- Cooperation between IL-4/STAT6 signaling and PPARs directs alternative macrophage activation
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- As shown in Figure 2E through 2G, macrophage‐derived foam cells released more EVs than normal macrophages
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Figure 3. Microultrasound imaging evaluation of carotid artery plaque. A, Representative microultrasound long-axis views of the left common carotid arteries in a mice model of unstable atherosclerotic lesions treated with vehicle or TRAM-34. B, Representative view of carotid blood flow obtained by pulse-wave Doppler assessment. C, Quantification of ultrasound-derived plaque intima-medial thickness (IMT), eccentric index, and maximal systolic velocity of blood flow in the vehicle- and TRAM-34-treated group (n=9 in each group). Statistical significance values (P) are indicated.
AMs were suspended in complete RPMI-1640 containing 10% heat-inactivated human AB serum and plated at a concentration of 1 × 106 cells/well into 12-well plates. After a 2-h incubation at 37° C, the nonadherent cells were removed by gentle washing and the remaining AM monolayer was cultured for 24 to 48 h in 1/0 ml of AIM-V medium (GIBCO) supplemented with 2% human AB serum. Cells were cultured in the presence or absence of 1 μg/ml Escherichia coli LPS (Sigma). Supernatants were harvested at either 24 h for determination of tumor necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) or 48 h for determination of interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β). Collected supernatants were centrifuged to remove residual cells and frozen at −80° C until use.
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In this article, we aimed at reviewing the recent progress in developing cytokine-neutralizing agents with a focus on platform technologies that directly capture and neutralize inflammatory cytokines (Figure 1). Based on their mechanisms of action, some anticytokine agents are “monoplex,” which bind with a specific cytokine for neutralization. In this category, cytokine-neutralizing antibodies (CNAs) represent the most dominant and rapidly growing class of anticytokine therapeutics. We provide a thorough review of cytokine-neutralizing antibodies currently in clinical use. In addition to free antibodies, biomaterial-conjugated antibodies are emerging with unique applications through their altered pharmacokinetic profiles and biodistribution in vivo. In contrast to monoplex platforms, “multiplex” anticytokine agents are able to concurrently neutralize multiple cytokines that reflect the multiplexity of the cytokine targets in diseases. In this category, two emerging technologies include glycosaminoglycan (GAG)-containing biomaterials and cell-membrane-coated nanoparticles. The former mimic the intracellular matrix for dynamic cytokine binding and neutralization, and the latter harness natural cell membranes as broad-spectrum cytokine-neutralizing agents. Herein, we have reviewed recent progress in the rational design of each anticytokine platform and discussed their application by highlighting the material structure–function relationships. Overall, we believe that therapeutic platforms featuring potent, dynamic, and safe cytokine neutralization ability are of great importance and have the potential for effective treatment of inflammatory diseases.
Receiver-operating characteristics (ROC) curve shows an optimal sensitivity and specificity for MAS, obtained by use of ferritin/ESR ratio. Comparison of ROC curves with other biomarkers is shown; table gives details of ferritin/ESR sensitivity and specificity. A cutpoint of ferritin/ESR ratio of 80 provides optimum sensitivity and specificity for MAS, as shown in the detailed analysis. MAS: macrophage activation syndrome; ESR: erythrocyte sedimentation rate; FSTL-1: Follistatin-like protein 1; sIL-2Rα: soluble interleukin-2 receptor-α.
Erythrocyte sedimentation rate (ESR) was derived from the clinical record. Serum ferritin levels were measured at the Children’s Hospital of Pittsburgh Clinical Laboratory using standard techniques.
Macrophage activation contributes to functional heterogeneity
Statement 9: Mal-HLH comes in 2 forms: “malignancy-triggered HLH” as a presenting feature of the malignancy at diagnosis or at relapse and “HLH during chemotherapy,” in most cases induced by infections. Differentiating these HLH subtypes is important, because the therapeutic approach differs markedly (strong consensus).
Broken lines show cytoplasm of histiocytes. White stars show nuclei of histiocytes. Arrows show intracytoplasmic phagocytized nuclei of hematopoietic precursors. White arrowhead shows ghost of phagocytized hematopoietic precursor.
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Fresh fasting plasma was separated by centrifugation from the peripheral blood obtained from healthy participants and mixed together. LDL was isolated from plasma by density gradient ultracentrifugation and then oxidized using 5 μmol/L copper sulfate. The oxidized LDL was dialyzed 3 times against 1 L of PBS (10 mmol/L, pH 7/0), sterilized with a 0/22‐μm filter, stored in sealed tubes at 4°C in the dark, and used within 2 months. The level of sample oxidation was measured using the thiobarbituric acid method, and its value was 9/2 nmol of malondialdehyde equivalents per milligram of LDL.
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Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of pathologic immune activation, occurring as either a familial disorder or a sporadic condition, in association with a variety of triggers. This immune dysregulatory disorder is prominently associated with cytopenias and a unique combination of clinical signs and symptoms of extreme inflammation. Prompt initiation of immunochemotherapy is essential for survival, but timely diagnosis may be challenging because of the rarity of HLH, its variable presentation, and the time required to perform diagnostic testing. Therapy is complicated by dynamic clinical course, high risk of treatment-related morbidity, and disease recurrence. Here, we review the clinical manifestations and patterns of HLH and describe our approach to the diagnosis and therapy for this elusive and potentially lethal condition.
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In gene profiling of patients with s-JIA, increased expression of components of the IL-1 and TLR pathways has been identified38,39, and IL-1 blockade with anakinra is effective in a significant proportion of patients. Reports suggest also that administration of anakinra is effective in treatment of established MAS/r-HLH38,40,41,42. However, initiation of anakinra treatment has occasionally been associated with triggering of MAS/r-HLH43. Moreover, IL-1 blockade is not effective in mice harboring nonfunctional mutations of Unc1344. More information is needed to fully understand the role of IL-1 and the potential of IL-1 blockade in MAS/r-HLH42. An IL-1–IL-6 synergy may be hypothesized in patients with s-JIA exposed to infections. IL-1 induces IL-6 production from a variety of cell types45,46,47. IL-6 strengthens TLR ligand-induced IL-1β production by peripheral blood mononuclear cells (Strippoli, et al, unpublished data), and blockade of IL-1 may affect biological effects of IL-6 in multiple myeloma48.
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Many patients with sHLH require <8 weeks of etoposide. Although patients may be continued on the full course (8 weeks) of etoposide in the absence of major toxicities, we recommend a weekly reevaluation of the need for continued etoposide therapy. Patients with residual disease after 8 weeks may benefit from maintenance therapy and, possibly, alloSCT.
Epigenetic Mechanisms in Monocytes/Macrophages Regulate Inflammation in Cardiometabolic and Vascular Disease
Only biallelic variations reported. Specific mutant site not reported for patients ≥18 years.
Over the past decade, multiple investigations have demonstrated that chronic inflammation often underlies the pathogenesis of cardiometabolic and vascular disease. Inflammatory responses, based on macrophage polarization, depend on the cell’s ability to respond to environmental changes and stimuli. Given the impact of epigenetic modifications on the dynamic regulation of macrophage phenotype, recent studies have examined the role of epigenetics in disease progression. This section will focus on the role of epigenetic modifications regulating macrophage phenotype in the pathogenesis of obesity, diabetic wound healing, atherosclerosis, and aortic aneurysms.
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Unliganded PPARδ interacts with BCL6, preventing it from repressing its target genes. Availability of PPARδ ligand or genetic deletion of PPARδ releases BCL6, allowing it to repress transcription of inflammatory genes.
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An untreated sample and a sample treated with isotypic immunoglobulin G were used to define EVs positive for exposed phosphatidylserine (PS) and CD45, respectively. Quantitative determination of EVs was performed using Trucount beads. We set the FL1 and FL4 photomultiplier tubes to identify the Trucount beads, and the proportion of sample flowing into Gallios was calculated by counting the detected Trucount beads.
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We conclude that macrophage activation syndrome in the setting of AOSD can be treated with anakinra
Treatment of malignancy-triggered HLH needs to balance HLH-specific and tumor-specific treatment. Corticosteroids are often used as first-line treatment to combat inflammation.
