Human milk oligosaccharide 2'-fucosyllactose supplementation improves gut barrier function and signaling in the vagal afferent pathway in mice.

2'-Fucosyllactose (2'-FL) is one of the predominant oligosaccharides found in human milk and has several well-established beneficial effects in the host. It has previously been shown that 2'-FL can improve the metabolic phenotype in high-fat (HF)-fed mice. Here we investigated whether dietary supplementation with 2'-FL was associated with improved intestinal barrier integrity, signaling in the vagal afferent pathway and cognitive function. Mice were fed either a low-fat (LF, 10% fat per kcal) or HF (45% fat per kcal) diet with or without supplementation of 2'-FL (10% w/w) in the diet for 8 weeks. Body weight, energy intake, fat and lean mass, intestinal permeability (ex vivo in Ussing chambers), lipid profiles, gut microbiome and microbial metabolites, and cognitive functions were measured. Vagal afferent activity was measured via immunohistochemical detection of c-Fos protein in the brainstem in response to peripheral administration of cholecystokinin (CCK). 2'-FL significantly attenuated the HF-induced increase in fat mass and energy intake. 2'-FL significantly reduced intestinal permeability and significantly increased expression of interleukin (IL)-22, a cytokine known for its protective role in the intestine. Additionally, 2'-FL led to changes in the gut microbiota composition and in the associated microbial metabolites. Signaling in the vagal afferent pathway was improved but there was no effect on cognitive function. In conclusion, 2'-FL supplementation improved the metabolic profiles, gut barrier integrity, lipid metabolism and signaling in the vagal afferent pathway. These findings support the utility of 2'-FL in the control of gut barrier function and metabolic homeostasis under a metabolic challenge.

Selective Identification of α-Galactosyl Epitopes in N-Glycoproteins Using Characteristic Fragment Ions from Higher-Energy Collisional Dissociation.

The α-galactosyl epitope is a terminal N-glycan moiety of glycoproteins found in mammals except in humans, and thus, it is recognized as an antigen that provokes an immunogenic response in humans. Accordingly, it is necessary to analyze the α-galactosyl structure in biopharmaceuticals or organ transplants. Due to an identical glycan composition and molecular mass between α-galactosyl N-glycans and hybrid/high-mannose-type N-glycans, it is challenging to characterize α-galactosyl epitopes in N-glycoproteins using mass spectrometry. Here, we describe a method to identify α-galactosyl N-glycopeptides in mice glycoproteins using liquid chromatography with tandem mass spectrometry with higher-energy collisional dissociation (HCD). The first measure was an absence of [YHM] ion peaks in the HCD spectra, which was exclusively observed in hybrid and/or high-mannose-type N-glycopeptides. The second complementary criterion was the ratio of an m/z 528.19 (Hex2HexNAc1) ion to m/z 366.14 (Hex1HexNAc1) ion (Im/z528/Im/z366). The measure of [Im/z528/Im/z366 > 0.3] enabled a clear-cut determination of α-galactosyl N-glycopeptides with high accuracy. In Ggta1 knockout mice, we could not find any α-galactosyl N-glycoproteins identified in WT mice plasma. Using this method, we could screen for α-galactosyl N-glycoproteins from mice spleen, lungs, and plasma samples in a highly sensitive and specific manner. Conclusively, we suggest that this method will provide a robust analytical tool for determination of α-galactosyl epitopes in pharmaceuticals and complex biological samples.

Synthesis and biological evaluation of a trisaccharide repeating unit derivative of Streptococcus pneumoniae 19A capsular polysaccharide.

Streptococcus pneumoniae (SP) is a common human pathogen associated with a broad spectrum of diseases and it is still a leading cause of mortality and morbidity worldwide, especially in children. Moreover, SP is increasingly associated with drug resistance. Vaccination against the pathogen may thus represent an important strategy to overcome its threats to human health. In this context, revealing the molecular determinants of SP immunoreactivity may be relevant for the development of novel molecules with therapeutic perspectives as vaccine components. Serogroup 19 comprises the immune-cross reactive types 19F, 19A, 19B and 19C and it accounts for a high percentage of invasive pneumococcal diseases, mainly caused by serotypes 19F and 19A. Herein, we report the synthesis and biological evaluation of an aminopropyl derivative of the trisaccharide repeating unit of SP 19A. We compare two different synthetic strategies, based on different disconnections between the three monosaccharides which make up the final trisaccharide, to define the best approach for the preparation of the trisaccharide. Synthetic accessibility to the trisaccharide repeating unit lays the basis for the development of more complex biopolymer as well as saccharide conjugates. We also evaluate the binding affinity of the trisaccharide for anti-19A and anti-19F sera and discuss the relationship between the chemical properties of the trisaccharide unit and biological activity.

