Phage-Encoded Sigma Factors Alter Bacterial Dormancy.

By entering a reversible state of reduced metabolic activity, dormant microorganisms are able to tolerate suboptimal conditions that would otherwise reduce their fitness. Dormancy may also benefit bacteria by serving as a refuge from parasitic infections. Here, we focus on dormancy in the Bacillota, where endospore development is transcriptionally regulated by the expression of sigma factors. A disruption of this process could influence the survivorship or reproduction of phages that infect spore-forming hosts with implications for coevolutionary dynamics. We characterized the distribution of sigma factors in over 4,000 genomes of diverse phages capable of infecting hosts that span the bacterial domain. From this, we identified homologs of sporulation-specific sigma factors in phages that infect spore-forming hosts. Unlike sigma factors required for phage reproduction, we provide evidence that sporulation-like sigma factors are nonessential for lytic infection. However, when expressed in the spore-forming Bacillus subtilis, some of these phage-derived sigma factors can activate the bacterial sporulation gene network and lead to a reduction in spore yield. Our findings suggest that the acquisition of host-like transcriptional regulators may allow phages to manipulate a complex and ancient trait in one of the most abundant cell types on Earth. IMPORTANCE As obligate parasites, phages exert strong top-down pressure on host populations with eco-evolutionary implications for community dynamics and ecosystem functioning. The process of phage infection, however, is constrained by bottom-up processes that influence the energetic and nutritional status of susceptible hosts. Many phages have acquired auxiliary genes from bacteria, which can be used to exploit host metabolism with consequences for phage fitness. In this study, we demonstrate that phages infecting spore-forming bacteria carry homologs of sigma factors, which their hosts use to orchestrate gene expression during spore development. By tapping into regulatory gene networks, phages may manipulate the physiology and survival strategies of nongrowing bacteria in ways that influence host-parasite coevolution.

Clinical and demographic characteristics predictive of treatment outcomes for certolizumab pegol in moderate to severe Crohn's disease: analyses from the 7-year PRECiSE 3 study.

BACKGROUND: Clinical factors were previously identified as predictors of short-term treatment efficacy in Crohn's disease (CD). The PRECiSE 3 (P3) 7-year trial provides an opportunity to study predictors of short- and long-term clinical remission among CD patients treated with certolizumab pegol (CZP). AIM: To identify factors that influence long-term remission of CD with CZP treatment. METHODS: Patients who had completed placebo-controlled studies (PRECiSE 1/PRECiSE 2, P1/P2) enrolled in P3 and received open-label CZP 400 mg every 4 weeks up to 7 years. Baseline predictors included, but were not limited to, smoking status, disease duration, prior inflammatory bowel disease (IBD) surgery, Harvey-Bradshaw Index (HBI), albumin, haematocrit and CZP exposure; association with time to initial remission (HBI ≤4) was tested for patients who received CZP in P1/P2; time to loss of remission/frequency of maintenance of remission was also tested. Univariate analyses and multivariate Cox or logistic regression models were used. RESULTS: Predictors for initial remission (N = 377) included age, haematocrit, prior IBD surgery and entry HBI (P < 0.05 for all). Predictors for loss of remission (N = 437) included HBI, serum albumin concentration, haematocrit, smoking status and exposure. Predictors of maintenance of remission (N = 437) included haematocrit, IBD surgery, HBI, disease duration, serum albumin concentration and exposure. Significant predictors were confirmed with stepwise multivariate regression models. CONCLUSIONS: These analyses identified several influential parameters for short-and long-term remission of Crohn's disease with certolizumab pegol treatment. The data yield valuable hypotheses regarding factors that influence certolizumab pegol treatment. More investigation is needed. (ClinicalTrials.gov identifier NCT00552058).

Long-term safety and efficacy of certolizumab pegol in the treatment of Crohn's disease: 7-year results from the PRECiSE 3 study.

