Safety, growth, and support to healthy gut microbiota by an infant formula enriched with functional compounds.

BACKGROUND & AIMS: Safety and growth adequacy of infant formulae enriched by functional ingredients need stringent evaluation by means of randomized controlled trials (RCTs), therefore we performed a double-blind RCT to evaluate an infant formula enriched with galacto-oligosaccharides, beta-palmitate, and acidified milk vs. a standard infant formula. METHODS: Weight, length, head circumference and fecal bacteria (Bifidobacteria, BIF/Clostridia, CLO) were measured in healthy full term infants, at baseline - as before 21 days of life - at 60 and 135 days thereafter. A group of 51 neonates received the enriched formula (ENR), 59 the standard one (ST). Parents were trained to daily register gastrointestinal diseases. RESULTS: All the infants grew homogeneously increasing the anthropometric parameters and complying with WHO and Italian standards: the mean (SD) difference in daily weight between ENR and ST groups was -0.74 (1.13) g/day, corresponding to a 90% CI of -2.62 to 1.13 g/day, well within the postulated interval of equivalence of -3.9 to +3.9 g/day. A statistical improvement in BIF concentration in the microbiota of infants fed by ENR was recorded. There was no between-group change in log10CLO, but log10BIF increase was higher at T2 vs. T0 in ENR (treatment × time interaction = 0.71, 95% CI 0.08-1.34, p = 0.028) than in ST neonates. This corresponds to estimated mean (95% CI) values of 8.37 (8.04-8.69) log10-units for ENR vs. 8.08 (7.77-8.39) log10-units for ST neonates. Gastrointestinal effects were mild and similar, with no statistical difference between two groups. CONCLUSION: Safety and growth ability of the enriched formula has been confirmed. A positive effect on neonatal gut microbiota, consisting of increased fecal BIF counts at T2 vs. baseline has been shown too. Nonetheless, larger RCTs are needed to estimate with greater precision the effective potential attributable to the enriched formula on neonatal microbiota, with particular reference to the mode of delivery.

Impact of Early Postnatal Nutrition on the NMR Urinary Metabolic Profile of Infant.

NMR-based metabolomics was used to compare the metabolic urinary profiles of exclusively breast-fed term infants (n = 11) with those of a double-blinded controlled trial with 49 formula-fed term newborns randomized to receive either an infant formula enriched by functional ingredients (n = 24) or a standard formula (n = 25). Anthropometric measurements and urine samples were taken at enrollment (within the first month of life), at around 60 days of life, and at the end of study period (average age of 130 days). The metabolic profiles were examined in relation to time and diet strategy. A common age-dependent modification of the urine metabolome was observed for the three types of nutrition, mainly characterized by similar temporal trends of choline, betaine, myoinositol, taurine, and citrate. Contrariwise, differences in the metabolic profiles were identified according to the type of diet (human versus formula milk), while no significant difference was observed between the two formulas. These modifications are discussed mainly in terms of the different milk compositions. Despite the low number of enrolled infants (n = 60), these findings pointed out the potential of the metabolomics approach for neonatal nutritional science, in particular to provide important contributions to the optimization of formula milk.

Lactobacillus paracasei CBA L74 interferes with gliadin peptides entrance in Caco-2 cells.

Several recent reports describe a role of probiotics as a therapeutic approach for celiac disease (CD). Two undigested A-gliadin peptides, P31-43 and P57-68, are central to CD pathogenesis, inducing an innate and an adaptive immune response, respectively. They enter enterocytes and localize to vesicular compartment to induce their toxic/immunogenics effects. In this article, we tested the effect of probiotic Lactobacillus paracasei (LP) CBA L74 (International Depository Accession Number LMG P-24778), its supernatant and LP-fermented cereals on gliadin peptides, P31-43 and P57-68, entrance in Caco-2 cells. Both LP CBA L74 and its supernatant inhibit P31-43 (intensity of fluorescence; FI: 75%) and P57-68 (FI: 50%) entrance in Caco2 cells, indicating that this biological effect is due to some product included in LP CBA L74 supernatant. This effect was present also after fermentation of cereals. This study describes a novel effect of probiotics in the prevention of undigested gliadin peptides toxic effects.

Lactobacillus paracasei CBA L74 metabolic products and fermented milk for infant formula have anti-inflammatory activity on dendritic cells in vitro and protective effects against colitis and an enteric pathogen in vivo.

