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Abstract Background The human noroviruses are a highly diverse group of viruses with a single-stranded RNA genome encoding a single major structural protein (VP1), which has a hypervariable domain (P2 domain) as the most exposed part of the virion. The noroviruses are classified on the basis of nucleotide sequence diversity in the VP1-encoding ORF2 gene, which divides the majority of human noroviruses into two genogroups (GI and GII). GII-4 noroviruses are the major aetiological agent of outbreaks of gastroenteritis around the world. During a winter season the diversity among the GII-4 noroviruses has been shown to fluctuate, driving the appearance of new virus variants in the population. We have previously shown that sequence data and in silico modelling experiments suggest there are two surface-exposed sites (site A and site B) in the hypervariable P2 domain. We predict these sites may form a functional variant-specific epitope that evolves under selective pressure from the host immune response and gives rise to antibody escape mutants. Results In this paper, we describe the construction of recombinant baculoviruses to express VLPs representing one pre-epidemic and one epidemic variant of GII-4 noroviruses, and the production of monoclonal antibodies against them. We use these novel reagents to provide evidence that site A and site B form a conformational, variant-specific, surface-exposed site on the GII-4 norovirus capsid that is involved in antibody binding. Conclusion As predicted by our earlier study, significant amino acid changes at site A and site B give rise to GII-4 norovirus epidemic variants that are antibody escape mutants.

Abstract Background Reverse transcription-polymerase chain reaction (RT-PCR) is the main method for laboratory diagnosis of norovirus-associated infectious intestinal disease (IID). However, up to 16% of healthy individuals in the community, with no recent history of IID, may be RT-PCR positive; so it is unclear whether norovirus is actually the cause of illness in an IID case when they are RT-PCR positive. It is important to identify the pathogen causing illness in sporadic IID cases, for clinical management and for community based incidence studies. The aim of this study was to investigate how faecal viral load can be used to determine when norovirus is the most likely cause of illness in an IID case. Methods Real-time RT-PCR was used to determine the viral load in faecal specimens collected from 589 IID cases and 159 healthy controls, who were infected with genogroup II noroviruses. Cycle threshold (Ct) values from the real-time RT-PCR were used as a proxy measure of viral load. Receiver-operating characteristic (ROC) analysis was used to identify a cut-off in viral load for attributing illness to norovirus in IID cases. Results One hundred and sixty-nine IID cases and 159 controls met the inclusion criteria for the ROC analysis. The optimal Ct value cut-off for attributing IID to norovirus was 31. The same cut-off was selected when using healthy controls, or IID cases who were positive by culture for bacterial pathogens, as the reference negative group. This alternative reference negative group can be identified amongst specimens routinely received in clinical virology laboratories. Conclusion We demonstrated that ROC analysis can be used to select a cut-off for a norovirus real time RT-PCR assay, to aid clinical interpretation and diagnose when norovirus is the cause of IID. Specimens routinely received for diagnosis in clinical virology laboratories can be used to select an appropriate cut-off. Individual laboratories can use this method to define in-house cut-offs for their assays, to provide the best possible diagnostic service to clinicians and public health workers. Other clinical and epidemiological information should also be considered for patients with Ct values close to the cut-off, for the most accurate diagnosis of IID aetiology.

Abstract Background Noroviruses are highly infectious pathogens that cause gastroenteritis in the community and in semi-closed institutions such as hospitals. During outbreaks, multiple units within a hospital are often affected, and a major question for control programs is: are the affected units part of the same outbreak or are they unrelated transmission events? In practice, investigators often assume a transmission link based on epidemiological observations, rather than a systematic approach to tracing transmission. Here, we present a combined molecular and statistical method for assessing: 1) whether observed clusters provide evidence of local transmission and 2) the probability that anecdotally|linked outbreaks truly shared a transmission event. Methods 76 healthcare associated outbreaks were observed in an active and prospective surveillance scheme of 15 hospitals in the county of Avon, England from April 2002 to March 2003. Viral RNA from 64 out of 76 specimens from distinct outbreaks was amplified by reverse transcription-PCR and was sequenced in the polymerase (ORF 1) and capsid (ORF 2) regions. The genetic diversity, at the nucleotide level, was analysed in relation to the epidemiological patterns. Results Two out of four genetic and epidemiological clusters of outbreaks were unlikely to have occurred by chance alone, thus suggesting local transmission. There was anecdotal epidemiological evidence of a transmission link among 5 outbreaks pairs. By combining this epidemiological observation with viral sequence data, the evidence of a link remained convincing in 3 of these pairs. These results are sensitive to prior beliefs of the strength of epidemiological evidence especially when the outbreak strains are common in the background population. Conclusion The evidence suggests that transmission between hospitals units does occur. Using the proposed criteria, certain hypothesized transmission links between outbreaks were supported while others were refuted. The combined molecular/epidemiologic approach presented here could be applied to other viral populations and potentially to other pathogens for a more thorough view of transmission.

Abstract Background Noroviruses are the most common cause of gastroenteritis outbreaks in industrialised countries. Gastroenteritis caused by Norovirus infection has been described as a highly seasonal syndrome, often referred to as "winter vomiting disease". Methods The Public Health Laboratory Service Communicable Disease Surveillance Centre has systematically collected reports of laboratory confirmed cases of Norovirus-gastroenteritis since 1995. We analysed these data for annual and seasonal trends and age distribution. Results A mid-summer peak in reported cases of Norovirus was observed in 2002, unlike all six previous years when there was a marked summer decline. Total reports from 2002 have also been higher than all previous years. From the first 10 months of 2002, a total of 3029 Norovirus diagnoses were reported compared the previous peak in 1996 of 2437 diagnoses for the whole 12-month period. The increase in 2002 was most marked in the 65 and older age group. Conclusion This surveillance data challenges the view that Noroviruses infections exclusively have wintertime seasonality.

A distinct CD83-expressing subset of γδ T cells are enriched in preterm infants with sepsis, providing insights into their functional maturation dynamics in settings of homeostasis and disease (León-Lara et al. https://doi.org/10.1084/jem.20231987)., (© 2024 Farber and Gray.)

