Regulation of human erythrocyte metabolism by insulin: cellular distribution of 6-phosphofructo-1-kinase and its implication for red blood cell function

Human erythrocytes are highly specialized cells whose function is oxygen transport. These cells' sole metabolic source of energy is the fermentation of glucose via glycolysis. They contain an active insulin receptor and respond to insulin by increasing phosphorylation of tyrosine residues in several proteins. However, no metabolic effects have yet been associated with activation of this receptor in human erythrocytes. Here, we show that insulin increases the rate of glycolysis in human erythrocytes. Lactate production increased 56 and 173% in the presence of 10 and 100 nM insulin, respectively. A higher insulin concentration (1000 nM) partially reversed the stimulation of glycolysis. These effects occur through activation of the key glycolytic enzyme 6-phosphofructo-1-kinase, which exhibits the same pattern of modulation by insulin as seen for glycolytic flux. This modulation also occurs physiologically since ex vivo experiments revealed 50% stimulation of 6-phosphofructo-1-kinase (PFK) activity following a high carbohydrate meal. Insulin increases phosphorylation of PFK and redistributes the enzyme in red blood cells, causing it to detach from the erythrocyte membrane: upon insulin stimulation, the amount of enzyme associated with the plasma decreases by 86%. Detachment is a common mechanism of enzyme activation. As a consequence, insulin prevents up to 68% of red cells hemolysis. These results show that insulin regulates erythrocyte glycolysis and viability and suggest that this regulation is associated to other erythrocyte functions such as oxygen transport. Finally, we suggest that this regulatory mechanism might be compromised in patients with diabetes mellitus.     

Fibrin-polyurethane composites for articular cartilage tissue engineering: a preliminary analysis

In this study we investigated the use of a fibrin hydrogel to improve the potential of a polyurethane (PU) scaffold-based system for articular cartilage tissue engineering. PU-only ("no-fibrin") and PU-fibrin ("fibrin") composites were cultured for up to 28 days and analyzed for DNA content, glycosaminoglycan (GAG) content, type II collagen content, GAG release, and gene expression of aggrecan, collagen I, and collagen II. The use of fibrin allowed for higher viable cell-seeding efficiency (10% higher DNA content on day 2 in fibrin versus no-fibrin composites) and more even cell distribution on seeding, a more than 3-fold increase in the percentage of newly synthesized GAG retained in the constructs, and 2- to 6-fold higher levels of type II collagen and aggrecan gene expression through day 14. Addition of aprotinin to the medium inhibited fibrin degradation, most noticeably in the center of the constructs, but had little effect on biochemical composition or gene expression. Short-term mechanical compression (0-10% sinusoidal strain at 0.1 Hz for 1 h, applied twice daily for 3 days) doubled the rate of GAG release from the constructs, but had little effect on gene expression, regardless of the presence of fibrin. Although further work is needed to optimize this system, the addition of fibrin hydrogel to encapsulate cells in the stiff, macroporous PU scaffold is a step forward in our approach to articular cartilage tissue engineering.     

Neutralizing and binding antibodies to IFN-beta: relative frequency in relapsing-remitting multiple sclerosis patients treated with different IFN-beta preparations.

The frequencies of anti-interferon-beta (IFN-beta) antibody development reported to date in patients treated with different IFN-beta preparations are not readily comparable mainly because of differences in underlying diseases and assay methods. Thus, the frequency of neutralizing antibody (NAb) and binding antibody (BAb) development was analyzed in a sample of sera derived from a homogeneous group of relapsing-remitting multiple sclerosis (RRMS) patients treated with different IFN-beta preparations. The frequency of developing NAb and BAb to IFN-beta varied according to the IFN-beta given. Specifically, the NAb seroconversion frequency was significantly higher in patients treated with Betaferon, Schering AG, Berlin, Germany (31.3%) than in patients treated with both preparations of recombinant IFN-beta 1a (Rebif, Serono, Geneva, Switzerland [7.4%] or Avonex, Biogen, Cambridge, MA [6.3%]). Analysis of BAb seroconversion frequency in the same patients revealed that different IFN-beta preparations may also have different capability to induce BAb development and that BAb are produced during IFN-beta therapy at a significantly higher rate than NAb. Our main conclusion is that different human IFN-beta preparations may possess different immunogenicities, leading to varying frequency of development of antibody to IFN-beta in RRMS.     

Chemokines and pro-inflammatory cytokines in Down's syndrome: an early marker for Alzheimer-type dementia?

