Evidence that human and equine erythrocytes could have significant roles in the transport and delivery of amino acids to organs and tissues.

Erythrocytes have a well-defined role in the gaseous exchange of oxygen and carbon dioxide in the mammalian body. The erythrocytes can contain more than half of the free amino acids present in whole blood. Based on measures showing that venous erythrocyte levels of amino acids are much less than arterial erythrocyte levels, it has previously been proposed that erythrocytes also play a role in the delivery of amino acids to tissues in the body. This role has been dismissed because it has been assumed that to act as an amino acid transport vehicle, the erythrocytes should release their entire amino acid content in the capillary beds at the target tissues with kinetic studies showing that this would take too long to achieve. This investigation set out to investigate whether the equine erythrocytes could rapidly take up and release smaller packages of amino acids when exposed to high or low external concentrations of amino acids, because it seemed very unlikely that cells would be able to release all of their amino acids without serious impacts on osmotic balance. Freshly prepared erythrocytes were placed in alternating solutions of high and low amino acid concentrations in PBS to assess the capacities of these cells to rapidly take up and release amino acids depending on the nature of the external environment. It was found that amino acids were rapidly taken up and released in small quantities in each cycle representing 15% of their total load in equine erythrocytes and 16% in human erythrocytes. The capacity for rapid uptake/release of amino acids by equine and human erythrocytes provided evidence to support the theory that mammalian erythrocytes have a significant role in transport of amino acids from the liver to tissues, muscles and organs.

Modelling of protein turnover provides insight for metabolic demands on those specific amino acids utilised at disproportionately faster rates than other amino acids.

The nitrogen balance is regulated by factors such as diet, physical activity, age, pathogenic challenges, and climatic conditions. A paradigm was developed from published recommended rates of protein intake (g/kg/day) with corresponding rates of endogenous protein turnover and excretion, to extrapolate amino acid balances under various conditions. The average proportions of amino acids in the ingested proteins representing a well-balanced diet were used to assess intake and an average human composition profile from five major high-turnover proteins in the body to assess endogenous protein turnover. The amino acid excretion profiles for urine and sweat were constructed for males and females from published data. The model calculated the nitrogen balances for a range of amino acids to determine the amino acid requirements to support daily exertion. Histidine, serine, glycine, and ornithine were in negative balances in males and females and this potential deficit was greater in the higher body-mass ranges. Conversely, leucine, isoleucine, and valine were conserved during nitrogen flux and resulted in positive balances. The model was run under a scenario of high demand for the synthesis of IgG during a response to an infectious challenge which indicated that these were increased requirements for tyrosine, threonine, and valine. It was concluded that these amino acids represent points of limitation to anabolic metabolism by restriction of their supply at critical times of demand. This would especially occur under conditions of fitness training, maintaining intensive exercise regimes, facilitating responses to pathogenic challenge, or recovery from injury.

Sex differences in amino acids lost via sweating could lead to differential susceptibilities to disturbances in nitrogen balance and collagen turnover.

Fluid collected during sweating is enriched with amino acids derived from the skin's natural moisturising factors and has been termed "faux" sweat. Little is known about sex differences in sweat amino acid composition or whether faux sweat amino acid losses affect nitrogen balance. Faux sweat collected by healthy adults (n = 47) after exercise, and at rest by chronic fatigue patients, was analysed for amino acid composition. Healthy females had higher total amino acid concentrations in sweat (10.5 ± 1.2 mM) compared with healthy males (6.9 ± 0.9 mM). Females had higher levels of 13 amino acids in sweat including serine, alanine and glycine. Higher hydroxyproline and proline levels suggested greater collagen turnover in females. Modelling indicated that with conservative levels of exercise, amino acid losses in females via faux sweat were triple than those predicted for urine, whereas in males they were double. It was concluded that females were more susceptible to key amino acid loss during exercise and/or hot conditions. Females reporting chronic fatigue had higher levels of methionine in faux sweat than healthy females. Males reporting chronic fatigue had higher levels of numerous amino acids in faux sweat compared to healthy males. Higher amino acid loss in faux sweat associated with chronic fatigue could contribute to a hypometabolic state. Depending on activity levels, climatic conditions and gender, amino acid losses in sweat and skin leachate could influence daily protein turnover where periods of continuously high turnover could lead to a negative net nitrogen balance.

