Distinct Tlr4-expressing cell compartments control neutrophilic and eosinophilic airway inflammation.

Allergic asthma is a chronic, inflammatory lung disease. Some forms of allergic asthma are characterized by T helper type 2 (Th2)-driven eosinophilia, whereas others are distinguished by Th17-driven neutrophilia. Stimulation of Toll-like receptor 4 (TLR4) on hematopoietic and airway epithelial cells (AECs) contributes to the inflammatory response to lipopolysaccharide (LPS) and allergens, but the specific contribution of TLR4 in these cell compartments to airway inflammatory responses remains poorly understood. We used novel, conditionally mutant Tlr4(fl/fl) mice to define the relative contributions of AEC and hematopoietic cell Tlr4 expression to LPS- and allergen-induced airway inflammation. We found that Tlr4 expression by hematopoietic cells is critical for neutrophilic airway inflammation following LPS exposure and for Th17-driven neutrophilic responses to the house dust mite (HDM) lysates and ovalbumin (OVA). Conversely, Tlr4 expression by AECs was found to be important for robust eosinophilic airway inflammation following sensitization and challenge with these same allergens. Thus, Tlr4 expression by hematopoietic and airway epithelial cells controls distinct arms of the immune response to inhaled allergens.

Role of the lipoxygenase pathway in RSV-induced alternatively activated macrophages leading to resolution of lung pathology.

Resolution of severe Respiratory Syncytial Virus (RSV)-induced bronchiolitis is mediated by alternatively activated macrophages (AA-Mφ) that counteract cyclooxygenase (COX)-2-induced lung pathology. Herein, we report that RSV infection of 5-lipoxygenase (LO)(-/-) and 15-LO(-/-) macrophages or mice failed to elicit AA-Mφ differentiation and concomitantly exhibited increased COX-2 expression. Further, RSV infection of 5-LO(-/-) mice resulted in enhanced lung pathology. Pharmacologic inhibition of 5-LO or 15-LO also blocked differentiation of RSV-induced AA-Mφ in vitro and, conversely, treatment of 5-LO(-/-) macrophages with downstream products, lipoxin A4 and resolvin E1, but not leukotriene B4 or leukotriene D4, partially restored expression of AA-Mφ markers. Indomethacin blockade of COX activity in RSV-infected macrophages increased 5-LO and 15-LO, as well as arginase-1 mRNA expression. Treatment of RSV-infected mice with indomethacin also resulted not only in enhanced lung arginase-1 mRNA expression and decreased COX-2, but also decreased lung pathology in RSV-infected 5-LO(-/-) mice. Treatment of RSV-infected cotton rats with a COX-2-specific inhibitor resulted in enhanced lung 5-LO mRNA and AA-Mφ marker expression. Together, these data suggest a novel therapeutic approach for RSV that promotes AA-Mφ differentiation by activating the 5-LO pathway.

New insights into innate immune mechanisms underlying allergenicity.

Allergic diseases, which have reached epidemic proportions, are caused by inappropriate immune responses to a relatively small number of environmental proteins. The molecular basis for the propensity of specific proteins to promote maladaptive, allergic responses has been difficult to define. Recent data suggest that the ability of such proteins to promote allergic responses in susceptible hosts is a function of their ability to interact with diverse pathways of innate immune recognition and activation at mucosal surfaces. This review highlights recent insights into innate immune activation by allergens--through proteolytic activity, engagement of pattern recognition receptors, molecular mimicry of TLR signaling complex molecules, lipid-binding activity, and oxidant potential--and the role of such activation in inducing allergic disease. A greater understanding of the fundamental origins of allergenicity should help define new preventive and therapeutic targets in allergic disease.

Topical interferon alpha 2b eye-drops for treatment of ocular surface squamous neoplasia: a dose comparison study.