The rapid expansion of commercially available fermented food products raises important safety issues particularly when infant food is concerned. In many cases, the activity of the microorganisms used for fermentation as well as what will be the immunological outcome of fermented food intake is not known. In this manuscript we used complex in vitro, ex-vivo and in vivo systems to study the immunomodulatory properties of probiotic-fermented products (culture supernatant and fermented milk without live bacteria to be used in infant formula). We found in vitro and ex-vivo that fermented products of Lactobacillus paracasei CBA L74 act via the inhibition of proinflammatory cytokine release leaving anti-inflammatory cytokines either unaffected or even increased in response to Salmonella typhimurium. These activities are not dependent on the inactivated bacteria but to metabolic products released during the fermentation process. We also show that our in vitro systems are predictive of an in vivo efficacy by the fermented products. Indeed CBA L74 fermented products (both culture medium and fermented milk) could protect against colitis and against an enteric pathogen infection (Salmonella typhimurium). Hence we found that fermented products can act via the inhibition of immune cell inflammation and can protect the host from pathobionts and enteric pathogens. These results open new perspectives in infant nutrition and suggest that L. paracasei CBA L74 fermented formula can provide immune benefits to formula-fed infants, without carrying live bacteria that may be potentially dangerous to an immature infant immune system.

Investigation of the degradation of cresols in the treatments with ozone.

The reaction between ozone and the three cresol isomers was investigated in pure water. Cresols were selected as model substrates representing an important component of humic material. Cresols carry both a hydroxyl and a methyl group, each theoretically increasing the reactivity of ozone with the aromatic ring. Direct comparison of the aromatic ring and the methyl group reactivities was made possible by the analysis of reaction products. The substrate degradation kinetics was studied by preparing aqueous solutions of each cresol and treating them with ozone for increasing time periods. It had been hypothesized that hydroxybenzaldehydes and hydroxybenzoic acids could be possible degradation intermediates of cresols. To verify this hypothesis, the degradation kinetics of three hydroxybenzaldehydes and two hydroxybenzoic acids were also studied. The reaction products were studied using gas chromatography (GC)-electron capture negative ionization (ECNI)-mass spectrometry (MS) analysis after direct derivatization of the samples with 5-chloro-2,2,3,3,4,4,5,5-octafluoro-1-pentyl chloroformate (ClOFPCF). This new analytical approach enables the extraction and analysis of highly polar polycarboxylic and hydroxycarboxylic acids, as well as highly polar aldehydes and hydroxy aldehydes that are difficult to extract and measure using conventional methods. As such, this new approach offered insights into ozone reaction intermediates that had been previously hypothesized, but not confirmed. Several highly hydrophilic degradation intermediates were identified, including malic, citraconic, itaconic, malonic, methylmuconic, and tartronic acid, but no hydroxybenzaldehydes were observed. The results support a 3-stage mechanism previously hypothesized, which involves ring-opening of the phenolic group, followed by the generation of several intermediates of increasing oxidation state, finally leading to relatively stable products, such as malonic and oxalic acids. We demonstrated that oxidation of the methyl group does not occur during cresol degradation.

Application of the novel 5-chloro-2,2,3,3,4,4,5,5-octafluoro-1-pentyl chloroformate derivatizing agent for the direct determination of highly polar water disinfection byproducts.

A novel derivatizing agent, 5-chloro-2,2,3,3,4,4,5,5-octafluoropentyl chloroformate (ClOFPCF), was synthesized and tested as a reagent for direct water derivatization of highly polar and hydrophilic analytes. Its analytical performance satisfactorily compared to a perfluorinated chloroformate previously described, namely 2,2,3,3,4,4,5,5-octafluoropentyl chloroformate (OFPCF). The chemical properties (reactivity, selectivity, derivatization products, and their chromatographic and spectral features) for ClOFPCF were investigated using a set of 39 highly polar standard analytes, including, among others, hydroxylamine, malic and succinic acids, resorcinol, hydroxybenzaldehyde, and dihydroxybenzoic acid. Upon derivatization, the analytes were extracted from the aqueous solvent and analyzed by gas chromatography (GC)-mass spectrometry (MS) in the electron-capture negative ionization (ECNI) mode. Positive chemical ionization (PCI)-MS was used for confirming the molecular ions, which were virtually absent in the ECNI mass spectra. ClOFPCF showed good reaction efficiency, good chromatographic and spectroscopic properties (better than with OFPCF), good linearity in calibration curves, and low detection limits (0.3-1 microg/L). A unique feature of the derivatizations with ClOFPCF, and, in general, highly fluorinated chloroformates, is their effectiveness in reacting with carboxylic, hydroxylic, and aminic groups at once, forming multiply-substituted non-polar derivatives that can be easily extracted from the aqueous phase and determined by GC-ECNI-MS. The entire procedure from raw aqueous sample to ready-to-inject hexane solution of the derivatives requires less than 10 min. Another benefit of this procedure is that it produced stable derivatives, with optimal volatility for GC separation, and high electron affinity, which allows their detection as negative ions at trace level. In addition, their mass spectra exhibits chlorine isotopic patterns that clearly indicate how many polar hydrogens of the analyte undergo derivatization. Finally, derivatization with ClOFPCF was used successfully to identify 13 unknown highly polar disinfection byproducts (DBPs) in ozonated fulvic and humic acid aqueous solutions and in real ozonated drinking water.