Diverse characteristics of the urinary excretion of amino acids in humans and the use of amino acid supplementation to reduce fatigue and sub-health in adults.

BACKGROUND: The excretion of amino acids in urine represents an important avenue for the loss of key nutrients. Some amino acids such as glycine and histidine are lost in higher abundance than others. These two amino acids perform important physiological functions and are required for the synthesis of key proteins such as haemoglobin and collagen. METHODS: Stage 1 of this study involved healthy subjects (n = 151) who provided first of the morning urine samples and completed symptom questionnaires. Urine was analysed for amino acid composition by gas chromatography. Stage 2 involved a subset of the initial cohort (n = 37) who completed a 30 day trial of an amino acid supplement and subsequent symptom profile evaluation. RESULTS: Analyses of urinary amino acid profiles revealed that three groups could be objectively defined from the 151 participants using k-means clustering. The amino acid profiles were significantly different between each of the clusters (Wilks' Lambda = 0.13, p < 0.0001). Cluster 1 had the highest loss of amino acids with histidine being the most abundant component. Cluster 2 had glycine present as the most abundant urinary amino acid and cluster 3 had equivalent abundances of glycine and histidine. Strong associations were observed between urinary proline concentrations and fatigue/pain scores (r = .56 to .83) for females in cluster 1, with several other differential sets of associations observed for the other clusters. CONCLUSIONS: Different phenotypic subsets exist in the population based on amino acid excretion characteristics found in urine. Provision of the supplement resulted in significant improvements in reported fatigue and sleep for 81% of the trial cohort with all females reporting improvements in fatigue. TRIAL REGISTRATION: The study was registered on the 18th April 2011 with the Australian New Zealand Clinical Trials Registry ( ACTRN12611000403932 ).

The clinical relevance of alloantibody in liver transplantation.

The transplanted liver appears resistant to antibody-mediated injury compared to other transplanted organs such as kidney or heart. However, a growing number of reports suggest that alloantibody to the liver is associated with poorer outcomes. The data surrounding this field are unclear, and their interpretation remains controversial. Mechanistically, there is not a clear explanation for the liver's resistance to antibody-mediated injury, and the pathological criteria for antibody-mediated rejection (AMR) remain ill-defined. Furthermore, treatment of AMR is non-uniform. The field would benefit from better outcome data based on measurement of antibody at the time of transplantation and at the time of rejection. Consensus opinion regarding antibody and the liver might emerge with better standardization of antibody measurement and pathological definition of AMR.

Health characteristics of inland waterway merchant marine captains and pilots.

BACKGROUND: Most published studies on seafarer health have focused on patterns of mortality, injury and communicable diseases. Little information is available regarding lifestyle-related cardio-metabolic disease in maritime populations. AIMS: To describe health characteristics of a population of US inland waterway merchant marine captains and pilots. METHODS: A cross-sectional study of the health characteristics of mariners required to complete the United States Coast Guard physical assessment at a regional medical centre from 2003-10. Variables collected included self-reported smoking status, body mass index, fasting lipids, glucose and triglyceride levels, blood pressure and treadmill time and maximal oxygen uptake as measured using the Bruce Protocol. Major medical conditions related to lifestyle and risk for metabolic syndrome were also assessed. RESULTS: There were 388 participants. The study population had high prevalence of obesity (61%), smoking (41%), high triglycerides (42%), low HDL cholesterol (47%), high blood pressure (42%), high fasting glucose (22%) and three or more features of the metabolic syndrome (39%). CONCLUSIONS: This population exhibited a high prevalence of chronic disease risk factors and could potentially benefit from health promotion programmes aimed at improving health and fitness.

Metabolic effects of static magnetic fields on Streptococcus pyogenes.