During ontogeny, γδ T cells emerge from the thymus and directly seed peripheral tissues for in situ immunity. However, their functional role in humans has largely been defined from blood. Here, we analyzed the phenotype, transcriptome, function, and repertoire of human γδ T cells in blood and mucosal and lymphoid tissues from 176 donors across the life span, revealing distinct profiles in children compared with adults. In early life, clonally diverse Vδ1 subsets predominate across blood and tissues, comprising naïve and differentiated effector and tissue repair functions, whereas cytolytic Vδ2 subsets populate blood, spleen, and lungs. With age, Vδ1 and Vδ2 subsets exhibit clonal expansions and elevated cytolytic signatures, which are disseminated across sites. In adults, Vδ2 cells predominate in blood, whereas Vδ1 cells are enriched across tissues and express residency profiles. Thus, antigenic exposures over childhood drive the functional evolution and tissue compartmentalization of γδ T cells, leading to age-dependent roles in immunity.

Influenza A virus (IAV) infection leads to the formation of mucosal memory CD4 T cells that can protect the host. An in-depth understanding of the signals that shape memory cell development is required for more effective vaccine design. We have examined the formation of memory CD4 T cells in the lung following IAV infection of mice, characterizing changes to the lung landscape and immune cell composition. IAV-specific CD4 T cells were found throughout the lung at both primary and memory time points. These cells were found near lung airways and in close contact with a range of immune cells including macrophages, dendritic cells, and B cells. Interactions between lung IAV-specific CD4 T cells and major histocompatibility complex (MHC)II+ cells during the primary immune response were important in shaping the subsequent memory pool. Treatment with an anti-MHCII blocking antibody increased the proportion of memory CD4 T cells found in lung airways but reduced interferon-γ expression by IAV-specific immunodominant memory CD4 T cells. The immunodominant CD4 T cells expressed higher levels of programmed death ligand 1 (PD1) than other IAV-specific CD4 T cells and PD1+ memory CD4 T cells were located further away from MHCII+ cells than their PD1-low counterparts. This distinction in location was lost in mice treated with anti-MHCII antibodies. These data suggest that sustained antigen presentation in the lung impacts the formation of memory CD4 T cells by regulating their cytokine production and location., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Respiratory failure in COVID-19 is characterized by widespread disruption of the lung's alveolar gas exchange interface. To elucidate determinants of alveolar lung damage, we performed epithelial and immune cell profiling in lungs from 24 COVID-19 autopsies and 43 uninfected organ donors ages 18-92 years. We found marked loss of type 2 alveolar epithelial (T2AE) cells and increased perialveolar lymphocyte cytotoxicity in all fatal COVID-19 cases, even at early stages before typical patterns of acute lung injury are histologically apparent. In lungs from uninfected organ donors, there was also progressive loss of T2AE cells with increasing age, which may increase susceptibility to COVID-19-mediated lung damage in older individuals. In the fatal COVID-19 cases, macrophage infiltration differed according to the histopathological pattern of lung injury. In cases with acute lung injury, we found accumulation of CD4+ macrophages that expressed distinctly high levels of T cell activation and costimulation genes and strongly correlated with increased extent of alveolar epithelial cell depletion and CD8+ T cell cytotoxicity. Together, our results show that T2AE cell deficiency may underlie age-related COVID-19 risk and initiate alveolar dysfunction shortly after infection, and we define immune cell mediators that may contribute to alveolar injury in distinct pathological stages of fatal COVID-19.

Tissue-resident immune cells span both myeloid and lymphoid cell lineages, have been found in multiple human tissues, and play integral roles at all stages of the immune response, from maintaining homeostasis to responding to infectious challenges to resolution of inflammation to tissue repair. In humans, studying immune cells and responses in tissues is challenging, although recent advances in sampling and high-dimensional profiling have provided new insights into the ontogeny, maintenance, and functional role of tissue-resident immune cells. Each tissue contains a specific complement of resident immune cells. Moreover, resident immune cells for each lineage share core properties, along with tissue-specific adaptations. Here we propose a five-point checklist for defining resident immune cell types in humans and describe the currently known features of resident immune cells, their mechanisms of development, and their putative functional roles within various human organs. We also consider these aspects of resident immune cells in the context of future studies and therapeutics.

Adaptive immune responses to SARS-CoV-2 infection have been extensively characterized in blood; however, most functions of protective immunity must be accomplished in tissues. Here, we report from examination of SARS-CoV-2 seropositive organ donors (ages 10 to 74) that CD4 + T, CD8 + T, and B cell memory generated in response to infection is present in the bone marrow, spleen, lung, and multiple lymph nodes (LNs) for up to 6 months after infection. Lungs and lung-associated LNs were the most prevalent sites for SARS-CoV-2–specific memory T and B cells with significant correlations between circulating and tissue-resident memory T and B cells in all sites. We further identified SARS-CoV-2–specific germinal centers in the lung-associated LNs up to 6 months after infection. SARS-CoV-2–specific follicular helper T cells were also abundant in lung-associated LNs and lungs. Together, the results indicate local tissue coordination of cellular and humoral immune memory against SARS-CoV-2 for site-specific protection against future infectious challenges.

Effective tolerogenic intervention in Rheumatoid Arthritis (RA) will rely upon understanding the evolution of articular antigen specific CD4 T cell responses. TCR clonality of endogenous CD4 T cell infiltrates in early inflammatory arthritis was assessed to monitor evolution of the TCR repertoire in the inflamed joint and associated lymph node (LN). Mouse models of antigen-induced breach of self-tolerance and chronic polyarthritis were used to recapitulate early and late phases of RA. The infiltrating endogenous, antigen experienced CD4 T cells in inflamed joints and LNs were analysed using flow cytometry and TCRβ sequencing. TCR repertoires from inflamed late phase LNs displayed increased clonality and diversity compared to early phase LNs, while inflamed joints remained similar with time. Repertoires from late phase LNs accumulated clones with a diverse range of TRBV genes, while inflamed joints at both phases contained clones expressing similar TRBV genes. Repertoires from LNs and joints at the late phase displayed reduced CDR3β sequence overlap compared to the early disease phase, however the most abundant clones in LNs accumulate in the joint at the later phase. The results indicate CD4 T cell repertoire clonality and diversity broadens with progression of inflammatory arthritis and is first reflected in LNs before mirroring in the joint. These observations imply that antigen specific tolerogenic therapies could be more effective if targeted at earlier phases of disease when CD4 T cell clonality is least diverse., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Al Khabouri, Benson, Prendergast, Gray, Otto, Brewer and Garside.)

