Neutrophil Heterogeneity Is Modified during Acute Lung Inflammation in Apoa1-/- Mice.

Neutrophils play important roles in inflammatory airway diseases. In this study, we assessed whether apolipoprotein A-I modifies neutrophil heterogeneity as part of the mechanism by which it attenuates acute airway inflammation. Neutrophilic airway inflammation was induced by daily intranasal administration of LPS plus house dust mite (LPS+HDM) to Apoa1-/- and Apoa1+/+ mice for 3 d. Single-cell RNA sequencing was performed on cells recovered from bronchoalveolar lavage fluid on day 4. Unsupervised profiling identified 10 clusters of neutrophils in bronchoalveolar lavage fluid from Apoa1-/- and Apoa1+/+ mice. LPS+HDM-challenged Apoa1-/- mice had an increased proportion of the Neu4 neutrophil cluster that expressed S100a8, S100a9, and Mmp8 and had high maturation, aggregation, and TLR4 binding scores. There was also an increase in the Neu6 cluster of immature neutrophils, whereas neutrophil clusters expressing IFN-stimulated genes were decreased. An unsupervised trajectory analysis showed that Neu4 represented a distinct lineage in Apoa1-/- mice. LPS+HDM-challenged Apoa1-/- mice also had an increased proportion of recruited airspace macrophages, which was associated with a reciprocal reduction in resident airspace macrophages. Increased expression of a common set of proinflammatory genes, S100a8, S100a9, and Lcn2, was present in all neutrophils and airspace macrophages from LPS+HDM-challenged Apoa1-/- mice. These findings show that Apoa1-/- mice have increases in specific neutrophil and macrophage clusters in the lung during acute inflammation mediated by LPS+HDM, as well as enhanced expression of a common set of proinflammatory genes. This suggests that modifications in neutrophil and macrophage heterogeneity contribute to the mechanism by which apolipoprotein A-I attenuates acute airway inflammation.

Effects of treatment with monacolin K, berberine and coenzyme Q10 on lipid metabolism in patients with moderate cardiovascular risk.

OBJECTIVE: The use of nutritional supplements to treat hypercholesterolemia is gradually increasing, however further studies on their efficacy and safety are required. PATIENTS AND METHODS: The present clinical trial included patients with moderate hypercholesterolemia and cardiovascular risk who were treated either with a nutraceutical preparation containing 3.75mg of monacolin K, 515mg of berberine and 50mg of coenzyme Q10 per tablet (Lipok®) or with a placebo. The clinical and laboratory variables were analyzed at baseline and at three and six months. None of the patients was diabetic, and none was being treated with lipid-lowering drugs or with any other nutritional supplements affecting lipid metabolism. RESULTS: In patients of the intervention group and of the placebo group, baseline LDL-C was 134.7mg/dL (14.4) and 138.7mg/dL (15.2), respectively. At three months after treatment start, LDL-C had decreased by 26.1mg/dL (-32.4 to 19.7) and increased by 4.5mg/dL (-1.5 to 10.5) in the respective groups. In the intervention group, a similar decrease in non-HDL-C and total cholesterol was observed, while no significant changes were observed in either group for HDL-C, triglycerides and lipoprotein(a). A good tolerance and safety profile was observed. CONCLUSION: In conclusion, this study demonstrates that the combination of monacolin K, berberine and coenzyme Q10 is effective and safe for treating hypercholesterolemia in patients with a moderate degree of excess LDL-C and cardiovascular risk.

Dissipation of a mix of priority PAHs in soils by using availability enhancers. Effect of aging and pollutant interactions.