BACKGROUND/AIMS To compare the effectiveness and side-effect profile of two doses of interferon alpha2b (IFNalpha2b) eye-drops (1 million international units (IU)/ml versus 3 million IU/ml) in the treatment of ocular surface squamous neoplasia (OSSN). METHODS Retrospective case series. RESULTS Thirty-five eyes were identified over an 11-year period (1996-2007). Twenty-one eyes (19 patients) with conjunctival intraepithelial neoplasia (CIN) were treated with 1 million IU/ml of topical IFN-alpha2b; 12 eyes (nine patients) with CIN were treated with 3 million IU/ml. Two patients with squamous cell carcinoma (SCC) were treated with topical interferon, one with 1 million IU/ml and one with 3 million IU/ml. Baseline demographic information was not statistically different between the two groups. In patients with CIN, topical therapy eliminated disease in 81% of eyes in the 1 million IU/ml group versus 92%, in the 3 million IU/ml group, p=0.41. The median time to OSSN resolution was 2.8 months in the 1 million IU/ml group and 1.9 months in the 3 million IU/ml group, p=0.55. Neither eye with SCC responded to interferon therapy. Topical therapy was well tolerated. After a median follow-up of 24 months, three recurrences were seen in eyes successfully treated with topical therapy. CONCLUSION In our study, there were no significant differences between the 1 million IU/ml group and the 3 million IU/ml group for the treatment of CIN.

Therapeutic starvation and autophagy in prostate cancer: a new paradigm for targeting metabolism in cancer therapy.

BACKGROUND: Autophagy is a starvation induced cellular process of self-digestion that allows cells to degrade cytoplasmic contents. The understanding of autophagy, as either a mechanism of resistance to therapies that induce metabolic stress, or as a means to cell death, is rapidly expanding and supportive of a new paradigm of therapeutic starvation. METHODS: To determine the effect of therapeutic starvation in prostate cancer, we studied the effect of the prototypical inhibitor of metabolism, 2-deoxy-D-glucose (2DG), in multiple cellular models including a transfected pEGFP-LC3 autophagy reporter construct in PC-3 and LNCaP cells. RESULTS: We found that 2DG induced cytotoxicity in PC-3 and LNCaP cells in a dose dependent fashion. We also found that 2DG modulated checkpoint proteins cdk4, and cdk6. Using the transfected pEGFP-LC3 autophagy reporter construct, we found that 2DG induced LC3 membrane translocation, characteristic of autophagy. Furthermore, knockdown of beclin1, an essential regulator of autophagy, abrogated 2DG induced autophagy. Using Western analysis for LC3 protein, we also found increased LC3-II expression in 2DG treated cells, again consistent with autophagy. In an effort to develop markers that may be predictive of autophagy, for assessment in clinical trials, we stained human prostate tumors for Beclin1 by immunohistochemistry (IHC). Additionally, we used a digitized imaging algorithm to quantify Beclin1 staining assessment. These data demonstrate the induction of autophagy in prostate cancer by therapeutic starvation with 2DG, and support the feasibility of assessment of markers predictive of autophagy such as Beclin1 that can be utilized in clinical trials. Prostate 68: 1743-1752 (c) 2008 Wiley-Liss, Inc. These data demonstrate the induction of autophagy in prostate cancer by therapeutic starvation with 2DG, and support the feasibility of assessment of markers predictive of autophagy such as Beclin1 that can be utilized in clinical trials.

HRPAP20: a novel calmodulin-binding protein that increases breast cancer cell invasion.

We previously reported the identification of HRPAP20 (hormone-regulated proliferation-associated protein 20), a novel hormone-regulated, proliferation-associated protein. In tumor cell lines, constitutive HRPAP20 expression enhanced proliferation and suppressed apoptosis, characteristics frequently associated with malignant progression. Here, we report that highly invasive breast cancer cell lines and human breast tumor specimens express elevated HRPAP20, which in transfection experiments in MCF-7 and MDA-MB-231 cells, increased invasion. Results from mechanistic studies revealed that HRPAP20 bound to calmodulin (CaM) via a conserved CaM-binding motif. Transfection of MCF-7 breast cancer cells with HRPAP20 harboring a mutated CaM-binding motif (HRPAP20K73A) inhibited its interaction with CaM and failed to increase invasion. Other experiments revealed that transfection with HRPAP20, but not HRPAP20K73A, increased secretion of matrix metalloproteinase-9 (MMP-9). Moreover, knockdown of HRPAP20 with small interfering RNA in MCF-7/HRPAP20 transfectants and wild-type MDA-MB-231 cells reduced invasion and inhibited secretion of MMP-9. Together these observations suggest that HRPAP20 may be an important regulator of breast tumor cell invasion by a CaM-mediated mechanism that leads to increased MMP-9 secretion. We conclude that dysregulation of HRPAP20 expression in tumor cells may contribute to the observed phenotypic changes associated with breast cancer progression.