Sensitive typing of reverse ABO blood groups with a waveguide-mode sensor.

Portable, on-site blood typing methods will help provide life-saving blood transfusions to patients during an emergency or natural calamity, such as significant earthquakes. We have previously developed waveguide-mode (WM) sensors for forward ABO and Rh(D) blood typing and detection of antibodies against hepatitis B virus and hepatitis C virus. In this study, we evaluated a WM-sensor for reverse ABO blood typing. Since reverse ABO blood typing is a method for detection of antibodies against type A and type B oligosaccharide antigens on the surface of red blood cells (RBCs), we fixed a synthetic type A or type B trisaccharide antigen on the sensor chip of the WM sensor. We obtained significant changes in the reflectance spectra from a WM sensor on type A antigen with type B plasma and type O plasma and on type B antigen with type A plasma and type O plasma, and no spectrum changes on type A antigen or type B antigen with type AB plasma. Signal enhancement with the addition of a peroxidase reaction failed to increase the sensitivity for detection on oligosaccharide chips. By utilizing hemagglutination detection using regent type A and type B RBCs, we successfully determined reverse ABO blood groups with higher sensitivity compared to a method using oligosaccharide antigens. Thus, functionality of a portable device utilizing a WM sensor can be expanded to include reverse ABO blood typing and, in combination with forward ABO typing and antivirus antibody detection, may be useful for on-site blood testing in emergency settings.

Fast, sensitive method for trisaccharide biomarker detection in mucopolysaccharidosis type 1.

Certain recessively inherited diseases result from an enzyme deficiency within lysosomes. In mucopolysaccharidoses (MPS), a defect in glycosaminoglycan (GAG) degradation leads to GAG accumulation followed by progressive organ and multiple system dysfunctions. Current methods of GAG analysis used to diagnose and monitor the diseases lack sensitivity and throughput. Here we report a LC-MS method with accurate metabolite mass analysis for identifying and quantifying biomarkers for MPS type I without the need for extensive sample preparation. The method revealed 225 LC-MS features that were >1000-fold enriched in urine, plasma and tissue extracts from untreated MPS I mice compared to MPS I mice treated with iduronidase to correct the disorder. Levels of several trisaccharides were elevated >10000-fold. To validate the clinical relevance of our method, we confirmed the presence of these biomarkers in urine, plasma and cerebrospinal fluid from MPS I patients and assessed changes in their levels after treatment.

Major human milk oligosaccharides are absorbed into the systemic circulation after oral administration in rats.

Human milk oligosaccharides (HMO) are involved in many biological functions influencing infant health. Although HMO act locally at the intestine, recent evidence has demonstrated that HMO are partially incorporated into the systemic circulation of breast-fed infants. In the last few years, a large amount of research has been conducted using preclinical models to uncover new biological functions of HMO. The aim of this study was to evaluate the absorption and urine excretion of HMO in rats. We administered a single oral dose of the following HMO: 2'-fucosyllactose (2'-FL), 6'-sialyllactose and lacto-N-neotetraose at different concentrations to adult rats. The time course of absorption of HMO into the bloodstream and their appearance in urine was studied. Our results showed that rats, similar to human infants, are able to effectively absorb a portion of HMO from the intestine into plasma and to excrete them in urine. On the basis of this, we also conducted a specific kinetic absorption study with 2'-FL, the most predominant HMO in human milk, in 9-11-d-old rat pups. Our results confirmed that a significant amount of 2'-FL was absorbed into the systemic circulation and subsequently excreted in urine during lactation in rats in a dose-depended manner. We also found basal levels of these HMO in plasma and urine of adult rats as well as rat pups as a natural result of nursing. Our data suggest that the rat may be a useful preclinical model that provides new insights into the metabolism and functions of HMO.

