Biphasic Inflammatory Response Research Articles

CD4 + T cells ferroptosis is associated with the development of sepsis in severe polytrauma patients

BackgroundImmunological disorder remains a great challenge in severe poly-trauma, in which lymphopenia is an important contributor. The purpose of present study is to explore whether ferroptosis, a new manner of programmed cell death (PCD), is involved in the lymphocyte depletion and predictive to the adverse prognosis of severe injuries. Patients and MethodsSevere polytrauma patients admitted from January 2022 to December 2022 in our trauma center were prospectively investigated. Peripheral blood samples were collected at admission (day 1), day 3 and day 7 from them. Included patients were classified based on whether they developed sepsis or not. Clinical outcomes, systematic inflammatory response, lymphocyte subpopulation, CD4 + T cell ferroptosis were collected, detected and analyzed. ResultsNotable lymphopenia was observed on the first day after severe trauma and failed to normalize on the 7th day if patients were complicated with sepsis, in which CD4 + T cell was the subset of lymphocyte that depleted most pronouncedly. Lymphocyte loss was significantly correlated with the acute and biphasic systemic inflammatory response. Ferroptosis participated in the death of CD4 + T cells, potentially mediated by the downregulation of xCT-GSH-GPX4 pathway. CD4 + T cells ferroptosis had a conducive predicting value for the development of sepsis following severe trauma. ConclusionsCD4 + T cells ferroptosis occurs early in the acute stage of severe polytrauma, which may become a promising biomarker and therapeutic target for post-traumatic sepsis.

Biphasic inflammatory response induced by intra-plantar injection of L-cysteine: Role of CBS-derived H2S and S1P/NO signaling

This study investigates the inflammatory response to intra-plantar injection of L-cysteine in a murine model. L-cysteine induces a two-phase response: an early phase lasting 6 h and a late phase peaking at 24 h and declining by 192 h. The early phase shows increased neutrophil accumulation at 2 h up to 24 h, followed by a reduction at 48 h. On the other hand, the late phase exhibits increased macrophage infiltration peaking at 96 h. Inhibition of cystathionine β-synthase (CBS), the first enzyme in the transsulfuration pathway, significantly reduces L-cysteine-induced edema, suggesting its dependence on CBS-derived hydrogen sulfide (H2S). Sequential formation of sphingosine-1-phosphate (S1P) preceding nitric oxide (NO) generation suggests the involvement of a CBS/S1P/NO axis in the inflammatory response. Inhibition of de novo sphingolipid biosynthesis, S1P1 receptor, and endothelial NO synthase (eNOS) attenuates L-cysteine-induced paw edema. These findings indicate a critical role of the CBS/H2S/S1P/NO signaling pathway in the development and maintenance of L-cysteine-induced inflammation. The co-presence of H2S and NO is necessary for inducing and sustaining the inflammatory response, as NaHS or L-arginine alone do not replicate the marked and prolonged inflammatory effect observed with L-cysteine. This study enhances our understanding of the complex molecular mechanisms of the interplay between NO and H2S pathways in inflammation and identifies potential therapeutic targets for inflammatory disorders.

Metformin: An Old Taboo yet a New Friend for Targeted Glucose Control in Critically Ill Patients