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Certain HLH triggers carry the inherent risk of acquired cellular immunodeficiency, such as Hodgkin lymphoma, T-cell lymphomas, or HIV. Additionally, HLH-directed treatment depletes leukocytes (T/B cells and granulocytes). For patients requiring such treatment, administration of broad antimicrobial prophylaxis against Pneumocystis jirovecii and fungi is recommended. Hospitalization in units with high efficiency particulate air–filtered air should be considered. We also suggest antiviral prophylaxis because of the severe T-cell depletion.
There is some evidence suggesting efficacy of IL-1 inhibition in overt MAS/r-HLH, supporting a role of IL-1 in maintaining MAS/r-HLH. While there is still no solid information on the role of IL-6 in overt MAS/r-HLH, data from animals suggest that IL-6 may contribute to triggering it. Some data point to major involvement of IFN-γ in p-HLH; however, a comprehensive analysis of the levels and the signature of this cytokine in patients with MAS/s-HLH is still needed. Incidentally, increased levels of neopterin, a marker of IFN-γ activation, have been reported in 5 patients with MAS/r-HLH; this finding needs confirmation in a larger number33. A better understanding of the interplay between these cytokines might allow identification of the most suitable therapeutic target, to dampen as rapidly as possible the cytokine storm leading to multiple organ damage in patients with severe MAS/r-HLH.
Testing should also be considered for HLH patients with unknown trigger. Importantly, pending results must not delay the clinical decision to treat HLH.
Emergencies in Pediatric Rheumatology 295anemia, an
The lungs are protected by a network of defense mechanisms that include mechanical factors, phagocytic effector cells, lymphocytes, and immunoregulatory cytokines (reviewed in Reference 40). The present study specifically evaluated the effects of marijuana and cocaine, as well as tobacco, on the function of AMs. AMs are the predominant lung leukocyte and act as the lung's resident phagocytic defense against both bacteria and fungi (5, 34). They secrete a variety of cytokines capable of regulating their own activity, as well as the activity of other immune effector cells. In addition, AMs are exposed to the highest possible concentrations of inhaled marijuana and cocaine, making them a likely target for drug-related effects. Our findings clearly demonstrate that habitual smoking of either substance significantly impairs the antibacterial and tumoricidal activities of human AMs, as well as, in the case of marijuana, their ability to produce inflammatory cytokines.
A model of secondary HLH caused by Epstein-Barr virus (EBV) infection has been obtained using humanized mice. Human hematopoietic CD34+ cells are introduced into immunodeficient mice, thus rebuilding a “humanized” immune system. Infection of these mice with EBV induces a condition resembling EBV-associated HLH in humans, with thrombocytopenia and hemophagocytosis. By 10 weeks postinfection, two-thirds of the infected mice die after exhibiting high and persistent viremia, leukocytosis, anemia, and thrombocytopenia16. Chronic typhoid fever following infection with Salmonella enterica has been proposed as a model of secondary HLH17. In this experimental model, an acute response is followed by a chronic inflammatory disease characterized by anemia, thrombocytopenia, reduced lymphocytes, increased neutrophils and monocytes, hypofibrinogenemia, hyperferritinemia, fever, and hepatosplenomegaly. Hemophagocytic macrophages were found in bone marrow and spleen.
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Serum levels of FSTL-1 were elevated at time of presentation of sJIA (mean 200/7 ng/ml) and decreased to normal (mean 133/7 ng/ml) over 24 months (p < 0/01). FSTL-1 levels were markedly elevated during acute MAS (mean 279/8 ng/ml) and decreased to normal following treatment (p < 0/001). FSTL-1 levels correlated with serum markers of inflammation, including sIL-2Rα and ferritin. Ferritin/ESR ratio was superior to ferritin, sIL-2Rα, and FSTL-1 in discriminating MAS from new-onset sJIA. PBMC from patients with FSTL-1 levels > 200 ng/ml showed altered expression of genes related to innate immunity, erythropoiesis, and natural killer cell dysfunction. Two patients with the highest FSTL-1 levels at disease onset (> 300 ng/ml) ultimately developed MAS.
In recent years, interest in adult HLH has increased markedly; as a result, HLH is more frequently diagnosed in adults. The dramatic therapeutic success in pediatric HLH has also positively affected the survival of adults with HLH. However, there are profound differences between adult and pediatric HLH; genetic HLH is rare in adults, pediatric diagnostic criteria are suboptimal, frequent (often occult) underlying malignancies or other conditions require a different diagnostic workup, and pediatric treatment regimens may have to be adapted on a case-by-case basis.
McClain; How I treat hemophagocytic lymphohistiocytosis
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Because of the impact of tuberculosis on world health, we focused additional experiments on macrophages exposed to M. tuberculosis. These experiments were designed to test the difference index and evaluate clues to pathogenesis. It was striking that the expression levels of genes encoding two proteins critical for host defense and adaptive immunity, IL-12 and IL-15, were differentially regulated.
The results of our bacterial killing assay suggest that, under the conditions studied, NO is one of the mechanisms by which AMs kill their targets. AMs from both NS and TS exhibited potent antibacterial activity that was significantly reduced in the presence of NGMMA. In contrast, AMs from CS exhibited much lower levels of antibacterial activity that were not affected by NGMMA.
A diagnosis of adult-onset Still’s disease (AOSD) was made. Prednisone doses of 20 mg BID were necessary to abate temperature. Because of her recent surgical instrumentation for vertebral tuberculosis, adding a biologic agent was feared. Weekly intramuscular gold salts were started1. She developed a rash and gold salts were stopped after 3 months. Prednisone was gradually tapered.
It is well known that integrins are a class of adhesion molecules and are highly organized plasma membrane proteins that establish close connections with the cytoskeleton. Consequently, it is unlikely that integrins are released from the donor cells to target cells in a manner similar to that of cytokines. Nevertheless, EVs generated by exocytosis or budding from cells provide a possible mechanism for the transfer of integrins between cells. In addition, the presence of integrins in EVs was reported in a previous study40 and was confirmed by our study. In this study, we revealed that FC‐EVs can release integrins to the surface of VSMCs. Because integrins can adhere to extracellular matrix components, such as fibronectin, and subsequently activate migration‐ and adhesion‐related pathways in cells, the transfer of integrins from foam cells to VSMCs by EVs might be a potential mechanism for the promotion of VSMC migration and adhesion.
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Emergencies in Pediatric Rheumatology 293PRS is con
The tumor cytotoxicity assay measures the ability of AMs to destroy [3H]thymidine-labeled tumor cells (Figure 4). AMs recovered from TS were as efficient as AMs from NS in their ability to kill tumor targets (41 to 79% versus 50 to 81% tumor lysis). In contrast, AMs isolated from MS were able to lyse only 24 to 40% of the tumor targets (p < 0/01 compared with NS), while AMs isolated from CS killed 15 to 65% of the tumor cells (p < 0/01 compared with NS). On average, effector cells from CS had the least antitumor activity, with AMs from five of the seven cocaine-smoking subjects destroying < 23% of the target cells. Interestingly, IFN-γ significantly increased the antitumor activity of AMs from CS (37 ± 5/6% increase) but had no significant effect on the tumoricidal activity of AMs from MS, TS, or NS (data not shown). No significant difference was noted in the susceptibility of the two different tumor target cell populations (K562 and NCI-H69) to AM-mediated cytotoxicity.
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Examination disclosed polyadenopathy, hepatosplenomegaly, and synovitis of all distal interphalangeal joints, most proximal interphalangeal joints and both wrists. She also had hypotension (blood pressure 80/40 mm Hg). Laboratory results (Table 1) disclosed pancytopenia and increased ferritin, mildly elevated liver enzymes (aspartate aminotransferase 61 U/l), hypofibrinogenemia (1/47 g/l), normal coagulation measurements, and normal triglycerides. A bone marrow biopsy disclosed moderate to severe hemophagocytosis (Figure 1). A diagnosis of macrophage activation syndrome (MAS) was made. Anakinra 100 mg subcutaneously daily was initiated, along with pulse methylprednisolone 1 g/day for 3 days, then prednisone 25 mg twice daily. A dramatic response was observed. Within 48 hours of anakinra treatment, blood count increased and fever abated.
One of the most important prognostic factor for atherosclerotic CVD is age, and the accumulation of genomic DNA mutations is a well-known feature of cellular aging. Recently, it has been demonstrated that somatic mutations leading to clonal expansion in the absence of other hematologic abnormalities, or clonal hematopoiesis of indeterminate potential predisposed to increase risk of atherosclerotic CVD.