Effect of a mixture of GOS/FOS® on calcium absorption and retention during recovery from protein malnutrition: experimental model in growing rats.

INTRODUCTION: During growth, protein deprivation impairs epiphyseal growth plate (EGP) height, bone volume (BV) and endochondral ossification. During catch-up growth, Ca availability becomes essential to ensure the extra amount needed to achieve optimal peak bone mass and strength. GOS and FOS improve mineral absorption in the colon. PURPOSE: The effect of a mixture of GOS/FOS® 9:1 added to a 0.5 %Ca (NCa) and a 0.3 %Ca (LCa) diets on Ca, P and Mg absorptions and bone mineralization, density and structure using an experimental model of growing rats recovering from early protein malnutrition was investigated. METHODS: To induce protein malnutrition, rats were fed a low protein diet: 4 % (LPD) during 1 week and then were randomly assigned to recovery groups (R) until day 50 (T = 50) as follows: R0.5 %: NCa; RP0.5 %: NCa + 5.3 % GOS/FOS®; R0.3 %: LCa and RP0.3 %: LCa + 5.3 % GOS/FOS®. Control groups received the 0.5 %Ca or 0.3 %Ca diet from weaning until day 40 or 50. RESULTS: Body weight and length increased in C groups throughout the study; both were arrested in all R during LPD consumption and increased immediately after re-feeding. Independently of dietary Ca content, LS counts, ß-glucosidase and Ca, P and Mg absorption increased, whereas cecum pH, ß-glucuronidase, urease and tryptophanase decreased in RP0.5 %: and RP0.3 %: as compared to the other studied groups (p < 0.01). Prebiotic consumption decreased CTX levels and increased femur Ca, Mg and P contents, total skeleton bone mineral content, proximal tibia and spine BMD, BV, EGP height and hypertrophic zone thickness, stiffness and elastic modulus as compared to recovery groups fed the prebiotic-free diets. CONCLUSION: Under the present experimental conditions, GOS/FOS® mixture induced colonic positive effects, which increased Ca, P and Mg absorption. Thus, consuming the prebiotic-containing diet resulted in an extra amount of minerals that improved bone development in growing rats recovering from protein malnutrition.

Direct evidence for the presence of human milk oligosaccharides in the circulation of breastfed infants.

BACKGROUND: It has been hypothesized that human milk oligosaccharides (HMOs) confer systemic health benefits to breastfed infants; however, plausible mechanisms for some effects, such as systemic immunomodulation, require HMOs to access the bloodstream of the developing infant. While small concentrations of HMOs have been detected in the urine of breastfed infants there are no published studies of these oligosaccharides accessing the plasma compartment of breastfed infants. Here we determined the relative fractions of several ingested HMOs in infant urine and plasma. Plasma from formula-fed infants was used as a control. METHODS: Using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry/tandem mass spectrometry (LC/MS/MS), and high performance liquid chromatography (HPLC), we analyzed the urine and plasma from 17 healthy formula-fed infants and 16 healthy breast-fed infants (and the milk from their mothers). RESULTS: Multiple HMOs were detected in the urine and plasma of breastfed infants, but not in formula-fed infants. Levels of 2'-fucosyllactose (2'FL), 3FL and lacto-N-neotetraose (LNnT) in both plasma (r = 0.98, p<0.001; r = 0.75, p = 0.002; r = 0.71, p = 0.004) and urine (r = 0.81, p<0.001; r = 0.56, p = 0.026; NS) correlated significantly with concentrations in the corresponding breast milk. The relative fractions of HMOs were low, 0.1% of milk levels for plasma and 4% of milk levels for urine. Within the breastfed cohort, there were significant differences between secretor and nonsecretor groups in levels of several fucosylated HMOs. CONCLUSION: At least some ingested HMOs are absorbed intact into the circulation and excreted in the urine and their concentrations in these fluids correlate with levels of the corresponding mother's milk. While relative fractions of absorbed HMOs were low, these levels have been shown to have biological effects in vitro, and could explain some of the postulated benefits of human milk.

