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Antecedents: The serum levels of some cytokines can be useful in the diagnosis of neonatal sepsis; the prognostic value of a cytokine profile has not, to our knowledge, been explored in this diseas...

Background Interleukin (IL) 16 and thymus and activation-regulated cytokine (TARC) are chemoattractant cytokines for eosinophils and T H 2 cells. Differential levels of these components in aspirin-exacerbated respiratory disease (AERD) and allergic rhinitis with asthma (ARwA) may be related to a different inflammatory response in both asthma phenotypes. Objective To assess the nasal lavage immunoreactivity of IL-16 and TARC cytokines. Methods We used multienzyme-linked immunosorbent assays to detect IL-5, IL-13, IL-16, IL-33, I-309/CCL1, TARC/CCL17, monocyte-derived chemokine/CCL22, periostin, and eosinophil cationic protein levels in nasal lavages from patients with AERD and patients with ARwA. Results The IL-13, IL-16, TARC, and periostin levels were significantly higher in patients with AERD compared with those of patients with ARwA. Correlation analysis of mediator levels in AERD revealed a possible role of IL-16 and TARC in eosinophil recruitment and activation. Conclusion IL-16, TARC, and periostin distinguish between patients with AERD and those with ARwA. These mediators, taken together rather than individually, may comprise good specific nasal markers in patients with AERD. The effects of IL-16 and TARC on T H 1, T H 2, and T-regulatory cell functions in AERD cannot be disregarded.

Aspirin-exacerbated respiratory disease (AERD) is an asthma phenotype characterized by nasal polyps, chronic hypertrophic eosinophilic sinusitis, asthma, and sensitivity to aspirin. Unawareness of this disease by patients and their physicians may have serious consequences because of the risk of near-fatal asthma. Its confirmative diagnosis can be established only by provocative aspirin challenge, which represents a potential risk for patient health. Purpose: Because CCL4 plays an important role in several pulmonary conditions, we tested its immunoreactivity in nasal lavages and sera from control subjects, and from aspirin-sensitive and non-aspirin-sensitive patients. Method: Immunoreactivity of CCL4 was measured in serum and nasal lavages from 30 healthy controls (HC), 23 patients with AERD, and 20 patients with aspirin-tolerant asthma (ATA). Additionally, a serum biobank contained sera from 90 HC, 83 patients with AERD, and 69 patients with ATA was employed. Results: The serum immunoreactivity of CCL4 could distinguish both types of asthma phenotypes. Conclusion: CCL4 may play an unexpected role in thephysiopathology of AERD.

Aspirin-exacerbated respiratory disease is a tetrad of nasal polyps, chronic hypertrophic eosinophilic sinusitis, asthma, and sensitivity to aspirin. Unawareness of this clinical condition by patients and physicians may have grave consequences because of its association with near-fatal asthma. The pathogenesis of aspirin-intolerant asthma is not related with an immunoglobin E mechanism, but with an abnormal metabolism of the lipoxygenase (LO) and cyclooxygenase (COX) pathways. At present, a diagnosis of aspirin sensitivity can be established only by provocative aspirin challenge, which represents a health risk for the patient. This circumstance has encouraged the search for aspirin intolerance-specific biomarkers. Major attempts have focused on mediators related with inflammation and eicosanoid regulation. The use of modern laboratory techniques including high-throughput methods has facilitated the detection of dozens of biological metabolites associated with aspirin-intolerant asthma disease. Not surprisingly, the majority of these is implicated in the LO and COX pathways. However, substantial amounts of data reveal the participation of many genes deriving from different ontologies. Biomarkers may represent a powerful, noninvasive tool in the diagnosis of aspirin sensitivity; moreover, they could provide a new way to classify asthma phenotypes.