Glucose management in critically ill adults and children has always been controversial. A few recent studies mention that the use of any drug other than insulin for glucose control in intensive care unit is not recommended anymore1. Increased levels of counter-regulatory hormones and insulin resistance at organ levels contribute immensely to the emergence of hyperglycemia in these patients. Consequently, in some patients higher doses of insulin are required for the maintenance of normoglycemia and in such scenarios incidence of hypoglycemia becomes a real concern. Moreover, insulin therapy might lead to hypokalaemia and hypomagnesaemia which in turns promote insulin resistance and higher blood glucose level (BGL). All these events make insulin administration unavoidable; thereby, beginning a vicious cycle with adverse outcomes. One of therapeutic options in this scenario is using insulin sensitizing agents as an adjunct therapy for glycemic control in critically ill patients. Different studies have shown that metformin, similar to insulin, is of anti-inflammatory and antioxidant properties, improves lipid profile, decreases nursing workload and lowers the incidence of adverse effects related to high-dose insulin therapy without being associated with the increased risk of lactic acidosis or hypoglycemia2-4. Panahi et al., in their study, showed that metformin therapy in hyperglycemic critically ill patients resulted in similar outcomes with insulin thersapy5. Also, there are some studies reporting that metformin limits ischemia reperfusion injury, modulates inflammation; it consequently contributes to the survival benefits probably through increasing adenosine receptor stimulation6-8. In sepsis, there is a biphasic inflammatory response; Systemic Inflammatory Response Syndrome (SIRS), as an initial hyperinflammatory phase, and Counterregulatory anti-inflammatory response syndrome as a later hypoactive phase. Therefore, anti-inflammatory drugs like metformin may be associated with the best results only if added prior to the initial hyperinflammatory response and might have detrimental effects if added during hypoactive phase9,10. Timing of metformin administration may be an important factor contributing to its effect in critically ill patients. Finally, in patients with refractoriness to insulin who require high doses of insulin, metformin could be used as a safe adjunct therapy to reach targeted glucose levels. Metformin plus insulin appears to lower the incidence of insulin resistance, insulin requirements while maintaining blood glucose level control, and consequently the incidence of adverse effects related to high-dose insulin therapy, particularly hypoglycaemia. Declined nursing workload is also considered a major benefit. However, two important items should be noted: first, timing of drug administration and second, characteristics of the patients like renal function, hypoperfusion status and monitoring of drug complications.

Carbon black nanoparticles induce biphasic gene expression changes associated with inflammatory responses in the lungs of C57BL/6 mice following a single intratracheal instillation

Inhalation of carbon black nanoparticles (CBNPs) causes pulmonary inflammation; however, time course data to evaluate the detailed evolution of lung inflammatory responses are lacking. Here we establish a time-series of lung inflammatory response to CBNPs. Female C57BL/6 mice were intratracheally instilled with 162μg CBNPs alongside vehicle controls. Lung tissues were examined 3h, and 1, 2, 3, 4, 5, 14, and 42days (d) post-exposure. Global gene expression and pulmonary inflammation were assessed. DNA damage was evaluated in bronchoalveolar lavage (BAL) cells and lung tissue using the comet assay. Increased neutrophil influx was observed at all time-points. DNA strand breaks were increased in BAL cells 3h post-exposure, and in lung tissues 2–5d post-exposure. Approximately 2600 genes were differentially expressed (±1.5 fold; p≤0.05) across all time-points in the lungs of exposed mice. Altered transcript levels were associated with immune-inflammatory response and acute phase response pathways, consistent with the BAL profiles and expression changes found in common respiratory infectious diseases. Genes involved in DNA repair, apoptosis, cell cycle regulation, and muscle contraction were also differentially expressed. Gene expression changes associated with inflammatory response followed a biphasic pattern, with initial changes at 3h post-exposure declining to base-levels by 3d, increasing again at 14d, and then persisting to 42d post-exposure. Thus, this single CBNP exposure that was equivalent to nine 8-h working days at the current Danish occupational exposure limit induced biphasic inflammatory response in gene expression that lasted until 42d post-exposure, raising concern over the chronic effects of CBNP exposure.

<b>Inflammatory response after session of resistance exercises in untrained volunteers

The present study aimed to investigate the interaction between the blood cells, inflammatory markers, oxidative stress parameters and delayed onset muscle soreness (DOMS) after a session of resistance exercise (SRE). The sample consisted of sixteen untrained men (26.4±5 years; 25.9±3 kg m -2 ). The SRE was composed of 4 sets of 10 repetitions maximum (extensor bench, squat and leg press) for each exercise. Complete blood cell count, C-reactive protein (CRP), creatine kinase (CK), lipid peroxidation and antioxidant capacity against peroxyl radicals were previously evaluated (baseline), and at 0, 30 and 120 min. after the SRE. DOMS was assessed 24 hours after the exercises. Immediately after the SRE, an increase of blood cell number was observed; returning to baseline after 30 min. However, after 120 min., neutrophils showed higher values than the baseline and 30 min. assessments. CK and CRP increased progressively throughout the experiment. LPO increased immediately and 120 min. after the SRE. Untrained volunteers presented an apparent biphasic inflammatory response after an acute SRE and the changes in oxidative stress, inflammatory markers and leukocytosis were best evidenced two hours after exercise.