A remediation strategy using three non-toxic availability enhancers (two cyclodextrins and a rhamnolipid biosurfactant) was applied to various soils artificially contaminated with a mix of Polycyclic Aromatic Hydrocarbons (PAHs) considered priority pollutants at two levels of contamination: only with 7 low molecular weight PAHs (LMW PAHs, 5 with 3-ring and 2 with 4-ring - fluoranthene and pyrene) or with 14 PAHs (from 3 to 6 rings). Natural attenuation of PAHs in all soils showed degradation capacity for the LMW PAHs, with a final content of LMW PAHs <5% of their initial concentration. Conversely, the rest of PAHs (high molecular weight PAHs, HMW) remained in the soils (61% - 83.5%), indicating abiotic dissipation of HMW PAHs due to formation of non-extractable residues in soils. The influence of the presence of HMW PAHs on the degradation of the 7 LMW PAHs was also tested, showing a general decrease in the time to obtain 50% dissipation (DT50), statistically significant for acenaphthene, acenaphthylene and fluorene. Availability enhancers showed different effects on PAHs dissipation. 2-hydroxypropyl-ß-cyclodextrin (HP) decreased DT50 of some of the lighter PAHs, whereas the rhamnolipid (RL) caused a slight DT50 increase due to its initial toxicity on native soil microorganisms, but showing later high degradation rate for LMW PAHs. On the contrary, randomly methylated-ß-cyclodextrin (RAMEB) slowed down PAHs degradation due to its high adsorption onto soil surface, blocking the desorption of PAHs from the soils. The high number of experimental factors not studied simultaneously before (soil type, co-contamination, availability enhancers and incubation time) allowed to conduct a statistical analysis which supported the conclusions reached. Principal Component Analysis separated the studied PAHs in 3 groups, in relation with their molecular weight and Kow. The first principal component was related with LMW PAHs, and separate the inefficient RAMEB from the other availability enhancers.

Food intake biomarkers for berries and grapes.

Grapes and berries are two types of widely consumed fruits characterized by a high content in different phytochemicals. However, their accurate dietary assessment is particularly arduous, because of the already wide recognized bias associated with self-reporting methods, combined with the large range of species and cultivars and the fact that these fruits are popularly consumed not only in fresh and frozen forms but also as processed and derived products, including dried and canned fruits, beverages, jams, and jellies. Reporting precise type and/or quantity of grape and berries in FFQ or diaries can obviously be affected by errors. Recently, biomarkers of food intake (BFIs) rose as a promising tool to provide accurate information indicating consumption of certain food items. Protocols for performing systematic reviews in this field, as well as for assessing the validity of candidate BFIs have been developed within the Food Biomarker Alliance (FoodBAll) Project. This paper aims to evaluate the putative BIFs for blueberries, strawberries, raspberries, blackberries, cranberries, blackcurrant, and grapes. Candidate BFIs for grapes were resveratrol metabolites and tartaric acid. The metabolites considered as putative BFI for berries consumption were mostly anthocyanins derivatives together with several metabolites of ellagitannins and some aroma compounds. However, identification of BFIs for single berry types encountered more difficulties. In the absence of highly specific metabolites reported to date, we suggested some multi-metabolite panels that may be further investigated as putative biomarkers for some berry fruits.

Sequential pulmonary immunization with heterologous recombinant influenza A virus tuberculosis vaccines protects against murine M. tuberculosis infection.

Tuberculosis (TB) infection affects a quarter of the global population resulting in a large burden of TB disease and mortality. The long-term control of TB requires vaccines with greater efficacy and durability than the current Mycobacterium bovis Bacille Calmette-Guérin (BCG). Pulmonary immunization may increase and prolong immunity at the site of Mycobacterium tuberculosis infection. We have investigated recombinant influenza A viruses (rIAVs) expressing the p25 CD4+ T cell epitope of M. tuberculosis Ag85B240-254 for single and sequential immunization against M. tuberculosis infection. Intranasal immunization with single dose of rIAV X31 (H3N2 strain) expressing the p25 epitope (X31-p25), induced p25-specific CD4+ T cells and conferred protection against aerosol delivery of M. tuberculosis infection in the lungs. To enhance this effect, prime-boost immunization with hetero-subtypic rIAVs was examined. Sequential immunization with X31-p25 and a second rIAV, PR8 (H1N1 strain) expressing the same epitope (PR8-p25), increased the frequency of p25-specific IFN-γ T cell responses and polyfunctional CD4+ T cells producing IFN-γ, IL-2, and TNF, compared to immunization with each rIAV alone. This combination resulted in protection against M. tuberculosis in both the lungs and spleen. Therefore, our study revealed that rIAV is not only an efficient vector to induce protective immunity in the lungs, but also has a potential use for sequential immunization with heterologous rIAV to boost the immunogenicity and improve the protection against M. tuberculosis.