The monocyte locomotion inhibitory factor (MLIF) is a heat-stable pentapeptide produced by Entamoeba histolytica in culture. This factor displays several anti-inflammatory properties ( i.e. , inhibition of locomotion and respiratory burst in monocytes, reduction of skin hypersensitivity and delay of mononuclear cells in human Rebuck skin windows) with inhibition of adhesion molecules, chemokines, and other genes including interleukin-1β (IL-1β). In animal models, it reduces carragenin-induced inflammation and delays the inflammatory process in murine collagen-induced arthritis (CIA). Objectives To test, in vitro , the anti-inflammatory capacity of MLIF on a promonocytic human cell line (U-937) cells and peripheral blood mononuclear cells (PBMC) from healthy subjects and from patients with rheumatoid arthritis (RA). Material and methods IL-1β gene expression was evaluated in cell cultures either in the presence of MLIF, lipopolysaccharide (LPS), or both. Relative gene expression and immunoreactivity of IL-1β were assayed in cells and supernatants, respectively. Results Amebic peptide was able to down-regulate LPS-induced expression of IL-1β, in U-937 cells without a detectable effect upon the bioavailability of the cytokine. In similar culture conditions, MLIF was capable to down-regulate baseline and LPS-induced expression of IL-β only in PBMC from patients with RA. Peptide effect on immunoreactivity of IL-1β was not statistically significant. Conclusions MLIF exerts, in primed cells, exquisite anti-inflammatory properties that deserve to be explored mechanistically.

Objective: Helicobacter pylori antigen or DNA in stool are meant to detect the bacteria; however, in children the colonization of the gastric mucosa by H pylori is usually weak and fecal excretion of antigen or DNA varies considerably, challenging the utility of these tests in this age group. The aim of the present study was to carry out a systematic review and meta-analysis to evaluate the performance of stool H pylori DNA and antigen tests for the diagnosis of infection in children. Methods: We conducted a systematic review and meta-analysis to assess the accuracy of stool tests for diagnosis of H pylori infection in children. We searched PubMed, EMBASE, and LILACS databases. Selection criteria included participation of at least 30 children and the use of a criterion standard for H pylori diagnosis. In a comprehensive search, we identified 48 studies. Results: Regarding antigen-detection tests, enzyme-linked immunosorbent assay (ELISA) monoclonal antibodies showed the best performance, with sensitivity and specificity of 97%, positive likelihood ratio (LR+) of 29.9, and negative likelihood ratio (LR-) of 0.03. ELISA polyclonal antibodies had sensitivity of 92%, specificity of 93%, LR+ of 16.2, LR- of 0.09, and high heterogeneity (P < 0.0001). One-step monoclonal antibody tests demonstrated sensitivity of 88%, specificity of 93%, LR+ of 10.6, and LR- of 0.11. For DNA detection, polymerase chain reaction-based test showed sensitivity of 80.8%, specificity of 98%, LR + of 17.1, and LR- of 0.18. Conclusions: Detection of H pylori antigen in stools with ELISA monoclonal antibodies is a noninvasive efficient test for diagnosis of infection in children. One-step tests showed low accuracy and more studies are needed to obtain a useful office-based screening test. The available molecular tests are still unreliable.

During carcinogenesis it is known that growth factors and cytokines from stromal and inflammatory cells from the microenvironment promote angiogenesis and lymphangiogenesis. However, the participation of macrophages and mast cells in these processes is not well understood. The aim of this study was to evaluate the relationship between mast cell and macrophage density with blood and lymphatic vessels in various stages of carcinoma of the uterine cervix. Tissue sections from archival paraffin-embedded samples from cases with cervical intraepithelial neoplasias (CIN) 1, 2, 3, carcinoma in situ, and invasive carcinoma were used. Immunohistochemical staining was done using the following antibodies: anti-LYVE-1; anti-CD31; anti-CD68, and anti-tryptase. Our results showed a significant increase in the number of macrophages in carcinoma in situ, a correlation between lymphatic vessels and macrophages in premalignant lesions CIN 2, and a correlation between mast cells and blood vessels in both CIN 2 and carcinoma in situ. In conclusion, our data underscore the importance of the recruitment of macrophages and mast cells in the development of tumor-associated blood and lymphatic capillaries.

