Results from the AAPM Task Group 324 respiratory motion management in radiation oncology survey.

PURPOSE: To quantify the clinical practice of respiratory motion management in radiation oncology. METHODS: A respiratory motion management survey was designed and conducted based on clinician survey guidelines. The survey was administered to American Association of Physicists in Medicine (AAPM) members on 17 August 2020 and closed on 13 September 2020. RESULTS: A total of 527 respondents completed the entire survey and 651 respondents completed part of the survey, with the partially completed surveys included in the analysis. Overall, 84% of survey respondents used deep inspiration breath hold for left-sided breast cancer. Overall, 83% of respondents perceived respiratory motion management for thoracic and abdominal cancer radiotherapy patients to be either very important or required. Overall, 95% of respondents used respiratory motion management for thoracic and abdominal sites, with 36% of respondents using respiratory motion management for at least 90% of thoracic and abdominal patients. The majority (60%) of respondents used the internal target volume method to treat thoracic and abdominal cancer patients, with 25% using breath hold or abdominal compression and 13% using gating or tracking. CONCLUSIONS: A respiratory motion management survey has been completed by AAPM members. Respiratory motion management is generally considered very important or required and is widely used for breast, thoracic, and abdominal cancer treatments.

Investigation of the Tissue Distribution and Physiological Roles of Indoleamine 2,3-Dioxygenase-2.

Indoleamine 2,3-dioxygenase-2 (IDO2) is 1 of the 3 enzymes that can catalyze the first step in the kynurenine pathway of tryptophan metabolism. Of the 2 other enzymes, tryptophan 2,3-dioxygenase is highly expressed in the liver and has a role in tryptophan homeostasis, whereas indoleamine 2,3-dioxygenase-1 (IDO1) expression is induced by inflammatory stimuli. Indoleamine 2,3-dioxygenase-2 is reportedly expressed comparatively narrow, including in liver, kidney, brain, and in certain immune cell types, and it does not appear to contribute significantly to systemic tryptophan catabolism under normal physiological conditions. Here, we report the identification of an alternative splicing pattern, including the use of an alternative first exon, that is conserved in the mouse Ido1 and Ido2 genes. These findings prompted us to assess IDO2 protein expression and enzymatic activity in tissues. Our analysis, undertaken in Ido2 +/+ and Ido2-/- mice using immunohistochemistry and measurement of tryptophan and kynurenine levels, suggested an even more restricted pattern of tissue expression than previously reported. We found IDO2 protein to be expressed in the liver with a perinuclear/nuclear, rather than cytoplasmic, distribution. Consistent with earlier reports, we found Ido2 -/- mice to be phenotypically similar to their Ido2+/+ counterparts regarding levels of tryptophan and kynurenine in the plasma and liver. Our findings suggest a specialized function or regulatory role for IDO2 associated with its particular subcellular localization.

Subcellular compartmentalisation of copper, iron, manganese, and zinc in the Parkinson's disease brain.

Elevated iron and decreased copper levels are cardinal features of the degenerating substantia nigra pars compacta in the Parkinson's disease brain. Both of these redox-active metals, and fellow transition metals manganese and zinc, are found at high concentrations within the midbrain and participate in a range of unique biological reactions. We examined the total metal content and cellular compartmentalisation of manganese, iron, copper and zinc in the degenerating substantia nigra, disease-affected but non-degenerating fusiform gyrus, and unaffected occipital cortex in the post mortem Parkinson's disease brain compared with age-matched controls. An expected increase in iron and a decrease in copper concentration was isolated to the soluble cellular fraction, encompassing both interstitial and cytosolic metals and metal-binding proteins, rather than the membrane-associated or insoluble fractions. Manganese and zinc levels did not differ between experimental groups. Altered Fe and Cu levels were unrelated to Braak pathological staging in our cases of late-stage (Braak stage V and VI) disease. The data supports our hypothesis that regional alterations in Fe and Cu, and in proteins that utilise these metals, contribute to the regional selectively of neuronal vulnerability in this disorder.

Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson's disease brain.