Protection against Chikungunya virus induced arthralgia following prophylactic treatment with adenovirus vectored interferon (mDEF201)

Recent outbreaks of Chikungunya virus (CHIKV) infection have resulted in millions of cases of disease with significant morbidity. No approved antiviral treatments exist for the prevention or treatment of this viral disease. Infection with CHIKV results in a high rate of symptomatic disease that primarily includes a debilitating arthralgia. To model this cardinal disease manifestation, adult DBA/1J mice were challenged with CHIKV by footpad injection. Viremia and hind limb virus titers increased ∼100-fold while spleen virus increased >1000-fold within 1day post-virus infection (dpi). Footpad swelling was measured over a 10-day period, with peak swelling observed between 6 and 7dpi. Histology of the hind leg at the site of virus challenge showed evidence of myositis and synovitis starting on 5dpi. Cytokine profiling of the hind limb at the site of inoculation revealed a biphasic inflammatory response represented by an increase in IL-6, MCP-1, IFN-γ, MIP-1α, RANTES, and IL-17. To investigate the prophylactic capacity of IFN, mice were treated with mDEF201, an adenovirus-vectored IFN-α. Intranasal administration of a single 107pfu/ml dose of mDEF201 administered 21days to 24h prior to infection, significantly reduced footpad swelling, virus titers in the hind leg and spleen, and several inflammatory cytokines. Efficacy was not observed when treatment was initiated 24h after virus challenge. This arthralgia model of CHIKV recapitulates relevant disease features commonly observed in human disease making it applicable to preclinical testing of therapies that target both viral replication and the associated joint disease.

Early Apoptosis of Macrophages Modulated by Injection of Yersinia pestis YopK Promotes Progression of Primary Pneumonic Plague

Yersinia pestis causes pneumonic plague, a disease characterized by inflammation, necrosis and rapid bacterial growth which together cause acute lung congestion and lethality. The bacterial type III secretion system (T3SS) injects 7 effector proteins into host cells and their combined activities are necessary to establish infection. Y. pestis infection of the lungs proceeds as a biphasic inflammatory response believed to be regulated through the control of apoptosis and pyroptosis by a single, well-conserved T3SS effector protein YopJ. Recently, YopJ-mediated pyroptosis, which proceeds via the NLRP3-inflammasome, was shown to be regulated by a second T3SS effector protein YopK in the related strain Y. pseudotuberculosis. In this work, we show that for Y. pestis, YopK appears to regulate YopJ-mediated apoptosis, rather than pyroptosis, of macrophages. Inhibition of caspase-8 blocked YopK-dependent apoptosis, suggesting the involvement of the extrinsic pathway, and appeared cell-type specific. However, in contrast to yopJ, deletion of yopK caused a large decrease in virulence in a mouse pneumonic plague model. YopK-dependent modulation of macrophage apoptosis was observed at 6 and 24 hours post-infection (HPI). When YopK was absent, decreased populations of macrophages and dendritic cells were seen in the lungs at 24 HPI and correlated with resolution rather than progression of inflammation. Together the data suggest that Y. pestis YopK may coordinate the inflammatory response during pneumonic plague through the regulation of apoptosis of immune cells.

Human Cytomegalovirus Induces a Biphasic Inflammatory Response in Primary Endothelial Cells

Contradictory studies report either pro- or anti-inflammatory endothelial cell (EC) responses to human cytomegalovirus (hCMV) infection, hindering the validation of a potential link between this virus and associated inflammatory pathologies. Clarifying this issue, we report that hCMV induces a biphasic response. Early after inoculation, hCMV promoted lymphocyte and, to a lesser extent, neutrophil capture under in vivo relevant shear stresses. In contrast, later stages of infection rendered EC refractory to basal, or cytokine-induced, leukocyte recruitment.

Cockroach Allergens Induce Biphasic Asthma-Like Pulmonary Inflammation in Outbred Mice

Objectives. The aim of this study is to define the kinetics of the pulmonary inflammatory response in cockroach allergen (CRA) sensitized and challenged outbred mice. Methods. Asthma-like pulmonary inflammation was induced with three pulmonary exposures to CRA, without the use of adjuvants. Mice were sacrificed at multiple time points and asthma-like pulmonary inflammation quantified. Results. Several pulmonary parameters showed a pronounced biphasic inflammatory response with an early stage (1.5 hours post challenge) and late stage (24 hours). The initial phase was characterized by the production of multiple inflammatory mediators, including CXC chemokines, and the recruitment of neutrophils to the lung. The number of pulmonary eosinophils decreased in the early phase but quickly rebounded. Both the early and late phases had increases in TNF production in addition to airways hyperreactivity. The model also demonstrated early production of mucin with clearance by 12 hours followed by new accumulation of mucin in the pulmonary epithelial cells. Eotaxins within the lung peaked at about 12 hours and the numbers of eosinophils in the lung remained constant throughout the 48 hours of the study. Conclusions. The pulmonary inflammatory parameters in response to a clinically relevant allergen define a biphasic response. These data may be used to investigate the pathogenesis of the disease and develop targeted therapies for the distinct phases.