Asthma is characterized by allergic airway inflammatory response involving extensive leukocyte infiltration. The stromal cell-derived factor (SDF)-1 or chemokine (C-X-C motif) ligand 12 (CXCL12) attracts a number of cells, including resting T lymphocytes, monocytes, CD34(+) stem cells, basophils, and mature eosinophils. To date, however, the potential role of CXCL12/SDF-1 in relation to leukocyte recruitment in asthma has not been previously examined, to our knowledge.Levels of CXCL12/SDF-1 in the BAL fluid (BALF) of 15 subjects with asthma and 13 healthy subjects were measured by enzyme-linked immunosorbent assay. Immunohistochemistry was performed to identify the cellular source of this chemokine.CXCL12/SDF-1 concentrations were significantly elevated in BALF from subjects with asthma compared with normal subjects (median 845 pg/mL, range, 296-1,700 pg/mL vs median 55 pg/mL, range 25-97 pg/mL; P.001). Concentrations of CXCL12/SDF-1 correlated with macrophages (r = 0.728, P.01), lymphocytes (r = 0.651, P.01), and eosinophils (r = 0.765, P.01). By immunohistochemistry, CXCL12/SDF-1 was localized to the airway epithelium and to a lesser extent to mononuclear cells.CXCL12/SDF-1 is released in high concentration in BALF of patients with asthma. The finding that concentrations of this chemokine correlated with leukocyte numbers in BALF suggests that this chemokine may contribute to the cell recruitment in asthma.

Monocyte locomotion inhibitory factor (MLIF), an amebic peptide with antiinflammatory properties, was evaluated in collagen-induced arthritis (CIA) to test its effects on the onset and acute inflammatory response of arthritis. DBA1/J mice at 8–10 weeks of age were divided into four groups (eight mice per group). The adjuvant group received Freund adjuvant, the CIA group was immunized with collagen II, the MLIF/CIA group received collagen II and MLIF, and the MLIF group received MLIF and Freund adjuvant. All groups were evaluated clinically. Seven weeks after the collagen injection, at the peak of the clinical arthritis score, limb specimens were collected and histological studies and gene expression analysis using microarrays were performed. MLIF administered weekly as a preventive scheme delayed and reduced the severity of acute arthritis. MLIF induced gene changes in functional categories including adhesion molecules, matrix metalloproteinases, and inflammatory cytokines. MLIF could be an interesting new molecule to investigate in the field of rheumatoid arthritis pathogenesis research for its potential to prevent inflammation.

The development of the allergic airway disease conveys several cell types, such as T-cells, eosinophils, mast cells, and dendritic cells, which act in a special and temporal synchronization. Cellular mobilization and its complex interactions are coordinated by a broad range of bioactive mediators known as chemokines. These molecules are an increasing family of small proteins with common structural motifs and play an important role in the recruitment and cell activation of both leukocytes and resident cells at the allergic inflammatory site via their receptors. Trafficking and recruitment of cell populations with specific chemokines receptors assure the presence of reactive allergen-specific T-cells in the lung, and therefore the establishment of an allergic inflammatory process. Different approaches directed against chemokines receptors have been developed during the last decades with promising therapeutic results in the treatment of asthma. In this review we explore the role of the chemokines and chemokine receptors in allergy and asthma and discuss their potential as targets for therapy.

The selective anti-inflammatory effects of the pentapeptide [Met-Gln-Cys-Asn-Ser] with anti-inflammatory properties are manifest in both in vitro and in vivo models. A synthetic structure considered by our group displays the same effects; nonetheless, this is not so for a scrambled version using the same amino acids in a different sequence [Gln-Cys-Met-Ser-Asn]. When the amino acid proline substituted glutamine as the second residue [i.e. Met-Pro-Cys-Asn-Ser], the anti-inflammatory effects of monocyte locomotion inhibitory factor (MLIF) were preserved, although with a weaker effect observed on the respiratory burst and enhanced inhibition of delayed hypersensitivity. The above motivates us to study the electronic structure and the physicochemical properties of these three peptides through a theoretical study at the Hartree–Fock and DFT (B3LYP) levels, with the aim of understanding this behavior from an electronic structure framework. Geometric analysis allowed us to identify a pharmacophore group at the acidic end of the molecule [i.e. …Cys-Asn-Ser]. The electrostatic charge of the pharmacophore group and the charge of the sulphur atom (S Cys 37) in the cysteine group are affected by the presence of electron-attracting groups. These results are further supported by a bond order study and an atomic charges analysis. The structural and physicochemical features studied here could be associated with the anti-inflammatory activity of these peptides.