Neuronal loss in numerous neurodegenerative disorders has been linked to protein aggregation and oxidative stress. Emerging data regarding overlapping proteinopathy in traditionally distinct neurodegenerative diseases suggest that disease-modifying treatments targeting these pathological features may exhibit efficacy across multiple disorders. Here, we describe proteinopathy distinct from classic synucleinopathy, predominantly comprised of the anti-oxidant enzyme superoxide dismutase-1 (SOD1), in the Parkinson's disease brain. Significant expression of this pathology closely reflected the regional pattern of neuronal loss. The protein composition and non-amyloid macrostructure of these novel aggregates closely resembles that of neurotoxic SOD1 deposits in SOD1-associated familial amyotrophic lateral sclerosis (fALS). Consistent with the hypothesis that deposition of protein aggregates in neurodegenerative disorders reflects upstream dysfunction, we demonstrated that SOD1 in the Parkinson's disease brain exhibits evidence of misfolding and metal deficiency, similar to that seen in mutant SOD1 in fALS. Our data suggest common mechanisms of toxic SOD1 aggregation in both disorders and a potential role for SOD1 dysfunction in neuronal loss in the Parkinson's disease brain. This shared restricted proteinopathy highlights the potential translation of therapeutic approaches targeting SOD1 toxicity, already in clinical trials for ALS, into disease-modifying treatments for Parkinson's disease.

TIGR4 strain causes more severe disease than WU2 strain in a mouse model of Streptococcus pneumoniae meningitis: a common pathogenic role for interferon-γ.

Streptococcus pneumoniae (S. pneumoniae) meningitis causes debilitating neurological symptoms and acute fatalities in patients, and long-term neurological sequelae in some survivors. Current vaccines do not protect against all 94 known S. pneumoniae capsular serotypes, many of which are capable of causing pneumococcal meningitis (PM). We here compare the pathogenic outcomes of two clinically virulent isolates of S. pneumoniae, serotype 3 strain WU2 and serotype 4 strain TIGR4, in a murine model of PM. At an identical infectious dosage of 103 CFU administered via the intracerebroventricular route, significantly greater mortality, interleukin (IL)1ß and IL6 production, and blood-brain barrier dysfunction occurred in TIGR4-induced PM compared to PM caused by WU2. Higher bacterial counts in the cerebrospinal fluid and nitrite/nitrate in serum were observed 40 h post inoculation with TIGR4 compared to mice infected with WU2. Similar to our previous findings in WU2 PM, interferon-γ was an essential driver of the pathogenesis of TIGR4 PM, suggesting that this cytokine may be a common pathogenic agent across a range of pneumococcal meningitides and, thus, a potential therapeutic target for intervention.

The kynurenine pathway and parasitic infections that affect CNS function.

The kynurenine pathway of tryptophan metabolism has been implicated in brain function, immunoregulation, anti-microbial mechanisms and pregnancy. Some of these actions are due to depletion of tryptophan and others to the formation of biologically active metabolites. This review focuses on the roles of the kynurenine pathway in host responses during two parasitic diseases of major health and economic importance, malaria and toxoplasmosis, with an emphasis on their impacts on CNS function. This article is part of the Special Issue entitled 'The Kynurenine Pathway in Health and Disease'.

Synergistic induction of CXCL10 by interferon-gamma and lymphotoxin-alpha in astrocytes: Possible role in cerebral malaria.

Cerebral malaria (CM) has a high mortality rate and incidence of neurological sequelae in survivors. Hypoxia and cytokine expression in the brain are two mechanisms thought to contribute to the pathogenesis of CM. The cytokines interferon (IFN)-γ and lymphotoxin (LT)-α and the chemokine CXCL10 are essential for the development of CM in a mouse model. Furthermore, serum IFN-γ protein levels are higher in human CM than in controls, and CXCL10 is elevated in both serum and cerebrospinal fluid in Ghanaian paediatric CM cases. Astrocytes actively participate in CNS pathologies, becoming activated in response to various stimuli including cytokines. Astrocyte activation also occurs in murine and human CM. We here determined the responsiveness of mouse and human astrocytes to IFN-γ and LT-α, with the aim of further elucidating the role of astrocytes in CM pathogenesis. Initially we confirmed that Ifn-γ and Cxcl10 are expressed in the brain in murine CM, and that the increased Cxcl10 expression is IFN-γ-dependant. IFN-γ induced CXCL10 production in human and murine astrocytes in vitro. The degree of induction was increased synergistically in the presence of LT-α. IFN-γ induced the expression of receptors for LT-α, while LT-α increased the expression of the receptor for IFN-γ, in the astrocytes. This cross-induction may explain the synergistic effect of the two cytokines on CXCL10 production. Expression of these receptors also was upregulated in the brain in murine CM. The results suggest that astrocytes contribute to CM pathogenesis by producing CXCL10 in response to IFN-γ and LT-α.