Analysis of the inflammatory response in HY-TCR transgenic mice highlights the pathogenic potential of CD4− CD8− T cells

Transgenic mice expressing a rearranged T cell receptor (TCR)-αβ prematurely at the double-negative stage develop an abnormal population of peripheral T cells that lack CD4 and CD8 expression and are hyper-reactive to anti-TCR antibody stimulation. One such example is the HY-TCR transgenic mice. These mice express a TCR transgenic specific for the HY antigen that is expressed in male but not in female mice. As a result, male mice have an abnormal population of HY+/CD4− 8− or HY+/CD4− CD8low T cells that are much lower in female mice. In this study, we investigated the potential patho/physiological function of these cells in vivo using a model of delayed-type hypersensitivity (DTH) reaction: the λ-carrageenan-induced paw edema. Interestingly, while both male and female HY-TCR mice develop a classical biphasic inflammatory response to λ-carrageenan, the degree of inflammation in the former was much higher than that in the latter. This was accompanied by a selective expansion of HY+/CD4− 8− and HY+/CD4− CD8low T cells in male mice and by a markedly increased production of typical DTH cytokines compared with cells from female mice. These results were specific since analysis of the inflammatory response of HY-TCR transgenic mice subjected to zymosan-induced peritonitis showed no differences between male and female mice. Together, these findings provide novel evidence for the pathological role of self-reactive CD4− CD8− T cells, previously described in several autoimmune strains and recently identified in patients suffering from autoimmune diseases such as systemic lupus erythematosus.

The PPAR-γ Agonist, Darglitazone, Restores Acute Inflammatory Responses to Cerebral Hypoxia–Ischemia in the Diabeticob/obMouse

Diabetes is an increased risk factor for stroke and results in increased brain damage in experimental animals and humans. The precise mechanisms are unclear, but our earlier studies in the db/db mice suggested that the cerebral inflammatory response initiating recovery was both delayed and diminished in the diabetic mice compared with the nondiabetic db/+ mice. In this study, we investigated the actions of the peroxisome proliferator-activated receptor (PPAR)-gamma agonist darglitazone in treating diabetes and promoting recovery after a hypoxic-ischemic (H/I) insult in the diabetic ob/ob mouse. Male ob/+ and ob/ob mice received darglitazone (1 mg/kg) for 7 days before induction of H/I. Darglitazone restored euglycemia and normalized elevated corticosterone, triglycerides, and very-low-density lipoprotein levels. Darglitazone dramatically reduced the infarct size in the ob/ob mice at 24 h of recovery compared with the untreated group (30+/-13% to 3.3+/-1.6%, n=6 to 8) but did not show any significant effect in the ob/+ mice. Microglial and astrocytic activation monitored by cytokine expression (interleukin-1beta and tumor necrosis factor-alpha) and in situ hybridization studies (bfl1 and glial fibrillary acidic protein) suggest a biphasic inflammatory response, with darglitazone restoring the compromised proinflammatory response(s) in the diabetic mouse at 4 h but suppressing subsequent inflammatory responses at 8 and 24 h in both control and diabetic mice.

Age-related decrease in proteasome expression contributes to defective nuclear factor-κB activation during hepatic ischemia/reperfusion