Mycobacterium interface keratitis after laser in situ keratomileusis.

PURPOSE: To report the clinical course, management, and outcome of infectious interface keratitis caused by mycobacterium species after laser in situ keratomileusis (LASIK). DESIGN: A small noncomparative interventional case series. PARTICIPANTS: Five eyes in four patients who underwent LASIK in different locations around the world and had culture-positive mycobacterium keratitis develop. INTERVENTION: The LASIK flap was lifted or amputated, samples were submitted for Ziehl-Neelsen acid-fast stain and Lowenstein-Jensen's agar cultures for diagnosis; topical treatment with fortified clarithromycin and amikacin was administered until clinical resolution. MAIN OUTCOME MEASURES: Time periods from onset to diagnosis and from diagnosis to clinical resolution, and the final visual acuity. RESULTS: Onset of symptoms of infection occurred after a mean of 20 days (range, 11 days-6 weeks) after LASIK or an enhancement procedure. Definitive diagnosis was obtained after a mean period of 4.5 weeks (range, 12 days-8 weeks) from onset. Keratitis resolved within 8.4 weeks (range, 1-18 weeks) of treatment with fortified clarithromycin and amikacin. Corticosteroids were found to worsen and prolong the course of disease. In four of five eyes the LASIK flap was amputated. CONCLUSIONS: Mycobacterial keratitis is a potentially vision-threatening complication after LASIK, characterized by a long latent period, delayed diagnosis, and a protracted course even under intensive specific antibiotic therapy. Inclusion of specific culture media and staining protocols for mycobacteria, along with aggressive treatment on diagnosis, including lifting or amputating the LASIK flap, culturing, topical fortified clarithromycin and amikacin, while avoiding corticosteroids, may significantly improve resolution of the infection and potentially improve the visual outcome.

A common single nucleotide polymorphism in the CD14 promoter decreases the affinity of Sp protein binding and enhances transcriptional activity.

CD14 is a pattern recognition receptor that plays a central role in innate immunity through recognition of bacterial lipoglycans, primarily LPS. Recently, our group has identified a common single nucleotide polymorphism, -159C-->T, in the CD14 proximal promoter. Homozygous carriers of the T allele have a significant increase in soluble CD14, but a decreased total serum IgE. This epidemiologic evidence led us to investigate the molecular basis for the effects of CD14/-159C-->T on CD14 regulation in monocytes and hepatocytes, the two major cell types known to express this gene in vivo. EMSA analysis showed that the T allele results in decreased affinity of DNA/protein interactions at a GC box that contains a binding site for Sp1, Sp2, and Sp3 transcription factors. In reporter assays, the transcriptional activity of the T allele was increased in monocytic Mono Mac 6 cells, which express low levels of Sp3, a member of the Sp family with inhibitory potential relative to activating Sp1 and Sp2. By contrast, both alleles were transcribed equivalently in Sp3-rich hepatocytic HepG2 cells. Our data indicate that the interplay between CD14 promoter affinity and the [Sp3]:[Sp1 + Sp2] ratio plays a critical mechanistic role in regulating transcription of the two CD14 alleles. Variation in a key gene of innate immunity may be important for the pathogenesis of allergy and inflammatory disease through gene-by-gene and/or gene-by-environment interactions.

Intracameral anesthesia: a report by the American Academy of Ophthalmology.

OBJECTIVE: This document describes the technique of intracameral anesthesia and examines the available evidence to address questions about its effectiveness, possible corneal endothelial and retinal toxicity, and the optimal and maximal dose. METHODS: A literature search conducted for the years 1968 to 2000 retrieved over 180 citations that matched the search criteria. Panel members and a methodologist reviewed this information, and it was evaluated for the quality of the evidence presented. RESULTS: Some studies report effectiveness of intracameral anesthesia while others report no effect. In those studies showing an effect, levels of pain in the groups that were compared were low. Short-term studies seem to indicate that preservative (methylparaben)-free lidocaine 1% is well tolerated by the corneal endothelium but that higher concentrations of lidocaine are toxic. There is some evidence of electroretinogram changes after exposure to lidocaine or bupivacaine. CONCLUSIONS: The ideal timing and placement of intracameral anesthesia has not been determined. Because topical anesthesia alone is effective, surgeons may elect to use intracameral anesthesia for incremental pain control in patients who cannot be adequately managed with topical alone. Appropriate patient selection is important when using this method of anesthesia. While short-term studies seem to indicate safety, long-term effects are unknown. Patient preferences for anesthesia are not well studied.