Interferon-γ-Induced Nitric Oxide Synthase-2 Contributes to Blood/Brain Barrier Dysfunction and Acute Mortality in Experimental Streptococcus pneumoniae Meningitis.

The proinflammatory cytokine interferon-gamma (IFNγ) recently was shown to play a crucial role in experimental pneumococcal meningitis (PM) pathogenesis, and we aimed in this study to investigate IFNγ-driven nitric oxide synthase-2 (NOS2)-mediated pathogenesis of murine PM. We demonstrate that costimulation of toll-like receptors and IFNγ receptors was synergistic for NOS2 expression in cultured murine microglia. Using an experimental PM model, wild-type mice treated with anti-IFNγ antibody, as well as IFNγ and NOS2 gene knockout (GKO) mice, were inoculated intracerebroventricularly with 10(3) colony-forming units of Streptococcus pneumoniae (WU2 strain). Mice were monitored daily during a 200-h disease course to assess survival rate and blood-brain barrier (BBB) permeability measured at 48 h. IFNγ deficiency was protective in PM, with an approximate 3-fold increase in survival rates in both antibody-treated and IFNγ GKO mice compared to controls (P < 0.01). At 48 h postinoculation, brain NOS2 mRNA expression was significantly increased in an IFNγ-dependent manner. Mortality was significantly delayed in NOS2 GKO mice compared to controls (P < 0.01), and BBB dysfunction was reduced by 54% in IFNγ GKO mice and abolished in NOS2 GKO. These data suggest that IFNγ-dependent expression of NOS2 in the brain contributes to BBB breakdown and early mortality in murine PM.

An effective, low-cost method for achieving and maintaining hypoxia during cell culture studies.

Here we report a simple new method for exposing cells to normoxic and hypoxic conditions using vacuum bags, normally employed for food storage, to establish and maintain low oxygen levels in vitro. Vacuum bags were gassed with a mixture containing specified levels of oxygen, then sealed, creating a hypoxic microenvironment for cells cultured in flasks placed therein. Oxygen levels in the gas mixture and culture medium in flasks inside the sealed bags equilibrated after two hours of incubation. The vacuum bags maintained low oxygen levels (either <2% or 5%) in medium for at least 4 days. Human fetal astrocytes grew normally in flasks for at least 4 days in a 5% oxygen/ 5% CO2/ 90% nitrogen atmosphere, but viability decreased at <2% oxygen. Vacuum bags can accommodate varying oxygen levels that would otherwise require systems with separate chambers or modules, but are less useful when repeated experimental manipulations of individual cultures are required.

Low efficiency IDO2 enzymes are conserved in lower vertebrates, whereas higher efficiency IDO1 enzymes are dispensable.

Indoleamine 2,3-dioxygenase (IDO) is a Trp-degrading enzyme that catalyzes the first step in the kynurenine pathway. Two IDO genes, IDO1 and IDO2, are found in vertebrates and the timing of the gene duplication giving rise to the genes has been controversial. In the present study, we report that several fishes and two turtles also have both IDO1 and IDO2. This represents definitive evidence for the gene duplication occurring before the divergence of vertebrates, with IDO1 having been lost in a number of lower vertebrate lineages. IDO2 enzymes have a relatively low affinity for l-Trp; however, Anolis carolinensis (lizard) IDO2 has an affinity for l-Trp comparable to mammalian IDO1 enzymes. We identified a Ser residue located in the distal heme pocket of IDO1 (distal-Ser) (corresponding to Ser167 of human IDO1) that is conserved in all IDO1 enzymes and the lizard IDO2. This residue is conserved as Thr (distal-Thr) in other IDO2 enzymes. Biochemical analyses, using IDO variants with either Ser or Thr substitutions, suggest that the distal-Ser change was crucial for the improvement in affinity for l-Trp in ancient IDO1. The ancestral IDO1 likely had a 'moderate' enzymatic efficiency for l-Trp, clearly higher than IDO2 but lower than mammalian IDO1. The distal-Ser of lizard IDO2 bestows a high affinity for l-Trp, however, this unique IDO2 has a low enzymatic efficiency because of its very low catalytic velocity. Thus, low efficiency IDO2 enzymes have been conserved throughout vertebrate evolution, whereas higher efficiency IDO1 enzymes are dispensable in many lower vertebrate lineages.