Hepatic ischemia/reperfusion (I/R) leads to liver injury and dysfunction through the initiation of a biphasic inflammatory response that is regulated by the transcription factor nuclear factor kappaB (NF-kappaB). We have previously shown that there is an age-dependent difference in the injury response to hepatic I/R in mice that correlates with divergent activation of NF-kappaB such that young mice have greater NF-kappaB activation, but less injury than old mice. In this study, we investigated the mechanism by which age alters the activation of NF-kappaB in the liver during I/R. Young (4-5 weeks) and old (12-14 months) mice underwent partial hepatic I/R. Livers were obtained for RNA microarray analysis and protein expression assays. Using microarray analysis, we identified age-dependent differences in the expression of genes related to protein ubiquitinylation and the proteasome. In old mice, genes that are involved in the ubiquitin-proteasome pathway were significantly down-regulated during I/R. Consistent with these findings, expression of a critical proteasome subunit, non-adenosine triphosphatase 4 (PSMD4), was reduced in old mice. Expression of the NF-kappaB inhibitory protein, IkappaB alpha, was increased in old mice and was greatly phosphorylated and ubiquitinylated. The data provide strong evidence that the age-related defect in hepatic NF-kappaB signaling during I/R is a result of decreased expression of PSMD4, a proteasome subunit responsible for recognition and recruitment of ubiquitinylated substrates to the proteasome. It appears that decreased PSMD4 expression prevents recruitment of phosphorylated and ubiquitinylated IkappaB alpha to the proteasome, resulting in a defect in NF-kappaB activation.

Atopic dermatitis and allergy in children: A dynamic relationship

Atopic dermatitis (AD) is the most common chronic skin disease in children. The defective barrier function of the skin in patients with AD allows foreign proteins to enter the body and interact with components of the innate and adaptive immune systems. The immune response characteristic of AD is complex with the majority of patients making IgE in response to ingested and/or inhaled antigens. Although IgE-mediated mechanisms may represent initial immune responses, they are only one element of a biphasic inflammatory response. Identifying the role of specific allergens in a patient’s skin disease is critical yet difficult because diagnostic tests are often not able to distinguish asymptomatic sensitization from true clinical allergy.

Endothelial nitric oxide synthase activation is critical for vascular leakage during acute inflammation in vivo

The role of endothelium-derived nitric oxide (NO) in acute inflammation is not known. Here, we examine acute inflammation in congenic endothelial NO synthase-deficient (eNOS-/-) mice. Intraplantar injection of carrageenan induces a biphasic inflammatory response. The early phase (0-6 h) is largely eliminated, and the secondary phase (24-96 h) is markedly reduced in eNOS-/- but not WT mice. Inhibition of phosphatidylinositol 3-kinase or hsp90, pathways upstream of eNOS activation, also reduces carrageenan-stimulated edema formation. To separate the ability of eNOS to regulate leukocyte trafficking vs. vascular permeability, zymosan-stimulated leukocyte infiltration and protein extravasation were assessed in WT and eNOS-/- mice. Zymosan increases inflammatory cell extravasation to the same extent in WT and eNOS-/- mice, whereas the extravasation of plasma protein is lower in eNOS-/- mice. Inhibition of phosphatidylinositol 3-kinase and hsp90 also blocks protein leakage, but not leukocyte influx. These data collectively support the critical role for eNOS in regulating the magnitude of the acute inflammatory response and show that eNOS is critical for regulating microcirculatory endothelial barrier function in vivo.

Multiorgan failure is an adaptive, endocrine-mediated, metabolic response to overwhelming systemic inflammation

Sepsis and other critical illnesses produce a biphasic inflammatory, immune, hormonal, and metabolic response. The acute phase is marked by an abrupt rise in the secretion of so-called stress hormones with an associated increase in mitochondrial and metabolic activity. The combination of severe inflammation and secondary changes in endocrine profile diminish energy production, metabolic rate, and normal cellular processes, leading to multiple organ dysfunction. This perceived failure of organs might instead be a potentially protective mechanism, because reduced cellular metabolism could increase the chances of survival of cells, and thus organs, in the face of an overwhelming insult. We propose that, first, multiple organ failure induced by critical illness is primarily a functional, rather than structural, abnormality. Indeed, it may not be failure as such, but a potentially protective, reactive mechanism. Second, the decline in organ function is triggered by a decrease in mitochondrial activity and oxidative phosphorylation, leading to reduced cellular metabolism. Third, this effect on mitochondria might be the consequence of acute-phase changes in hormones and inflammatory mediators.