Immune response dynamics in coronavirus disease 2019 (COVID-19) and their severe manifestations have largely been studied in circulation. Here, we examined the relationship between immune processes in the respiratory tract and circulation through longitudinal phenotypic, transcriptomic, and cytokine profiling of paired airway and blood samples from patients with severe COVID-19 relative to heathy controls. In COVID-19 airways, T cells exhibited activated, tissue-resident, and protective profiles; higher T cell frequencies correlated with survival and younger age. Myeloid cells in COVID-19 airways featured hyperinflammatory signatures, and higher frequencies of these cells correlated with mortality and older age. In COVID-19 blood, aberrant CD163 + monocytes predominated over conventional monocytes, and were found in corresponding airway samples and in damaged alveoli. High levels of myeloid chemoattractants in airways suggest recruitment of these cells through a CCL2-CCR2 chemokine axis. Our findings provide insights into immune processes driving COVID-19 lung pathology with therapeutic implications for targeting inflammation in the respiratory tract., Competing Interests: Declaration of interests J.Z., M.S., and S.M. have competing interests with IsoPlexis. The remaining authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Memory T cells respond rapidly in part because they are less reliant on a heightened levels of costimulatory molecules. This enables rapid control of secondary infecting pathogens but presents challenges to efforts to control or silence memory CD4 T cells, for example in antigen-specific tolerance strategies for autoimmunity. We have examined the transcriptional and functional consequences of reactivating memory CD4 T cells in the absence of an adjuvant. We find that memory CD4 T cells generated by infection or immunisation survive secondary activation with antigen delivered without adjuvant, regardless of their location in secondary lymphoid organs or peripheral tissues. These cells were, however, functionally altered following a tertiary immunisation with antigen and adjuvant, proliferating poorly but maintaining their ability to produce inflammatory cytokines. Transcriptional and cell cycle analysis of these memory CD4 T cells suggests they are unable to commit fully to cell division potentially because of low expression of DNA repair enzymes. In contrast, these memory CD4 T cells could proliferate following tertiary reactivation by viral re-infection. These data indicate that antigen-specific tolerogenic strategies must examine multiple parameters of Tcell function, and provide insight into the molecular mechanisms that may lead to deletional tolerance of memory CD4 T cells., (© 2020 The Authors. Immunology published by John Wiley & Sons Ltd.)

T cell protective immunity is associated with multifunctional memory cells that produce several different cytokines. Currently, our understanding of when and how these cells are generated is limited. We have used an influenza virus mouse infection model to investigate whether the cytokine profile of memory T cells is reflective of primary responding cells or skewed toward a distinct profile. We found that, in comparison to primary cells, memory T cells tended to make multiple cytokines simultaneously. Analysis of the timings of release of cytokine by influenza virus-specific T cells, demonstrated that primary responding CD4 T cells from lymphoid organs were unable to produce a sustained cytokine response. In contrast CD8 T cells, memory CD4 T cells, and primary responding CD4 T cells from the lung produced a sustained cytokine response throughout the restimulation period. Moreover, memory CD4 T cells were more resistant than primary responding CD4 T cells to inhibitors that suppress T cell receptor signaling. Together, these data suggest that memory CD4 T cells display superior cytokine responses compared to primary responding cells. These data are key to our ability to identify the cues that drive the generation of protective memory CD4 T cells following infection., (© 2019 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Immunological memory provides rapid protection to pathogens previously encountered through infection or vaccination. CD4 T-cells play a central role in all adaptive immune responses. Vaccines must, therefore, activate CD4 T-cells if they are to generate protective immunity. For many diseases, we do not have effective vaccines. These include human immunodeficiency virus (HIV), tuberculosis and malaria, which are responsible for many millions of deaths each year across the globe. CD4 T-cells play many different roles during the immune response coordinating the actions of many other cells. In order to harness the diverse protective effects of memory CD4 T-cells, we need to understand how memory CD4 T-cells are generated and how they protect the host. Here we review recent findings on the location of different subsets of memory CD4 T-cells that are found in peripheral tissues (tissue resident memory T-cells) and in the circulation (central and effector memory T-cells). We discuss the generation of these cells, and the evidence that demonstrates how they provide immune protection in animal and human challenge models., (© 2018 The Authors. Immunology Published by John Wiley & Sons Ltd.)

The desaturation of myristoyl-CoA to myristoleoyl-CoA was measured in microsomal preparations of hen liver. The desaturation was maximal at pH 7.4. The enzymatic activity was linear with time up to 10 min and proportional to microsomal protein concentrations. The initial velocity was linear with substrate concentrations between 13 and up to 200 microM. A decrease in desaturation activity was observed at substrate concentrations greater than 266 microM. There was an absolute requirement for reduced pyridine nucleotide (NADH), while a maximum activity was observed at a myristoyl-CoA:NADH mole ratio of 1. Competitive inhibition studies of myristoyl-CoA desaturation suggest that the inhibitors, stearyl and oleyl-CoA, were more effective than palmitoyl-CoA. Free CoA did not inhibit the delta(9)-desaturase system. The desaturation of myristoyl-CoA was stimulated by bovine serum albumin and reduced by cytoplasmic proteins. The effect of cytoplasmic proteins on the enzymatic reaction was completely abolished by trypsin digestion and boiling for 30 min. On the basis of these data, it was concluded that 9,10-desaturation of acyl-CoA derivatives containing 14-18 carbon fatty acyl chains is catalyzed by the same enzyme.

