Growth, tolerance, and DHA and ARA status of healthy term infants receiving formula with two different ARA concentrations: Double-blind, randomized, controlled trial.

Circulating docosahexaenoic acid (DHA) and arachidonic acid (ARA) in total red blood cells (RBC) are considered indicators of fatty acid status. In this study, healthy term infants received study formula through 120 days of age. All study formulas had 17 mg DHA/100 kcal. Investigational formulas had 1) 25 g ARA/100 kcal and no added prebiotic blend (ARA-25; n = 29) or 2) 34 mg ARA/100 kcal and a prebiotic blend (1:1 ratio; 4 g/L) of polydextrose and galactooligosaccharides (PDX/GOS; n = 20). The control formula had 34 mg ARA/100 kcal and no added prebiotic blend (Control: n = 31). Fatty acids in total RBCs and plasma phospholipids (PPLs) at 120 days and buccal epithelial PLs at 14 and 120 days of age were assessed by capillary column gas chromatography. The calculated 90% confidence interval (CI) of each investigational formula relative to the Control for total RBC ARA (ARA-25: 93-105%; PDX/GOS: 96-110%) and total RBC DHA (ARA-25: 95-113%; PDX/GOS: 94-113%) fell within the pre-specified equivalence limit (85-118%), establishing study formula equivalence with respect to ARA and DHA. At day 120, total RBC and buccal epithelia PL ARA (µg/ml) were not significantly correlated (r = 0.041; p = 0.732); correlation in total RBC and buccal epithelia PL DHA was low, albeit significant (r = 0.324; p = 0.006). Consequently, buccal epithelial may not provide a suitable substitute for RBC when assessing fatty acid status and availability. The present RBC data suggest availability of DHA for central nervous system development and function is equivalent among infants receiving formulas that had 34 or 25 mg/100 kcal ARA and 17 mg/100 kcal DHA.

DHA and ARA addition to infant formula: Current status and future research directions.

Docosahexaenoic acid (DHA) and arachidonic acid (ARA) are present in breast milk and play important roles in early infant development. A supply of these fatty acids in infant formula (typically following breast milk as a model with ARA > DHA) is thought to be important since endogenous synthesis is insufficient to maintain tissue levels equivalent to breast-fed infants. Intervention studies assessing the impact of DHA- and ARA-supplemented formulas have resulted in numerous positive developmental outcomes (closer to breast-fed infants) including measures of specific cognition functions, visual acuity, and immune responses. A critical analysis of outcome assessment tools reveals the essentiality of selecting appropriate, focused techniques in order to provide accurate evaluation of DHA- and ARA-supplemented formulas. Future research directions should encompass in-depth assessment of specific cognitive outcomes, immune function, and disease incidence, as well as sources of experimental variability such as the status of fatty acid desaturase polymorphisms.

Immediate and extended effects of abrasion on stratum corneum natural moisturizing factor.

BACKGROUND: Natural moisturizing factor (NMF), principally comprised of hygroscopic amino acids and derivatives that absorb moisture from the surrounding environment, serves as the primary humectant of the stratum corneum (SC). Acute barrier disruption has been shown to differentially affect the concentration of NMF in the SC. This study measured the recovery kinetics of NMF after mechanical damage of the SC, which is not well understood. METHODS: The study population included 20 healthy female volunteers (18-72-year old) with no history of dermatological disorders. Transepidermal water loss (TEWL), erythema, and SC water and NMF were measured at all sites before abrasion, 30 min following abrasion, and 1-3, 6, 8, and 10 days following abrasion. Measurements obtained from the abraded site were compared with those obtained from an untreated site. RESULTS: As expected, both TEWL and erythema increased significantly with abrasion. Erythema and TEWL values remained higher at the abraded site for 2 and 6 days, respectively, after abrasion. No changes in NMF component levels in the SC were observed at 30 min after abrasion. One day following abrasion, reduced levels of glycine, histidine pH4, trans-urocanic acid (tUca) pH4, and tUca pH8 were observed. In addition, a significantly lower level of serine was observed at the abraded site 2 and 6 days following abrasion. Within 8 days after abrasion, these components returned to levels comparable to those observed in untreated skin. Throughout the study, no differences were observed in the level of water in the SC. CONCLUSION: These results demonstrate that acute barrier disruption induced by mechanical abrasion has relatively little impact on biochemical events responsible for NMF generation. Though reductions in certain NMF components were observed, abrasion had no measureable effect on SC water content over the duration of the study. This implies that the reduced NMF components may not contribute substantially to water retention in the SC. The reduced components belong to a group of NMF molecules thought to be principally derived through degradation of S-100 proteins in the epidermis. NMF components measured in this study that are derived from sweat and/or urea cycling were not impacted. These data imply that while abrasion elicits clinical signs of barrier disruption within the SC, effects on its biochemical constituents and ability to retain water are relatively minor.