Lung oxidative response after acute coal dust exposure

Coal dust exposure can induce an acute alveolar and interstitial inflammation that can lead to chronic pulmonary diseases. The objective of this study was to describe the acute and later effects of acute coal dust exposure in lung parenchyma and the involvement of reactive oxygen species in coal dust effects. Forty-eight male Wistar rats (200–250mg) were separated into four groups: 48h, 7 days, 30 days, and 60 days after coal dust instillation. Gross mineral coal dust (3mg/0.5mL saline) was administered directly in the lungs of the treatment group by intratracheal instillation. Control animals received only saline solution (0.5mL). Lipid peroxidation was determined by the quantity of thiobarbituric acid-reactive species (TBARS), oxidative damage to protein was obtained by the determination of carbonyl groups, the total radical-trapping antioxidant parameter (TRAP) was estimated by luminol chemoluminescence emission, catalase activity was measured by the rate of decrease in hydrogen peroxide, and superoxide dismutase activity was assayed by the inhibition of adrenaline autooxidation. Histological evaluation of coal dust-treated rats demonstrated an inflammatory infiltration after 48h of the exposure. Initially, this was a cellular infiltration suggestive of lymphocyte infiltration with lymphoid hyperplasia that remained until 7 days after induction. This initial response was followed by a chronic inflammatory infiltration characterized by aggregates of macrophages 30 days after induction. This inflammatory response tended to resolve 60 days after induction, being similar to that of control animals. During both the acute and chronic phases of lung inflammation we observed a decrease in the TRAP in the lung of coal dust-exposed animals compared to that in control animals. We also observed an activation of superoxide dismutase 60 days after coal dust exposition. TBARS were increased 60 days after coal dust exposure and protein carbonyl groups increased at all times after coal dust exposure (48h, 7days, 30 days, and 60 days). These data suggested a biphasic inflammatory response and the involvement of oxidative damage in coal dust-induced lung damage.

Inflammatory joint disease: clinical, histological, and molecular parameters of acute and chronic inflammation and tissue destruction.

AbstractPeptidoglycan-polysaccharide complexes from Group A streptococcal cell walls (SCW) induce a biphasic inflammatory response in rats that resembles rheumatoid arthritis (RA) in humans (1–3). Initiation of synovitis is dependent on the dissemination, deposition, and persistence of the SCW in the synovium. The subsequent infiltration of leukocytes into the synovial tissue coincides with the development of an acute inflammatory response. The clinical acute phase is transient and as the erythema and swelling subside, mononuclear cells are recruited into the synovial tissue as the chronic, erosive arthritis evolves. Similar to RA, susceptibility to the development of SCW-arthritis is gender-biased (4) and under genetic control with neuroendocrine and hormonal influences (5–7). Moreover, the mechanisms responsible for the development of SCW-arthritis are susceptible to regulation by pharmacologic agents and biological response modifiers and as such, this model is considered a valid preclinical model of RA.KeywordsSodium Dodecyl SulfateEthylene Diamine Tetraacetic AcidEthylene Diamine Tetraacetic AcidRNase Protection AssayArthritis InductionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Mycobacterium terrae isolated from indoor air of a moisture-damaged building induces sustained biphasic inflammatory response in mouse lungs.

Occupants in moisture-damaged buildings suffer frequently from respiratory symptoms. This may be partly due to the presence of abnormal microbial growth or the altered microbial flora in the damaged buildings. However, the specific effects of the microbes on respiratory health and the way they provoke clinical manifestations are poorly understood. In the present study, we exposed mice via intratracheal instillation to a single dose of Mycobacterium terrae isolated from the indoor air of a moisture-damaged building (1 X 10(7), 5 X 10(7), or 1 X 10(8) microbes). Inflammation and toxicity in lungs were evaluated 2 hr later. The time course of the effects was assessed with the dose of 1 X 10(8) bacterial cells for up to 28 days. M. terrae caused a sustained biphasic inflammation in mouse lungs. The characteristic features for the first phase, which lasted from 6 hr to 3 days, were elevated proinflammatory cytokine [i.e., tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6)] levels in the bronchoalveolar lavage fluid (BALF). TNF-alpha was produced in the lungs more intensively than was IL-6. Neutrophils were the most abundant cells in the airways during the first phase, although their numbers in BALF remained elevated up to 21 days. The characteristics of the second phase, which lasted from 7 to 28 days, were elevated TNF-alpha levels in BALF, expression of inducible nitric oxide synthase in BAL cells, and recruitment of mononuclear cells such as lymphocytes and macrophages into the airways. Moreover, total protein, albumin, and lactate dehydrogenase concentrations were elevated in both phases in BALF. The bacteria were detected in lungs up to 28 days. In summary, these observations indicate that M. terrae is capable of provoking a sustained, biphasic inflammation in mouse lungs and can cause a moderate degree of cytotoxicity. Thus, M. terrae can be considered a species with potential to adversely affect the health of the occupants of moisture-damaged buildings.

