology , Animals , Female , Interferon-gamma/biosynthesis , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Nitric Oxide/biosynthesis
17.
Clin Exp Immunol ; 115(3): 491-7, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10193423

ABSTRACT

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.


Subject(s)
Candida albicans/immunology , Interferon-gamma/pharmacology , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Nitric Oxide/biosynthesis , Animals , Antibodies, Monoclonal/pharmacology , Candida albicans/pathogenicity , Female , In Vitro Techniques , Interleukin-1/biosynthesis , Interleukin-10/antagonists & inhibitors , Interleukin-10/physiology , Mice , Mice, Inbred BALB C , Neutralization Tests , Recombinant Proteins , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/physiology
18.
Eur J Immunol ; 29(2): 643-9, 1999 02.
Article in English | MEDLINE | ID: mdl-10064081

ABSTRACT

We reported recently that interleukin (IL)-12 and IL-18 synergistically increased the fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by inducing the production of interferon (IFN)-gamma by natural killer (NK) cells. To confirm these findings in vivo, we examined the effect of combined treatment using these two cytokines on the course of experimentally induced pulmonary and disseminated cryptococcosis in mice. IL-12 and IL-18 were used at subtherapeutic doses (0.005 and 2 microg/mouse/day, respectively). A single administration of either cytokine was not effective in protecting mice against the infection, while combined treatment significantly prolonged survival time of infected mice and reduced the lung and brain loads of organisms. These protective effects were associated with elevated IFN-gamma and reduced IL-4 levels in bronchoalveolar lavage fluid. Finally, depletion of NK and gammadelta T cells, but not of CD4+ T cells, by administration of specific antibodies, significantly reduced the production of IFN-gamma in lungs by IL-12/IL-18 treatment during the 7 days of infection. Our results demonstrated that IL-12 and IL-18 protected mice against cryptococcal infection in a synergistic manner by enhancing the local production of IFN-gamma by NK and gammadelta T cells in the early phase of infection and by suppressing the production of IL-4 in lungs.


Subject(s)
Cryptococcosis/immunology , Cryptococcus neoformans/immunology , Cytotoxicity, Immunologic , Interleukin-12/immunology , Interleukin-18/immunology , Killer Cells, Natural/immunology , T-Lymphocytes/immunology , Animals , Cryptococcosis/drug therapy , Female , Interferon-gamma/immunology , Interleukin-12/pharmacology , Interleukin-12/therapeutic use , Interleukin-18/pharmacology , Interleukin-18/therapeutic use , Mice , Receptors, Antigen, T-Cell, gamma-delta/immunology
19.
J Mol Microbiol Biotechnol ; 1(1): 87-92, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10941789

ABSTRACT

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.


Subject(s)
Gammaproteobacteria/physiology , Water Microbiology , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Classification , Gammaproteobacteria/classification , Gammaproteobacteria/genetics , Pressure , Seawater/microbiology , Shewanella/classification , Shewanella/genetics
20.
Extremophiles ; 2(2): 93-9, 1998 May.
Article in English | MEDLINE | ID: mdl-9672683

ABSTRACT

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.


Subject(s)
Bacteria/enzymology , Oxidoreductases/isolation & purification , Oceans and Seas , Oxidoreductases/metabolism
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