SUMMARY Axenically grown Entamoeba histolytica produces a pentapeptide (Met-Gln-Cys-Asn-Ser) with several anti-inflammatory properties, including the inhibition of human monocyte locomotion ( Monocyte Locomotion Inhibitory Factor (MLIF)). A construct displays the same effects as the native material. It remains to be seen if MLIF is used, or even produced in vivo by the tissue-invading parasite. If MLIF were to be relevant in invasive amoebiasis, immunizing against it could diminish this parasite advantage and prevent lesions. KLHlinked MLIF mixed with Freund’s adjuvant was too aggressive an immunizing material to answer this question. However, immunization with a tetramer of MLIF (but not a scrambled version of MLIF) around a lysine core (MLIF‐MAPS), that displays increased antigenicity, yet lacks excessive innate immunity activation, completely protects gerbils against amoebic abscess of the liver caused by the intraportal injection of virulent E. histolytica. Liver abscesses caused by Listeria monocytogenes were not prevented. Invasive E. histolytica may produce the parent protein of MLIF in vivo , and if appropriately cleaved, it may play a role in invasive amoebiasis. MLIF may join new vaccination strategies against amoebiasis.

Axenically grown Entamoeba histolytica produces a pentapeptide (Met-Gln-Cys-Asn-Ser) with anti-inflammatory properties that, among others, inhibits the in vitro and in vivo locomotion of human monocytes, sparing polymorphonuclear leucocytes from this effect [hence the name originally given. Monocyte Locomotion Inhibitory Factor (MLIF)]. A synthetic construct of this peptide displays the same effects as the native material. We now added MLIF to resting and PMA-stimulated cells of a human monocyte cell line and measured the effect upon mRNA and protein expression of pro-inflammatory chemokines (RANTES, IP-10, MIP-1alpha, MIP-1beta, MCP-1, IL-8, I-309 and lymphotactin) and the shared CC receptor repertoire. The constitutive expression of these chemokines and the CC receptors was unaffected, whereas induced expression of MIP-1alpha, MIP-1beta, and I-309, and that of the CCR1 receptor--all involved in monocyte chemotaxis--was significantly inhibited by MLIF. This suggests that the inhibition of monocyte functions by MLIF may not only be exerted directly on these cells, but also--and perhaps foremost--through a conglomerate down-regulation of endogenous pro-inflammatory chemokines.

SUMMARY Eosinophils elaborate a number of proinflammatory mediators, including immunoregulatory cytokines and chemokines. Interleukin (IL)-4 and RANTES are important cytokines that have previously been shown to be expressed by mature eosinophils. We hypothesized that de novo synthesis of IL-4 and RANTES occurs in nascent eosinophils, leading to storage of newly produced proteins in crystalloid granule-like structures. Cytokine mRNA and protein expression were examined in cultured eosinophil colonies, which were derived from purified cord blood CD34 + cells and generated in semisolid media (methylcellulose) in the presence of recombinant human (rh)IL-3 and rhIL-5. Cytokine mRNA profiles were analysed by the reverse transcription‐polymerase chain reaction (RT‐PCR) to determine transcription of IL-4 and RANTES in cells on days 0, 7, 14, 21 and 28 of culture. The expression of translated cytokine products and granule major basic protein (MBP) was confirmed, from day 23 onwards, for colonies cultured in semisolid media, by immunofluorescent labelling and confocal laser-scanning microscopy (CLSM). We found that mRNA sequences encoding IL-4 and RANTES were expressed in freshly prepared, nondifferentiated CD34 + cells. Furthermore, RANTES mRNA localized to carbol chromotrope 2Rpositive colony cells, as assessed using in situ RT‐PCR on day 21 of culture in semisolid media, and was found to gradually decrease (relative to b2-microglobulin) in rhIL-3- and rhIL-5-treated colony cells (comprising >90% eosinophil-like cells) up to day 28. Immunoreactivity for IL-4 and RANTES co-localized with MBP in maturing colony eosinophils on day 23 of culture in semisolid media, as judged by CLSM. These results suggest that synthesis and storage of immunoregulatory cytokines, essential for processes associated with adaptive immunity, occurs in nascent eosinophils during their growth and differentiation.