Direct desaturation of free myristic acid by hen liver microsomal Delta(9)-desaturase without prior activation to myristoyl-CoA by the addition of adenosine triphosphate (ATP) and CoA was observed when the incubation medium was mixed at mixing speeds of >250 rpm in the presence of fatty acid-binding proteins (FABP). Desaturation was linear with time and proportional to the microsomal protein concentration. Desaturation was maximal at pH 7.9. The greatest desaturation rate was observed at a mixing speed of 500 rpm in the presence of FABP. Desaturation decreased at mixing speeds of >500 rpm. Data suggest that when myristic acid is bound to FABP in the form of protein-monomer complexes, its activation to the CoA derivative is not necessary for it to be desaturated by the Delta(9)-desaturase when using mixing rates of >250 rpm. Myristic acid-FABP complexes serve as substrates for the Delta(9)-desaturase at mixing rates of >250 rpm. Desaturation was reduced by bovine serum albumin and alpha-bromohexadecanoate, and no desaturation was observed in the absence of FABP. These findings suggest that FABP may regulate the accessibility of fatty acids in the desaturation reaction to the active site of the desaturase rather than just protect the membrane-bound desaturase from the cytotoxic effect of free fatty acids.

The antioxidant activity of 3-dehydroshikimic acid (DHS), an intermediate in the biosynthesis of aromatic amino acids, was evaluated in three assay systems: bulk oil (lard), liposomes, and a 10% corn oil-in-water emulsion. Upon initiation of peroxidation in the liposome or emulsion systems, DHS exhibited weak antioxidant activity. In contrast, DHS displayed strong antioxidant activity in lard, suppressing peroxidation with activity comparable to that of tert-butylhydroquinone, propyl gallate, and gallic acid and superior to that of alpha-tocopherol. Two major DHS oxidation products, gallic acid and protocatechuic acid, were identified by gas chromatography/mass spectral analysis of lard extracts; both compounds are effective antioxidants in the bulk oil system. In the liposome system, DHS remained intact throughout the assay period. A small amount of gallic acid was observed in extracts of the emulsion; however, protocatechuic acid was not detected. A mechanism to explain the different activities of DHS in the three lipid systems is proposed.

Organosulfur compounds and sodium bisulfite significantly inhibited (P < 0.05) heterocyclic aromatic amine (HAA) formation in model systems containing phenylalanine, creatinine, and glucose. There was, however, no inhibition by the same compounds in a model system containing only phenylalanine and creatinine. Diallyl disulfide (DAD) and dipropyl disulfide (DPD) concentrations in the model systems were significantly decreased (P < 0.05) after heating for 10 min at 180 degrees C. Only very low concentrations of sulfhydryl groups (4.19 and 4.00 micromol) were produced on heating DAD and DPD for 30 min. Reaction of glucose and DAD produced several sulfur-containing compounds. After 10 min of heating at 180 degrees C, HAA formation in the control model systems was increased significantly, and DAD was an effective inhibitor during this heating period. Tetrahydrothiophene-3-one (THT) and tetrahydrothiophene (THP); two products resulting from the interaction of glucose and DAD, had no direct influence on HAA formation in the model systems.

The effects of garlic and selected organosulfur compounds (diallyl disulfide, dipropyl disulfide, diallyl sulfide, allyl methyl sulfide, allyl mercaptan, cysteine, and cystine) on the formation of heterocyclic aromatic amines (HAAs) in fried ground beef patties were evaluated. Minced garlic cloves (ca. 4.8 to 16.7%, wt/wt) or organosulfur compounds (0.67 mmol) were added directly to ground beef. Patties (100 g) were fried at 225 degrees C (surface temperature) for 10 min per side. Two patties were fried for each replication, and five replicates were analyzed for each treatment. For each replicate, four subsamples were analyzed (two unspiked subsamples for concentration and two spiked subsamples for the recovery of HAA standards). The volatile sulfur compounds significantly (P < 0.05) reduced concentrations of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by reductions of 46 to 81%, while average reductions of 35, 22, and 71%, were achieved with cystine, cysteine, and whole garlic, respectively. The volatile sulfur compounds reduced concentrations of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by 34 to 67%, while reductions of 25, 19, and 63% (P < 0.05) were achieved with cystine, cysteine, and whole garlic, respectively. These studies confirm that garlic and some organosulfur compounds have the potential to reduce HAA formation incooked beef patties.

Monoclonal antibodies (MAb) were produced to hexanal-bovine serum albumin conjugates. An indirect competitive ELISA was developed with a detection range of 1-50 ng of hexanal/mL. Hexanal conjugated to three different proteins was recognized, whereas free hexanal and the native proteins were not detected. The antibody cross-reacted with pentanal, heptanal, and 2-trans-hexenal conjugated to chicken serum albumin (CSA) with cross-reactivities of 37.9, 76.6, and 45.0%, respectively. There was no cross-reactivity with propanal, butanal, octanal, and nonanal conjugated to CSA. The hexanal content of a meat model system was determined using MAb and polyclonal antibody-based ELISAs and compared with analysis by a dynamic headspace gas chromatographic (HS-GC) method and a thiobarbituric acid reactive substances (TBARS) assay. Both ELISAs showed strong correlations with the HS-GC and TBARS methods. ELISAs may be a fast and simple alternative to GC for monitoring lipid oxidation in meat.

The effect of vitamin E and oleoresin rosemary on heterocyclic aromatic amine (HAA) formation in fried ground beef patties was studied. Patties were fried at three temperatures (175 degrees C, 200 degrees C, 225 degrees C) for 6 and 10 min/side to determine the conditions for optimum HAA formation. HAAs were isolated by solid phase extraction and quantitated by HPLC. Greatest concentrations were generated when patties were fried at 225 degrees C for 10 min/side, 31.4 ng/g 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 5.8 ng/g 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Vitamin E, when used at two concentrations (1% and 10% based on fat content) and added directly to the ground beef patties, reduced PhIP concentrations in the cooked patties by 69% and 72%, respectively. Smaller but more variable reductions were achieved for MeIQx. Comparable inhibition of HAA formation was achieved by the direct addition of vitamin E (1% based on fat content) to the surface of the patties before frying. Concentrations of five HAAs studied were all significantly reduced (P<0.006), with average reductions ranging from 45% to 75%. Oleoresin rosemary, when used at two concentrations (1% and 10% based on fat content), reduced PhIP formation by 44%.