Influence of HLA class I, HLA class II and KIRs on vertical transmission and chronicity of hepatitis C virus in children.

BACKGROUND & AIM: There is evidence that maternal viral load of HCV during delivery influences the risk for Mother-to-child transmission (MTCT), but this does not explain all cases. We study the role of the immunogenetic profile (HLA, KIRs and KIR-ligand binding) of mothers and children in HCV-MTCT and in chronicity in the children. METHODOLOGY: 79 HCV-RNA (+) mothers and their 98 children were included. 24 children were infected, becoming chronic in 8 cases and clearing in 16. HLA-class-I and II and KIRs were determined by Luminex. RESULTS: MTCT study: The presence of HLA-C1-ligand in mothers and/or their children reduces the risk of transmission (mothers: Pc = 0.011, children: P = 0.033), whereas the presence of HLA-C2C2-ligand in mothers increases it (Pc = 0.011). In children KIR2DL3-HLA-C1 is a protector factor (Pc = 0.011). Chronicity in children study: Maternal DQA1*01 allele (Pc = 0.027), KIR2DS1 (Pc = 0.011) or KIR3DS1 (Pc = 0.011) favours chronicity in the child. The presence of the DQB1*03 allele (Pc = 0.027) and KIR2DS3 (P = 0.056) in the child and homozygosity for KIR3DL1/3DL1 (Pc = 0.011) and for the HLA-Bw4/Bw4 ligand (P = 0.027) is associated with viral clearance, whereas the presence of HLA-Bw6 ligand (P = 0.027), the binding of KIR3DS1-HLA-Bw4 (P = 0.037) and heterozygosity for KIR3DL1/3DS1 (Pc = 0.011) favour viral chronicity. Mother/child allele matching: In the joint HLA analysis, matching was greater between mothers and children with chronic infection vs those who had cleared the virus (67%±4.1 vs 57%±1.2, P = 0.003). CONCLUSIONS: The HLA-C1 ligand in the mother is related to MTCT, while several genetic factors of the mother or child are involved in the chronification or clearance of infection in the child. Matching allelic data is considered to be an indicator of HCV chronicity in the child and can be used as a potential prognostic test. This implies that NK cells may play a previously undocumented role in protecting against MTCT and that both NK cell immunity and adaptive T-cell responses may influence viral clearance in infected children.

Effects of the presence of a composted biosolid on the metal immobilizing action of an urban soil.

The influence of a composted biosolid from urban wastewater treatment on the retention and solubility of Cu, Pb or Zn added to a soil was studied by batch adsorption/desorption experiments, equilibrating both materials and their mixtures with solutions containing various metal concentrations. The composted biosolid adsorbed less Cu or Pb and slightly more Zn than the soil, and thus caused a noticeable decrease in the retention of Cu or Pb and an increase in Zn adsorption by soil-biosolid mixtures, but these effects in the mixtures were not additive for any metal. The pH effects were studied by means of (log metal concentration)/pH diagrams. It was shown that Cu behaviour was different from that of the other metals: the relation between pH and Cu concentrations suggested similar solubilities in the presence of the biosolid and the mixtures, whereas the biosolid-free soil gave data located on a region of the diagram corresponding to slightly lower solubility. In the case of Pb or Zn, the data for the biosolid were located in a region of the diagram corresponding to clearly lower solubilities than those for the biosolid/soil mixtures. It was concluded that the biosolid has little effect on metal solubility when it is mixed with the soil in the proportions used here.

Skipping of exon 30 in C5 gene results in complete human C5 deficiency and demonstrates the importance of C5d and CUB domains for stability.