Eosinophils have been shown to express the gene encoding regulated upon activation, normal T‐cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up‐regulated by a number of agonists, including complement‐dependent factors (C3b/iC3b) and interferon‐γ (IFN‐γ). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil‐specific agonists. We analysed RANTES mRNA expression by reverse‐transcription polymerase chain reaction (RT‐PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum‐coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN‐γ (5 ng/ml) transiently and significantly (P < 0·05, n = 3) depleted relative amounts of RANTES PCR product (compared with β2‐microglobulin) after 1–4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN‐γ incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil‐active cytokines, interleukin‐3 (IL‐3), IL‐4, IL‐5 and granulocyte–macrophage colony‐stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN‐γ on RANTES mRNA was reversed by cycloheximide, suggesting that IFN‐γ may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN‐γ may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released bioactive proteins.

The CC chemokine RANTES is synthesized, stored, and upregulated in response to interferon-gamma (IFN-gamma) in human peripheral blood eosinophils. In this report, we propose that RANTES is rapidly mobilized from eosinophil crystalloid granules during agonist-induced degranulation. We stimulated purified eosinophils (>99%) from atopic asthmatics with 500 U/mL IFN-gamma to analyze the kinetics of mobilization and release of RANTES (0 to 240 minutes). We used subcellular fractionation, immunogold analysis, two-color confocal laser scanning microscopy (CLSM), and enzyme-linked immunosorbent assay (ELISA) to trace the movement of eosinophil-derived RANTES from intracellular stores to release. RANTES was rapidly mobilized (10 minutes) and released after 120 minutes of stimulation (80 +/- 15 pg/mL per 2 x 10(6) cells). RANTES appeared to be stored in at least two intracellular compartments: the matrix of crystalloid granules, detected by major basic protein and eosinophil peroxidase activities, and a specialized small secretory vesicle present in light membrane fractions. The extragranular RANTES was mobilized more rapidly than that of crystalloid granules during IFN-gamma stimulation. This effect was not observed in eosinophils treated with IFN-alpha, interleukin-3 (IL-3), IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or genistein followed by IFN-gamma. Our findings suggest that RANTES may be mobilized and released by piecemeal degranulation upon stimulation, involving transport through a putative pool of small secretory vesicles.

Eosinophils, prominent cells in asthmatic inflammation, have been shown to synthesize, store, and release an array of up to 18 cytokines and growth factors, including interleukin-6 (IL-6). In this report, we show that IL-6 immunofluorescence localizes to the matrix of the crystalloid granule in peripheral blood eosinophils from atopic asthmatics using confocal laser scanning microscopy (CLSM). Granule localization of IL-6 was confirmed using dot-blot analysis and enzyme-linked immunosorbent assay (ELISA) on subcellular fractions of highly purified eosinophils produced from density centrifugation across a 0% to 45% Nycodenz gradient. IL-6 was found to coelute with eosinophil crystalloid granule marker proteins, including eosinophil peroxidase (EPO), major basic protein (MBP), arylsulfatase B, and β-hexosaminidase. Immunoreactivity to IL-6 colocalized with granule-associated IL-2 and IL-5 in subfractionated eosinophils. We also made the novel and compelling observation that interferon γ (IFNγ), a Th1-type cytokine, stimulated an early elevation in eosinophil IL-6 immunoreactivity. A 2.5-fold enhancement of IL-6 immunoreactivity in eosinophil granules was observed within 10 minutes of IFNγ treatment (500 U/mL), as determined by subcellular fractionation and CLSM. These findings suggest that IFNγ has short-term effects on human eosinophil function and imply that a physiologic role exists for Th1-type cytokine modulation of Th2-type responses in these cells.