IRGM3 contributes to immunopathology and is required for differentiation of antigen-specific effector CD8+ T cells in experimental cerebral malaria.

Gamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection with Plasmodium species. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used the Plasmodium berghei ANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA for Irgm1 and Irgm3 was found in the brains and spleens of infected mice at times of peak IFN-γ production. Irgm3-/- but not Irgm1-/- mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8+ T cells within the brain. Although antigen-specific proliferation of transferred CD8+ T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred into Irgm3-/- recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens of Irgm3-/- mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology in Irgm3-/- mice occurs due to impaired generation of CD8+ effector function. This defect is nonintrinsic to CD8+ T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.

Efficient tryptophan-catabolizing activity is consistently conserved through evolution of TDO enzymes, but not IDO enzymes.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) enzymes have independently evolved to catalyze the first step in the catabolism of tryptophan (L-Trp) through the kynurenine pathway. TDO is found in almost all metazoan and many bacterial species, but not in fungi. We show that TDO enzymes have high catalytic-efficiency for L-Trp catabolism, regardless of their biological origin, suggesting that TDO has been an L-Trp-specific degrading enzyme throughout its evolution. Meanwhile, IDO was initially discovered in mammals, and subsequently has been found in lower vertebrates, several invertebrates, fungi and a number of bacterial species. Some lineages have independently generated multiple IDO paralogues through gene duplications. Interestingly, only mammalian IDO1s and fungal "typical" IDOs have high affinity and catalytic efficiency for L-Trp catabolism, comparable to TDOs. We show that invertebrate IDO enzymes have low affinity and catalytic efficiency for L-Trp catabolism. We suggest that the phylogenetic distribution of "low catalytic-efficiency IDOs" indicates the ancestral IDO also had low affinity and catalytic efficiency for L-Trp catabolism. IDOs with high catalytic-efficiency for L-Trp-catabolism may have evolved in certain lineages to fulfill particular biological roles. The low catalytic-efficiency IDOs have been well conserved in a number of lineages throughout their evolution, although it is not clear that the enzymes contribute significantly to L-Trp catabolism in these species. Investigation of other substrates and functions of the ancestral IDO and low catalytic efficiency IDOs may identify additional biological roles for these enzymes.

Mechanisms of murine cerebral malaria: Multimodal imaging of altered cerebral metabolism and protein oxidation at hemorrhage sites.

Using a multimodal biospectroscopic approach, we settle several long-standing controversies over the molecular mechanisms that lead to brain damage in cerebral malaria, which is a major health concern in developing countries because of high levels of mortality and permanent brain damage. Our results provide the first conclusive evidence that important components of the pathology of cerebral malaria include peroxidative stress and protein oxidation within cerebellar gray matter, which are colocalized with elevated nonheme iron at the site of microhemorrhage. Such information could not be obtained previously from routine imaging methods, such as electron microscopy, fluorescence, and optical microscopy in combination with immunocytochemistry, or from bulk assays, where the level of spatial information is restricted to the minimum size of tissue that can be dissected. We describe the novel combination of chemical probe-free, multimodal imaging to quantify molecular markers of disturbed energy metabolism and peroxidative stress, which were used to provide new insights into understanding the pathogenesis of cerebral malaria. In addition to these mechanistic insights, the approach described acts as a template for the future use of multimodal biospectroscopy for understanding the molecular processes involved in a range of clinically important acute and chronic (neurodegenerative) brain diseases to improve treatment strategies.

The kynurenine pathway of tryptophan degradation is activated during osteoblastogenesis.