The deficiency of complement C5 is rare and frequently associated with severe and recurrent infections, especially caused by Neisseria spp. We observed the absence of component C5 in the serum of 3 siblings from a Brazilian family with history of consanguinity. The patients had suffered from recurrent episodes of meningitis and other less severe infections. Sera from these patients were unable to mediate hemolytic activity either by the classical or alternative pathways and presented extremely low levels of C5 protein (1.3, 0.9 and 1.0 microg/ml-normal range: 45-190 microg/ml). Hemolytic activity could be restored by the addition of purified C5 to deficient serum. Sequencing of sibling C5 cDNA revealed a homozygous 153 bp deletion that corresponds precisely to exon 30. The parents carried the same deletion but only in one allele. Sequencing of the corresponding region in the genomic DNA revealed a C to G substitution within intron 30 and, most significantly, the substitution of GAG(4028) for GAA(4028) at the 3' end of exon 30 which is most likely responsible for skipping of exon 30. The resulting in-frame deletion in the C5 mRNA codes for a mutant C5 protein lacking residues 1289-1339. These residues map to the CUB and C5d domains of the C5 alpha chain. This deletion is expected to produce a non-functional and unstable C5 protein which is more susceptible to degradation.

Constitutive expression of Bcl-2 in the haematopoietic compartment alters the metabolism of iron and increases resistance to mycobacterial infection.

Mice expressing a vav-bcl-2 transgene were tested for their resistance to an experimental infection with Mycobacterium avium. When compared with control littermates, transgenic mice exhibited an increase in the resistance to infection which was independent of B or T lymphocytes and did not require the production of gamma interferon. Macrophages from both control and transgenic mice showed equal permissiveness to M. avium growth in vitro. Finally, transgenic mice expressed diminished circulating iron levels which correlated with the increased resistance to infection.

Deficiency of the human complement regulatory protein factor H associated with low levels of component C9.

We identified a 4-year-old Brazilian boy from a family of Japanese descent and history of consanguinity, who suffered from severe recurrent pneumonia. He carries factor H (FH) deficiency associated with reduced levels of component C9 and low serum levels of C3 and factor B. His mother also presented low levels of these proteins and factor I, while his father and sister had only lower levels of FH. Western blot assays confirmed the complete absence of FH and FHL-1 polypeptides in this patient. Sequencing of the proband's FH cDNA revealed a homozygous G453A substitution, encoding an Arg(127)His change. His mother, father and sister are heterozygous for this substitution. Despite the absence of FH in the plasma, this protein was detected in the patient's fibroblasts, suggesting that Arg(127) may be important for FH secretion. Low concentrations of C9 were detected in the proband serum but no mutations in the patient's C9 gene or promoter have been identified, suggesting that this is a consequence of uncontrolled complement activation and high C9 consumption.

Estimating the extractability of potentially toxic metals in urban soils: a comparison of several extracting solutions.

Metals released by the extraction with aqua regia, EDTA, dilute HCl and sequential extraction (SE) by the BCR protocol were studied in urban soils of Sevilla, Torino, and Glasgow. By multivariate analysis, the amounts of Cu, Pb and Zn liberated by any method were statistically associated with one another, whereas other metals were not. The mean amounts of all metals extracted by HCl and by SE were well correlated, but SE was clearly underestimated by HCl. Individual data for Cu, Pb and Zn by both methods were correlated only if each city was considered separately. Other metals gave poorer relationships. Similar conclusions were reached comparing EDTA and HCl, with much lower values for EDTA. Dilute HCl extraction cannot thus be recommended for general use as alternative to BCR SE in urban soils.

Interleukin-12 primes CD4+ T cells for interferon-gamma production and protective immunity during Mycobacterium avium infection.

Interleukin-12 (IL-12) is a crucial cytokine for the generation of a protective immune response against Mycobacterium avium infection. In contrast to infected control mice, IL-12-deficient mice were unable to control bacterial proliferation and their spleen T cells were almost unresponsive in vitro to specific antigens of M. avium. Susceptibility of mice deficient in IL-12 was similar to that of interferon-gamma (IFN-gamma)-deficient mice. These data indicate a crucial role of IL-12 in the development of a T-cell population able to produce IFN-gamma and to mediate protection against M. avium infection. Treatment of M. avium-infected mice with IL-12 induced CD4+ T cells with enhanced capacity to produce IFN-gamma as well as to confer increased protection against M. avium.