Occurrence and mammalian cell toxicity of iodinated disinfection byproducts in drinking water.

An occurrence study was conducted to measure five iodo-acids (iodoacetic acid, bromoiodoacetic acid, (Z)-3-bromo-3-iodo-propenoic acid, (E)-3-bromo-3-iodo-propenoic acid, and (E)-2-iodo-3-methylbutenedioic acid) and two iodo-trihalomethanes (iodo-THMs), (dichloroiodomethane and bromochloroiodomethane) in chloraminated and chlorinated drinking waters from 23 cities in the United States and Canada. Since iodoacetic acid was previouslyfound to be genotoxic in mammalian cells, the iodo-acids and iodo-THMs were analyzed for toxicity. A gas chromatography (GC)/negative chemical ionization-mass spectrometry (MS) method was developed to measure the iodo-acids; iodo-THMs were measured using GC/high resolution electron ionization-MS with isotope dilution. The iodo-acids and iodo-THMs were found in waters from most plants, at maximum levels of 1.7 microg/L (iodoacetic acid), 1.4 microg/L (bromoiodoacetic acid), 0.50 microg/L ((Z)-3-bromo-3-iodopropenoic acid), 0.28 microg/L ((E)-3-bromo-3-iodopropenoic acid), 0.58 microg/L ((E)-2-iodo-3-methylbutenedioic acid), 10.2 microg/L (bromochloroiodomethane), and 7.9 microg/L (dichloroiodomethane). Iodo-acids and iodo-THMs were highest at plants with short free chlorine contact times (< 1 min), and were lowest at a chlorine-only plant or at plants with long free chlorine contact times (> 45 min). Iodide levels in source waters ranged from 0.4 to 104.2 microg/L (when detected), but there was not a consistent correlation between bromide and iodide. The rank order for mammalian cell chronic cytotoxicity of the compounds measured in this study, plus other iodinated compounds, was iodoacetic acid > (E)-3-bromo-2-iodopropenoic acid > iodoform > (E)-3-bromo-3-iodo-propenoic acid > (Z)-3-bromo-3-iodo-propenoic acid > diiodoacetic acid > bromoiodoacetic acid > (E)-2-iodo-3-methylbutenedioic acid > bromodiiodomethane > dibromoiodomethane > bromochloroiodomethane approximately chlorodiiodomethane > dichloroiodomethane. With the exception of iodoform, the iodo-THMs were much less cytotoxic than the iodo-acids. Of the 13 compounds analyzed, 7 were genotoxic; their rank order was iodoacetic acid >> diiodoacetic acid > chlorodiiodomethane > bromoiodoacetic acid > E-2-iodo-3-methylbutenedioic acid > (E)-3-bromo-3-iodo-propenoic acid > (E)-3-bromo-2-iodopropenoic acid. In general, compounds that contain an iodo-group have enhanced mammalian cell cytotoxicity and genotoxicity as compared to their brominated and chlorinated analogues.

Fully immunocompetent CD8+ T lymphocytes are present in autologous haematopoietic stem cell transplantation recipients despite an ineffectual T-helper response.