Next, the medium was ultracentrifuged at 100 000g at 4°C for 1 hour. After that, the EV pellets were resuspended in DMEM in the same volume as the collected culture media or another medium at the indicated volume. The protein concentrations of the EV preparations were quantified using a MicroBCA Protein Assay Kit (Thermo Scientific). Both circulating and in vitro EVs were stored at 4°C and used to treat cells or to perform other experiments within 48 hours. All steps for isolation of the EVs that were to be used for cell treatments were performed using sterile techniques.
A series of findings have revealed that cytokines secreted by foam cells are important for VSMC proliferation and migration into the intima and for extracellular matrix production, in that matrix production is responsible for the formation of fibrous plaques. Nevertheless, the forms of communication between macrophage‐derived foam cells and VSMCs have not been fully defined.
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To determine whether FSTL-1 might improve the sensitivity of diagnosis of MAS, FSTL-1 levels were compared to known markers. Serum FSTL-1 levels were compared with ferritin, sIL-2Rα, and ESR in all patients at all timepoints of disease as well as in patients at onset and resolution of MAS (Figure 2). Serum FSTL-1 was highly correlated with both sIL-2Rα (Figure 2A, 2B) and with ferritin (Figure 2C, 2D), but correlated least well with ESR (Figure 2E, 2F). Of the 7 patients with active MAS, 6 showed an ESR of 30 or less despite markedly elevated FSTL-1 levels. The mean ESR in the MAS group was 26 mm/h, while the mean ESR in the pretreatment (sJIA presentation) group was 90 mm/h. Low ESR at onset of MAS is a well-established laboratory finding in sJIA, due to fibrinogen consumption and decreased production of fibrinogen2.
Figure S2. U937 FC‐EVs promote VSMC migration and adhesion. A, Foam cell formation for U937 cells. B through D, Data for wound‐healing and cell‐adhesion assays on VSMCs after indicated treatment. B, Representative photograph of VSMC migration in the wound‐healing assay. C, Quantification of VSMC migration. D, Result of VSMC‐adhesion experiments. FC‐EVs, foam cell–derived extracellular vesicles; NM‐EVs, normal macrophage–derived extracellular vesicles; VSMC, vascular smooth muscle cell.
Although HLH appears to be a disease of excessive immune activation, the ideal form of immune suppression/anti-inflammatory therapy remains unknown. Although somewhat responsive to corticosteroids and clearly responsive to etoposide or anti-T-cell serotherapy (ATG or alemtuzumab), HLH remains difficult to treat. In the future, a variety of rationally designed immunosuppressive agents are likely to come into clinical use for transplantation or autoimmune disorders. Some of these agents may also prove to be useful for the treatment of HLH.
After starting therapy, patients should be monitored closely for signs of improvement as well as potential complications and toxicities. An example strategy is outlined in Figure 6. Patients may follow a highly unpredictable and dynamic clinical course, which may require customization of therapy. For patients who respond well, with resolution of symptoms and normalization of inflammatory markers, therapy may be weaned per protocol. However, dexamethasone doses and etoposide frequency may need to be increased in response to disease reactivation (see “Salvage therapy”).
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The Histiocyte Society opened a new trial in 2004, HLH-2004, which is currently enrolling patients. The major modifications from HLH-94 were to move cyclosporine dosing to the beginning of induction and add hydrocortisone to intrathecal therapy.
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The pretransplant conditioning regimen in malignancy-associated HLH (Mal-HLH) may be guided by disease-specific protocols, as provided by local standards, which includes MAC to optimally control the underlying disease. In patients in whom HLH-causing mutations are detected, HLA typing of close relatives should also integrate screening for the same gene mutations, to avoid a stem cell source with the same pathogenic biallelic mutation(s).
The absorbance of the plates was read at 450 nm on a microplate reader. A titration of purified FSTL-1 was used to generate a standard curve from which serum concentration of samples was calculated. Serum levels of sIL-2Rα were measured using a Quantikine ELISA kit (R&D Systems) according to the manufacturer’s instructions.
Although macrophage hemophagocytosis is considered highly specific for MAS/r-HLH, the pathogenic role of macrophages is a matter of debate. Macrophages have been proposed to have a causal role, to be secondarily activated, or even to have antiinflammatory activity2. Although cells staining positive for CD68, a macrophage cell marker, are increased during the development of HLH in prf−/− mice, a causal role of macrophages in the pathogenesis of HLH was not established in this experimental model6. On the other hand, nonlymphoid cells are highly expanded and may play a role in the production of IFN-γ in the repeated CpG model of secondary HLH18. Macrophages have been demonstrated to produce IFN-γ under different kinds of stimuli28,29. Macrophages producing TNF-α and IL-6 have been found in livers of patients with HLH secondary to infections or MAS/r-HLH21. A characteristic of MAS/r-HLH is the presence of macrophage-expressing CD163, which is traditionally correlated with antiinflammatory scavenger activity of fully differentiated macrophages30. Liver biopsies of patients with HLH secondary to infections or MAS/r-HLH also show infiltrating CD68+ macrophages, which are often considered highly proinflammatory21. Immunohistochemistry of bone marrow aspirates from patients with MAS/r-HLH in s-JIA shows similar numbers of CD68+ and CD163+ macrophages (Bracaglia and Strippoli, unpublished data). However, the phagocytic activity and production of inflammatory cytokines by macrophages from patients with MAS/r-HLH in s-JIA have not been reported.
If hemophagocytic activity is not proven at the time of presentation, further search for hemophagocytic activity is encouraged. If the bone marrow specimen is not conclusive, material may be obtained from other organs. Serial marrow aspirates over time may also be helpful. The following findings may provide strong supportive evidence for the diagnosis: spinal fluid pleocytosis (mononuclear cells) and/or elevated spinal fluid protein and histological picture in the liver resembling chronic persistent hepatitis (biopsy). Other abnormal clinical and laboratory findings consistent with the diagnosis are cerebromeningeal symptoms, lymph node enlargement, jaundice, edema, skin rash, hepatic enzyme abnormalities, hypoproteinemia, hyponatremia, and elevated very low-density lipoprotein (VLDL↑)/low high-density lipoprotein (HDL↓).
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Matrix A. A(k1,k2) defines state of tumour cell at the (k1,k2) element. A(k1,k2)=0: proliferating tumour cells.
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HLH has to be considered when cytopenia is unduly prolonged after chemotherapy, fever persists in spite of antibiotic treatment, and other HLH parameters are present. These patients benefit from anti-inflammatory treatment with corticosteroids (prednisolone 1-2 mg/kg or dexamethasone 5-10 mg/m2) and, possibly, IVIG 1/6 g/kg over 2 to 3 days. Etoposide should be used sparingly, because bone marrow recovery is central for immune reconstitution. Ongoing monitoring is required to detect recurrent malignant disease as a potential alternate HLH trigger.
Primary HLH (p-HLH) is caused by mutations of genes coding for proteins involved in granule exocytosis, including perforin, Munc13-4, syntaxin11, and Munc18-25. In the absence of a genetic cause or familial inheritance, secondary HLH occurs in response to infections, associated with malignancies (particularly lymphoma), or in the context of rheumatic disease (MAS/r-HLH). Studies in patients with p-HLH, as well as in murine models of p-HLH6,7,8,9,10,11, support the hypothesis that a defective cytotoxic CD8+ T cell response and an alteration of the antigen-presenting cell (APC)–CD8+ T cell crosstalk leads to defective silencing of the immune response and abnormal T cell activation2,3,6,9,12. The end result of this process is uncontrolled production of proinflammatory cytokines by macrophages and T lymphocytes, leading to organ damage and hematologic alterations13,14. Indeed, characteristic of HLH, although not necessary for the diagnosis, is the presence of macrophages phagocyting blood cells in liver or bone marrow15.