Liquid human milk fortifier significantly improves docosahexaenoic and arachidonic acid status in preterm infants.

We report the fatty acid composition of mother׳s own human milk from one of the largest US cohorts of lactating mothers of preterm infants. Milk fatty acid data were used as a proxy for intake at enrollment in infants (n=150) who received human milk with a powder human milk fortifier (HMF; Control) or liquid HMF [LHMF; provided additional 12mg docosahexaenoic acid (DHA), 20mg arachidonic acid (ARA)/100mL human milk]. Mothers provided milk samples (n=129) and reported maternal DHA consumption (n=128). Infant blood samples were drawn at study completion (Study Day 28). Human milk and infant PPL fatty acids were analyzed using capillary column gas chromatography. DHA and ARA were within ranges previously published for US term and preterm human milk. Compared to Control HMF (providing no DHA or ARA), human milk fortified with LHMF significantly increased infant PPL DHA and ARA and improved preterm infant DHA and ARA status.

Immediate and extended effects of sodium lauryl sulphate exposure on stratum corneum natural moisturizing factor.

OBJECTIVES: Natural moisturizing factor (NMF) serves as the primary humectant of the stratum corneum (SC), principally comprised of hygroscopic amino acids and derivatives that absorb moisture. Barrier disruption has been shown to differentially affect the levels of specific NMF components, though the kinetics of NMF component restoration following disruption have not been examined. Here, we investigated the impact of barrier disruption caused by surfactant exposure on a subset of NMF components immediately following exposure and out to 10 days post-exposure. METHODS: Volunteers wore patches containing either 1% w/v sodium lauryl sulphate (SLS) or distilled water on their forearms for 24 h. Measurements of transepidermal water loss, erythema, SC water content and a subset of SC NMF and lipid components were obtained at both sites before treatment, the day of patch removal, and 1, 2, 3, 6, and 10 days following treatment. RESULTS: Most measured NMF components decreased in response to SLS exposure. Exceptions were increases in lactate, ornithine and urea, and no difference in proline levels. In the days following exposure, reduced levels of several NMF components continued at the SLS site; however, all measured NMF components demonstrated equivalence to the vehicle control within 10 days. Histidine pH 7, lactate, ornithine and urea were the first to achieve levels equivalent to the vehicle control site, normalizing within 1 day after patch removal. CONCLUSION: Results imply that NMF components derived from sweat and urea cycling are least impacted by SLS exposure whereas NMF components derived from degradation of filaggrin and/or other S-100 proteins are most impacted. This implies the restoration of the processes responsible for S-100 protein processing into free amino acids takes several days to return to normal. Further examination of the enzymes involved in S-100 protein processing following barrier disruption would provide insight into the pathway(s) for NMF restoration during SC recovery.

Isolation, cloning and characterization of Polistes dominulus venom phospholipase A1 and its isoforms.

Polistes dominulus is known to be an allergenically important social wasp. Its venom has four major allergens (ziv. phospholipase A1, hyaluronidase, antigen 5 and a serine-protease). Amino acid sequences of its serine-protease and Antigen 5 have been published. In this paper, the partial amino acid sequence of its venom phospholipase native protein is reported. Also, we give an account to the complete nucleotide sequence of Polistes dominulus venom phospholipase A1 gene, its isoforms and their complete deduced amino acid sequences. Their similarity to the other phospholipases A1 of the family: Vespidae is discussed.

Immunologic characterization of the recombinant fire ant venom allergen Sol i 3.