BACKGROUND: Reduced CD4 T lymphocytes counts can be observed in HIV infection and in patients undergoing autologous haematopoietic stem cell transplantation (ASCT). Nevertheless, whereas opportunistic infections (OI) are frequent in HIV-infected individuals with low cell counts, OI are uncommon in ASCT patients. METHODOLOGY/PRINCIPAL FINDINGS: To verify whether this observation could be secondary to intrinsic HIV-correlated T cell defects, we performed in-depth immunologic analyses in 10 patients with comparable CD4 counts in whom lymphopenia was secondary either to HIV-infection or ASCT-associated immunosuppressive therapy and compared them to age-matched healthy subjects. Results showed the presence of profound alterations in CD4+ T lymphocytes in both groups of patients with respect to healthy controls. Thus, a low percentage of CCR7+ CD4+ T cells and a compensative expansion of CD45RA-CCR7- CD4+ T cells, a reduced IL-2/IFN-gamma cytokine production and impaired recall antigens-specific proliferative responses were detected both in ASCT and HIV patients. In stark contrast, profound differences were detected in CD8+ T-cells between the two groups of patients. Thus, mature CD8+ T cell prevailed in ASCT patients in whom significantly lower CD45RA-CCR7- cells, higher CD45RA+CCR7- CD8+ cells, and an expansion of CCR7+CD8+ cells was detected; this resulted in higher IFN-gamma +/TNFalpha production and granzyme CD8+ expression. The presence of strong CD8 T cells mediated immune responses justifies the more favorable clinical outcome of ASCT compared to HIV patients. CONCLUSION/SIGNIFICANCE: These results indicate that CD8 T cells maturation and functions can be observed even in the face of a profound impairment of CD4+ T lymphocytes in ASCT but not in HIV patients. Primary HIV-associated CD8 defects or an imprinting by an intact CD4 T cell system in ASCT could justify these results.

Occurrence, synthesis, and mammalian cell cytotoxicity and genotoxicity of haloacetamides: an emerging class of nitrogenous drinking water disinfection byproducts.

The haloacetamides, a class of emerging nitrogenous drinking water disinfection byproduct (DBPs), were analyzed for their chronic cytotoxicity and for the induction of genomic DNA damage in Chinese hamster ovary cells. The rank order for cytotoxicity of 13 haloacetamides was DIAcAm > IAcAm > BAcAm > TBAcAm > BIAcAm > DBCAcAm > CIAcAm > BDCAcAm > DBAcAm > BCAcAm > CAcAm > DCAcAm > TCAcAm. The rank order of their genotoxicity was TBAcAm > DIAcAm approximately equal to IAcAm > BAcAm > DBCAcAm > BIAcAm > BDCAcAm > CIAcAm > BCAcAm > DBAcAm > CAcAm > TCAcAm. DCAcAm was not genotoxic. Cytotoxicity and genotoxicity were primarily determined by the leaving tendency of the halogens and followed the order I > Br > > Cl. With the exception of brominated trihaloacetamides, most of the toxicity rank order was consistent with structure-activity relationship expectations. For di- and trihaloacetamides, the presence of at least one good leaving halogen group (I or Br but not Cl) appears to be critical for significant toxic activity. Log P was not a factor for monohaloacetamides but may play a role in the genotoxicity of trihaloacetamides and possible activation of dihaloacetamides by intracellular GSH and -SH compounds.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G: a possible role in the resistance to HIV of HIV-exposed seronegative individuals.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), a human cytidine deaminase, is a potent inhibitor of HIV replication. To explore a possible role of this protein in modulating in vivo susceptibility to HIV infection, we analyzed APOBEC3G expression in HIV-exposed seronegative individuals, HIV-seropositive patients, and healthy control subjects. The results showed that the expression of APOBEC3G is significantly increased in peripheral blood mononuclear cells (PBMCs)--mainly CD14(+) cells--and in cervical tissues of HIV-exposed seronegative individuals. Higher APOBEC3G expression correlated with a reduced susceptibility of PBMCs to in vitro infection with the HIV-1(Ba-L) R5 strain. APOBEC3G could be important in modulating in vivo susceptibility to sexually transmitted HIV infection.

Programmed cell death of myelin basic protein-specific T lymphocytes is reduced in patients with acute multiple sclerosis.

We investigated the apoptosis of myelin basic protein (MBP)-specific T lymphocytes in multiple sclerosis (MS) patients with acute (AMS) or stable (SMS) MS by evaluating the expression of apoptosis markers on peripheral cells. Cells of healthy controls (HC) were evaluated as well. Results showed that mitogen-stimulated apoptosis was comparable among patients and controls, whereas MBP-stimulated CD4+ and CD8+ 7-AAD+ and 7-AAD+ Fas+ cell (apoptotic cells) were significantly reduced in AMS patients. A reduction of the apoptotic rate of myelin-specific CD4+ and CD8+ T lymphocytes could be involved in the immune-mediated destruction of the myelin sheath seen in AMS patients.