The relation of the level of serum anti-TF, -Tn and -alpha-Gal IgG to survival in gastrointestinal cancer patients.

OBJECTIVE: To evaluate the relation of the level of serum anti-TF, -Tn and -αGal carbohydrate antibodies to survival in gastrointestinal cancer patients. METHODS: The level of anti-TF (Thomsen-Friedenreich antigen), -Tn and -αGal IgG was analysed in the serum of patients with gastric (n = 83) and colorectal (n = 51) cancers in the long-term follow-up, using ELISA with polyacrylamide glycoconjugates. To evaluate overall survival and the risk of death, the Kaplan-Meier method and the Cox proportional hazards model were used in the univariate analysis of patients groups. RESULTS: A significantly better survival was observed: (1) in patients with an increased level of anti-TF antibodies (all, stage III, T2-4, N1-2 and G3; P = 0.004-0.038, HR = 0.16-0.46); and (2) in patients with an increased level of anti-Tn antibodies (G1-2 tumors; P = 0.034-0.042, HR = 0.34-0.47). A significantly worse survival was observed in gastrointestinal, gastric and colorectal groups with an increased level of serum anti-αGal antibodies. This association depended on the patho-morphology of tumors (all, stages I-II, III, T2-4, N0, N1-2 and G1-2; P = 0.006-0.048, HR = 1.99-2.33). In the combined assessment of the anti-TF and -αGal antibodies level of the whole gastrointestinal group (n = 53), P = 0.002, HR = 0.25, 95% CI 0.094-0.655. In the follow-up, the survival time was shorter in patients whose level of anti-αGal antibodies rose (P = 0.009-0.040, HR = 2.18-4.27). The level of anti-TF antibodies inversely correlated with neutrophil/lymphocyte ratio (NLR, r = - 0.401, P = 0.004, n = 49). Patients with a higher level of anti-αGal antibodies and NLR values demonstrated a significantly worse survival (P = 0.009, HR = 2.98, n = 48). CONCLUSIONS: The preoperative levels of anti-TF, -Tn and -αGal antibodies and their dynamics are of prognostic significance. The method for the determination of circulating anti-carbohydrate antibodies may be a useful supplement in clinical outcome assessment.

IgE production to α-gal is accompanied by elevated levels of specific IgG1 antibodies and low amounts of IgE to blood group B.

IgE antibodies to gal-α-1,3-gal-ß-1,4-GlcNAc (α-gal) can mediate a novel form of delayed anaphylaxis to red meat. Although IgG antibodies to α-gal (anti-α-gal or anti-Gal) are widely expressed in humans, IgE anti-α-gal is not. We explored the relationship between the IgG and IgE responses to both α-gal and the related blood group B antigen. Contradicting previous reports, antibodies to α-gal were found to be significantly less abundant in individuals with blood group B or AB. Importantly, we established a connection between IgE and IgG responses to α-gal: elevated titers of IgG anti-α-gal were found in IgE-positive subjects. In particular, proportionally more IgG1 anti-α-gal was found in IgE-positive subjects against a background of IgG2 production specific for α-gal. Thus, two types of immune response to α-gal epitopes can be distinguished: a 'typical' IgG2 response, presumably in response to gut bacteria, and an 'atypical', Th2-like response leading to IgG1 and IgE in addition to IgG2. These results suggest that IgE to a carbohydrate antigen can be formed (probably as part of a glycoprotein or glycolipid) even against a background of bacterial immune stimulation with essentially the same antigen.

A first total synthesis of a hybrid-type ganglioside associated with amyotrophic lateral sclerosis-like disorder.

The hybrid ganglioside X1, which was identified in the bovine brain, was synthesized for the first time. Ganglioside X1 is believed to be involved in the development of amyotrophic lateral sclerosis-like disorders in patients with neurological disorders after treatment with bovine brain gangliosides. A convergent approach using two branched glycan units, the GM2-core trisaccharide and the lacto-ganglio tetrasaccharide, efficiently provided the highly branched heptasaccharide part of ganglioside X1, which was conjugated with the ceramide part to produce the protected ganglioside X1. Global deprotection delivered homogenous ganglioside X1, with which serum from the patient was reacted.