Interleukin-6 regulates the phenotype of the immune response to a tuberculosis subunit vaccine.

We investigated the role of interleukin-6 (IL-6) in the development of the immune response to a subunit vaccine against tuberculosis consisting of the culture filtrate proteins of Mycobacterium tuberculosis emulsified in the adjuvant dimethyldioctadecylammonium bromide (DDA). C57Bl/6 mice immunized with this vaccine developed a strong T helper 1 (Th1) response characterized by an increased production of interferon-gamma (IFN-gamma) secreted by CD4+ T cells. Neutralization of IL-6 during in vivo priming resulted in marked reduction in the ability of T cells to secrete IFN-gamma and IL-2 and to proliferate. IL-6 gene-disrupted mice primed with the vaccine showed a decrease in the number of IFN-gamma-producing cells and an increase in IL-4-secreting cells as compared to control mice. In contrast, neutralization of IL-6 during a boost of the vaccine in previously primed mice did not affect the development of IFN-gamma-producing cells but still increased the number of IL-4-producing cells. Our work shows that IL-6 plays a major role in the priming but not in the later expression of a Th1 response to a tuberculosis vaccine.

Inherited complete factor I deficiency associated with systemic lupus erythematosus, higher susceptibility to infection and low levels of factor H.

Here we describe two new cases of complete deficiency of factor I (fI) in two sisters from a consanguineous Brazilian family. The eldest sibling (20-year-old) developed systemic lupus erythematosus (SLE) early during childhood while the youngest had been committed on several occasions owing to repeated infections although she was asymptomatic for auto-immune diseases. We also detected lower concentrations of C3 and factor B in both sisters. Biological functions dependent on complement activation such as the production of opsonins and killing of phagocytozed micro-organisms, chemotactic factors and haemolytic activity were all significantly reduced in both probands. Consistent with the absence of fI and low levels of fH, a deregulated production of C3b was observed by bidimensional electrophoresis in sera of both the probands.

Hereditary human complement C3 deficiency owing to reduced levels of C3 mRNA.

An 8-year-old son (L.A.S.) of consanguineous parents, presented recurrent bacterial infections, vasculitis and extremely low levels of serum C3 (0.15 microg/ml). The classical and alternative pathway haemolytic activities and the generation of opsonins and chemotactic factors derived from the activation of the complement system were markedly affected in the proband's serum. An in vitro addition of purified C3 restored the classical pathway-dependent haemolytic activity of his serum. Autoradiographs of the proband's lipopolysaccharide (LPS)-stimulated and 35S-labelled fibroblast supernatants after that the SDS-PAGE revealed no C3 alpha or beta chains. The amount of C3 mRNA synthesized by the proband's fibroblasts, as evaluated by reverse transcription-polymerase chain reaction (RT-PCR) assays, was greatly reduced.

Deficiency of human complement factor I associated with lowered factor H.

Deficiencies of factor I and/or factor H result in an increased consumption of C3 and higher susceptibility to recurrent infections. Here we describe a case of human factor I deficiency and lowered factor H levels. C3 concentration was 50% lower than normal, the classical pathway-dependent hemolytic activity was reduced to almost 30% of normal, and alternative pathway-dependent activity was completely absent. The killing by peripheral leukocytes of Candida albicans treated with deficient serum and the production of complement-dependent chemotactic factors were reduced in the proband's serum when compared with normal serum. Finally, we observed that C3 antigen present in the proband's serum has a different electrophoretic mobility than native C3 (most likely C3b), confirming the deregulation of complement activation due to the lack of regulatory proteins factors I and H. The impaired complement system described in this case, the first of its kind described in a Chile, explains the higher susceptibility to infections found in the proband.

Differences in resistance of C57BL/6 and C57BL/10 mice to infection by Mycobacterium avium are independent of gamma interferon.