Distinct patterns of HIV-specific memory T lymphocytes in HIV-exposed uninfected individuals and in HIV-infected patients.

BACKGROUND: Repeated exposure to HIV is not always associated with infection and multiple cohorts of HIV-exposed but seronegative individuals (ESN) have been described. HIV-specific CD4 and CD8 T lymphocytes are detected both in HIV patients and in ESN; we verified whether different patterns of HIV-specific memory T lymphocytes would be detected in individuals in whom exposure to HIV results or does not result in infection. METHODS: Gag-specific T cells were analysed in 15 ESN, 14 HIV patients, and 15 healthy controls using extensive flow cytometry analysis. RESULTS: Data confirmed that gag-specific T lymphocytes are present in ESN. Gag-specific T cells mainly secrete interleukin-2 in ESN and interferon-gamma in HIV patients. In addition the CD4/CD8 and the memory/naive ratios are altered, central memory (45RA-/CCR7+) CD4 and CD8 T lymphocytes are more abundant, and terminally differentiated (45RA+/CCR7- and 27-/28-) CD8 T lymphocytes are augmented in ESN individuals. CONCLUSIONS: Exposure to HIV occurs in high risk seronegative individuals; the observation that naive cells and CM are skewed in ESN indicate that this exposure is robust enough to modulate the CM/EM ratio. The increase in late effectors and in natural killer cells seen in ESN suggests a role for these cells in preventing actual infection.

Interleukin-10 and interleukin-6 gene polymorphisms as risk factors for Alzheimer's disease.

In the pathogenesis of Alzheimer disease (AD), it has been proposed that the anti-inflammatory interleukins such as IL-10 regulate beta-amyloid-induced microglial inflammatory responses inhibiting the proinflammatory cytokine IL-6. Since the promoters of the IL-10 and IL-6 genes show single nucleotide polymorphisms (SNPs) (IL-10: -1082 G --> A; IL-6: -174 G --> C), we investigated these SNPs and cytokine production by peripheral blood mononuclear cells in 65 AD patients and 65 controls (HC). In AD there was a significant increase of the -1082A IL-10 allele (P=0.009) and a decrease of -1082GG genotype (P=0.019). The frequency of the GG IL-6 genotype in AD was lower and the C allele significantly higher (P <0.005). The co-occurrence of IL-10 A and IL-6 C alleles significantly raised the odds ratio (OR 11.2, confidence interval: CI 1.3-97.3; P <0.05) independently of apolipoprotein E4 (adjusted OR 10.3, CI 1-108; P <0.05). Only amyloid-stimulated IL-10 production differed between the groups (P=0.023). These results raise questions regarding the inflammatory theory in AD, pointing to a pivotal role of IL-10 and IL-6 and a selective alteration in this network.

Human alpha defensin in HIV-exposed but uninfected individuals.

Human alpha defensins 1, 2, and 3 are produced by CD8 T cells of HIV-infected long-term nonprogressors and have an antiviral activity. alpha Defensins were examined in peripheral blood mononuclear cells (PBMCs), cervical-vaginal mononuclear cells (CVMCs), and cervical biopsies of 9 HIV-1-exposed but uninfected women (ESNs), 10 HIV-infected patients (HIV), and 13 low-risk healthy controls (HCs). Results showed that, whereas alpha defensin production and alpha defensin-expressing CD8 lymphocytes were comparable in ESNs and HIV patients, constitutive alpha defensin production by peripheral CD8 and CVMCs was augmented in ESNs compared with HCs (P = 0.001 and P = 0.058, respectively); alpha defensin mRNA was increased in PBMCs of ESNs; unstimulated, alpha defensin-expressing peripheral and mucosal CD8 lymphocytes were 10-fold higher in ESNs compared with HCs (P = 0.003 and P = 0.01, respectively); and alpha defensin mRNA and alpha defensin-expressing cells were augmented in cervical biopsies of ESN compared with HCs (mRNA:P = 0.03). The differences were reduced upon in vitro mitogen stimulation. A robust constitutive production of alpha defensin is seen in HIV-exposed uninfected individuals; these peptides could have a role in the potentially protective immune response that characterizes ESNs.