Hexanal content is a widely used index of lipid oxidation in foods. The objectives of this study were to develop antibodies to hexanal-lysine adducts, devise an ELISA, and characterize antibody specificity. Hexanal was made immunogenic by covalent attachment to lysine side chains of bovine serum albumin via reductive alkylation. Polyclonal antibodies had antiserum titers as high as 6.15 x 10(5). A competitive indirect ELISA was developed with a detection limit of 0.7 ng of hexanal/mL. Antibodies were carrier-independent, reacting with hexanal conjugates of several proteins but not with the corresponding native proteins. Cross-reactivities with chicken serum albumin conjugates of n-heptanal, n-pentanal, and n-octanal were 86. 3, 11.8, and 2.2%, respectively. Antibodies reacted strongly with hexanal-modified lysine and hexanal-modified epsilon-aminocaproic acid but did not recognize free amino acids or free hexanal. It may be feasible to use this ELISA to monitor lipid oxidation in food provided hexanal is alkylated to a carrier protein prior to analysis.

Montmorency and Balaton tart cherries were lyophilized and sequentially extracted with hexane, ethyl acetate, and methanol. Methanolic extracts of dried Balaton and Montmorency tart cherries (Prunus cerasus) inhibited lipid peroxidation induced by Fe(2+) at 25 ppm concentrations. Further partitioning of this methanol extract with EtOAc yielded a fraction that inhibited lipid peroxidation by 76% at 25 ppm. Purification of this EtOAc fraction afforded eight polyphenolic compounds, 5,7,4'-trihydroxyflavanone (1), 5,7, 4'-trihydroxyisoflavone (2), chlorogenic acid (3), 5,7,3', 4'-tetrahydroxyflavonol-3-rhamnoside (4), 5,7,4'-trihydroxyflavonol 3-rutinoside (5), 5,7,4'-trihydroxy-3'methoxyflavonol-3-rutinoside (6), 5,7,4'-trihydroxyisoflavone-7-glucoside (7), and 6, 7-dimethoxy-5,8,4'-trihydroxyflavone (8), as characterized by (1)H and (13)C NMR experiments. The antioxidant assays revealed that 7-dimethoxy-5,8,4'-trihydroxyflavone (8) is the most active, followed by quercetin 3-rhamnoside, genistein, chlorogenic acid, naringenin, and genistin, at 10 microM concentrations.

As indicated by an Fe(II)-induced liposome peroxidation bioassay, the EtOAc extract of tart cherries (Prunus cerasus) was found to have strong antioxidant activity. Purification of this extract afforded chlorogenic acid methyl ester (1) and three novel compounds, 2-hydroxy-3-(o-hydroxyphenyl) propanoic acid (2); 1-(3', 4'-dihydroxycinnamoyl)-cyclopenta-2,5-diol (3), and 1-(3', 4'-dihydroxycinnamoyl)-cyclopenta-2,3-diol (4), as determined by their spectral data. At a 20-microM concentration, the antioxidant activities of compounds 3 and 4 were comparable to the antioxidant activities of caffeic acid, whereas compound 1 showed activity similar to chlorogenic acid. Also, these compounds showed antioxidant activities similar to the commercial antioxidants tert-butylhydroquinone and butylated hydroxytoluene. However, compound 2 was not active when tested at a 100-microM concentration.

1. Oxidation of meat and membrane from broilers fed on a diet containing 500 mg/kg rosemary and sage extracts was compared to meat and membrane oxidation from broilers receiving a control diet (not enriched with antioxidants) and a diet enriched in alpha-tocopheryl acetate (200 mg/kg). 2. After 9 d of refrigerated storage, thiobarbituric acid reactive substances of white meat from broilers fed on the control and the alpha-tocopheryl acetate-enriched diets were 0.51 and 0.25 mg malonaldehyde/kg meat, respectively. Values for meat from broilers fed on the diets containing the rosemary and sage extracts were in the range 0.30 to 0.35 mg malonaldehyde/kg meat, significantly lower than those from birds fed on the control diet. A similar trend was observed in the dark meat but differences were not significant at 9 d of storage. Similar trends were observed in raw samples stored at -20 degrees C for up to 4 months and in samples cooked at 70 degrees C and kept stored under refrigeration for up to 4 d. 3. The meat from broilers fed on the diet containing spice extracts had smaller concentrations of total cholesterol oxidation products (COPS) than meat from the control group (P < 0.05). Supplemental alpha-tocopheryl acetate reduced the COPS concentrations to a greater extent than did spice extracts (P < 0.05). 4. A similar trend was observed in microsomal fraction isolates, in which the rate of metmyoglobin/hydrogen peroxide-catalysed lipid peroxidation was lower in animals receiving spice extracts than in those fed on the basal diet.

1. The susceptibility to oxidation of meat from broilers receiving 200 g/kg oats in the diet was compared to the stability of meat from broilers receiving a control diet (10 mg/kg of alpha-tocopheryl acetate) and a diet enriched in alpha-tocopheryl acetate (200 mg/kg). 2. After 9 d of refrigerated storage thiobarbituric acid-reactive substances (TBARS) values in raw dark meat (thigh) were 0.51 and 0.19, respectively, for broilers fed on the control and the alpha-tocopheryl acetate-enriched diets. Meat from broilers fed on the diet containing oats had a lower TBARS value than the control group (P < 0.05), but higher than the group receiving the supplemented diet. The same trend was observed in white meat (breast). Similar effects were observed after the frozen storage of meats, although the antioxidant effect of oats was more evident (P < 0.05) than in the raw meat. 3. Cholesterol oxidation products (COPS) in broiler meat were also influenced by the dietary regimen. COPS concentration in white and dark meat, respectively, from broilers fed on the oat diet were 43% and 32% lower than those in the control samples. 4. Supplemental alpha-tocopheryl acetate reduced COPS concentration by approximately 60%. The availability of natural antioxidants and their possible synergistic effects suggest an interesting way of improving meat stability.