Previous studies demonstrating that both Δ9-THS and marijuana smoke can reduce the antibacterial activity of AMs were performed solely with animal cells and animal models (12, 35– 37). Our results are the first to demonstrate that these effects occur in the lungs of habitual smokers. In the case of MS, we observed two distinct deficiencies in their response to S. aureus: reduced phagocytosis and reduced bacterial killing. These results differ somewhat from a previous study in which we found cells from MS to be defective in their ability to kill Candida albicans but normal in their phagocytic activity (29). This discrepancy is likely due to differences in the way AMs interact with bacteria and fungi. Phagocytosis is a complex process involving a host of different cell membrane receptors, cytoskeletal elements, and intracellular signaling pathways (38). While the pathways responsible for the phagocytosis of S. aureus and C. albicans have not been fully elucidated, they likely differ in many respects. We found that AMs from MS, although defective in their ability to take up S. aureus, were normal in their ability to phagocytose another fungus, Candida pseudotropicalis (data not shown).
Emergencies in Pediatric Rheumatology
The present study was designed to assess the in vivo effects of regular use of marijuana and cocaine on the immune function of human AMs. AMs are one of the central mediators of lung immunity and, because of their location within the alveolus, are exposed to high concentrations of these drugs. We recruited healthy nonsmokers or long-term smokers of tobacco alone, marijuana alone, or cocaine alone and recovered AMs from their lungs by bronchoscopy with bronchoalveolar lavage (BAL). These effector cells were analyzed for their antibacterial activity, their ability to kill tumor cells, and their ability to produce both inflammatory and immunosuppressive cytokines. Our results show that regular smoking of marijuana or cocaine, but not tobacco, dramatically alters the function of AMs. The ultimate outcome of these effects may be an enhanced susceptibility to infectious disease, cancer, and AIDS in both marijuana and cocaine users.
The macrophage then exhibits the antigens from the destroyed bacterium. Macrophages engulf the agglutinate.
VSMCs were placed in 96‐well plates and grown to 80% confluence and then treated with FBS‐free medium containing 20 μg/mL of FC‐EVs for 0 minutes, 15 minutes, 30 minutes, 1 hour, or 2 hours. The cells were washed with PBS and fixed with methanol for 5 minutes and then washed 3 times with PBS and blocked with 2% bovine serum albumin for 2 hours at 37°C. The integrins on the cell surfaces were evaluated using primary monoclonal antibodies for integrin β1 (ab52971; Abcam) or integrin α5 (ab150361; Abcam), followed by binding of an HRP‐conjugated secondary antibody (1:2000). The bound antibodies were quantified using a TMB peroxidase EIA substrate kit (Thermo Scientific), and the intensity of the color developed at an optical density at 450 nm was evaluated. Five parallel wells were set up for each condition.
Two months after the diagnosis of MAS, the patient was feeling well with anakinra 100 mg daily and prednisone 5 mg twice daily. She had no more fever and only mild residual joint pain and no synovitis. Ten months after the initiation of anakinra, she is well and has minimal morning stiffness but no other symptoms.
Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc analysis. Data presented as mean ± SD *P ≤ 0/05, **P ≤ 0/01, ***P ≤ 0/001.
Our study demonstrates that serum levels of FSTL-1 are elevated at the onset of sJIA and decline over the course of treatment. This is also, to our knowledge, the first reported association of MAS with a protein produced by cells of the mesenchymal lineage. These results support our earlier studies suggesting that FSTL-1 is a mediator of the inflammatory mechanisms that underlie arthritis, and specifically may be related to the IL-1β and innate immune pathways in sJIA12,13,14. We have also demonstrated for the first time that FSTL-1 is elevated in MAS and reverts to normal following treatment of MAS.
Host–pathogen relationships are characterized by the complex interplay between host defense mechanisms and attempts to circumvent these defenses by microorganisms (1, 2). Macrophages play key roles in host defense by recognizing, engulfing, and killing microorganisms (3). Among the microorganisms recognized by macrophages, bacteria are an important and highly diverse class of human pathogens. Bacterial pathogens that overcome host defenses ensure their ability to survive and propagate (4). Therefore, a thorough understanding of the normal host response to bacteria provides a foundation to understand bacterial tactics for evading these responses and thus disease prevention.
Effects%20of%20Microgravity%20and%20Space%20Flight%20on%20Macrophages PowerPoint PPT Presentation
Freshly elutriated human monocytes (>95% pure) were purchased from Advanced Biotechnologies (Columbia, MD). Monocytes were cultured at a density of 2 × 107 cells/10 ml of DMEM (Invitrogen) with 20% FCS (Intergen, Purchase, NY), 10% human serum (Nabi, Boca Raton, FL), and 50 μg/ml gentamicin (Invitrogen) in Primaria T-25 flasks (Becton Dickinson) for 5 days at 37°C, 5% CO2. On days 5 and 7, half of the media was removed and replaced with media lacking FCS. Media on the cultured macrophages was changed to 5 ml of DMEM with 1% human serum on day 9, 1 hour before experiments were begun.
These models based on genetic alterations in the granule exocytic pathway are models of p-HLH and therefore represent tools to investigate cellular and molecular mechanisms of diseases caused by genetic mutations. However, the findings may not be directly translated to the setting of secondary HLH triggered by infections or presenting in the context of rheumatic diseases. Four models of secondary HLH have been published in the last year, of which 2 are triggered by living pathogens (Table 1).
KCa3/1 protein expression during macrophage differentiation and polarization was assessed by immunofluorescence and Western blotting (Figure 1A through 1C). Low KCa3/1 levels were present in the undifferentiated human monocytes. Compared with monocytes, significant upregulation of KCa3/1 expression was found in macrophage colony-stimulating factor–induced M0 macrophages. Macrophage polarization into the M1 and M2 subtypes is associated with inflammation and plaque stability. Using human monocyte-derived macrophages, we then investigated the expression of KCa3/1 during macrophage polarization into the M1 or M2 subtypes. M0 macrophages were stimulated with IFN-γ and LPS or with IL-4 for 48 hours to induce M1 or M2 polarization, respectively. Here, we found that IFN-γ and LPS stimulation increased KCa3/1 expression 3/2-fold, whereas IL-4 treatment increased KCa3/1 expression 2/1-fold.
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Concerning genetic background, recent evidence suggests a potential role of the same genes involved in p-HLH. One study showed heterozygosity for low-penetrance variants of perforin in some patients with MAS/r-HLH24; another reported the association between a specific Munc13-4 haplotype and development of MAS/r-HLH in patients with s-JIA34. To our knowledge, no information is available on other genes involved in p-HLH. One could hypothesize that hypomorphic variants in each of these genes, as well as in other unknown genes of the cytotoxic pathways, may contribute. The genetic background may also affect the intensity of the response to TLR ligands. A recent report demonstrated an association of an interferon regulatory factor-5 (IRF-5) polymorphism with susceptibility to MAS/r-HLH in Japanese patients with s-JIA35. IRF-5 is one of the transcription factors that mediate TLR signaling. Although this observation needs confirmation in white subjects, it points to an additional potential contribution of the genetic background.
Special approaches of tumor biology and chemotaxis PowerPoint PPT Presentation
Preliminary examination included a detailed respiratory and drug use questionnaire, a medical history, a physical examination, urine drug screen and pregnancy (female subjects only) tests, platelet count, coagulation studies, and HIV serology. All volunteers provided written informed consent in accordance with the policies of the UCLA School of Medicine Human Subject Protection Committee.
Overview of the HLH-94 treatment protocol. Note that dose and frequency adjustments of this protocol are advised for adult patients (see Statement 7).
Figure 5. FC‐EVs activate the ERK and Akt pathways. A, A representative Western blot result of the phosphorylated forms of ERK and Akt and the total ERK and Akt from VSMCs that had been treated with FC‐EVs for the indicated time. B, The line graph of the results of the Western blot. The phosphorylation level was calculated as the ratio of the integrated optical density value of the phosphorylated protein to that of corresponding total protein. C and D, The results of the Western blot for VSMCs treated with the normal macrophage–derived EVs. FC‐EVs indicates foam cell–derived extracellular vesicles; P, phosphorylated; T, total; VSMC, vascular smooth muscle cell.
Biology - Biology Topic 1 Notes PowerPoint PPT Presentation
However, they were unable to reduce the inflammation in vivo. This lack of efficacy in vivo was attributable to the high density of the hydrogel cross-linking, which limited cytokine diffusion into the gel matrix and diminished the neutralization efficacy. This result suggested that when designing such conjugates, hydrogel cross-linking density needs to be optimized to maintain gel-like properties while maximizing cytokine influx for effective neutralization. Meanwhile, tuning the residence time of cytokine-neutralizing antibodies could be an effective strategy in regulating the inflammatory response associated with acute injuries.