After infection with a low-virulence strain of Mycobacterium avium, C57BL/6 and C57BL/10 mice had clear differences in the control of the infection in their livers and spleens. This difference in susceptibility was not associated with differences in the H-2 complex. It was dependent on the activity of CD4(+) T cells but unrelated to the ability of these cells to secrete gamma interferon or to the development of delayed-type hypersensitivity responses at 3 weeks of infection. It was associated with lower total numbers of CD4(+) cells present in infected spleens and was related to an earlier induction of protective T cells, as measured by adoptive-transfer assays. These data further strengthen the notion of gamma-interferon-independent mechanisms of protection against mycobacteria.

Murine alpha-2-macroglobulin increase during inflammatory responses and tumor growth.

OBJECTIVE AND DESIGN: To determine the alpha-2-macroglobulin (alpha2M) levels in mice during acute and chronic inflammatory responses. MATERIALS AND METHODS: Inflammation was induced by one of the following stimuli: carrageenin, zymosan, lipopolysacharide, thioglycollate, bacilli Calmette Guerin, PPD (in pre-immunized and non-immunized animals) and tumor cells. The concentration of alpha2M was determined in plasma or peritoneal liquid by electroimmunoassay. RESULTS: In all the treatments employed, the plasma levels of alpha2M were higher than in untreated animals. This increase varied from 9%, 24 h after injection up a maximum of 66% 72 h post-injection. When compared to animals injected only with saline, the increases were significant 48 h after treatment with either zymosan or LPS, and 72 h after treatment with either thioglycollate or carrageenin. Treatment with BCG triggers an increase in alpha2M levels after 24 h (18.60%) and 48 h (27.90%). Immunized mice presented higher levels of this protein than non-immunized animals after challenge with PPD. The growth of Ehrlich tumor cells in the peritoneal cavity was directly correlated with the local levels of alpha2M which increased 3.5 fold, 10 days after injection. CONCLUSIONS: These results strongly indicate that in mice, the concentration of alpha2M can increase during acute and chronic inflammatory reactions with kinetics dependent on the particular kind of inflammatory agent.

Improved clearance of Mycobacterium avium upon disruption of the inducible nitric oxide synthase gene.

Mice genetically deficient in the inducible NO synthase gene (iNOS-/-) were used to study the role played by NO during infection by Mycobacterium avium. iNOS-/- macrophages were equally able to restrict M. avium growth in vitro following stimulation by IFN-gamma and TNF-alpha as macrophages from wild-type mice. In vivo, the infection progressed at similar rates in wild-type and NO-deficient mice during the first 2 mo of infection, but the latter mice were subsequently more efficient in clearing the mycobacteria than the former. The increased resistance of iNOS-/- mice was associated with higher IFN-gamma levels in the serum and following in vitro restimulation of spleen cells with specific Ag, increased formation of granulomas and increased survival of CD4+ T cells. We show that NO is not involved in the antimycobacterial mechanisms of M. avium-infected macrophages and, furthermore, that it exacerbates the infection by causing the suppression of the immune response to the pathogen.

Resistance of virulent Mycobacterium avium to gamma interferon-mediated antimicrobial activity suggests additional signals for induction of mycobacteriostasis.

The cytokine gamma interferon (IFN-gamma) plays a major role in the control of Mycobacterium avium infections. We assessed whether the progressive growth of virulent strains of M. avium was associated with alterations in the production of this cytokine as evaluated by reverse transcription-PCR and detection of immunoreactive cytokine in the serum and in spleen homogenates. We found that IFN-gamma was induced during infection by a virulent strain of M. avium to similar or even higher extents than the levels found during infections by a less virulent strain whose growth was controlled. IFN-gamma produced during infection by both mycobacterial strains was partly derived from T cells and led to activation of macrophages, namely, those that were infected. Concomitant with the development of the infection with the virulent strain of M. avium there was an extensive depletion of lymphocytes in the spleen. Thymectomy alone promoted the proliferation of the virulent, but not of the less virulent, strain of M. avium. Our data indicate that virulent strains of M. avium resist the antimicrobial mechanisms of IFN-gamma-activated macrophages and raise the possibility that a second, T-cell-dependent signal is required for the effective control of mycobacterial replication inside macrophages.