This experiment was conducted to study the effect of dietary vegetable oil on lipid oxidation in rabbit muscle. A control diet with no added fat and two diets with olive or sunflower oil (30 g/kg) were used. Within each treatment, one group was fed a low level of alpha-tocopheryl acetate (10 mg/kg diet), and the other a supplemental level (200 mg/kg). Rabbits were fed experimental diets from weaning (20 d) to slaughter (69 d). The supplemental level of dietary alpha-tocopheryl acetate produced higher alpha-tocopherol concentration in muscle (P < 0.006) and lower lipid oxidation (P < 0.004). Rabbits that received sunflower oil had higher concentrations of thiobarbituric acid reactive substances than rabbits that consumed olive oil (P < 0.05). Moreover, a significant effect due to fat inclusion in the diet was found. Muscles from rabbits fed diets not enriched with fat had higher susceptibility to lipid oxidation (P < 0.005) and higher concentration of (n-3) fatty acids in polar lipids (P < 0.04) than those from rabbits fed fat-enriched diets. A second experiment was conducted and confirmed the higher lipid oxidation in the muscle of rabbits fed diets not enriched with fat than in that of rabbits fed diets containing sunflower oil (28 g/kg) (P < 0.003) as well as in diets with identical digestible energy. In this experiment, alpha-tocopheryl acetate was at the lower level (10 mg/kg feed). Inclusion of oils rich in oleic (olive oil) or linoleic acid (sunflower oil) in rabbit diets reduces lipid oxidation in muscles.

Lipid oxidation is one of the primary mechanisms of quality deterioration in foods and especially in meat products. The changes in quality are manifested by adverse changes in flavor, color, texture and nutritive value, and the possible production of toxic compounds. Lipid oxidation in muscle systems is initiated at the membrane level in the intracellular phospholipid fractions. How this occurs has still not been resolved, although it is generally believed that the presence of transition metals, notably iron, is pivotal in facilitating the generation of species capable of abstracting a proton from an unsaturated fatty acid. This paper provides an overview of how lipid oxidation affects the quality and shelf life of meat and meat products, and how shelf life can be extended through dietary vitamin E supplementation above requirement levels. The formation of cholesterol oxidation products and the possible role of lipids and their oxidation products in the formation of heterocyclic aromatic amines are also discussed.

Oxidation of lipids is a major cause of deterioration in the quality of muscle foods and can directly affect many quality characteristics such as flavor, color, texture, nutritive value, and safety of the food. Lipid oxidation in muscle systems is initiated at the membrane level in the intracellular phospholipid fractions. In the processing of muscle foods, one of the most important questions concerns the methods used to delay the initiation of oxidation and loss of quality. Vitamin E is a major lipid-soluble antioxidant, and one of its primary functions is to maintain and protect biological membranes against lipid peroxidation. Dietary vitamin E supplementation above requirement levels is effective in reducing lipid oxidation. This review focuses on the antioxidant function of vitamin E and how supplementation of the diet of pigs with vitamin E influences the rate of lipid peroxidation, color, water-holding capacity, and cholesterol oxidation in pig meat.

The effect of oxidized corn oil and vitamin E (α-tocopheryl acetate) in pig diets on the oxidative stability of muscle lipids and on the surface colour characteristics of fresh and previously frozen pork chops in refrigerated storage was investigated. Lipid oxidation (TBARS values) and surface redness (Hunter 'a' values) were significantly influenced (P < 0·01) by dietary α-tocopheryl acetate levels but not by degree of oxidation of dietary corn oil. Lipid oxidation and colour deterioration during refrigerated storage were greater in previously frozen chops compared to fresh chops. TBARS values were lower and Hunter 'a' values higher in pork chops from pigs fed 100 and 200 mg α-tocopheryl acetate/kg diet compared to pigs fed 10 mg/kg diet after 2, 4, 6 and 8 days of refrigerated storage. Hunter 'a' values were significantly correlated (P < 0·01) with the logarithm of TBARS values. The results suggest that oxidation of myoglobin precedes oxidation of muscle lipids in pork chops stored at 4°C., (Copyright © 1994. Published by Elsevier Ltd.)

Female athymic nude mice were implanted subcutaneously with human breast carcinoma MDA-MB231. Seven to ten days later, the mice were divided into groups and fed a purified diet containing the following types of fat (% of diet): (i) 20% corn oil (CO); (ii) 15% CO:5% fish (menhaden) oil (FO); (iii) 10% CO:10% FO; (iv) 5% CO:15% FO; (v) 1% CO:19% FO; and (vi) 1% CO:19% FO plus antioxidants (alpha-tocopherol acetate, 2000 IU/kg diet and tertiary butyl-hydroquinone, 2% of total fat). The linoleic acid levels (% of diet) of the groups were 12.0, 9.1, 6.2, 3.3, 0.9 and 0.9%, respectively. After 6-8 wk, the carcinomas were assessed for tumor volume (cm3) and assayed for thiobarbituric acid reactive substances (TBARS). Human breast carcinoma growth was suppressed in mice consuming FO diets without antioxidants as compared to mice fed CO; the greater the amount of dietary FO fed, the greater the carcinoma growth suppression (P < 0.05). The addition of antioxidants to the FO diet significantly (P < 0.05) reversed the FO-induced carcinoma growth suppression. Concentrations of TBARS in the human breast carcinomas were increased in all the FO (without antioxidants) fed mice, compared to mice fed CO; the level of increase in TBARS was directly related to the increase in the level of FO fed (P < 0.05). The addition of antioxidants to the FO diet significantly (P < 0.05) reduced the concentration of TBARS in the breast carcinomas.(ABSTRACT TRUNCATED AT 250 WORDS)

Smoked foods including turkey, pork, chicken, beef and fish products were screened for the presence of carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons (PAHs). Eighteen commercial liquid smoke flavourings and seasonings were also analysed. Total PAH concentrations in smoked meat products ranged from 2.6 micrograms/kg in a cooked ham sample to 29.8 micrograms/kg in grilled pork chops, while those in fish products ranged from 9.3 micrograms/kg in smoked shrimp to 86.6 micrograms/kg in smoked salmon. Total concentrations of the carcinogenic PAHs (benzo[a]anthracene, benzo[b]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, and indeno[1,2,3-c,d]pyrene) ranged from non-detectable in several meat products to 7.4 micrograms/kg in grilled pork chops, and from 0.2 micrograms/kg in trout to 16.0 micrograms/kg in salmon. In liquid smoke flavourings and seasonings, total PAH concentrations ranged from 6.3 to 43.7 micrograms/kg, with the carcinogenic PAHs ranging from 0.3 to 10.2 micrograms/kg.