While normal human eosinophils are destroyed in vitro by virulent Entamoeba histolytica, notwithstanding the presence of antibodies and complement, activated eosinophils promptly destroy the parasite although dying also at the end of the process. To study the possible in vivo participation of eosinophils in invasive amebiasis, we compared the induction of experimental amebic abscess of the liver (AAL) in gerbils (Meriones unguiculatus) previously made eosinophilic through Toxocara canis antigen injection and in normal control gerbils. After intraportal inoculation of 10(5) ameba trophozoites (6 and 24 hr), the ratio of gerbils with AAL, as well as the number and size of the microabscesses was comparable in eosinophilic and control gerbils. However, at 96 hr the number and size of the microabscesses were significantly smaller (p < 0.05) in eosinophilic gerbils. On the other hand the actuarial AAL survival curve up to 45 days post-amebic inoculation was significantly (p < 0.05) shifted to the right in controls. These results suggest that antigen-induced eosinophilia may exert a protective effect against AAL in gerbils.

Entamoeba histolytica produces monocyte locomotion inhibitory factor (MLIF), a pentapeptide with in vitro and in vivo anti-inflammatory properties. MLIF may interfere with leukocyte migration, disturbing the balance of pro- and anti-inflammatory cytokines secreted by CD4(+) T lymphocytes. We evaluated the effect of MLIF on expression of pro- and anti-inflammatory cytokines in human CD4(+) T lymphocytes. Regulatory cytokines [interleukin-1 beta (IL-1beta), IL-2, interferon gamma (IFN-gamma), IL-5, IL-6, and IL-10] were studied by enzyme-linked immunosorbent assay method in CD4(+)-cell supernatant fluids. Proinflammatory cytokines were produced per se by MLIF (IL-1beta, IL-2, and IFN-gamma) and also anti-inflammatory cytokines (IL-5, IL-6, and IL-10) with 1-phorbol-12 myristate-13 acetate + MLIF; the IL-1beta, IFN-gamma, IL-5 and IL-6 production was inhibited but not that of IL-10 which disclosed increase in its expression. MLIF disturbs the pro- and anti-inflammatory balance, and it induces inhibition of IL-1beta (principal proinflammatory cytokine) and increases IL-10 (prototype of an anti-inflammatory cytokine).

Entamoeba histolytica in culture produces a pentapeptide (MQCNS). This oligopeptide inhibits the in vitro and in vivo locomotion of human monocytes, hence its denomination Monocyte Locomotion Inhibitory Factor (MLIF). The original isolated peptide and its synthetic construct display similar effects, among others, being inhibition of the respiratory burst in monocytes and neutrophils, decrease of Dinitrochlorobenzene (DNCB) skin hypersensitivity in guinea pigs and gerbils, and delay of mononuclear leukocytes in human Rebuck skin windows with inhibition of vascular cell Very late antigen (VLA)-4 and Vascular adhesion molecules (VCAM) in endothelia and monocytes. The MLIF molecular mechanism of action is unknown, but data reveal its implication in Nuclear factor-kappa B (NF-κB) and Mitogenactivated protein kinase (MAPK) pathways. This could explain MLIF multiplicity of biological effects. On the other hand, the amebic peptide has been useful in treating experimental amebiasis of the liver. The amebic peptide is effective in reducing inflammation induced by carragenin and arthritis in a Collagen-induced arthritis (CIA) model. Microarray data from experimental arthritis revealed an MLIF gene expression profile that includes genes that are involved in apoptosis, cell adhesion, extracellular matrix, and inflammation / chemotaxis. MLIF could be involved in unsuspected biological factions because there is increasing data on the peptide effect on several cell activities. This review also presents uses of MLIF as described in patents.