Individuals sensitized to fire ant stings show immunoglobulin (Ig)E antibodies against the venom protein Sol i 3. We determined the full-length complementary DNA (cDNA) sequence of this protein and expressed recombinant Sol i 3 in immunogenic form. The complete cDNA of Sol i 3 was obtained by reverse transcription polymerase chain reaction (RT-PCR) and PCR + 1 reactions using gene-specific oligonucleotides, and oligonucleotides designed from the amino acid sequence of this protein. The encoding cDNA is 705 bp in length corresponding to 235 amino acids. The first 22 amino acids are a leader sequence. The protein with an added C-terminal hexahistidine tag was expressed in insect cells using a baculovirus system. The recombinant protein was secreted into the supernatant and affinity purified with a cobalt chelating resin. The recombinant fire ant venom allergen Sol i 3 showed similar IgE binding activity to the native protein in radioallergosorbent test (RAST) and RAST inhibition assays. It was produced in both a glycosylated and an unglycosylated form. A three-dimensional reconstruction of Sol i 3 was compared with the experimentally determined structure of the related allergen Ves v 5. This model is supported by results of circular dichroism spectroscopy.

Expression systems for production of recombinant allergens.

Recombinant allergenic proteins have been produced in a variety of different expression systems. This review gives examples of and compares prokaryotic expression systems, such as Escherichia coli, and eukaryotic systems including the yeasts, Saccharomyces cerevisiae and Pichia pastoris, baculovirus-insect cell systems, mammalian cell systems and plant systems. Important factors to consider in choosing an expression system are discussed and examples of applications are given.

Occupational allergy to bumblebees: allergens of Bombus terrestris.

BACKGROUND: With the increase in commercial vegetable production in greenhouses, occupational sensitization to bumblebee venom is becoming more common. Studies using sera from subjects thus sensitized allow evaluation of the allergenic specificity of bumblebee sensitization. OBJECTIVE: The purposes of this study were to determine the degree of species group specificity of bumblebee venom allergens in sera of allergic patients and to investigate the structural basis of this specificity. METHODS: Allergens were purified from bumblebee venom, studied serologically by direct binding and inhibition techniques, and characterized by enzyme analysis and amino acid sequencing. Three-dimensional models of the phospholipases were constructed and analyzed. RESULTS: Bombus terrestris venom contains phospholipase A(2), venom protease, hyaluronidase, and acid phosphatase allergens. The protease and phospholipase A(2) allergens contain IgE-reactive epitopes that are different from those seen in Bombus pennsylvanicus, a North American species. Bumblebee phospholipase A(2) is only 53% identical to honeybee phospholipase A(2). The results of 3-dimensional modeling are consistent with the immunologic observations. CONCLUSIONS: Patients with primary bumblebee sensitization should be diagnosed and treated with venom from the appropriate species group of bumblebees. Bumblebee venom phospholipase A(2) and protease are antigenically distinct from honeybee venom proteins. There are significant species group-specific epitopes on bumblebee venom proteins.

Essential fatty acids in visual and brain development.

Essential fatty acids are structural components of all tissues and are indispensable for cell membrane synthesis; the brain, retina and other neural tissues are particularly rich in long-chain polyunsaturated fatty acids (LC-PUFA). These fatty acids serve as specific precursors for eicosanoids, which regulate numerous cell and organ functions. Recent human studies support the essential nature of n-3 fatty acids in addition to the well-established role of n-6 essential fatty acids in humans, particularly in early life. The main findings are that light sensitivity of retinal rod photoreceptors is significantly reduced in newborns with n-3 fatty acid deficiency, and that docosahexaenoic acid (DHA) significantly enhances visual acuity maturation and cognitive functions. DHA is a conditionally essential nutrient for adequate neurodevelopment in humans. Comprehensive clinical studies have shown that dietary supplementation with marine oil or single-cell oil sources of LC-PUFA results in increased blood levels of DHA and arachidonic acid, as well as an associated improvement in visual function in formula-fed infants matching that of human breast-fed infants. The effect is mediated not only by the known effects on membrane biophysical properties, neurotransmitter content, and the corresponding electrophysiological correlates but also by a modulating gene expression of the developing retina and brain. Intracellular fatty acids or their metabolites regulate transcriptional activation of gene expression during adipocyte differentiation and retinal and nervous system development. Regulation of gene expression by LC-PUFA occurs at the transcriptional level and may be mediated by nuclear transcription factors activated by fatty acids. These nuclear receptors are part of the family of steroid hormone receptors. DHA also has significant effects on photoreceptor membranes and neurotransmitters involved in the signal transduction process; rhodopsin activation, rod and cone development, neuronal dendritic connectivity, and functional maturation of the central nervous system.