Biphasic ocular inflammatory response to endotoxin-induced uveitis in the mouse.

To examine the kinetics and mechanisms of endotoxin-induced uveitis in the mouse. C3H/HeN mice were injected subcutaneously with 0.3 mg of Salmonella typhimurium lipopolysaccharide (LPS) in 0.1 mL of phosphate-buffered saline solution or phosphate-buffered saline solution alone in 3 separate experiments; mice were killed after 1, 3, 5, and 7 days. In 2 other separate experiments, mice were killed 1, 3, 6, and 24 hours after LPS injection. All eyes were collected for histological examination, immunohistochemical analyses, aqueous protein level determination, and reverse transcriptase-polymerase chain reaction for ocular interleukin (IL)1alpha, IL-6, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor messenger RNA (mRNA). Enzyme-linked immunosorbent assay was used to measure tumor necrosis factor alpha and IL-6 levels in aqueous and serum samples. Results were consistent for all experiments. Numbers of ocular inflammatory cells and levels of aqueous protein peaked 1 and 5 days after LPS injection. Control mice did not develop inflammation. Serum and aqueous IL-6 and ocular IL-6 mRNA levels peaked at 1 day and subsided at 3 days. However, ocular IL-1alpha, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor mRNA appeared, peaked, and subsided at 3, 5, and 7 days, respectively. Predominant infiltrating cells were neutrophils at 1 day and macrophages at 5 days. Although no ocular inflammatory cells were detected before 24 hours after LPS injection, tumor necrosis factor alpha mRNA was noticed at 1 hour, peaked at 3 hours, and disappeared at 6 hours and granulocyte-macrophage colony-stimulating factor mRNA was spotted only at 3 hours after LPS injection. The ocular inflammatory response to C3H/ HeN mouse endotoxin-induced uveitis is biphasic for 7 days. The first wave appears at day 1 and subsides by day 3. A second, higher peak appears at day 5. The 2 inflammatory waves are related to the kinetics of the different cytokines released in the eye. This is in contrast to the rat monophasic endotoxin-induced uveitis model, which has only one peak of intense inflammation associated with cytokine release. A biphasic inflammatory response associated with cytokine release lasting several days is observed in C3H/HeN mice with endotoxin-induced uveitis. Because human anterior uveitis has a tendency to be recurrent in nature, this might be a better experimental model.

Cytokine gene expression in different strains of mice with endotoxin-induced uveitis (EIU)

PURPOSE: The kinetics of various cytokines in the eye plays a critical role in endotoxin-induced uveitis (EIU). This study examined the cytokine kinetics and susceptibility of EIU in four mice strains. METHODS: Four strains of TLR-4 or Toll-like receptor-4 (Lps, lipopolysaccharide-susceptible) gene-positive mice (C3H/HeN of H-2 k , C57/B6 of H-2 b , Balb/C of H-2 d , and 129/J of H-2 b ) were injected subcutaneously with either lipopolysaccharide (LPS) in phosphate-buffered saline (PBS) or PBS alone in two repeated experiments. Mice were sacrificed 1, 3, 6, 24 (1 d), 72 (3 d), 120 (5 d), or 168 (7 d) hours after LPS injection. Ocular histology and reverse transcriptase-polymerase chain reaction (RT-PCR) to detect ocular interleukin-1a (IL-1a), IL-6, tumor necrosis factor-a (TNF-a), and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA were performed. Serum IL-1a, IL-1ß, IL-6, and TNF-a levels were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: No ocular inflammation was present in any mice within six hours after LPS injection. Only the C3H/HeN mice developed a biphasic ocular inflammatory response (1 d and 5 d), during which all proinflammatory cytokine messages were expressed. In the other three strains with minimal (129/J and Balb/C) to mild (C57/B6) EIU that peaked at 1 d, IL-6 mRNA was barely detectable in C57/B6 and Balb/C; GM-CSF mRNA was also present in C57/B6. Serum IL-1a, IL-1ß, IL-6, and TNF-a were high in all EIU mice within six hours after LPS injection. Control mice did not develop uveitis or measurable cytokine messages. CONCLUSION: In the most susceptible strain, C3H/HeN, EIU was biphasic and correlated to multiple proinflammatory cytokines released in the eye. The less susceptible mice strains exhibited a monophasic response to LPS that may result from no cytokine cascade.