The induction and activation of PPARδ by apoptotic cells made it an ideal candidate to serve as a molecular switch for controlling macrophage deactivation. This was indeed the case as apoptotic cells were unable to enhance their own clearance or suppress expression of proinflammatory cytokines in PPARδ-null macrophages.
In the absence of any additional stimulation, AMs produced minimally detectable levels of inflammatory cytokines (data not shown). However, as shown in Figure 5, exposure to 1 μg/ml of LPS stimulated production of high levels of TNF-α, IL-6, and GM-CSF. In contrast to AMs from TS, which produced cytokine levels similar to those produced by AMs from NS, AMs from MS produced significantly less TNF-α (p = 0/05), IL-6 (p < 0/01), and GM-CSF (p < 0/01). While cytokine production tended to be the most variable in AMs from CS, the latter produced no significant differences in the average amount of cytokines when compared with AMs from NS. The pattern of cytokine production was different with respect to the potent inhibitory cytokine TGF-β. First, in contrast to the other cytokines, TGF-β was not stimulated by the presence of LPS. Second, AMs from MS produced levels of TGF-β that were similar to those produced by AMs from NS and TS. Cells from CS tended to produce less TGF-β, but this difference did not reach statistical significance when compared with NS (p = 0/2). Because cytokines are important regulators of macrophage function, we hypothesized that the decrement in inflammatory cytokine production seen in cells from MS might correlate with their decrement in effector function. However, linear correlation analysis failed to find a significant correlation between the production of any single cytokine and the corresponding effector cell activity for any of the groups studied. The sample sizes were too small to allow multivariate analysis.
Hemophagocytic lymphohistiocytosis: report of 122 children from the International Registry. FHL Study Group of the Histiocyte Society.
Structural Biology of HIV PowerPoint PPT Presentation
Elevated pretreatment serum FSTL-1 levels in sJIA are associated with dysregulated gene expression suggestive of occult MAS, and may have utility in predicting progression to overt MAS. Ferritin/ESR ratio may be superior to ferritin alone in discriminating overt MAS from new-onset sJIA.
The reported data show that FC‐EVs have significantly different effects on VSMCs than an equivalent amount of NM‐EVs. These results implied that the contents of FC‐EVs may differ from those of NM‐EVs. Consequently, we performed a proteomic experiment to define the protein profile of FC‐EVs. The proteomic experiments were performed on 2 FC‐EV and 2 NM‐EV samples analyzed in duplicate using nano–liquid chromatography and tandem mass spectrometry.
A Juvenile Rheumatoid Arthritis
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MB207 Molecular Cell Biology PowerPoint PPT Presentation
However, we cannot completely exclude that it is not a direct effect of TRAM-34 treatment on VSMCs in advanced lesions. In endothelial cells, the blocking KCa3/1 channels acts as a double-edge sword.
We developed a Trucount tube–based flow cytometry method to detect and quantify circulating EVs. Because exposed PS is a marker of EVs, we identified EVs by labeling with annexin V. It is known that circulating EVs originate from multiple sources and that they carry some markers of the cells of origin. On the basis that atherosclerosis is a progressive chronic inflammatory disease,38 we focused our interest on leukocyte‐, monocyte‐, and macrophage‐derived EVs and identified them by labeling with CD45.
Cell Biology Lectures PowerPoint PPT Presentation
EVs were then isolated from the CellTracker‐labeled foam cells and used to treat VSMCs for 1 hour. VSMCs were washed twice, fixed for 10 minutes, stained with Dil (red fluorescence to label the lipid membranes) and DAPI (blue fluorescence to label the nuclei), and prepared for confocal laser scanning microscopic imaging.
Moreover, we found that macrophage‐derived foam cells released a higher number of EVs than normal macrophages. Because EVs mediate intercellular communication, we proposed that intimal foam cells may modulate the function of VSMCs via EVs during the progression of atherosclerosis. In this study, we extensively assessed the effects of foam cell–derived EVs (FC‐EVs) on VSMC adhesion and migration.
Patients with CNS HLH need close posttransplantation follow-up. We recommend examination of spinal fluid within 100 days of HCT, even in asymptomatic patients. Follow-up MRIs are recommended if pretransplantation abnormalities were present. In some patients with mixed or full hematopoietic donor chimerism, HLH disease activity in the CSF can be effectively treated with intrathecal therapy during the early posttransplantation months. CNS disease is subsequently controlled as donor immune reconstitution progresses.
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Intracellular killing assays were performed in the presence and absence of NGMMA to measure the role of NO as an effector molecule involved in bacterial killing. As shown in Figure 3, inhibition of NO synthase significantly reduced the capacity for AMs from NS to kill S. aureus (p < 0/01). A similar effect, although slightly less prominent, was observed when NGMMA was added to the AMs from TS (p ⩽ 0/05). On the other hand, the presence of NGMMA did not significantly affect the killing activity of AMs from either CS or MS. Approximately 20% of the input bacteria were killed by AMs from CS both in the presence and absence of this NO inhibitor.
Pediatric Rheumatology Fellowship P
Macrophage/CD4+ CCR5+ Th1 lymphocytes cross-talk during MTB/HIV. Reinforced HIV replication and mycobacterial dissemination IL-1b/TNF-a HIV MTB TLR-2 La.
Statement 5: Lymphoma as a hidden trigger of HLH may be difficult to detect. Use of positron emission tomography–guided imaging, repetitive tissue sampling, and consultation with a lymphoma reference pathologist, are recommended (strong consensus).
Wounds on the backs of C57BL/6 wild-type mice were inflicted by 6 mm punch biopsy and treated with hydrogel discs for 5 days. RNA was isolated from whole wound tissue, gene expression was analyzed, and expression was calculated and compared with an unwounded skin. Each symbol represents one wound. Bars represent mean ± SD. Analysis of variance (ANOVA) was performed with multiple comparisons versus PEG/PEG using Bonferroni t-test or Dunnett’s method: ***P ≤ 0/001, **P ≤ 0/01, *P ≤ 0/05 (b and c). Unpaired t test: **P ≤ 0/01, *P ≤ 0/05.
Medical%20Virology%20Immunology PowerPoint PPT Presentation
Immunologic and genetic workup of HLH. Rapid immunologic testing (which may be performed in 1-3 days) may support a diagnosis of HLH and provide etiologic data, whereas gene sequencing (typically requiring 3-8 weeks) may define the underlying genetic cause. Measurement of NK cytotoxic function and sCD25 may also support the diagnosis of HLH but is not included in the diagram because abnormalities in these assays do not suggest specific genetic lesions. Of note, whereas an abnormal test suggests an underlying gene abnormality, a normal immunologic test does not preclude genetic testing. Genetic testing should be pursued until biallelic (or hemizygous) mutations are found or until all 5 genes (or 7, in the case of males) are assayed. LYST gene sequencing is not currently commercially available.
Figure 6. FC‐EVs influence VSMCs by conveying proteins. A, The result of confocal microscopy of VSMCs. VSMCs were stained with fluorescent dyes (red Dil for the lipid membranes and blue DAPI for the nuclei) and preincubated with FC‐EVs that had been labeled with CellTracker (green). B and C, The protein levels of integrins β1 and α5, respectively, on the surfaces of VSMCs after treatment with FC‐EVs for the indicated time, as indicated by the cell enzyme‐linked immunosorbent assay experiments. D and E, The protein levels of integrins β1 and α5 on surfaces of VSMCs following treatment with FC‐EVs after the protein expression in VSMCs was blocked with cycloheximide.
Biology 211 Anatomy PowerPoint PPT Presentation
The samples were lysed using RIPA buffer in the presence of protease inhibitors and phosphatase inhibitors. The lysates (30 μg proteins per lane) were subjected to electrophoresis on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes according to standard procedures. The membranes were blocked for 1 hour and incubated with the primary antibody overnight at 4°C, followed by incubation with the corresponding HRP‐conjugated secondary antibody for 1 hour. Antibody binding was detected using a Pierce Super Signal West Pico Kit (Thermo Scientific). All antibodies were diluted according to the manufacturers' protocols. The sources of the primary antibodies were as follows: anti–total ERK (ab17942; Abcam), anti–phosphorylated ERK (ab4819; Abcam), anti–total Akt (4691; Cell Signaling Technology), and anti–phosphorylated Akt (2965; Cell Signaling Technology).