This study aimed to develop a simple experimental system utilising bacterial cells to investigate the dose responses resulting from exposures to static magnetic flux densities ranging from 0.05 to 0.5 T on viability, bacterial metabolism and levels of DNA damage in Streptococcus pyogenes. Exposure of S. pyogenes to a field of 0.3 T at 24 degrees C under anaerobic conditions resulted in a significant (P < 0.05) decrease in growth rate, with an increased mean generation time of 199 +/- 6 min compared to the control cells at 165 +/- 6 min (P < 0.05). Conversely, exposure to magnetic fields of 0.5 T significantly accelerated the growth rate at 24 degrees C compared to control cells, with a decreased mean generation time of 147 +/- 4 min (P < 0.05). The patterns of metabolite release from cells incubated in phosphate buffered saline (PBS) at 24 degrees C and exposed to different magnetic flux densities (0.05-0.5 T) were significantly (P < 0.05) altered, compared to non-exposed controls. Concentrations of metabolites, with the exception of aspartic acid (r = 0.44), were not linearly correlated with magnetic flux density, with all other r < 0.20. Instead, "window" effects were observed, with 0.25-0.3 T eliciting the maximal release of the majority of metabolites, suggesting that magnetic fields of these strengths had significant impacts on metabolic homeostasis in S. pyogenes. The exposure of cells to 0.3 T was also found to significantly reduce the yield of 8-hydroxyguanine in extracted DNA compared to controls, suggesting some possible anti-oxidant protection to S. pyogenes at this field strength.

Detection of Anaplasma platys and Babesia canis vogeli and their impact on platelet numbers in free-roaming dogs associated with remote Aboriginal communities in Australia.

OBJECTIVE: To detect Anaplasma platys and Babesia canis vogeli infection, using polymerase chain reaction (PCR)-based assays, in free-roaming dogs associated with eight Aboriginal communities in remote areas of Australia and to determine the impact of infection through the assessment of platelet numbers. PROCEDURES: Blood samples from 215 dogs were screened by PCR for A platys and B canis vogeli using established genus-specific DNA primers for the 16S and 18S rRNA genes respectively. Both A platys DNA and B canis vogeli DNA were confirmed from the screening PCR either by sequencing or by the use of species-specific primers. Peripheral blood films from 92 of the 215 dogs were used to estimate platelet numbers through an indirect method. RESULTS: Of 215 dogs, 69 (32%) were positive for A platys, 22 (10%) for B canis vogeli and 24 (11%) for both. The two organisms were detected singularly and as coinfection in all communities. For the 92 dogs in which peripheral blood films were examined, the mean estimated platelet counts for the non-infected dogs was 318 x 10(9)/L, those infected with A platys alone was 256 x 10(9)/L, those with B canis vogeli alone was 276 x 10(9)/L and those infected with both parasites was 169 x 10(9)/L. In young dogs, infection produced significantly decreased mean platelet counts when compared to uninfected dogs. Thrombocytopenia (< 200 x 10(9)/L) was detected in 18 (51%) dogs infected with A platys alone, 3 (33%) dogs infected with B canis vogeli alone, 13 (72%) dogs coinfected, and 8 (27%) uninfected dogs. CONCLUSIONS: A platys and B canis vogeli infection, either singularly or together, was widespread in free roaming dogs associated with remote Aboriginal communities in the Northern Territory and north-western New South Wales. Moreover, both A platys and B canis vogeli infections were associated with a reduction in mean platelet numbers in dog populations, particularly in young dogs. The fact that 51% of dogs infected with A platys alone and 72% dogs coinfected were thrombocytopenic compared to 27% of uninfected dogs suggests that the organism alone or in combination with B canis vogeli has the potential to cause thrombocytopenia and perhaps contribute to a clinical bleeding disorder in infected dogs.

Ovine placental eluate immunoglobulins recognise isologous and third party acid-treated trophoblast microvesicle antigens in vitro.

Placental microvesicles were prepared from ovine placentae and immunoglobulins eluted with 0.5 M glycine buffer pH 2.5. The ability of eluate immunoglobulins to re-associate with isologous (self) and third party acidified microvesicles was tested by ELISA. Ovine placental immunoglobulins re-associated with isologous and third party acidified microvesicles suggesting that at least 2 types of antigenic epitopes I and II maybe expressed on the ovine placentae. Type I antigens may be present on placentae of all ovines while type II epitopes may be paternally derived, hence unique to each pregnancy. Analysis by SDS PAGE revealed the heavy and light chains of IgG at 57 and 27 kDa, respectively, together giving a relative molecular weight of 158 kDa. Results suggest that immunoglobulins produced to placental microvesicle antigens may be directed to some but not all antigenic epitopes expressed on the trophoblast, possibly defining a mechanism by which the foetus evades maternal immunological rejection.