Impaired synthesis of DHA in patients with X-linked retinitis pigmentosa.

Many patients with X-linked retinitis pigmentosa (XLRP) have lower than normal blood levels of the long-chain polyunsaturated omega3 fatty acid docosahexaenoic acid (DHA; 22:6omega3). This clinical trial was designed to test whether down-regulation of DHA biosynthesis might be responsible for these reduced DHA levels. DHA biosynthesis was assessed in five severely affected patients with XLRP and in five age-matched controls by quantifying conversion of [U-(13)C]alpha-linolenic acid (alpha-LNA) to [(13)C]DHA. Following oral administration of [U-(13)C]alpha-LNA, blood samples were collected at designated intervals for 21 days and isotopic enrichment of all omega3 fatty acids was determined by gas chromatography/mass spectroscopy. Activity of each metabolic step in the conversion of alpha-LNA to DHA was determined by comparison of the ratios of the integrated concentration of (13)C-product to (13)C-precursor in plasma total lipid fractions. The ratio of [(13)C]DHA to [(13)C]18:3omega3 (the entire pathway) and that of [(13)C]20:5omega3 to [(13)C]20:4omega3 (Delta(5)-desaturase) were significantly lower in patients versus controls (P = 0.03 and 0.05, respectively). The estimated biosynthetic rates of [(13)C]20:5omega3, [(13)C]22:5omega3, [(13)C]24:5omega3, [(13)C]24:6omega3, and [(13)C]22:6omega3 were significantly lower in XLRP patients (42%, 43%, 31%, 18%, and 32% of control values, respectively; P < 0.04), supporting down-regulation of Delta(5)-desaturase in XLRP. The disappearance of (13)C-labeled fatty acids from plasma was not greater in XLRP patients compared with controls, suggesting that XLRP was not associated with increased rates of fatty acid oxidation or other routes of catabolism.Thus, despite individual variation among both patients and controls, the data are consistent with a lower rate of Delta(5)-desaturation, suggesting that decreased biosynthesis of DHA may contribute to lower blood levels of DHA in patients with XLRP.

Detection of immunoglobulin antibodies in the sera of patients using purified latex allergens.

BACKGROUND: Latex allergy is largely an occupational allergy due to sensitization to natural rubber latex allergens present in a number of health care and household products. Although several purified allergens are currently available for study, information on the usefulness of these purified, native or recombinant allergens in the demonstration of specific immunoglobulin (Ig) E in the sera of patients is lacking. OBJECTIVE: To evaluate the purified latex allergens and to demonstrate specific IgE antibody in the sera of health care workers and spina bifida patients with clinical latex allergy. METHODS: Two radioallergosorbent and an enzyme-linked immunosorbent assay (ELISA) using latex proteins Hev b 1, 2, 3, 4, 6 and 7 along with two glove extracts and Malaysian nonammoniated latex (MNA) were evaluated to demonstrate IgE in the sera of health care workers and spina bifida with latex allergy and controls with no history of latex allergy. RESULTS: ELISA using the purified latex allergens demonstrated specific IgE in 32-65% health care workers and 54-100% of spina bifida patients with latex allergy. The corresponding figures for RAST were 13-48 and 23-85 for RAST-1 and 19-61 and 36-57 for RAST-2. These results were comparable with the results obtained with glove extracts and crude rubber latex proteins. CONCLUSIONS: When used simultaneously, latex proteins Hev b 2 and Hev b 7 reacted significantly with specific serum IgE in 80% of health care workers and 92% of spina bifida patients with latex allergy by ELISA technique, while this combination gave lower positivity when the RASTs were used. By the addition of Hev b 3, specific IgE was detected in all spina bifida patients with latex allergy. Both RASTs failed to show specific IgE in the control subjects, while the ELISA showed significant latex-specific IgE in 22% of controls.