Difructose anhydride III promotes absorption of the soluble flavonoid alphaG-rutin in rats.

A highly soluble quercetin glycoside, alphaG-rutin, is a glucose adduct of insoluble rutin. We examined the effects of difructose anhydride III (DFAIII; di-D-fructofuranosyl 1,2':2,3'-dianhydride) on intestinal absorption of alphaG-rutin by rat experiments. alphaG-rutin, rutin, quercetin, and the quercetin conjugate appeared in the portal blood after an intubation of alphaG-rutin (100 micromol), DFAIII effected higher portal concentrations of alphaG-rutin in portal cannulated rats. In ligated jejunal and ileal loops of rats, alphaG-rutin, rutin, quercetin, and the quercetin conjugate appeared in the intestinal mesenteric blood at both 30 and 60 min in both loops; the concentration of alphaG-rutin was 1.5-3 times higher when absorbed in the presence DFAIII. In the isolated mucosae of the jejunum and ileum, mucosal-to-serosal passage of alphaG-rutin increased with the addition of 100 micromol of DFAIII. These results indicate that alphaG-rutin is absorbed as the intact form and that DFAIII stimulates its absorption in the small intestine.

[A prompt method to quantitative assay of alpha-Gal on pig cell surface without injury].

OBJECTIVE: To develop a method for quantitative assay of alpha-Gal on pig cell surface. METHODS: After centrifugation of the anticoagulated blood samples, the white blood cell layer was collected for addition of FITC-BSIB4 and analysis using FCM as well as Laser Scan Confocal Microscope (Bio-Rad). RESULTS: With human blood group B as negative control, and mouse myeloma SP2/0 cells as positive control. the peripheral blood cells of two Chinese pigs were all positive, there were about 9.16 x 10(5) alpha-Galactosyl epitopes expressed on the cell-membrane of granulocytes 1.37 x 10(5) on the monocytes of Banna Minipig Inbred Line (BMI), and 1.16 x 10(6) on the granulocyte and 2.45 x 10(5) on the monocytes of Sichuan White Pig (SWP). The levels of alpha-Galactosyl expression on the surface of blood cells in BMI and SWP differed in various cells, the sequence from high to lower expression levels being on the platelets, granulocytes, monocytes, erthrocytes in each species of pig. CONCLUSION: The method is useful for quantitative assay of alpha-Gal on pig cell surface, and the assay may be used for alpha-Gal for on-site large-scaled screening of living pigs.

Assessment of relations between clinical outcome of ischemic stroke and activity of inflammatory processes in the acute phase based on examination of selected parameters.

BACKGROUND AND PURPOSES: Inflammatory factors play an important role in the pathogenesis of ischemic stroke. They may influence circulation during the acute phase of stroke and enhance the ischemic region. MATERIALS AND METHODS: We examined 51 patients--36 patients in the early stage of stroke, i.e. the first 24 h after onset. Of these, 15 patients had infection and 21 had no infection during the week preceding stroke. There were 15 patients with noninflammatory diseases in the control group. We analyzed parameters of inflammation such as: activity of serum chitotriosidase by fluorimetric assay, C-reactive proteins (CRP), number of white body cells (WBCs), IgG and fibrinogen. We also assessed the neurological stage according to the Scandinavian Stroke Scale (SSS). RESULTS: In our study, we observed a statistically significant difference (p < 0.05) in the activity of most parameters of inflammation. This difference could be seen in the levels of CRP, number of WBCs and the activity of chitotriosidase, apart from IgG and fibrinogen, between the control group and groups with versus without infection. A significantly increased level of CRP (p < 0.0005) and fibrinogen (p > 0.01) was found on the first day in the stroke group as compared to the control group. The neurological stage on day 4 after stroke, assessed according to the SSS, was significantly worse in the group of patients with infection before stroke than in stroke patients without infection (p < 0.008). CONCLUSION: These results suggest the importance of active inflammatory processes in the pathogenesis of stroke. We observed increased activity of chitotioridase, a parameter of the inflammatory process, in stroke. This study is one more proof that inflammatory processes caused by infection may influence the occurrence of stroke and worsen its outcome. It could be another step towards understanding immunological processes during the acute phase of stroke. The study may also help establish new diagnostic and therapeutic strategies and could be a useful tool for prophylaxis.