BACKGROUND: The efficacy and safety of certolizumab pegol (CZP) in moderate-to-severe Crohn's disease were demonstrated in two 26-week double-blind studies (PRECiSE 1 & 2). AIM: To report the safety and efficacy outcomes of long-term, CZP therapy from PRECiSE 3, in which patients received treatment up to 7 years treatment. METHODS: Patients completing PRECiSE 1 or 2 were eligible to enter PRECiSE 3 in which they received CZP 400 mg, open-label, every 4 weeks (without additional induction therapy) for up to 7 years, for up to 91 doses from study start. Safety (adverse events, including infections and malignancies) and efficacy (Harvey-Bradshaw Index, faecal calprotectin, C-reactive protein) were prospectively monitored. Remission was analysed using observed cases, last observation carried forward imputation and nonresponder imputation. RESULTS: A total of 595 patients entered the study; 117 (20%) completed 7 years. Discontinuation rates were 29.2%, 13.6%, 16.1%, 7.9%, 5.0%, 4.5% and 3.9% (years 1-7 respectively). During 1920 patient-years of exposure to CZP, no new safety signals were observed. Incidence rates (new cases/100 patient-years) for serious infections and malignant neoplasms were 4.37 and 1.06 respectively. No lymphoproliferative malignancies were reported. Clinical remission rates were ≥68% at each year (observed cases); rates by last observation carried forward and nonresponder imputation were 58% and 45% at year 1, 56% and 26% at year 3 and 55% and 13% at year 7 respectively. CONCLUSION: Certolizumab pegol was well tolerated in the long-term treatment of Crohn's disease, with sustained remission in some patients continuing in the study for up to 7 years. ClinicalTrials.gov identifier NCT00552058.

Detrimental role of the airway mucin Muc5ac during ventilator-induced lung injury.

Acute lung injury (ALI) is associated with high morbidity and mortality in critically ill patients. At present, the functional contribution of airway mucins to ALI is unknown. We hypothesized that excessive mucus production could be detrimental during lung injury. Initial transcriptional profiling of airway mucins revealed a selective and robust induction of MUC5AC upon cyclic mechanical stretch exposure of pulmonary epithelia (Calu-3). Additional studies confirmed time- and stretch-dose-dependent induction of MUC5AC transcript or protein during cyclic mechanical stretch exposure in vitro or during ventilator-induced lung injury in vivo. Patients suffering from ALI showed a 58-fold increase in MUC5AC protein in their bronchoalveolar lavage. Studies of the MUC5AC promoter implicated nuclear factor κB in Muc5ac induction during ALI. Moreover, mice with gene-targeted deletion of Muc5ac⁻/⁻ experience attenuated lung inflammation and pulmonary edema during injurious ventilation. We observed that neutrophil trafficking into the lungs of Muc5ac⁻/⁻ mice was selectively attenuated. This implicates that endogenous Muc5ac production enhances pulmonary neutrophil trafficking during lung injury. Together, these studies reveal a detrimental role for endogenous Muc5ac production during ALI and suggest pharmacological strategies to dampen mucin production in the treatment of lung injury.

Environmental genomics and human health.

Most diseases represent several or many specific pathophysiologic processes that can only be fully understood by focusing on the genetic and environmental contributions to etiology and pathogenesis. Environmental health research and genomic research are logical, even necessary, partners. Despite the difficulties inherent in teasing apart environmental contributions to human disease, a number of studies have shown both that non-genetic factors are significant components of disease risk and that environmental exposures, particularly during fetal development, can profoundly affect subsequent genetic expression. A comparative genomics approach among species using microarray analysis and bioinformatics approaches that allow the hypothetical creation of pathways should allow one to determine the functional homology of these different regulatory genes. However, performing such analyses across species requires a considerable collaborative effort between laboratories having expertise in human conditions and a working knowledge with each model system being compared. The vision of environmental genomics is to define the gene-environment underpinnings of human disease in ways that can lead to improved human health. Improved exposure assessment technology would allow researchers to decipher the environmental and genetic risk factors for disease development and progression, specifically the interaction between environmental exposures and gene sequence differences. It would provide a means to determine very early pathophysiologic measures of disease initiation, thus allowing for better screening and intervention strategies.

Identification and characterization of a loss-of-function human MPYS variant.

MPYS, also known as STING and MITA, is an interferon (IFN)ß stimulator essential for host defense against RNA, DNA viruses and intracellular bacteria. MPYS also facilitates the adjuvant activity of DNA vaccines. Here, we report identification of a distinct human MPYS haplotype that contains three non-synonymous single nucleotide polymorphisms (SNPs), R71H-G230A-R293Q (thus, named the HAQ haplotype). We estimate, in two cohorts (1,074 individuals), that ∼3% of Americans are homozygous for this HAQ haplotype. HAQ MPYS exhibits a > 90% loss in the ability to stimulate IFNß production. Furthermore, fibroblasts and macrophage cells expressing HAQ are defective in Listeria monocytogenes infection-induced IFNß production. Lastly, we find that the loss of IFNß activity is due primarily to the R71H and R293Q SNPs in HAQ. We hypothesize that individuals carrying HAQ may exhibit heightened susceptibility to viral infection and respond poorly to DNA vaccines.