Essential fat requirements of preterm infants.

The interest in factors that modify early infant development has led investigators to focus on n-3 and n-6 long-chain polyunsaturated fatty acids (LCPUFAs) in the past 2 decades. The presence of docosahexaenoic acid (DHA) and arachidonic acid (AA) in breast milk, compared with their absence from infant formulas available in the United States, has prompted clinical trials designed to examine whether LCPUFA enrichment of infant formula has beneficial effects on maturational events of the visual system. These trials have shown significant functional advantages of LCPUFA supplementation for preterm infants, whereas benefits for full-term infants remain controversial. The growth and safety of preterm infants was not compromised by LCPUFA enrichment, although these issues remain to be resolved in clinical trials with full-term infants.

Impact of early dietary intake and blood lipid composition of long-chain polyunsaturated fatty acids on later visual development.

BACKGROUND: In contrast to human milk, current infant formulas in the United States do not contain omega3 and omega6 long-chain polyunsaturated fatty acids. This may lead to suboptimal blood lipid fatty acid profiles and to a measurable diminution of visual function in developing term infants. The need for docosahexaenoic acid and arachidonic acid supplementation in the infant diet was evaluated in a double-blind, randomized clinical trial. METHODS: Healthy term infants were randomized to diets of (1) commercial formula, (2) docosahexaenoic acid-enriched formula (0.35% of total fatty acids), or (3) docosahexaenoic acid- (0.36%) and arachidonic acid- (0.72%) enriched formula. Eighty-seven infants completed the 17-week nutritional trial, and 58 were observed until 52 weeks of life. A reference group was exclusively breast fed for at least 17 weeks (n = 29). Outcome measures included electroretinographic responses, visual evoked potentials, and blood fatty acid analysis in infants at birth and at 6, 17, and 52 weeks of age. RESULTS: Commercial formula-fed infants had 30% to 50% lower content of docosahexaenoic acid in total red blood cell lipids during the 17-week feeding trial compared with breastfed infants. Significant differences persisted at the 1-year follow-up. Arachidonic acid content was consistently reduced in the commercial formula group by 15% to 20%. Infants fed long-chain polyunsaturated fatty acid-enriched formulas had docosahexaenoic acid and arachidonic acid blood lipid profiles resembling those of human milk-fed infants. Infants receiving this enriched formula had more mature electroretinographic responses than commercial formula-fed infants at 6 weeks of age. Human milk-fed and docosahexaenoic acid-enriched formula-fed infants had better visual acuity than commercial formula-fed infants at both 17 and 52 weeks of age. Early (17-week) fatty acid profiles in blood lipids were correlated with later (52-week) visual function development in study infants. CONCLUSIONS: Results from this clinical trial demonstrate that long-chain polyunsaturated fatty acid supplementation of formula in term infants produces blood lipid fatty acid profiles that are similar to those observed in breast-fed infants. This supplementation leads to better visual function later in life (i.e., 1 year of age) than that shown by infants fed commercial formula.

A randomized controlled trial of early dietary supply of long-chain polyunsaturated fatty acids and mental development in term infants.

The effects of dietary docosahexaenoic acid (DHA) supply during infancy on later cognitive development of healthy term infants were evaluated in a randomized clinical trial of infant formula milk supplemented with 0.35% DHA or with 0.36% DHA and 0.72% arachidonic acid (AA), or control formula which provided no DHA or AA. Fifty-six 18-month-old children (26 male, 30 female) who were enrolled in the trial within the first 5 days of life and fed the assigned diet to 17 weeks of age were tested using the Bayley Scales of Infant Development, 2nd edition (BSID-II) (Bayley 1993) at the Retina Foundation of the Southwest, Dallas, TX. These children had also been assessed at 4 months and 12 months of age for blood fatty-acid composition, sweep visual evoked potential (VEP) acuity, and forced-choice preferential looking (FPL) acuity (Birch et al. 1998). Supplementation of infant formula with DHA+AA was associated with a mean increase of 7 points on the Mental Development Index (MDI) of the BSID-II. Both the cognitive and motor subscales of the MDI showed a significant developmental age advantage for DHA- and DHA+AA-supplemented groups over the control group. While a similar trend was found for the language subscale, it did not reach statistical significance. Neither the Psychomotor Development Index nor the Behavior Rating Scale of the BSID-II showed significant differences among diet groups, consistent with a specific advantage of DHA supplementation on mental development. Significant correlations between plasma and RBC-DHA at 4 months of age but not at 12 months of age and MDI at 18 months of age suggest that early dietary supply of DHA was a major dietary determinant of improved performance on the MDI.