Type I interferons and IL-12: convergence and cross-regulation among mediators of cellular immunity.

Therapeutic use of type I IFN (IFN-alpha/beta) has become common. Many of the diverse diseases targeted are marked by pathogenetic abnormalities in cell-mediated immunity (CMI), these cellular immune responses either causing injury to the host, lacking sufficient vigor for virus or tumor clearance, or both. In general, therapeutic efficacy is limited. It is thus notable that the pleiotropic effects of type I IFN on CMI remain poorly understood. We characterized the effects of type I IFN on the production of IL-12, the central immunoregulatory cytokine of the CD4(+) T cell arm of CMI. We show that type I IFN are potent inhibitors of IL-12 production by human monocytes/macrophages. The underlying mechanism involves transcriptional inhibition of the IL-12p40 gene, marked by down-regulation of PU.1 binding activity at the upstream Ets site of the IL-12p40 promoter. Type I IFN have previously been shown to be able to substitute for IL-12 in driving IFN-gamma production from T and NK cells. The ability of IFN-alpha/beta to suppress IL-12 production while up-regulating IFN-gamma production suggests a possible mechanistic basis for the difficulties of employing these cytokines in diseases involving abnormalities of CMI.

Natural measles causes prolonged suppression of interleukin-12 production.

Among vaccine-preventable diseases, measles is the preeminent killer of children worldwide. Infection with measles virus (MV) is associated with prolonged suppression of cell-mediated immune responses, a phenomenon that is thought to underlie the susceptibility to secondary infections that accounts for most measles-related mortality. Interleukin (IL)-12 is critical for the orchestration of cellular immunity. MV specifically ablates IL-12 production by monocyte/macrophages in vitro through binding to CD46, a complement regulatory protein that is an MV receptor. To address the effect of MV on IL-12 responses in vivo, cytokine production was examined in Gambian patients with measles. IL-12 production by peripheral blood monocytes from such patients is markedly suppressed, which provides a unifying mechanism for many of the immunologic abnormalities associated with measles. This suppression is prolonged, with significant, stimulus-specific inhibition of IL-12 production demonstrable months after recovery from acute infection. However, despite this suppression, IL-12 responsiveness remains intact.

IL-12 suppression during experimental endotoxin tolerance: dendritic cell loss and macrophage hyporesponsiveness.

Endotoxin tolerance, the transient, secondary down-regulation of a subset of endotoxin-driven responses after exposure to bacterial products, is thought to be an adaptive response providing protection from pathological hyperactivation of the innate immune system during bacterial infection. However, although protecting from the development of sepsis, endotoxin tolerance also can lead to fatal blunting of immunological responses to subsequent infections in survivors of septic shock. Despite considerable experimental effort aimed at characterizing the molecular mechanisms responsible for a variety of endotoxin tolerance-related phenomena, no consensus has been achieved yet. IL-12 is a macrophage- and dendritic cell (DC)-derived cytokine that plays a key role in pathological responses to endotoxin as well as in the induction of protective responses to pathogens. It recently has been shown that IL-12 production is suppressed in endotoxin tolerance, providing a likely partial mechanism for the increased risk of secondary infections in sepsis survivors. We examined the development of IL-12 suppression during endotoxin tolerance in mice. Decreased IL-12 production in vivo is clearly multifactorial, involving both loss of CD11c(high) DCs as well as alterations in the responsiveness of macrophages and remaining splenic DCs. We find no demonstrable mechanistic role for B or T lymphocytes, the soluble mediators IL-10, TNF-alpha, IFN-alphabeta, or nitric oxide, or the NF-kappaB family members p50, p52, or RelB.

Treatment of conjunctival and corneal intraepithelial neoplasia with topical interferon alpha-2b.