BACKGROUND: People with Down's syndrome (DS) show early Alzheimer-like dementia. It has been suggested that the pro-inflammatory cytokine class plays a role in Alzheimer's disease (AD). The study aims at verifying whether pro-inflammatory cytokines in DS are correlated with age, affective symptoms and intellectual decline to a different degree than in subjects with non-DS learning disabilities. METHODS: Cases: 19 subjects with DS; controls: sex- and age-matched individuals with learning disabilities caused by perinatal ischaemic damage. The level of mental retardation was assessed according to DSM-IV; psychopathological symptoms were measured by the Assessment and Information Rating Profile. Serum levels of cytokines were determined with ELISA. RESULTS: DS patients showed higher levels of cytokines and chemokines, with the exception of RANTES; but the only significant difference detected was for MIP-1alpha. A correlation between the degree of mental retardation and IL-6, and between MIP-lalpha and age was found in patients with DS, but not in controls. CONCLUSIONS: The data obtained suggest a possible involvement of chemokines in the inflammatory and degenerative processes similar to AD in DS. Further longitudinal research is required to confirm these findings.     

Expression of human CD81 in transgenic mice does not confer susceptibility to hepatitis C virus infection

We previously demonstrated that hepatitis C virus (HCV) binds to human CD81 through the E2 glycoprotein. Therefore, expression of the human CD81 molecule in transgenic mice was expected to provide a new tool to study HCV infection in vivo, as the chimpanzee is the only species currently available as a laboratory animal model that can be infected with HCV. We produced transgenic mice expressing the human CD81 protein in a wide variety of tissues. We confirmed binding of recombinant E2 glycoprotein to the liver tissue as well as to thymocytes and splenic lymphocytes in the transgenic mice. We inoculated chimpanzee plasma infected with HCV into these animals. None of these transgenic animals showed evidence of viral replication. Furthermore, human CD81 transgenic mice that lack expression of endogenous mouse CD81 were also resistant to HCV infection. We conclude that expression of human CD81 alone is insufficient to confer susceptibility to HCV infection in the mouse. The presence of additional possible factors for HCV infection is discussed.     

Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP. Two coding substitutions were found in comparison with the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429]PLAP, into wild-type Chinese hamster ovary cells. We determined the k(cat) and K(m), of the PLAP S, F, and D allozymes using the non-physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples.     

Economy of locomotion in high-altitude Tibetan migrants exposed to normoxia

High-altitude Tibetans undergo a pattern of adaptations to chronic hypoxia characterized, among others, by a more efficient aerobic performance compared with acclimatized lowlanders. To test whether such changes may persist upon descent to moderate altitude, oxygen uptake of 17 male Tibetan natives lifelong residents at 3500–4500 m was assessed within 1 month upon migration to 1300 m. Exercise protocols were: 5 min treadmill walking at 6 km h⁻¹ on increasing inclines from +5 to +15% and 5 min running at 10 km h⁻¹ on a +5% grade. The data (mean ± s.e.m. ) were compared with those obtained on Nepali lowlanders. When walking on +10, +12.5 and +15% inclines, net     of Tibetans was 25.2 ± 0.7, 29.1 ± 1.1 and 31.3 ± 0.9 ml kg⁻¹ min⁻¹, respectively, i.e. 8, 10 and 13% less ( P < 0.05) than that of Nepali. At the end of the heaviest load, blood lactate concentration was lower in Tibetans than in Nepali (6.0 ± 0.9 versus 8.9 ± 0.6 m m ; P < 0.05) . During running,     of Tibetans was 35.1 ± 0.8 versus 39.3 ± 0.7 ml kg⁻¹ min⁻¹ (i.e. 11% less; P < 0.01). In conclusion, during submaximal walking and running at 1300 m, Tibetans are still characterized by lower aerobic energy expenditure than control subjects that is not accounted for by differences in mechanical power output and/or compensated for by anaerobic glycolysis. These findings indicate that chronic hypoxia induces metabolic adaptations whose underlying mechanisms still need to be elucidated, that persist for at least 1 month upon descent to moderate altitude.     

Identifying recent HIV infections using the avidity index and an automated enzyme immunoassay

We evaluated a procedure for identifying recent HIV infections, using sequential serum samples from 47 HIV-positive persons for whom the seroconversion date could be accurately estimated. Each serum sample was divided into two aliquots: one diluted with phosphate-buffered saline and the other diluted with 1 M guanidine. We assayed the aliquots with the automated AxSYM HIV1/2gO test (Abbott Diagnostics Division), without modifying the manufacturer's protocol. We then calculated the avidity index (AI): the ratio of the sample/cutoff value for the guanidine aliquot to that of the phosphate-buffered saline aliquot. We analyzed 216 serum samples: 34 samples were collected within 6 months of seroconversion (recent seroconversions), and 182 were collected after 6 months. The mean AIs, by time from seroconversion, were 0.68 +/- 0.16 (within 6 months) and 0.98 +/- 0.10 (after 6 months) (P < 0.0001). AI of <0.90 correctly identified 88.2% of recent infections but misclassified as recent infections 13.2% of serum samples collected afterward. The probability of an infection being classified as recent and having AI of > or = 0.90 would be 0.7% in a population with 5% recent infections. AI can identify with a certain degree of accuracy recent HIV infections, and being a quantitative index, it provides different levels of sensitivity and specificity, depending on the selected cutoff value. The standard assay procedure is not modified. This test is simple and inexpensive and could be used for surveillance, decision-making in treatment, and prevention.