Absorption and urinary excretion of quercetin, rutin, and alphaG-rutin, a water soluble flavonoid, in rats.

Quercetin, rutin, alphaG-rutin (a water soluble flavonoid), and a mixture of rutin and alphaG-rutin were administered to rats by a single gastric intubation, and their absorption and urinary excretion were examined. The plasma and 24 h urinary levels of aglycons (quercetin and tamarixetin/isorhamnetin) were measured by HPLC after deconjugation with beta-glucuronidase/sulfatase treatment. alphaG-rutin was absorbed more rapidly than quercetin or rutin, and the plasma concentrations of quercetin and tamarixetin/isorhamnetin reached the highest peak level 30 min after dosing. Quercetin, rutin, and the mixture of rutin and alphaG-rutin showed the first peak level 8 h, 8 h, and 30 min after dosing, respectively. The area under the concentration-time curve (AUC) for quercetin in rats administered alphaG-rutin was approximately 4.5- and 2-fold higher than those in rats administered quercetin and rutin, respectively, and was almost the same as that in rats administered a mixture of rutin and alphaG-rutin. The highest 24 h urinary excretion was observed in alphaG-rutin-administered rats. These results suggest that alphaG-rutin is absorbed more efficiently than either quercetin or rutin and that a high plasma concentration can be maintained by supplying rutin and alphaG-rutin in combination.

Micromolar A and B blood group active trisaccharides abolish human complement haemolytic activity assayed by erythrocytes.

BACKGROUND: At present, there is really no satisfactory treatment of severe haemolytic transfusion reactions involving the ABO system other than the use of steroids that at best are palliative in their effects. In contrast, the use of micromolar concentrations of A or B blood group active trisaccharides that are inexpensive and readily available may prevent lysis by generating soluble immune complexes (ICs) that consume complement. The purpose of this study was to determine the total lytic activity of human serum and to estimate the extent to which trisaccharides can exhaust this capacity. METHODS: We measured complement consumption by (ICs) formed between anti-blood group antibodies and A or B blood group active sugars on erythrocytes (solid phase) or soluble components carrying trisaccharides (fluid phase) in AB serum. A direct complement-mediated lysis (DCL) assay measured the solid-phase reaction and an indirect complement consumption assay (CCA) allowed determination of the fluid-phase reaction. In CCA, the residual lytic activity of AB serum was measured following preincubation with various ICs. RESULTS: Based on over 4000 data points a new mathematical model of complement consumption was formulated. Its predictions deviated by less than 6% in DCL and 9% in CCA when compared with the experimentally accessible data. The new model describes the dynamics of complement consumption including the soluble phase of the reaction. CONCLUSIONS: The mathematical model extrapolation predicts that: (1) in undiluted human serum up to 40% of the physiological erythrocyte concentration could be lysed (when antibodies did not limit lysis); and (2) a 40 microM (or less) concentration of blood group active trisaccharides per patient is sufficient to block the haemolytic complement activity.

Evaluating porcine RBC and platelet alpha-galactosyl expression.

BACKGROUND: Naturally occurring human xenoreactive antibodies bind and agglutinate porcine RBCs. STUDY DESIGN AND METHODS: To determine if xenoantigen expression on RBCs of individual pigs of different breeds and blood groups is variable, and if it correlates with platelet (PLT) expression, we measured adsorption of affinity-purified antibodies to alpha-galactosyl (alphaGal) by RBCs or PLTs from 22 pigs representing four breeds. Hemagglutination of RBCs from these pigs was also performed with pools of human group OAB, A, B, and AB sera, as well as with anti-alphaGal-depleted pooled OAB human serum. RESULTS: There was significant variation in alphaGal expression on RBCs and PLTs among pigs. PLT alphaGal expression did not correlate with RBC alphaGal. RBCs from all pigs were agglutinated by pooled group O, AB, A, or B sera, whereas titers were reduced by 87 percent with anti-alphaGal-depleted serum and by 82 percent with AB sera from two volunteers. Agglutination titers were higher against RBCs from the five highest RBC alphaGal expressers compared with those from the five lowest RBC alphaGal expressers (92 +/- 12 vs. >160, p < 0.05, where 160 was the maximum dilution tested). CONCLUSION: Hemagglutination is a feasible alternative for rapid identification of pigs with RBCs carrying less alphaGal.