Human placental immunoglobulins show unique re-association patterns with isologous and third party acid treated trophoblast microvesicles in vitro.

OBJECTIVE: To study re-association pattern of human placental eluate immunoglobulins with acid treated isologous and third party trophoblast derived placental microvesicles. DESIGN: Laboratory based experimentation. SETTING: Biological Sciences Department and Discipline for Reproductive Medicine University of Newcastle, Australia and the Department of Biochemistry, University of Nairobi, Kenya. RESULTS: Placental eluate immunoglobulins re-associated with isologous and third party acidified microvesicles in three distinct patterns. I: eluate immunoglobulins re-associated more strongly with isologous and third party acid treated placental microvesicles, II: eluate immunoglobulins re-associated strongly with isologous but weakly with third party acid treated placental microvesicles, III: eluate immunoglobulins did not show preferential re-association with isologous and third party acid treated placental microvesicles. CONCLUSION: Two types of antigenic epitopes I and II may be expressed on the human placentae. Type I antigens may be present on all human placentae while type II epitopes may be paternally derived hence unique to each pregnancy. Also, immunoglobulins produced to placental microvesicle antigens may be directed to some but not all antigenic epitopes expressed on the human placental trophoblast.

Immunoglobulin G bound to ovine placenta is eluted by surgical cannulation and acid perfusion in situ.

OBJECTIVE: To elute placental bound immunoglobulin G (IgG) in situ. DESIGN: Laboratory based experimentation. SETTING: Biological Sciences Department, The University of Newcastle Australia and the Department of Biochemistry, University of Nairobi, Kenya. SUBJECTS: Twelve pregnant ewes 10 to 15 days before the onset of natural parturition. RESULTS: Placental eluates were rich in IgG, and IgG2. The relative molecular weight of placental IgG was estimated at 158kDa by gel filtration chromatography. Analysis of eluate by SDS PAGE revealed the heavy and light chains of IgG at 57 and 27kDa respectively together giving a relative molecular weight of 168kDa. CONCLUSION: Placental bound IgG may be crucial in immunology of pregnancy and together with the cognate antigen thereof may be useful as models for the study of maternal-fetal interaction in human pregnancy and in the development of experimental immunotherapy to immunologically compromised pregnancies in humans and livestock.

Plasma cholesterol levels and Irlen syndrome: preliminary study of 10- to 17-yr.-old students.

The preliminary study investigated metabolic anomalies in children and teenagers with Irlen Syndrome, particularly in relation to the levels of n-3 and n-6 essential fatty acids, plasma cholesterol levels, and the relative abundance of plasma saturated fatty acids. The experimental group involved 13 subjects with Irlen Syndrome (M=13.3 yr., SD=2.5 yr.), with a comparison group of 16 age- and sex-matched controls (M=13.8 yr., SD=2.4 yr.). The Irlen Syndrome group were selected from people referred for help with reading and writing problems. The control group were primarily recruited from the general public. All subjects were screened for symptoms of the syndrome using the Scotopic Sensitivity Syndrome Screening Manual. Samples of whole blood were collected and plasma extracted. Metabolites were compared using the Student t test. There were no differences in n-3 and n-6 essential fatty acids between Irlen Syndrome and control groups, although the former group had lower mean levels in most of these essential fatty acids. Total plasma cholesterol level was significantly decreased for the Irlen Syndrome group, and there was a significant increase in the relative abundance of the odd-chain fatty acid, heptadecanoic acid. The differences in heptadecanoic acid may have implications for altered membrane function and neurotransmission. The differences in plasma cholesterol levels, as well as heptadecanoic acid, may also point to the presence of viral or bacterial infection.

Detection of Ehrlichia platys in dogs in Australia.