Axenically grown Entamoeba histolytica produces a pentapeptide (Met-Gln-Cys-Asn-Ser) with anti-inflammatory properties that, among others, inhibits the in vitro and in vivo locomotion of human monocytes, sparing polymorphonuclear leucocytes from this effect [hence the name originally given. Monocyte Locomotion Inhibitory Factor (MLIF)]. A synthetic construct of this peptide displays the same effects as the native material. We now added MLIF to resting and PMA-stimulated cells of a human monocyte cell line and measured the effect upon mRNA and protein expression of pro-inflammatory chemokines (RANTES, IP-10, MIP-1alpha, MIP-1beta, MCP-1, IL-8, I-309 and lymphotactin) and the shared CC receptor repertoire. The constitutive expression of these chemokines and the CC receptors was unaffected, whereas induced expression of MIP-1alpha, MIP-1beta, and I-309, and that of the CCR1 receptor--all involved in monocyte chemotaxis--was significantly inhibited by MLIF. This suggests that the inhibition of monocyte functions by MLIF may not only be exerted directly on these cells, but also--and perhaps foremost--through a conglomerate down-regulation of endogenous pro-inflammatory chemokines.

Background: In allergic inflammation and asthma, eosinophils are major effector cells. They have been shown to synthesize at least 23 cytokines, some of which are stored intracellularly in their unique crystalloid granules together with cationic granule protein. Little is known about the synthesis and storage of cytokines relative to cationic granule proteins in maturing eosinophils during eosinophilopoiesis. Objective: Our purpose was to analyze the expression of eosinophil-derived mediators, major basic protein (MBP), eosinophil cationic protein (ECP), IL-6, and RANTES, during early stages of eosinophil maturation in CD34 + cell-derived colonies. Methods: Purified human cord blood CD34 + cells were grown in methylcellulose cultures in the presence of recombinant human IL-3 and IL-5. By confocal laser scanning microscopy, the coexpression of eosinophil granular proteins MBP and ECP was determined concurrently with IL-6 and RANTES during eosinophil maturation on days 16, 19, 23, and 28 of culture. Results: Immunoreactivity against MBP, ECP, IL-6, and RANTES was not detectable in freshly purified CD34 + cells. Maturing eosinophils (>95%) exhibited positive immunostaining for all these proteins between days 16 and 28 of culture. At early stages of culture, discrete immunostaining was observed around the periphery but not in the center of granular structures. By day 28 cultured eosinophil-like cells showed evidence of the acquisition of crystalloid granule-like structures, analogous to those observed in mature peripheral blood eosinophils. Conclusions: Eosinophils express and store cytokines simultaneously with cationic granule proteins during the process of maturation. We propose that the storage of cytokines during the development of eosinophils is an early event and it may be integral to inflammatory responses involving these cells. The results of this study suggest a potential immunoregulatory function for maturing eosinophils. (J Allergy Clin Immunol 2000;105:1178-84.)

Eosinophils synthesize and release a number of cytokines and chemokines, including RANTES, a potent chemoattractant particularly for memory T cells and eosinophils. Long–term (>12 h) incubation with interferon–γ (IFN–γ) has been shown to activate eosinophils and induce expression of membrane receptors. We hypothesized that IFN–γ mobilizes intracellular RANTES in eosinophils in advance of mediator release. Highly purified peripheral blood eosinophils were obtained from asthmatics and stimulated with IFN–γ at 500 U/ml for time course analysis up to 2h. By specific ELISA, RANTES was detected in supernatants (80±15 pg per 2×106 cells) following 120min of stimulation. Immunoreactive RANTES in resting cells (5×107 eosinophils) was detected in two intracellular compartments in studies of subcellular fractionation by density gradient centrifugation. After 10 min IFN–γ stimulation, RANTES immunoreactivity was confined to crystalloid granules. RANTES was redistributed from secretory granules to light–membrane fractions after 60 min of IFN–γ incubation. Our data suggest that rapid mobilization and release of RANTES occurs from stimulated eosinophils. These findings may have important implications for the role of IFN–γ in activating human eosinophils, particularly in severe chronic asthma or viral exacerbation of asthmatic inflammation, where this cytokine may play a role.