Adalimumab for the treatment of fistulas in patients with Crohn's disease.

OBJECTIVE: To evaluate the efficacy of adalimumab in the healing of draining fistulas in patients with active Crohn's disease (CD). DESIGN: A phase III, multicentre, randomised, double-blind, placebo controlled study with an open-label extension was conducted in 92 sites. PATIENTS: A subgroup of adults with moderate to severely active CD (CD activity index 220-450) for >or=4 months who had draining fistulas at baseline. INTERVENTIONS: All patients received initial open-label adalimumab induction therapy (80 mg/40 mg at weeks 0/2). At week 4, all patients were randomly assigned to receive double-blind placebo or adalimumab 40 mg every other week or weekly to week 56 (irrespective of fistula status). Patients completing week 56 of therapy were then eligible to enroll in an open-label extension. MAIN OUTCOME MEASURES: Complete fistula healing/closure (assessed at every visit) was defined as no drainage, either spontaneous or with gentle compression. RESULTS: Of 854 patients enrolled, 117 had draining fistulas at both screening and baseline (70 randomly assigned to adalimumab and 47 to placebo). The mean number of draining fistulas per day was significantly decreased in adalimumab-treated patients compared with placebo-treated patients during the double-blind treatment period. Of all patients with healed fistulas at week 56 (both adalimumab and placebo groups), 90% (28/31) maintained healing following 1 year of open-label adalimumab therapy (observed analysis). CONCLUSIONS: In patients with active CD, adalimumab therapy was more effective than placebo for inducing fistula healing. Complete fistula healing was sustained for up to 2 years by most patients in an open-label extension trial.

Bakery flour dust exposure causes non-allergic inflammation and enhances allergic airway inflammation in mice.

BACKGROUND: Baker's asthma is one of the most commonly reported occupational lung diseases in countries where fresh bread is baked daily in large quantities, and is characterized by rhinitis, bronchial hyperresponsiveness, and reversible airflow obstruction. Epidemiological studies have identified pre-existing atopy as an important risk factor for developing baker's asthma, yet the aetiology and pathogenesis of baker's asthma remain poorly understood. OBJECTIVE: We sought to develop a mouse model of baker's asthma that could be used to characterize the development and progression of baker's asthma. METHODS: We were unable to sensitize mice to bakery flour dust or flour dust extract. We assessed total inflammatory cells, cellular differential, total serum IgE and the pro-inflammatory cytokine response to oropharyngeally instilled bakery flour dust or flour dust extract by itself or in the context of ovalbumin (OVA) sensitization and challenge. RESULTS: Both bakery flour dust and flour dust extract consistently elicited a neutrophilic inflammation in a Toll-like receptor 4-independent manner; suggesting that endotoxin is not playing a role in the inflammatory response to flour dust. Moreover, bakery flour dust and dust extract significantly enhance the inflammatory response in OVA-sensitized and challenged mice. CONCLUSIONS: Bakery flour dust and flour dust extract are strongly pro-inflammatory and can cause non-allergic airway inflammation and can enhance allergen-mediated airway inflammation.

Medical options for treating perianal Crohn's disease.

Perianal Crohn's disease can cause significant morbidity for patients affected by the disease. However, diagnostic modalities and treatment options have progressed changing the goals of treatment from fistula "improvement" to complete cessation of drainage. Fistula closure and fibrosis of the fistula track is achieved in some patients. Furthermore, treatment has become a combined effort between medical physicians and surgeons. Simple disease can be treated with medical therapy alone consisting of antibiotics and immunomodulators. Infliximab should be added to refractory simple disease or simple disease with the presence of inflammation. If complex fistula disease is evident a surgical evaluation should also be done to determine if intervention is indicated. Complex disease should be treated with antibiotics, immunomodulators and biologic therapy from the onset. This review will summarise current data regarding medical options for treatment of fistulising Crohn's disease.

Carcinoid tumors are 15 times more common in patients with Crohn's disease.