RBC T activation and hemolysis in a neonatal intensive care population: implications for transfusion practice.

BACKGROUND: Reports of transfusion-associated hemolysis in infants with T-activated RBCs have led to the suggestion that infants should be screened and provided with low-titer anti-T blood components. T-activated RBCs react with the lectins Arachis hypogea and Glycine soja; variants of T (Th and Tx) and Tk also react with A. hypogea, but not G. soja. Although Tk is not a true variant of T, for the purposes of this study, all RBCs that are reactive with A. hypogea but are not reactive with G. soja are called "T variants." STUDY DESIGN AND METHODS: A prospective study was carried out to examine T and T variant activation and transfusion-associated hemolysis in a neonatal intensive care population and to determine if antibodies to T and T variant are detectable in donor plasma. A total of 2041 samples from 375 infants were tested for T and T variant activation utilizing a lectin panel. Three hundred donor plasma samples were tested for antibodies to T and T variant. RESULTS: Forty-eight of 375 infants (12.8%) had T- and T-variant-activated RBCs. Of these, 13 of 48 (27%) developed at least one episode of sepsis and 9 of 48 (19%) developed necrotizing enterocolitis (NEC) at some point during their inpatient stay. T activation was not always temporally associated with the onset of NEC or sepsis. The remaining 26 of 48 (54%) were healthy infants receiving convalescent care in the neonatal intensive care units and showed no evidence of either NEC or sepsis. Twelve (of 375) additional infants (3.2%) who developed NEC and 100 (27%) who developed sepsis showed no RBC T activation. Twenty-three of 48 (48%) infants with T-activated RBCs received standard blood components, but no transfusion-associated hemolysis occurred. Donor plasma samples contained T but not T variant antibodies. CONCLUSION: T variant activation of RBCs occurs in healthy neonates as well as in infants with NEC and sepsis, but T activation appears rare. Transfusion- associated hemolysis was not seen. The provision of specially prepared blood components for infants with NEC is unnecessary.

Pig hematopoietic cell chimerism in baboons conditioned with a nonmyeloablative regimen and CD154 blockade.

BACKGROUND: In an attempt to induce mixed hematopoietic chimerism and transplantation tolerance in the pig-to-primate model, we have infused high-dose porcine peripheral blood progenitor cells (PBPC) into baboons pretreated with a nonmyeloablative regimen and anti-CD154 monoclonal antibody (mAb). METHODS: Group 1 baboons (n=2) received a nonmyeloablative regimen including whole body irradiation, pharmacological immunosuppression, porcine hematopoietic growth factors, and immunoadsorption of anti-Galalpha1,3Gal (Gal) antibody before infusion of high doses of PBPC (2.7-4.6x10(10) cells/kg). In group 2 (n=5), cyclosporine was replaced by anti-CD154 mAb. Group 3 (n=3) received the group 1 regimen plus anti-CD154 mAb. RESULTS: In group 1, pig chimerism was detected in the blood by flow cytometry (FACS) for 5 days (with a maximum of 14%), and continuously up to 13 days by polymerase chain reaction (PCR). In group 2, pig chimerism was detectable for 5 days by FACS (maximum 33%) and continuously up to 28 days by PCR. In group 3, initial pig chimerism was detectable for 5 days by FACS (maximum 73%). Two of three baboons showed reappearance of pig cells on days 11 and 16, respectively. In one, in which no anti-Gal IgG could be detected for 30 days, pig cells were documented in the blood by FACS on days 16-22 (maximum 6% on day 19) and pig colony-forming cells were present in the blood on days 19-33, which we interpreted as evidence of engraftment. Microchimerism was continuous by PCR up to 33 days. CONCLUSIONS: These results suggest that there is no absolute barrier to pig hematopoietic cell engraftment in primates, and that this may be facilitated if the return of anti-Gal IgG can be prevented.