This study revealed, for the first time, the proteome of the NM‐EVs and FC‐EVs. Meanwhile, the work reported demonstrated that FC‐EVs can stimulate the migration and adhesion of VSMCs as well as transfer integrins (receptors for fibronectin and laminin) to the surface of VSMCs and activate pathways in the cell.
After the ultrasound detection at 9 weeks after surgery, the mice were euthanized, and the left common carotid arteries were isolated after pressure perfusion. Compared with the vehicle-treated group, the animals that received TRAM-34 injection showed an obviously lower incidence of fresh left common carotid arterial lumen thrombus associated with severe plaque burden (1/9 versus 6/9; P=0/016; Figure 4A). Plaque composition was assessed with special stains and immunocytochemistry coupled with quantitative image analysis (Figure 4B and 4C). TRAM-34 treatment obviously decreased the intimal surface area (149 217±54 524 versus 102 766±37 009 μm2; P=0/025) relative to the vehicle-treated mice. Although lipid deposits make plaques more prone to rupture, collagen fibers stabilize plaques. Areas of lipid deposits and collagen fibers within a plaque were defined using oil red O and adjacent Sirius-stained tissue sections, respectively, and the ratio between these 2 areas was calculated. TRAM-34-treated mice showed reduced lipid contents (15/6±4/9% versus 22/2±6/6%; P=0/014) compared with vehicle-treated control mice. No changes in the collagen contents were observed after TRAM-34 treatment (35/2±6/2% versus 36/4±8/2%, P=0/363). Consequently, TRAM-34-treated mice displayed increased collagen/lipid ratios compared with control mice (2/46±0/67 versus 1/67±0/43; P=0/005). TRAM-34 treatment increased vascular smooth muscle cells (VSMCs) content (21/9±4/8% versus 17/2±5/6%; P=0/037) and reduced macrophages (21/0±5/3% versus 31/3±8/1%; P=0/003) in carotid plaque lesions.
Use of marijuana and “crack” cocaine is on the rise in the United States (1), as is public acceptance of marijuana for medicinal use in patients with cancer and AIDS (2, 3). Understanding the health effects of these drugs is therefore more important than ever. While marijuana is known to produce chronic bronchitis and a cellular alveolitis in chronic users (4, 5), and cocaine can induce “crack lung” in cases of extreme abuse (6), relatively little is known about the effects of these inhaled drugs on pulmonary host defenses.
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If a patient is treated successfully and then weaned off of therapy because he is deemed to not need HCT, he should be monitored closely. Because the distinction between primary and secondary HLH is increasingly blurred, blanket recommendations for off-therapy patients are difficult to make and most follow-up decisions will have to be individualized. It appears that many patients who relapse do so within 1 year. In the absence of complicating medical issues, monthly follow-up once off therapy appears advisable during the first year, followed by annual follow-up thereafter.
To determine whether KCa3/1 channels are involved in the polarization process, macrophages were also treated with TRAM-34 during IFN-γ and LPS or IL-4 stimulation. Surface marker expression and cytokine profiles during macrophages polarization were evaluated by flow cytometry and real-time polymerase chain reaction. M1 macrophages are characterized by higher cell surface marker levels, including CD80 and CD197. M2 macrophages differed from M1 in that they expressed higher levels of CD163 and CD206. Our results showed that IFN-γ plus LPS significantly increased the expression of CD80 and CD197 with no effect on CD163 and CD206, whereas IL-4 only significantly upregulated CD163 and CD206 levels. We also revealed that coculture with TRAM-34 significantly reduced IFN-γ and LPS-induced CD80 and CD197 expression. In contrast, TRAM-34 treatment only slightly decreased IL-4-induced CD163 and CD206 expression, and there was no statistically significant difference (Figure 2A; Figure I in the online-only Data Supplement). Consistent with these observations, blocking KCa3/1 significantly attenuated proinflammatory cytokine and chemokine mRNA expression in M1 macrophages, including CCR7, tumor necrosis factor-α, and IL-12p35. However, M2 macrophage markers, such as CXCR4, CD206, and IL-10, were unaltered or only marginally decreased with TRAM-34 cotreatment (Figure 2B). Moreover, similar results were obtained in KCa3/1 siRNA-treated THP-1 cells, which displayed similar KCa3/1 channel expression patterns during differentiation and polarization (Figure II in the online-only Data Supplement).
The difference index represents how expression levels induced by the three bacteria differ from the average expression level induced by all bacteria studied: large values are assigned to genes whose expression levels exhibit the greatest differences in specific bacterial infections. Multiple time course experiments were used (M. tuberculosis, two repeats; E. coli, three repeats; and S. aureus, two repeats). For every gene within each profile, the responses of macrophages to each of the bacterial species in question were compared with the average response to all bacterial species studied. Statistically significant differences were identified by using Student's t test (P < 0/005).
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Human monocytes were isolated from peripheral blood of 12 healthy volunteers and cultured in RPMI (Roswell Park Memorial Institute) medium supplemented with 10% fetal bovine plasma. We obtained informed consent from all subjects and a license from the ethical committee. Macrophage differentiation of human monocytes was induced by treatment with 100 ng/mL macrophage colony-stimulating factor. Human monocytes-derived macrophages were treated with 100 ng/mL IFN-γ and 10 ng/mL lipopolysaccharide (LPS) for M1 differentiation. M2 polarization was induced by incubating macrophages with 15 ng/mL IL-4.
Macrophage activation syndrome - English
HLH has been reported primarily with lymphomas or leukemias of the T or NK cell lineages, but associations with anaplastic large cell lymphoma, early B lineage lymphoblastic leukemia, myeloid leukemias, mediastinal germ cell tumors, and rarely other solid tumors are also found. Many of these patients simultaneously have a bacterial, viral, or fungal infection that may serve as a trigger for the HLH in the context of a dysfunctional immune system that has occurred because of chemotherapy for the malignancy or perhaps cytokine production by the malignant cells. CTs of the chest and abdomen, as well as the bone marrow aspirate and biopsy, at the time of presentation are helpful to evaluate possible underlying malignancy.
Macrophage gene expression was measured at 1, 2, 6, 12, and 24 h after the introduction of bacteria or latex beads. One hundred ninety-one genes met criteria for significant changes (see Materials and Methods) on exposure to six of eight bacteria studied. The genes are ordered along the y axis by category, with the degree of change indicated by color intensity in the color bar. The display has been described in detail elsewhere (8).
IL-12 plays a fundamental role in generating T helper 1 (Th1) immune responses (22) and is critical for host resistance to tuberculosis infection in mice and in humans (23–27). Repression of IL-12 production may enhance the survival of M. tuberculosis against the innate immune response or the developing adaptive immune response. This notion is supported by two observations. First, supplemental IL-12 enhances the ability of normal mice to clear a mycobacterial infection (24, 28). Second, exogenous IL-12 therapy has rescued two patients, one with a pulmonary Mycobacterium abscessus infection and one with disseminated M. tuberculosis infection, whose infections were refractory to antibiotics and supplemental IFN-γ (29, 30). Our findings support the idea that both IL-12 and IL-15 could be useful therapies for clinical tuberculosis, suggested by the animal models (24, 28, 31).
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IL-6 was assayed using a standard ELISA kit (Genzyme, Cambridge, MA) according to the manufacturer's protocol. Samples were run in duplicate with cytokine standards and measured on a microplate reader (Spectra/SLT-Lab Instruments, Salzburg, Austria).
It is well known that a large number of soluble molecules in culture media may influence VSMC behavior. Unfortunately, there is no perfect method to isolate EVs from media without incorporating any soluble molecules. To eliminate the interference of the soluble molecules contained in EV preparations, we treated VSMCs with the foam cell culture media and EV‐depleted culture media (foam cell culture media without EVs) in the migration and adhesion experiments. As shown in Figure 3A through 3C, removal of EVs from the media resulted in significant decreases in the capability of the foam cell culture media to promote VSMC migration (28/7% decrease) and adhesion (17/9% decrease).