BACKGROUND: The coexistence of intestinal neoplasms with Crohn's disease (CD) has been reported, but the evidence of an increased risk of carcinoid tumor with Crohn's disease has been mixed. We present 4 patients with CD with associated carcinoid tumor. METHODS: The charts of 111 patients with CD who had undergone resection between June 2001 and March 2005 were reviewed. The number of incidental carcinoid tumors in patients who underwent an appendectomy was used as a control. RESULTS: Four cases of carcinoid tumor discovered in patients at resection for CD were identified. None had metastatic disease or carcinoid syndrome. These included 1 cecal (1 mm), 2 appendiceal (3 and 7 mm), and 1 transverse colon (7 mm) carcinoid tumors. None of the carcinoid tumors were identified in regions of active Crohn's disease. The incidence of carcinoid tumor in patients with Crohn's disease was 4 of 111 (3.6%). In comparison, 3 of 1199 patients (0.25%) who had appendectomies were identified as having appendiceal carcinoid tumor. Crohn's disease was associated with an increased incidence of carcinoid tumor; OR 14.9 (95% CI 2.5-102.5), P<0.0001. CONCLUSIONS: There was a significantly increased incidence of carcinoid tumor in our Crohn's patients compared to the control patients. None of the carcinoid tumors developed in areas of Crohn's disease. This suggests that the development of carcinoid tumors may be secondary to distant proinflammatory mediators, rather than a local inflammatory effect from adjacent Crohn's disease. Patients with CD may be at increased risk of developing a carcinoid tumor.

Genetic regulation of rejection and survival following human lung transplantation by the innate immune receptor CD14.

We have developed the hypothesis that genetic polymorphisms which alter the expression or function of innate immune receptors contribute to the marked interindividual differences in the onset and severity of lung transplant rejection. In this analysis, we considered the effects of a common promotor polymorphism of the lipopolysaccharide receptor CD14 associated with increased transcriptional activity upon the development of posttransplant rejection and graft survival. Genotyping was performed in 226 lung transplant recipients well characterized with regards to clinical outcomes. An earlier onset of acute rejection, bronchiolitis obliterans syndrome (BOS) and worse posttransplant graft survival due to greater BOS related deaths was evident in patients with the CD14 -159 TT genotype (TT). The adverse effect upon graft survival of the TT genotype remained significant in a multivariate Cox model (Hazard Ratio 1.65, 95% CI, 1.03-2.64, p-value = 0.04) after adjusting for other important covariates. Furthermore, TT patients have significantly greater sCD14, TNF-alpha and IFN-gamma in the peripheral blood implying a heightened state of innate immune activation drives the development of increased post-transplant rejection. Inhibition of innate immune activation through CD14 represents a novel and potentially important therapeutic target to prevent post-transplant rejection and improve outcomes after human lung transplantation.

Non-reporting and inconsistent reporting of race and ethnicity in articles that claim associations among genotype, outcome, and race or ethnicity.

BACKGROUND: The use of race as a category in medical research is the focus of an intense debate, complicated by the inconsistency of presumed independent variables, race and ethnicity, on which analysis depends. Interpretation is made difficult by inconsistent methods for determining the race or ethnicity of a participant. The failure to specify how race or ethnicity was determined is common in the published literature. HYPOTHESIS: Criteria by which they assign a research participant to racial or ethnic categories are not reported by published articles. METHODS: Methods were reviewed for assigning race and ethnicity of research participants in 268 published reports reporting associations among race (or ethnicity), health outcome and genotype. RESULTS: Of the 268 published reports reviewed, it was found that 192 (72%) did not explain their methods for assigning race or ethnicity as an independent variable. This was despite the fact that 180 (67%) of those reports reached conclusions about associations among genetics, health outcome and race or ethnicity. CONCLUSIONS: More attention needs to be given to the definition of race and ethnicity in genetic studies, especially in those diseases where health disparities are known to exist.

FasL gene therapy: a new therapeutic modality for head and neck cancer.