The mechanisms involved in the anabolic effect of interferon gamma (IFNγ) on bone have not been carefully examined. Using microarray expression analysis, we found that IFNγ upregulates a set of genes associated with a tryptophan degradation pathway, known as the kynurenine pathway, in osteogenic differentiating human mesenchymal stem cells (hMSC). We, therefore, hypothesized that activation of the kynurenine pathway plays a role in osteoblastogenesis even in the absence of IFNγ. Initially, we observed a strong increase in tryptophan degradation during osteoblastogenesis with and without IFNγ in the media. We next blocked indoleamine 2,3-dioxygenase-1 (IDO1), the most important enzyme in the kynurenine pathway, using a siRNA and pharmacological approach and observed a strong inhibition of osteoblastogenesis with a concomitant decrease in osteogenic factors. We next examined the bone phenotype of Ido1 knockout (Ido1(-/-)) mice. Compared to their wild-type littermates, Ido1(-/-) mice exhibited osteopenia associated with low osteoblast and high osteoclast numbers. Finally, we tested whether the end products of the kynurenine pathway have an osteogenic effect on hMSC. We identified that picolinic acid had a strong and dose-dependent osteogenic effect in vitro. In summary, we demonstrate that the activation of the kynurenine pathway plays an important role during the commitment of hMSC into the osteoblast lineage in vitro, and that this process can be accelerated by exogenous addition of IFNγ. In addition, we found that mice lacking IDO1 activity are osteopenic. These data therefore support a new role for the kynurenine pathway and picolinic acid as essential regulators of osteoblastogenesis and as potential new targets of bone-forming cells in vivo.

Tryptophan-catabolizing enzymes - party of three.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that have independently evolved to catalyze the first step in tryptophan catabolism via the kynurenine pathway (KP). The depletion of tryptophan and formation of KP metabolites modulates the activity of the mammalian immune, reproductive, and central nervous systems. IDO and TDO enzymes can have overlapping or distinct functions depending on their expression patterns. The expression of TDO and IDO enzymes in mammals differs not only by tissue/cellular localization but also by their induction by distinct stimuli. To add to the complexity, these genes also have undergone duplications in some organisms leading to multiple isoforms of IDO or TDO. For example, many vertebrates, including all mammals, have acquired two IDO genes via gene duplication, although the IDO1-like gene has been lost in some lower vertebrate lineages. Gene duplications can allow the homologs to diverge and acquire different properties to the original gene. There is evidence for IDO enzymes having differing enzymatic characteristics, signaling properties, and biological functions. This review analyzes the evolutionary convergence of IDO and TDO enzymes as tryptophan-catabolizing enzymes and the divergent evolution of IDO homologs to generate an enzyme family with diverse characteristics not possessed by TDO enzymes, with an emphasis on the immune system.

Cerebral malaria: gamma-interferon redux.

There are two theories that seek to explain the pathogenesis of cerebral malaria, the mechanical obstruction hypothesis and the immunopathology hypothesis. Evidence consistent with both ideas has accumulated from studies of the human disease and experimental models. Thus, some combination of these concepts seems necessary to explain the very complex pattern of changes seen in cerebral malaria. The interactions between malaria parasites, erythrocytes, the cerebral microvascular endothelium, brain parenchymal cells, platelets and microparticles need to be considered. One factor that seems able to knit together much of this complexity is the cytokine interferon-gamma (IFN-γ). In this review we consider findings from the clinical disease, in vitro models and the murine counterpart of human cerebral malaria in order to evaluate the roles played by IFN-γ in the pathogenesis of this often fatal and debilitating condition.

Human indoleamine 2,3-dioxygenase-2 has substrate specificity and inhibition characteristics distinct from those of indoleamine 2,3-dioxygenase-1.

Indoleamine 2,3-dioxygenase-2 (IDO2) is one of three enzymes (alongside tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase (IDO1)) that catalyse dioxygenation of L-tryptophan as the first step in the kynurenine pathway. Despite the reported expression of IDO2 in tumours, some fundamental characteristics of the enzyme, such as substrate specificity and inhibition selectivity, are still to be clearly defined. In this study, we report the kinetic and inhibition characteristics of recombinant human IDO2. Choosing from a series of likely IDO2 substrates, we screened 54 tryptophan derivatives and tryptophan-like molecules, and characterised the 8 with which the enzyme was most active. Specificity of IDO2 for the two isomers of 1-methyltryptophan was also evaluated and the findings compared with those obtained in other studies on IDO2 and IDO1. Interestingly, IDO2 demonstrates behaviour distinct from that of IDO1 in terms of substrate specificity and affinity, such that we have identified tryptophan derivatives that are mutually exclusive as substrates for IDO1 and IDO2. Our results support the idea that the antitumour activity of 1-Me-D-Trp is unlikely to be related with competitive inhibition of IDO2, and also imply that there are subtle differences in active site structure in the two enzymes that may be exploited in the development of specific inhibitors of these enzymes, a route which may prove important in defining their role(s) in cancer.