C, The distribution of the EV diameters as indicated by dynamic light scattering analysis. D, Western blot analysis for the EVs and EV‐depleted supernatant. E and F, Representative density plots of the flow cytometric analysis of PS‐ and CD45‐positive NM‐EVs and FC‐EVs. G, Quantification of the numbers of PS‐ and CD45‐positive EVs.
Through characterization of human tissue samples and animal models, a complex picture of cellular processes has been shown to play pivotal roles in monocyte/macrophage inflammation in CVD. In this brief review, we will discuss recent findings that provide mechanistic insight into the role of epigenetics in the regulation of macrophage phenotype and function in the setting of inflammation. Additionally, we will highlight the impact of epigenetic modifications on maladaptive inflammation in cardiometabolic and vascular disease.
Evolutionary Biology PowerPoint PPT Presentation
Although the term MAS is commonly used, particularly by rheumatologists, the condition has recently been linked to secondary hemophagocytic lymphohistiocytosis (HLH). Indeed, similarities in clinical and laboratory features, genetic background, and pathogenesis with primary HLH are becoming more evident. The term rheumatologic-HLH (rheuma-HLH or r-HLH) has been recently proposed4.
Various triggers have been reported, among them the use of antituberculous agents and gold salts5. These might have been triggers in our patient, but the use of antituberculous drugs was longstanding, and gold salts were stopped before the initiation of the syndrome. The prednisone taper with secondary increase of systemic inflammation might also have been responsible. The diagnosis of MAS is sometimes difficult. Diagnostic criteria have been described in children with systemic juvenile idiopathic arthritis6,7. Our case fulfilled these criteria, but there are no diagnostic criteria for adults. It is probable that AOSD and MAS represent a continuum of disease with varying degrees of severity, and that MAS is underdiagnosed in AOSD4,8.
This sequence also reflects the common clinical observation that hemophagocytosis may not be found in initial biopsies and yet may linger in later biopsies, even when other disease parameters appear to be improving. Second, these categories reflect the uniqueness of HLH as a syndrome. We have divided most of the acute features of HLH as either indicating immune activation or immune-mediated pathology because this reflects the essence of this disease process. Although patients with a variety of inflammatory conditions display immune activation, normal cytotoxic immune regulatory mechanisms tend to dampen immune activation before unusual or paradoxical immunopathology (category 3) develops. The combination of acute systemic immune activation and the specific findings listed in category 3 is largely what distinguishes HLH from other inflammatory disorders. Organ-specific infectious or autoimmune processes (such as hepatitis, meningitis, or aplastic anemia) do not tend to display the systemic inflammatory and/or multiorgan features of HLH.
The data were analyzed by using a custom-built Oracle (Redwood Shores, CA) database, chipdb. Fluorescence intensities were normalized to median array intensities for all conditions tested on cells from a single donor, floored at 50, and fold-change was calculated relative to duplicate time-0 controls.
Immunology PowerPoint PPT Presentation
Many CVDs have been recognized as inflammatory conditions characterized by the infiltration of monocytes that promote localized inflammation. Circulating blood monocytes originate in the bone marrow and are a heterogeneous leukocyte population with various subsets that differ in surface markers, gene-expression profile, and function. In humans, 3 main subsets can be distinguished on the basis of the expression of the cell surface markers CD14 and CD16.
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To determine whether markedly elevated serum FSTL-1 at disease onset might represent a subset of patients with a unique gene expression profile, PBMC gene expression was compared between patients with pretreatment FSTL-1 levels > 200 ng/ml versus < 200 ng/ml. FSTL-1 level of 200 ng/ml was selected as a division point for this analysis because it is midway between the median disease-onset FSTL-1 values for patients without MAS and the median FSTL-1 values at MAS presentation. Statistical analysis (p < 0/05 by Welch’s t test) yielded a total of 2660 significant genes, with 318 genes upregulated > 1/7-fold and 135 genes that were downregulated > 2-fold.
Interestingly, the proteomic data showed that numerous EV markers were contained in FC‐EVs. These markers included tetraspanin CD9, VAMP8, HSP7C, HSP90B, HSP90A, HSP60, HSP10, HSP70‐13, HSP70‐4, PCD6IP, MFGE8, LAMP1, integrin β1, integrin α5, integrin αM, and integrin β2. The proteomic analysis also showed some monocyte and macrophage markers, including CD14 and CD45.
Figure 1 | Major therapeutic platforms that directly capture and neutralize inflammatory cytokines. Monoplex platforms such as cytokine-neutralizing antibodies and antibodies conjugated with biomaterials are designed primarily with one specific cytokine as the target. Meanwhile, multiplex platforms such as glycosaminoglycan (GAG)-containing biomaterials and cell-membrane-coated nanoparticles are designed for concurrently neutralizing multiple cytokines that reflect the multiplexity of cytokines involved in inflammatory diseases.
VSMCs were plated in 6‐well plates using DMEM containing 10% FBS and cultured until cell monolayers formed. Monolayers were wounded by manual scraping with a 10‐μL micropipette tip and then washed. The cells were then incubated with medium containing 1% FBS alone or combined with the indicated concentrations of circulating or cell‐derived EVs or other treatment factors for 36 hours. The cells were fixed with methanol, stained with crystal violet, and photographed using an inverted microscope. The cells that migrated past the wound edge were quantified in 3 high‐power fields (left field, middle field, and right field).
FC‐EVs indicates foam cell–derived extracellular vesicles; VSMC, vascular smooth muscle cell
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Because primary graft loss is common, we monitor total donor engraftment weekly in the early months after HCT. If total engraftment declines, especially approaching 50%-60% in the early posttransplantation months, it is possible to halt the decline in donor chimerism by the reduction or discontinuation of immunosuppression for GVHD prophylaxis. When this approach does not favorably impact declining donor chimerism, escalating doses of donor lymphocyte infusions may stabilize or increase donor chimerism. Full donor chimerism after transplantation is not required to suppress HLH disease in the majority of patients. Although very low levels of donor cells have been detected in healthy long-term survivors of HCT for HLH, the “safe” level of persistent total blood, or subset, chimerism is not known.
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To evaluate the sensitivity and specificity of FSTL-1 for differentiating overt MAS from new-onset sJIA, we analyzed the levels of FSTL-1, sIL-2Rα, and ferritin in our patients. Because many patients with new-onset (pretreatmemt) sJIA have elevated FSTL-1, ferritin, and sIL-2Rα, but nearly all patients with MAS had decreased ESR, we compared the ratio of FSTL-1, sIL-2Rα, and ferritin to ESR for patients with overt MAS and pretreatment sJIA. We performed ROC analysis for all of these and found that both ferritin/ESR ratio and sIL-2Rα/ESR ratio were strongest in discriminating active MAS from new-onset sJIA (Figure 3; sIL-2Rα/ESR is not shown, but was equivalent in area under the ROC curve to ferritin/ESR). A ferritin/ESR ratio > 80 provided statistically optimal sensitivity and specificity (100%) in our sample.
PATHOGEN AND HOST TARGET DISCOVERY IN BRUCELLA ABORTUS INFECTED MACROPHAGES PowerPoint PPT Presentation
Edema, purpura, dyspnea, diarrhea, diffuse bleeding, icterus, and an overall sepsis-like appearance are extremes of severe HLH with onset of organ failure. Mild initial signs, including recurrent fevers, lymphadenopathy, organomegaly, rash, and arthralgias, may progress with an unexpected rapidity and severity. These signs and symptoms, despite adequate antimicrobial therapy and/or without detectable infectious focus, along with a dramatic clinical progression serve as red flags for possible HLH. No single clinical or laboratory parameter has sensitivity and specificity to allow an unambiguous HLH diagnosis. Close clinical observation with repeated physical examinations and laboratory assessments are mandatory for diagnosis.
The results showed that FC‐EVs markedly promoted VSMC migration and adhesion, with increases of 44/6% and 18/6%, respectively, relative to the control, whereas NM‐EVs had neither of these effects (Figure 3A through 3C). In the wound‐healing experiments (Figure 3A and 3B), FC‐EVs promoted VSMC migration 39/7% more than NM‐EVs. The results from the adhesion assay were similar. As seen in Figure 3C, FC‐EVs promoted VSMC adhesion 18/7% more than NM‐EVs. We also confirmed these experiments using THP‐1 and U937 cell models (Figures S1 and S2). We also found that FC‐EVs promoted endothelial cell migration and adhesion (Figure S3).