Fesselin: a novel synaptopodin-like actin binding protein from muscle tissue.

A novel actin binding protein has been isolated from chicken gizzard muscle. When isolated, a pair of proteins with apparent molecular weights of 79 kDa and 103 kDa are obtained; both proteins have a pI near 9.3. Peptide mapping indicates that these proteins are related. Antibodies against this protein cross-reacted with proteins from other smooth muscle containing tissues as well as skeletal and heart muscle. Traces of cross-reactive material were also detected in brain and kidney tissue. The affinity of this protein for actin is ca. 1x10(6) M(-1). Interestingly, this actin binding protein is a potent actin-bundling agent. A partial sequence analysis confirmed that there were no previously reported homologues in smooth muscle. However, considerable homology was found with the protein synaptopodin that is found in nervous tissue and kidney but is absent from muscle tissue. It is likely that the new protein is a member of the synaptopodin family. We call the smooth muscle actin binding protein fesselin.

Fatty acid profile of buccal cheek cell phospholipids as an index for dietary intake of docosahexaenoic acid in preterm infants.

Cheek cells (buccal epithelia) were utilized as a noninvasive index of fatty acid status in a study of the effects of n-3 long chain polyunsaturated fatty acid supplementation on visual function in preterm infants. The fatty acid profile of cheek cell phospholipids was directly correlated with the dietary docosahexenoic acid (DHA) intake of infants receiving: (i) primarily human milk; (ii) n-3 fatty acid-deficient, corn oil-based, commercial formula (CO); (iii) alpha-linolenic acid-enriched, soy oil-based, commercial formula; or (iv) experimental formula enriched with soy and marine oils providing a DHA level equivalent to that in human milk. In a subset of infants with complete cheek cell fatty acid profiles and visual function assessments, preterm infants at both 36 wk (n = 63) and 57 wk (n = 45) postconceptional age had significantly (P < 0.0005) reduced cheek cell phospholipid DHA levels in the n-3-deficient, CO-fed group compared to the other diet groups. The DHA content in cheek cell phospholipids was highly correlated (P < 0.0005) with that of both red blood cell lipids and plasma phospholipids at the 36- and 57-wk time points. The DHA content in cheek cell lipids of infants at 36 wk was significantly correlated with electroretinographic responses (r = -0.29; P < 0.03) and visual acuity (r = -0.31; P < 0.02) as measured by visual-evoked potentials (VEP). Cheek cell DHA was highly correlated (r= -0.57; P < 0.0005) with VEP acuity at the 57-wk time point. These results suggest that the fatty acid profile of cheek cells is a valid index of essential fatty acid status, can be monitored frequently, and is associated with functional parameters in infants.

A novel locus (RP24) for X-linked retinitis pigmentosa maps to Xq26-27.

Two genetic loci, RP2 and RP3, for X-linked retinitis pigmentosa (XLRP) have been localized to Xp11.3-11.23 and Xp21.1, respectively. RP3 appears to account for 70% of XLRP families; however, mutations in the RPGR gene (isolated from the RP3 region) are identified in only 20% of affected families. Close location of XLRP loci at Xp and a lack of unambiguous clinical criteria do not permit assignment of genetic subtype in a majority of XLRP families; nonetheless, in some pedigrees, both RP2 and RP3 could be excluded as the causative locus. We report the mapping of a novel locus, RP24, by haplotype and linkage analysis of a single XLRP pedigree. The RP24 locus was identified at Xq26-27 by genotyping 52 microsatellite markers spanning the entire X chromosome. A maximum LOD score of 4.21 was obtained with DXS8106. Haplotype analysis assigned RP24 within a 23-cM region between the DXS8094 (proximal) and DXS8043 (distal) markers. Other chromosomal regions and known XLRP loci were excluded by obligate recombination events between markers in those regions and the disease locus. Hemizygotes from the RP24 family have early onset of rod photoreceptor dysfunction; cone receptor function is normal at first, but there is progressive loss. Patients at advanced stages show little or no detectable rod or cone function and have clinical hallmarks of typical RP. Mapping of the RP24 locus expands our understanding of the genetic heterogeneity in XLRP and will assist in development of better tools for diagnosis.