OBJECTIVE: To describe the detection of Ehrlichia platys in free-roaming dogs in Central Australia. PROCEDURE: Blood samples were collected from four dogs and examined for bacterial 16S ribosomal DNA using Polymerase Chain Reaction (PCR)-based assays. The three positive samples obtained were then sequenced and identification of the PCR product carried out. As a result of all three samples being identical to or closely related to part of the 16S rRNA gene of E. platys, blood samples were subsequently obtained from a further 24 dogs. These samples were screened using a PCR-assay to determine the presence of Ehrlichia DNA using genus-specific primers. The positive samples obtained from the screening process were then subjected to a further PCR-assay using E. platys specific primers. RESULTS: Of 28 dogs sampled, Ehrlichia DNA was detected in the blood of 13 dogs. Sequencing of the amplicons obtained indicated a high homology with the 16S rRNA gene for E. platys. When the E. platys-specific PCR was performed for 10 of those dogs, the 678 bp product obtained from the PCR amplification confirmed the identification as part of the 16S rRNA gene of E. platys in all 10 dogs. CONCLUSION: This study reports for the first time Ehrlichia carriage by dogs in Australia. It also indicates the usefulness of the PCR technique in rapidly and accurately identifying diseases that are otherwise difficult to detect. By using universal primers directed against bacterial 16S ribosomal DNA and sequencing analysis, the detection of potentially pathogenic Ehrlichia organisms that had not previously been found in Australia has been made possible.

A biochemical analysis of people with chronic fatigue who have Irlen Syndrome: speculation concerning immune system dysfunction.

This study investigated the biological basis of visual processing disabilities in adults with Chronic Fatigue Syndrome. The study involved 61 adults with symptoms of Chronic Fatigue Syndrome who were screened for visual processing problems (Irlen Syndrome) and divided into two groups according to the severity of symptoms of Irlen Syndrome. Significant variations were identified in blood lipids and urine amino and organic acids of the two groups, which may be indicative of activation of the immune system due to some infective agent. It was suggested that metabolic profiling may help the development of more valid diagnostic categories and allow more investigation of immune system dysfunction as a possible causal factor in a range of learning and behaviour disorders.

Immunological Role of the Placenta, Blocking Factors and NK Cells in Post-implantation Pregnancy.

The unexpected failure of the mother to immunologically reject the foetus is partly thought to result from immunological properties of the placenta. The placental trophoblast produces immunosuppressive factors including progesterone and blocking antibodies which together down-regulate maternal immune responses to the foetoplacental unit. This article reviews the post implantation immunology of pregnancy emphasizing the roles of placenta, blocking factors and natural killer (NK) cells.

Ovine and Human Placental IgG inhibit Human Natural Killer Cell Cytotoxicity in vitro.

To determine the effect of ovine and human placental IgG on human Natural Killer (NK) cell cytotoxicity in vitro placental IgG was eluted at acidic pH and purified by ion exchange and subsequently by affinity chromatography on protein G and protein A sepharose columns. These antibodies were analysed for presense of IgG by immuno-electrophoresis and relative purity determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). The effect of these antibodies on human NK cell cytotoxicity was investigated by slChromium Release Assay using human K562 cells as targets and human peripheral blood lymphocytes as effector cells. Both ovine and human placental IgG inhibited human NK cell cytotoxicity in a dose dependent manner. Placental IgG may down-regulate the cytotoxic effects of NK cells in vivo by competitively excluding the binding of NK cells to their respective targets on the trophoblast. Alternatively, these antibodies may themselves be toxic to NK cells. Either way, the presence of these antibodies on the placental trophoblast may prevent the binding of NK cells and subsequent immunological rejection of the fetal allograft. Also, ovine placental IgG may be functionally similar to its human counterpart and may therefore be suitable as a model for the study of maternal fetal interaction during pregnancy in humans.

Recurrent pregnancy losses and the role of immunotherapy.

Post implantation pregnancy losses are psychologically and economically stressful to the childbearing population. The etiology in the vast majority of cases is unknown but is partly thought to result from a break-down of the maternal tolerance to the fetoplacental unit. Immunologically based therapy remains controversial but no alternative therapy is available at the moment. This article reviews the conceived immunological basis of recurrent pregnancy losses, discussing the controversies arising, and recommending the use of intravenous immunoglobulin, IVIg, in well controlled experiments for further clinical trials.

Free radicals in chronic fatigue syndrome: cause or effect?

We have demonstrated that certain morphological and biochemical changes occur in chronic fatigue syndrome (CFS) and in rheumatoid arthritis (RA). These changes in RA can be explained by the well-established inappropriate increase in free radical generation. The similar changes in CFS suggest a similar explanation and a possible role for free radicals in the aetiology of this condition.