Two experiments were carried out to ascertain if supplementation of a semipurified diet to Swiss-ICR mice with either ascorbic acid (AA), vitamin E (Vit E) or a combination of the two would modulate the carcinogenic effects of N-nitrosopyrrolidine (NPyr) and of its probable precursors (nitrite-N0(2) and pyrrolidine-Pyr) in Experiment I or of NPyr in Experiment II. Results indicated that neither AA nor Vit E modulated the carcinogenic effects of NPyr or of its probable precursors (NO(2) and Pyr). Results verified a previous report from our laboratory showing that NPyr increased the number of malignant tumors by some 5-8 fold over controls. There was a lower incidence of tumors in the control group on the semi-purfied diet than in the groups given NO(2) and Pyr, although both treatments had a low frequency of malignant tumors (1 63 versus 5 72 survivors). Results support our earlier study suggesting that neither NO(2) nor Pyr alone or in combination together contribute to cancer-at least in the laboratory mouse., (Copyright © 1992. Published by Elsevier Ltd.)

The catalytic effect of haem proteins (haemoglobin and myoglobin) and inorganic iron (FeSO(4)) on lipid oxidation in a muscle model system was investigated. Haem proteins had a greater prooxidant effect than inorganic iron in raw and heated pork muscle residue when these prooxidants were present at levels approaching those in red meats. The rate of lipid oxidation catalysed by each prooxidant increased as the iron concentration increased over a range of 1-17 μg/g muscle residue. The relative prooxidant effects of haem protein and inorganic iron were not affected by the mode of addition of the prooxidants to the muscle residue (addition in a concentrated form or dispersed in water), or by the method of cooking (fast versus slow cooking)., (Copyright © 1993. Published by Elsevier Ltd.)

Peroxidation studies indicated that phospholipids, microsomes and mitochondria from cured pork samples are less susceptible to metmyoglobin/hydrogen peroxide-catalyzed peroxidation than their counterparts from nitrite-free pork samples. The reaction of phospholipids and polyunsaturated fatty acid ethyl esters with dinitrogen trioxide increased their stability to peroxidative changes. Phospholipids from cured pork and those lipids reacted with dinitrogen trioxide were capable of nitrosating a secondary amine. These data, together with infrared analyses, indicate that nitrite or dinitrogen trioxide reacts with unsaturated lipids to form nitro-nitroso derivatives, thus stabilizing the lipids toward peroxidation changes. This mechanism can, in part, explain the antioxidant role of nitrite in cured meats., (Copyright © 1992. Published by Elsevier Ltd.)

Purified corn and fish oil diets with different types and concentrations of antioxidants were evaluated for oxidation products. In addition, a determination of different organ and carcass oxidation product levels was performed. Peroxide value and thiobarbituric acid assays were performed on the diets immediately after mixing (0 h) and 24, 48 and 72 h after being fed to mice. The AIN-recommended level of antioxidant addition (butylated hydroxytoluene, 0.02 g/100 g oil) and even the addition of 100 times this level (2 g/100 g oil), although decreasing the level of oxidation products, failed to totally prevent oxidative deterioration in diets high in fish oil. Furthermore, other antioxidants added in excess to the fish oil diets also failed to completely suppress oxidative deterioration of the diets and, in addition, when fed daily to mice for a period of 4 wk, caused an accumulation of lipid peroxidation products in certain organs (e.g., heart, skeletal muscle, mammary glands) and in the carcass. These results provide evidence that in the preparation of fish oil diets, the addition of antioxidants at the AIN-recommended level, or even levels substantially higher, does not completely suppress oxidative deterioration of experimental diets.

The Maillard reaction, which involves Amadori rearrangement as a key step, also results in sugar fragmentation and free radical formation. The imidazoquinoline meat mutagens (2-amino-3-methylimidazo[4,5-f]-quinoline, or IQ, and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, or MeIQ) are formed from a reaction mixture containing alkylpyridine free radicals and creatinine. The imidazoquinoxaline meat mutagens (2-amino-3,4-dimethylimidazo[4,5-f]-quinoxaline, or MeIQx, and 2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline, or 4,8-DiMeIQx) may be produced by reacting a mixture containing dialkylpyrazine free radicals and creatinine. Two different pathways for free radical formation are proposed. One involves bimolecular ring formation from the enaminol form of the glycoaldehyde alkylimine and is followed by oxidative formation of the free radical. The other pathway involves formation of N,N1-dialkylpyrazinium ions from glyoxal monoalkylimine followed by reduction to produce the free radicals. The respective intermediates (glycoaldehyde alkylimine and glyoxal monoalkylamine) are formed by reacting glycoaldehyde and glyoxal with amino compounds. The glycoaldehyde system reacts faster and produces more free radicals than the glyoxal system. The reactions help to explain the formation of imidazoquinoxaline meat mutagens and their predominance in fried fish and why these mutagens are present in larger quantities in fried ground beef than the imidazoquinoline-type meat mutagens. These two pathways may not be the only mechanisms involved in formation of meat mutagens, but other free radical reactions may also contribute to meat mutagenicity and are mentioned briefly.(ABSTRACT TRUNCATED AT 250 WORDS)

Sixty-four pigs, approximately 3 weeks old, were fed diets containing 3% beef tallow or 3% soya oil with either a basal (10-50 mg/kg diet) or supplemented (200 mg/kg diet) level of α-tocopheral acetate. In pigs fed the soya oil diet the neutral and polar lipid fractions of muscle tissue and the total lipid fraction of adipose tissue had significantly (P < 0·05) higher C18:2/C18:1 ratios when compared to pigs fed the tallow diet. Muscle from pigs fed the soya oil diet was significantly more susceptible (P < 0·05) to lipid oxidation than muscle from pigs fed the tallow diet. In pigs receiving the α-tocopherol supplemented diet, α-tocopherol levels were approximately 3·3, 2·8 and 2-times higher in plasma, muscle and adipose tissue, respectively, when compared to pigs fed the basal level of α-tocopherol acetate. α-Tocopherol supplementation significantly increased (P < 0·05) the oxidative stability of muscle from pigs fed both the tallow and soya oil diets., (Copyright © 1991. Published by Elsevier Ltd.)