OBJECTIVE: To evaluate the role of topical interferon alfa-2b (IFNalpha2b) in the treatment of conjunctival and corneal intraepithelial neoplasia (CIN). DESIGN: Noncomparative case series. PARTICIPANTS: Five patients with histologically proven CIN or recurrences of proven CIN were studied prospectively. INTERVENTION: After histologic confirmation, patients were given topical recombinant IFNalpha2b (INTRON A, Schering Plough, Kenilworth, NJ) 1 million IU/ml four times a day. Patients were continued on interferon until complete resolution of the tumor had occurred. MAIN OUTCOME MEASURES: Patients were followed clinically and photographically for evidence of tumor resolution. RESULTS: All patients had complete resolution of the CIN lesion on IFNalpha2b. The mean time to clinical resolution was 11.6 weeks (range, 4-22 weeks). The mean follow-up was 17.6 months (range, 7-28 months). One patient had a clinical recurrence of his corneal CIN 1 year after tumor resolution. This patient was retreated, resulting in clinical resolution within 6 weeks, and has been tumor free for 8 months of follow-up.

Interferon-beta in multiple sclerosis: altering the balance of interleukin-12 and interleukin-10?

Interferon-beta is a remarkably pleiotropic molecule. Antiviral, pro- and antiproliferative, pro- and antiapoptotic, and complex immunoregulatory activities have all been described. The precise mechanism(s) that underlie the beneficial effects of interferon-beta in multiple sclerosis remain poorly understood; this has hindered progress in the search for more effective therapies. An increasing body of literature supports the hypothesis that interferon-beta-mediated changes in the production and activities of the immunoregulatory cytokines interleukin-12 and interleukin-10 are important to the therapeutic benefits of interferon-beta in multiple sclerosis. These data are reviewed here.

Complement and IL-12: yin and yang.

Interleukin 12 (IL-12) is central to the orchestration of cell-mediated immune responses in the innate as well as the adaptive immune system. Recent studies of the pathogenesis of diseases as disparate as measles and asthma have suggested that the complement system, itself at the interface of innate and adaptive immunity, is a biologically relevant regulator of IL-12 production. These data are reviewed here.

The germless theory of allergic disease: revisiting the hygiene hypothesis.

Rising rates of allergic disease accompany the healthier benefits of a contemporary westernized lifestyle, such as low infant mortality. It is likely that these twinned phenomena are causally related. The hygiene hypothesis states that allergy and increased longevity are both consequences of reducing infectious stressors during early childhood for millennia. Mechanistic explanations for the hygiene hypothesis have typically invoked the T-helper-type 1/2 (T(H)1/T(H)2) model. Here, we discuss why we favour a broader 'counter-regulatory' model--one that might also explain the increasing incidence of autoimmune disease in westernized countries.

Identification of complement factor 5 as a susceptibility locus for experimental allergic asthma.

The prevalence and severity of allergic asthma continue to rise, lending urgency to the search for environmental triggers and genetic substrates. Using microarray analysis of pulmonary gene expression and single nucleotide polymorphism-based genotyping, combined with quantitative trait locus analysis, we identified the gene encoding complement factor 5 (C5) as a susceptibility locus for allergen-induced airway hyperresponsiveness in a murine model of asthma. A deletion in the coding sequence of C5 leads to C5-deficiency and susceptibility. Interleukin 12 (IL-12) is able to prevent or reverse experimental allergic asthma. Blockade of the C5a receptor rendered human monocytes unable to produce IL-12, mimicking blunted IL-12 production by macrophages from C5-deficient mice and providing a mechanism for the regulation of susceptibility to asthma by C5. The role of complement in modulating susceptibility to asthma highlights the importance of immunoregulatory events at the interface of innate and adaptive immunity in disease pathogenesis.

Traumatic cataract presenting with unilateral nasal hemianopsia.

A 56-year-old man developed a nasal field defect in his left eye 3 months after a traumatic accident. An examination showed a posterior subcapsular cataract in the left eye with no neurologic deficits. Humphrey 24-2 visual field testing revealed a nasal hemianopsia in the left eye. After cataract extraction and intraocular lens implantation, the patient's visual field returned to normal. This case shows that a cataract can present with a localized visual field deficit, which may be corrected by cataract extraction.