Higher numbers of eosinophil/basophil colony-forming units (Eo/B CFU) are observed in blood of atopic individuals, and can be enhanced in atopic asthmatics by allergen-inhalation challenge. It is known that mature basophils and eosinophils synthesize cytokines relevant to allergic inflammation. To investigate the potential role of growth factors in allergic disease we examined the expression of the hemopoietic cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-5, in differentiating Eo/B colony cells from normal and atopic individuals, and from atopic asthmatics before and after allergen-inhalation challenge. Peripheral blood was collected from two normal and 12 atopic individuals, and also from 25 atopic asthmatics before and 24 h after allergen challenge. Nonadherent mononuclear cells were isolated and grown in semisolid growth medium. Eo/B colonies were selected and cytospins were prepared for immunocytochemical analysis of colony cells. Eo/B colonies, especially carbol chromotrope 2R+ cells, selected at Days 10, 14, and 18 from atopic donors contained messenger RNA for GM-CSF by combined in situ reverse transcription-polymerase chain reaction and cytochemistry, and demonstrated time-dependent expression of GM-CSF by immunocytochemistry (P = 0.007). Atopic individuals demonstrated a higher percentage of cells expressing GM-CSF than did normal subjects under all growth conditions when examined at Day 14 (P = 0. 04). Atopic asthmatics challenged with inhaled allergen who demonstrated a dual airway response, an increase in the number of blood eosinophils (P = 0.0001), and an increase in the number of Eo/B CFU (P = 0.02) also demonstrated a significant increase in the percentage of colony cells expressing immunostainable GM-CSF (P = 0. 0009), but only a variable effect on those expressing IL-5, 24 h after allergen. These results suggest that GM-CSF expression by differentiating Eo/Bs may provide an additional stimulus in vivo to enhance Eo/B progenitor differentiation in atopic and asthmatic individuals, especially after allergen challenge. The concept of microenvironmental differentiation, where blood progenitor cells may aid in their own differentiation, is supported by these ex vivo findings.

Background Neonatal sepsis is a worldwide public health issue in which, depending on the studied population, marked variations concerning its risk and prognostic factors have been reported. The aim of this study was to assess risk and prognostic factors for neonatal sepsis prevailing at a medical unit in southeastern Mexico. Thus, we used a historic cohort design to assess the association between a series of neonates and their mothers, in addition to hospital evolution features and the risk and prognosis of neonatal sepsis (defined by Pediatric Sepsis Consensus [PSC] criteria) in 11,790 newborns consecutively admitted to a Neonatology Service in Mérida, Mexico, between 2004 and 2007. Results Sepsis was found in 514 of 11,790 (4.3 %) newborns; 387 of these cases were categorized as early-onset (72 h) (24.7 %). After logistic regression, risk factors for sepsis included the following: low birth weight; prematurity; abnormal amniotic fluid; premature membrane rupture (PMR) at >24 h; respiratory complications, and the requirement of assisted ventilation, O2 Inspiration fraction (IF) >60 %, or a surgical procedure. Some of these factors were differentially associated with early- or late-onset neonatal sepsis. The overall mortality rate of sepsis was 9.5 %. A marked difference in the mortality rate was found between early- and late-onset sepsis (p >0.0001). After Cox analysis, factors associated with mortality in newborns with sepsis comprised the following: prematurity; low birth weight; low Apgar score; perinatal asphyxia, and the requirement of any invasive medical or surgical procedure. Conclusions The incidence of neonatal sepsis in southeastern Mexico was 4.3 %. A different risk and prognostic profile between early- and late-onset neonatal sepsis was found.