The Fe-heme structure of met-indoleamine 2,3-dioxygenase-2 determined by X-ray absorption fine structure.

Multiple-scattering (MS) analysis of EXAFS data on met-indoleamine 2,3-dioxygenase-2 (IDO2) and analysis of XANES have provided the first direct structural information about the axial donor ligands of the iron center for this recently discovered protein. At 10K, it exists in a low-spin bis(His) form with Fe-Np(av)=1.97Å, the Fe-NIm bond lengths of 2.11Å and 2.05Å, which is in equilibrium with a high-spin form at room temperature. The bond distances in the low-spin form are consistent with other low-spin hemeproteins, as is the XANES spectrum, which is closer to that of the low-spin met-Lb than that of the high-spin met-Mb. The potential physiological role of this spin equilibrium is discussed.

The kynurenine pathway contributes to long-term neuropsychological changes in experimental pneumococcal meningitis.

Pneumococcal meningitis is a lethal form of bacterial infection in the central nervous system that often causes lifelong neurological sequelae, despite therapeutic advances. The contemporary view is that the inflammatory response to infection contributes to the functional disabilities among survivors of this disease. We previously have established a mouse model of neurobehavioural deficits, using an automated IntelliCage™ system that revealed long-term behavioural and cognitive deficits in C57BL/6J female mice cured of meningitis by ceftriaxone treatment. We now have investigated the roles of two kynurenine pathway enzymes, indoleamine dioxygenase-1 (IDO1) and tryptophan dioxygenase-2 (TDO2), in the pathomechanisms of pneumococcal meningitis. Since tryptophan metabolism has long been implicated in behavioural and cognitive modulation through the production of neuroactive compounds, we hypothesised that preventing the actions of these enzymes through gene knockout would be beneficial in mice subjected to pneumococcal infection. We found no significant effect of IDO1 or TDO2 on mortality. Post-meningitic wild-type mice showed long-term diurnal hypoactivity and nocturnal hyperactivity when they were exposed to an Intellicage adaptation test throughout both the light and dark phases. These changes were not apparent in IDO1(-/-) survivors, but were present in the TDO2(-/-) survivors. Both IDO1(-/-) and TDO2(-/-) survivors were not protected against developing long-term cognitive deficits as measured in IntelliCage-based patrolling or reversal tasks. Collectively, these observations suggest (i) involvement of the kynurenine pathway in causing some behavioural sequelae of pneumococcal meningitis and (ii) that this pathway might operate synergistically with, or independently of, other pathways to cause other aspects of neurological sequelae.

The pro-inflammatory cytokine interferon-gamma is an important driver of neuropathology and behavioural sequelae in experimental pneumococcal meningitis.

Interferon-gamma is known to play a complex modulatory role in immune defence during microbial infections. Its actions in pneumococcal meningitis, however, remain ill-defined. Here, a pathological role for IFN-γ was demonstrated using a murine model of pneumococcal meningitis, in that C57BL/6J mice deficient in this pro-inflammatory cytokine (IFN-γ(-/-)) showed less severe acute and long-term neuropathology following intracerebral challenge with Streptococcus pneumoniae. The absence of IFN-γ significantly lengthened the survival of mice that otherwise would have developed fatal clinical signs within two days of CNS infection. Compared to their wild-type counterparts, IFN-γ(-/-) mice showed a diminished inflammatory response (attenuated levels of pro-inflammatory cytokines in the cerebrospinal fluid) and milder brain pathologies (less BBB permeability to protein and brain haemorrhage) during the acute phase of disease. Following a full regime of antibiotic treatment, we found substantial brain injuries in the wild-type mice 10days after infection. IFN-γ(-/-) mice, however, showed decreased neuronal damage in both hippocampus and cortex. In the longer term (≈10weeks p.i.), the wild-type mice that had survived meningitis due to antibiotic treatment had neurobehavioural abnormalities including diurnal hypoactivity, nocturnal hyperactivity and impaired performance in a discrimination reversal task. IFN-γ(-/-) mice, concomitantly tested in the automated IntelliCage platform, had reduced behavioural and cognitive disorders compared to wild-type mice. Both IFN-γ(-/-) and wild-type survivors of pneumococcal meningitis showed impaired working memory in the IntelliCage-based complex patrolling task. These observations indicate an association between IFN-γ-driven acute brain pathology and the long-term neurological sequelae resulting from pneumococcal meningitis.