In this study, we investigated the in vitro and in vivo efficacy of Fas ligand (FasL) gene therapy for the treatment of head and neck cancer. Three head and neck squamous cell carcinoma (HNSCC) cell lines (SCC-1, SCC-12, and SCC-14a) were treated with the Fas agonist CH-11, a monoclonal antibody to the Fas receptor, or with a replication-incompetent adenovirus (AdGFPFasL) expressing a modified murine Fas ligand gene fused to green fluorescent protein (GFP). A replication-incompetent adenovirus containing the GFP gene alone was used as a control for viral transduction toxicity (AdGFP). Cell death was quantified using a tetrazolium-based (MTS) assay. Cells were analyzed by flow cytometry to determine the expression of adenoviral and Fas receptors on the surface of the cells. Our results showed that the head and neck cancer cell lines are resistant to cell death induction when treated with the anti-Fas monoclonal antibody CH-11. This resistance can be overcome with AdGFPFasL, which was able to induce cell death in all three cell lines. Apoptosis induction was demonstrated using Western blotting by evaluating poly(ADP-ribose) polymerase, and caspase 9 cleavages. In addition, intratumoral injections of AdGFPFasL into SCC-14a xenografts induced significant growth suppression of tumors, indicating that FasL gene therapy may provide a new efficient therapeutic modality for HNSCC that is worthy of a clinical trial.

Review article: The medical treatment of Crohn's perianal fistulas.

Perianal fistulas are a frequent manifestation of Crohn's disease. The correct application of the newer diagnostic and therapeutic agents for treating perianal Crohn's disease are beginning to be better defined. In general, a combined medical and surgical approach is preferred. The perianal disease process should first be fully delineated with endoscopy and either MRI or EUS before treatment is begun. Patients are then stratified into one of three groups: simple fistulas and no proctitis; simple fistulas and concomitant proctitis; and complex fistulas. Patients with simple fistulas and no proctitis can be treated medically with a combination of antibiotics and an immunosuppressive agent (azathioprine or mercaptopurine). Patients with simple fistulas and concomitant proctitis should have infliximab added to their treatment plan. Complex fistulas require surgical intervention first prior to medical treatment. A combination of antibiotics, immunosuppressive therapy and infliximab are then initiated to facilitate fistula healing.

Subchronic endotoxin inhalation causes persistent airway disease.

The endotoxin component of organic dusts causes acute reversible airflow obstruction and airway inflammation. To test the hypothesis that endotoxin alone causes airway remodeling, we have compared the response of two inbred mouse strains to subchronic endotoxin exposure. Physiological and biological parameters were evaluated after 1 day, 5 days, or 8 wk of exposure to endotoxin [lipopolysaccharide (LPS)] in endotoxin-sensitive (C3HeB/FeJ) and endotoxin-resistant (C3H/HeJ) mice. After 5 days or 8 wk of LPS exposure, only C3HeB/FeJ had elevated airway hyperreactivity to inhaled methacholine. Only the C3HeB/FeJ mice had significant inflammation of the lower respiratory tract after 1 day, 5 days, or 8 wk of LPS exposure. Stereological measurements of small, medium, and large airways indicated that an 8-wk exposure to LPS resulted in expansion of the submucosal area only in the C3HeB/FeJ mice. Cell proliferation as measured by bromodeoxyuridine incorporation contributed to the expansion of the submucosa and was only significantly elevated in C3HeB/FeJ mice actively exposed to LPS. C3HeB/FeJ mice had significantly elevated levels of interleukin-1beta protein in whole lung lavage after 1 day and 5 days of LPS exposure and significantly elevated protein levels of total and active transforming growth factor-beta1 in whole lung lavage fluid after 5 days of LPS exposure. Our findings demonstrate that subchronic inhalation of LPS results in the development of persistent airway disease in endotoxin-responsive mice.

The role of TLR4 in endotoxin responsiveness in humans.

Despite the tremendous inter-individual variability in the response to inhaled toxins, we simply do not understand why certain people develop disease when challenged with environmental agents and others remain healthy. To address this concern, we investigated whether the Toll-4 (TLR4) gene, that has been shown to affect lipopolysaccharide (LPS) responsiveness in mice, underlies the variability in airway responsiveness to inhaled LPS in humans. Here we show that common, co-segregating missense mutations (Asp299Gly and Thr399Ile) in the extracellular domain of the TLR4 receptor are associated with a significantly blunted response to inhaled LPS in 83 humans. Transfection of THP-1 cells demonstrates that the Asp299Gly mutation (but not the Thr399Ile mutation) interrupts TLR4-mediated LPS signaling. Moreover, the wild-type allele of TLR4 rescues the LPS hyporesponsive phenotype in either primary airway epithelial cells or alveolar macrophages obtained from individuals with the TLR4 mutations. Our findings provide the first genetic evidence that common mutations in TLR4 are associated with differences in LPS responsiveness in humans, and demonstrate that gene sequence changes can alter the ability of the host to respond to environmental stress.