Expression of two isoforms of Lep d 2, the major allergen of Lepidoglyphus destructor, in both prokaryotic and eukaryotic systems.

BACKGROUND: The dust mite Lepidoglyphus destructor is a major cause of allergic diseases among farmers. We have previously cloned and sequenced two isoforms of the major allergen Lep d 2 (formerly designated Lep d 1) and found significant homology to group 2 allergens of the house dust mite species Dermatophagoides. We now report on the production and characterization of recombinant Lep d 2. OBJECTIVE: We have expressed both isoforms in two different expression systems; a eukaryotic system, baculovirus in insect cells and a prokaryotic system, E. coli. We have compared the two systems in regard to production yields and immunoreactivity of the recombinant allergens. METHODS: The complete cDNA including the natural leader sequence was cloned into the pBlueBacIII transfer vector, and the rLep d 2 was produced as a secreted protein in baculovirus. For the expression in E. coli, the cDNA was cloned into the pET vector, and the rLep d 2 was produced with six C-terminal histidine residues. The purified recombinant allergens were tested for immunoreactivity with 10 sera from subjects allergic to Lepidoglyphus destructor and were compared with native Lep d 2 using inhibition immunoblotting. The ability of the recombinant allergens to release histamine from basophils was evaluated using a histamine release assay. RESULTS: Both expression systems produced immunoreactive recombinant allergens. They inhibited the binding of human sera to native Lep d 2 confirming their retained IgE binding properties. The yield of pure recombinant protein from the prokaryotic system was approximately 1 mg/L compared to the eukaryotic system which produced up to 4 mg/L in an adherent cell culture system. CONCLUSIONS: We have produced recombinant Lep d 2 in prokaryotic and eukaryotic expression systems which are comparable to the native allergen. Recombinant Lep d 2 might now be included in more extensive clinical studies to confirm its usefulness in the in vitro and the in vivo diagnosis of Lepidoglyphus destructor.

Visual acuity and the essentiality of docosahexaenoic acid and arachidonic acid in the diet of term infants.

The need for a dietary supply of docosahexaenoic acid (DHA) and arachidonic aid (AA) in term infants was evaluated in a double-masked randomized clinical trial of the effects of supplementation of term infant formula with DHA (0.35% of total fatty acids) or with DHA (0.36%) and AA (0.72%) on visual acuity development. One hundred and eight healthy term infants were enrolled in the study; 79 were exclusively formula-fed from birth (randomized group) and 29 were exclusively breast-fed (gold standard group). Infants were evaluated at four time points during the first 12 mo of life for blood fatty acid composition, growth, sweep visual evoked potential (VEP) acuity, and forced choice preferential looking acuity. Supplementation of term infant formula with DHA or with DHA and AA during the first 4 mo of life yields clear differences in total red blood cell (RBC) lipid composition. Supplementation of term infant formula with DHA or with DHA and AA also yields better sweep VEP acuity at 6, 17, and 52 wk of age but not at 26 wk of age, when acuity development reaches a plateau. The RBC lipid composition and sweep VEP acuity of supplemented infants was similar to that of human milk-fed infants, whereas the RBC lipid composition and sweep VEP acuity of unsupplemented infants was significantly different from human milk-fed infants. Differences in acuity among diet groups were too subtle to be detected by the forced choice preferential looking protocol. Infants in all diet groups had similar rates of growth and tolerated all diets well. Thus, early dietary intake of preformed DHA and AA appears necessary for optimal development of the brain and eye of the human infant.