Emergencies in Pediatric Rheumatology 2870.5% of asy
A subset of tissue macrophages, such as splenic red pulp macrophages and osteoclasts, become highly specialized in their location and function. The splenic red pulp macrophages are adapted for clearance and recycling of senescent red blood cells, whereas osteoclasts are critical for remodeling of bone throughout adult life.
Correspondence to Junbo Ge, Department of Cardiology, Zhongshan Hospital, Fudan University, 180 Fenglin Rd, Shanghai 200032, China. E-mail [email protected]; or Juying Qian, Department of Cardiology, Zhongshan Hospital, Fudan University, 180 Fenglin Rd, Shanghai 200032, China.
Supported by NIH/NIDA Grants DA03018 and DA08254 (to Drs. Tashkin and Roth) and NIH Grant NS33432 (to Dr. Baldwin).
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Integrative Pediatric Rheumatology: How to help parents make the best decisions for their children
Resolution of injury, apoptosis and macrophages. Differential Macrophage Activation Macrophages in.
The recommendations are based on expert opinion supported by best available evidence from studies supporting the individual statements. They were initially proposed and discussed by e-mail and then selected and structured in a telephone conference. After further refinement by e-mail, each statement was discussed, revised, and voted on in face-to-face meeting, followed by final refinement by e-mail.
Emergencies in Pediatric Rheumatology - Français
Serum FSTL-1 levels during the course of sJIA. Sera were assayed by ELISA from sJIA patients pretreatment and 24 months after initiation of therapy and compared to matched controls. The interior line represents the median; upper and lower boundaries of the box represent the 75th and 25th percentiles; top and bottom whiskers represent the 95th and 5th percentiles. Dots represent individual values for patients’ FSTL-1. Two patients whose disease onset was with MAS were not included. B. Paired sera collected from 4 patients during and after an episode of MAS were assayed (p = 0/0096). FSTL-1: Follistatin-like protein 1; sJIA: systemic juvenile idiopathic arthritis; MAS: macrophage activation syndrome.
Figure 4. Histopathologic analysis of carotid lesions in vehicle- and TRAM-34-treated animal models. A, Representative gross anatomy of the left common carotid arterial segments at 9 weeks after surgery. B, Representative carotid artery cross-sections from vehicle- and TRAM-34-treated mice were stained with hematoxylin and eosin (H&E), Sirius red, or oil red O. C, Quantification of the intimal surface area, plaque collagen, and lipid content (n=3–6 in each group). Plaque stability was assessed by the collagen to lipid ratio. D, Representative photographs of sections of the carotid artery that were immunostained with either the smooth muscle cell–specific marker smooth muscle actin (α-SMA), the macrophage-specific marker Mac3, or the T cell–specific marker CD3. E, Quantification of α-SMA, Mac3, and CD3 in carotid lesions. Plaque stability was assessed with the forming α-SMA to Mac-3 ratio (n=6 in each group). F, Plasma cytokine levels for interleukin (IL)-1β, IL-2, IL-6, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MIP)-1α, MCP-1, and IL-10 (n=9 in each group). Statistical significance values (P) are indicated.
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- Epigenetic Regulation of Monocyte/Macrophage Phenotype
This study of host–pathogen interactions has provided insights into host defenses and pathogen-specific manipulations of those defenses that should have practical applications. Understanding the macrophage activation program, and the bacterial components that elicit it, will be useful in designing vaccines and cytokine therapies that engage the innate immune system in a targeted fashion. The expression data described here should provide a foundation for further studying the pathogenesis of these and other infectious agents such as fungi, viruses, and parasites.
The Western blot analysis (Figure 2D) and proteomics data (Table 1) described above showed that integrins β1 and α5 are contained in FC‐EVs. In this study, we chose these 2 proteins to verify that EVs transport proteins into VSMCs. On the basis that integrins are membrane proteins, we conducted a cell enzyme‐linked immunosorbent assay experiment to survey the progression of the transport of integrins from EVs to VSMCs. After treatment with FC‐EVs, the levels of integrins β1 and α5 on the VSMC surfaces were increased in a time‐dependent manner, with a significant increase after 1 hour. Ultimately, there were 26/3% and 24/9% increases in integrins β1 and α5, respectively, in VSMCs after 2 hours of treatment (Figure 6B and 6C).
Many patients will be admitted to the ICU because of delay in diagnosis or complications of the disease. Although individual findings in HLH may mimic features of sepsis and multiorgan dysfunction, close follow-up is needed to define the diagnostic criteria for HLH. The degree of abnormality of inflammatory markers may help to distinguish these disorders.
Reaching the Threshold: A Multilayer Pathogenesis of Macrophage Activation Syndrome
Patients may develop pulmonary dysfunction that leads to urgent admission to the intensive care unit. In a review of the radiographic abnormalities in 25 patients, 17 had acute respiratory failure with alveolar or interstitial opacities, with fatal outcomes in 88% of those cases.
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Figure 5. M1 and M2 phenotypic polarization of macrophages within atherosclerotic lesions. A, Carotid atherosclerotic lesions from vehicle- and TRAM-34-treated mice were probed with specific antibodies against the macrophage marker Mac3 and coprobed with antibodies against M1 markers (CD36 and FABP4). B, Atherosclerotic lesions were stained for M2 markers (Arg1 and Pgc1). C, Atherosclerotic lesions were stained for KCa3/1 channels. D, Quantification of the expression of CD36, FABP4, Arg1, Pgc1, and KCa3/1 in carotid lesions (n=6 in each group). Statistical significance values (P) are indicated. FABP indicates fatty acid–binding protein.
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AMs (2 × 106 cells) were suspended at a 1:1 ratio with S. aureus in a final volume of 1/0 ml of HBSS assay buffer. This suspension was then incubated with gentle rocking at 37° C in the presence or absence of 250 ng/ml of N G-monomethyl-l-arginine monoacetate (NGMMA; Calbiochem, La Jolla, CA), an inhibitor of nitric oxide (NO) synthase. A 0/1-ml aliquot of the suspension was removed at 0, 30, 60, and 120 min, diluted in 0/9 ml of sterile water, and sonicated with two 10-s pulses (60 W) using a Branson Sonifier 450 (Branson Ultrasonics, Danbury, CT). This sonication protocol effectively lysed 100% of the AMs without lysing the bacteria. The sonicate was then serially diluted and 0/1 ml was plated in each of duplicate trypticase soy agar plates. Plates were incubated at 37° C, bacterial colonies allowed to grow overnight, and the number of colonies counted the next day. Results are expressed as a direct ratio N/N0, where (N) is the number of colonies counted at each time point and (N0) is the number of colonies counted at time zero.
In a rodent model of deep partial-thickness burns, the conjugates were effective in attenuating the acute inflammation and reducing the secondary necrosis. This was validated further by the demonstration that much fewer immune cells infiltrated into the region where the polymer–antibody conjugates were retained. Notably, in polymer–antibody conjugates, the polymer backbone could affect the antibody–cytokine binding affinity, depending on the use of polymers. For example, anti-IL-1β conjugated with HA or carboxymethylcellulose (CMC) showed association kinetics comparable to that of the free antibody.
Media was removed at 4 h, and cells were washed with Hanks' balanced salt solution (HBSS, Invitrogen) and fed with 5 ml of DMEM with 1% human serum and 50 μg/ml gentamicin. Staphylococcus aureus strain ISP794, derived from strain 8325, and Listeria monocytogenes strain EGD were grown overnight in brain–heart infusion broth, pelleted, and resuspended in 0/5× vol of 20% glycerol to make frozen stocks that were thawed for the experiments. M. tuberculosis Erdman stain, obtained from Tuberculosis Research Materials and Vaccine Testing Contract, and Mycobacterium bovis BCG (ATCC no. 35734) were grown in Middlebrook 7H9 broth with 0/5% glycerol, 0/05% Tween 80, and ADC enrichment (Becton Dickinson). A 6- to 7-day-old culture was used for infection. Escherichia coli strain sd-4 (ATCC no. 11143) was grown in Luria–Bertani medium with streptomycin, and frozen stocks were created as described (6). The Salmonella typhi (Quailes strain), S. typhimurium (ATCC no. 14028) and enterohemorrhagic E. coli O157:H7 (EHEC) were provided as frozen stocks and thawed on the day of the experiment.