Human breast carcinoma cell lines MCF-7 and MDA-MB231 were transplanted s.c. to female athymic nude mice at 3-4 weeks of age. At 7-10 days after transplantation, the mice were divided into groups and fed for 6-8 weeks one of the following semi-purified diets containing different amounts and types of fat, i.e. 5% corn oil, 20% corn oil, 20% butter, 19% beef tallow/1% corn oil and 19% fish (Menhaden) oil/1% corn oil. In addition experiments, the fish oil diets were supplemented with antioxidants (vitamin E, 8 g or 2000 IU/kg diet plus tertiary butyl hydroquinone, TBHQ, 4 g/kg diet) or ferric citrate (3 g/kg diet). Tumor peroxidation product levels were assessed by measuring 2-thiobarbituric acid reactants (TBA assay). At the termination of the studies (6-8 weeks of diet feeding) mean human breast carcinoma volume (MCF-7 and MDA-MB231) was the largest in mice fed the 20% corn oil diet, intermediate in mice fed the butter or beef tallow diets and the least in mice fed the fish oil diet. The difference in mean tumor volumes among mice fed the 20% corn oil diet and those fed the fish oil diet was significant (P less than 0.01). When comparing low (5% corn oil) and high (20% corn oil) fat diets, numerical increases in human breast carcinoma volume (MCF-7 and MDA-MB231) were consistently observed in the high-fat diet groups but these differences were not always significant. Tumor lipid peroxidation product levels were determined on the MDA-MB231 tumors; tumor lipid peroxidation levels were significantly (P less than 0.01) increased only in mice fed the fish oil diets. Supplementation of the fish oil diets with antioxidants (vitamin E + TBHQ) significantly reduced the level of tumor peroxidation products and significantly increased tumor volume (P less than 0.05). When tumor lipid peroxidation product levels in the fish oil plus antioxidant fed mice were reduced to the level of that observed in the tumors of the corn oil fed mice, no significant differences in tumor volumes were observed in these two groups. In contrast, supplementation of the fish oil diets with ferric citrate, significantly (P less than 0.05) increased tumor lipid peroxidation product levels and decreased tumor volume. Thus, the type of dietary fat can clearly affect the growth of human breast carcinomas (MCF-7 and MDA-MB231) maintained in athymic nude mice.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of tetrasodium pyrophosphate (TSPP) (0, 0·25, 0·5% w/w) alone or in combination with salt (NaCl) (0, 0·5, 1·0% w/w) on water-holding capacity (WHC), pH, the ratio of absorbance at 250 nm over the absorbance at 260 nm (R-values) and 150m CaCl extractable protein (EP) was studied in prerigor and postrigor sternomandibularis homogenates over time. The 0 h samples were defined as when the NaCl was incorporated with the muscle. R-values verified that 0 h samples were in a prerigor or postrigor state. In prerigor homogenates, increasing phosphate concentration increased the time required to reach ultimate pH. Ultimate pH values of prerigor homogenates containing phosphate were lower (P < 0·05) than homogenates without phosphate and similarly treated postrigor homogenates. After six hours, no differences (P > 0·10) were noted in EP or WHC at different phosphate concentrations when averaged over NaCl concentrations in prerigor homogenates. With increasing phosphate concentration of postrigor homogenates, there was an increase (P < 0·05) in pH and EP at the initial sampling time. However, 0 and 0·25% phosphate WHC values could not be differentiated (P > 0·10). Results of this study indicate no advantages, after six hours post mortem, to using TSPP alone or in combination with NaCl in prerigor meat homogenates at concentrations added in this study., (Copyright © 1990. Published by Elsevier Ltd.)

Granuflex Paste and Granuflex E extend the range of indications for using Granuflex products in pressure sore therapy. These dressings enable ulcers of any degree of severity to be treated, although they perform better on sores on limbs than on sacral sores.

The effect of salt and pH on the colour stability (oxymyoglobin) in the sarcoplasmic extract (SPE) prepared from pre- and post-rigor beef muscle was studied in the presence or absence of mitochondria and microsomes during 96 h at 4°C. The sarcoplasmic extract from the post-rigor meat (pH 5·4) or from the pre-rigor meat adjusted to pH 5·4 contained more oxymyoglobin (MbO(2)) than the pre- or post-rigor SPE maintained at pH 7·4. The presence or absence of mitochondria and microsomes in the SPE at pH 5·4 had little effect on the percentage of MbO(2). At pH 7·4, however, the percentage of MbO(2) decreased in the SPE in the presence of mitochondria, whereas the percentage of MetMb was lower in the presence of microsomes. The relative proportion of MbO(2) decreased and that of metmyoglobin (MetMb) increased with increasing salt concentration at low pH (more so in the absence of subcellular organelles) and with storage period in the SPE from both pre- and post-rigor meat. However, at pH 7·4, high levels of salt (2-4%) helped to maintain a high percentage of MbO(2) in SPE in the absence of subcellular organelles, especially the mitochondria. The first-order interactions of pH value × subcellular organelles (P < 0·01) and pH value × salt concentration (P < 0·01) accounted for about 55% of the total variation in the percentage of MbO(2) in the SPE. It is suggested that depressing microbial growth may be the operative mechanism by which added salt stabilizes the colour in pre-rigor minced meat rather than by enzymic effects of subcellular organelles., (Copyright © 1990. Published by Elsevier Ltd.)

Experiments were designed to investigate the effects of dietary α-tocopherol supplementation for 2 weeks prior to slaughter on plasma and muscle α-tocopherol levels and on the oxidative stability of raw and cooked pig muscle during refrigerated storage at 4°C. Plasma and muscle α-tocopherol levels of the pigs on the α-tocopherol supplemented diet (200 mg α-tocopherol acetate/kg feed) were ∼2·5-fold higher than those of the pigs on the control diet (30 mg α-tocopherol acetate/kg feed). Dietary supplementation with α-tocopherol significantly (p < 0·01) improved the oxidative stability of both raw and cooked muscle after storage at 4°C for up to 8 days. α-Tocopherol stabilized the membrane-bound lipids against metmyoglobin/H(2)O(2)-initiated oxidation and also significantly (p < 0·05) improved the oxidative stability of rendered fat., (Copyright © 1990. Published by Elsevier Ltd.)