Airway inflammation and responsiveness in prostaglandin H synthase-deficient mice exposed to bacterial lipopolysaccharide.

Bacterial lipopolysaccharide (LPS) is a risk factor for exacerbation of asthma and causes airway inflammation. The aim of this study was to examine the effects of disruption of prostaglandin (PG) H synthase (PGHS)-1 and PGHS-2 genes on pulmonary responses to inhaled LPS. PGHS-1(-/-), PGHS-2(-/-), and wild-type (WT) mice were exposed to 4 to 6 microg/m(3) LPS via aerosol. Enhanced pause (PenH), a measure of bronchoconstriction, was assessed using a whole-body plethysmograph before and immediately after a 4-h LPS exposure. Bronchoalveolar lavage (BAL) was performed after LPS exposure to assess inflammatory cells, cytokines/chemokines (tumor necrosis factor-alpha, interleukin-6, and macrophage inflammatory protein-2), and PGE(2). The degree of lung inflammation was scored on hematoxylin-and-eosin-stained sections. PGHS-1 and PGHS-2 protein levels were determined by immunoblotting. All mice exhibited increased PenH and methacholine responsiveness after LPS exposure; however, these changes were much more pronounced in PGHS-1(-/-) and PGHS-2(-/-) mice relative to WT mice (P < 0.05). There were no significant differences in inflammation as assessed by BAL fluid (BALF) cells or lung histology between the genotypes despite reduced BALF cytokines/chemokines and PGE(2) in PGHS-1(-/-) and PGHS-2(-/-) mice relative to WT mice (P < 0.05). PGHS-2 was upregulated more in PGHS-1(-/-) mice compared with WT mice after LPS exposure. We conclude that: (1) airway inflammation and hyperresponsiveness are dissociated in PGHS-1(-/-) and PGHS-2(-/-) mice exposed to LPS; (2) the balance of PGHS-1 and PGHS-2 is important in regulating the functional respiratory responses to inhaled LPS; and (3) neither PGHS-1 nor PGHS-2 is important in regulating basal lung function or the inflammatory responses of the lung to inhaled LPS.

Diagnosis and treatment of perianal fistulas in Crohn disease.

Perianal fistulas occur in up to 43% of patients with Crohn disease. Diagnostic evaluation to determine the location and type of fistulas and the presence or absence of rectal inflammation is required. A combined medical and surgical approach to the management of such patients is the optimal treatment plan. Perianal abscesses must be drained. Superficial, low transsphincteric, and low intersphincteric fistulas are usually treated with fistulotomy and antibiotics. High transsphincteric, suprasphincteric, and extrasphincteric fistulas are usually treated with noncutting setons, antibiotics, and azathioprine or 6-mercaptopurine and, in many cases, infliximab.

Genes other than TLR4 are involved in the response to inhaled LPS.

For several decades, the mouse strains C3H/HeJ and C57BL/10ScNCr have been known to be hyporesponsive to endotoxin or lipopolysaccharide (LPS). Recently, mutations in Toll-like receptor (TLR) 4 have been shown to underlie this aberrant response to LPS. To further determine the relationship between TLR4 and responsiveness to LPS, we genotyped 18 strains of mice for TLR4 and evaluated the physiological and biological responses of these strains to inhaled LPS. Of the 18 strains tested, 6 were wild type for TLR4 and 12 had mutations in TLR4. Of those strains with TLR4 mutations, nine had mutations in highly conserved residues. Among the strains wild type for TLR4, the inflammatory response in the airway induced by inhalation of LPS showed a phenotype ranging from very sensitive (DBA/2) to hyporesponsive (C57BL/6). A broad spectrum of airway hyperreactivity after inhalation of LPS was also observed among strains wild type for TLR4. Although the TLR4 mutant strains C3H/HeJ and C57BL/10ScNCr were phenotypically distinct from the other strains with mutations in the TLR4 gene, the other strains with mutations for TLR4 demonstrated a broad distribution in their physiological and biological responses to inhaled LPS. The results of our study indicate that although certain TLR4 mutations can be linked to a change in the LPS response phenotype, additional genes are clearly involved in determining the physiological and biological responses to inhaled LPS in mammals.