Modeling the MHC class I pathway by combining predictions of proteasomal cleavage, TAP transport and MHC class I binding.

Epitopes presented by major histocompatibility complex (MHC) class I molecules are selected by a multi-step process. Here we present the first computational prediction of this process based on in vitro experiments characterizing proteasomal cleavage, transport by the transporter associated with antigen processing (TAP) and MHC class I binding. Our novel prediction method for proteasomal cleavages outperforms existing methods when tested on in vitro cleavage data. The analysis of our predictions for a new dataset consisting of 390 endogenously processed MHC class I ligands from cells with known proteasome composition shows that the immunological advantage of switching from constitutive to immunoproteasomes is mainly to suppress the creation of peptides in the cytosol that TAP cannot transport. Furthermore, we show that proteasomes are unlikely to generate MHC class I ligands with a C-terminal lysine residue, suggesting processing of these ligands by a different protease that may be tripeptidyl-peptidase II (TPPII).

Vaccination with minigenes encoding for novel 'self' antigens are effective in DNA-vaccination against neuroblastoma.

The induction of T-cell mediated immunity against neuroblastoma is a challenge due to poor immunogenicity of this malignancy. Here, we demonstrate the induction of protective immunity in a syngeneic murine neuroblastoma model following vaccination with minigenes comprising of three novel natural MHC class I ligands. First, after immunoprecipitation of MHC class I from NXS2 cells, peptides were eluted and examined in tandem-MS analysis which lead to the identification of three novel natural MHC class I peptide ligands, TEALPVKLI from ribonucleotide reductase M2, NEYIMSLI from Ser/Thr protein phosphatase 2A and FEMVSTLI with unknown origin. Second, we constructed two different minigenes, one encoding for the three novel epitopes and the second for three known mTH derived epitopes with high predicted binding affinity to MHC class I by cloning them into the mammalian expression vector pCMV-3FUB. This lead to constructs with an ubiquitin-tag upstream the inserted epitopes in order to facilitate proteasomal degradation. Furthermore the epitopes were separated by a spacer peptide (AAY), which proved to be a preferential proteasome cleavage site. Third, we demonstrate the induction of protective immunity against neuroblastoma using an attenuated strain of Salmonella typhimurium as a carrier harboring pCMV 3FUb vectors encoding for the two minigenes. These findings establish proof of concept that disruption of self tolerance against neuroblastoma associated epitopes may be an effective adjuvant therapeutic strategy.

Bioconversion of glucose and fructose to sorbitol and gluconic acid by untreated cells of Zymomonas mobilis.

The bioconversion of glucose and fructose to gluconic acid and sorbitol, respectively, by the enzymes glucose-fructose oxidoreductase (GFOR) and glucono-delta-lactonase (GL), contained in untreated cells of Zymomonas mobilis ATCC 29191, was investigated in batch runs with glucose plus fructose concentrations (S0) varying from 100 to 750 g l-1 in equimolar ratio. When S0 was increased to 650 g l-1, the yields were improved, reaching a maximum of 91% for both products, with productivities of 1.6 and 1.5 g g-1 cell h-1 for gluconic acid and sorbitol, respectively. Above this level (S0 = 750 g l-1), no further improvement in yields was observed and productivities decreased due to the longer process time. The high yields of bioconversion runs with S0 > or = 650 g l-1 are a consequence of the sequential inhibition of the normal metabolism of Z. mobilis by substrates and products, resulting in preferential utilization of substrates via the GFOR/GL system.

Ketone body transport in the human neonate and infant.

Using a continuous intravenous infusion of D-(-)-3-hydroxy[4,4,4-2H3]butyrate tracer, we measured total ketone body transport in 12 infants: six newborns, four 1-6-mo-olds, one diabetic, and one hyperinsulinemic infant. Ketone body inflow-outflow transport (flux) averaged 17.3 +/- 1.4 mumol kg-1 min-1 in the neonates, a value not different from that of 20.6 +/- 0.9 mumol kg-1 min-1 measured in the older infants. This rate was accelerated to 32.2 mumol kg-1 min-1 in the diabetic and slowed to 5.0 mumol kg-1 min-1 in the hyperinsulinemic child. As in the adult, ketone turnover was directly proportional to free fatty acid and ketone body concentrations, while ketone clearance declined as the circulatory content of ketone bodies increased. Compared with the adult, however, ketone body turnover rates of 12.8-21.9 mumol kg-1 min-1 in newborns fasted for less than 8 h, and rates of 17.9-26.0 mumol kg-1 min-1 in older infants fasted for less than 10 h, were in a range found in adults only after several days of total fasting. If the bulk of transported ketone body fuels are oxidized in the infant as they are in the adult, ketone bodies could account for as much as 25% of the neonate's basal energy requirements in the first several days of life. These studies demonstrate active ketogenesis and quantitatively important ketone body fuel transport in the human infant. Furthermore, the qualitatively similar relationships between the newborn and the adult relative to free fatty acid concentration and ketone inflow, and with regard to ketone concentration and clearance rate, suggest that intrahepatic and extrahepatic regulatory systems controlling ketone body metabolism are well established by early postnatal life in humans.

Insulin pump therapy in young children with type 1 diabetes.

Six 17- to 53-month-old diabetic children had marked metabolic instability characterized by chronic hyperglycemia and frequent or severe hypoglycemia with conventional management that included twice daily insulin injections, diet, and home blood glucose monitoring. Because of the metabolic instability, all were given continuous subcutaneous insulin infusions (CSII) via portable externally worn infusion pump. During 6 months of CSII, there was marked improvement: hemoglobin A1 decreased from 192% +/- 8% (SD) to 152% +/- 31% of the normal mean (P less than 0.02), and hypoglycemic episodes decreased in both severity and frequency. CSII was incorporated into the children's treatment with no appreciable adverse psychologic effects or interference with normal activities. CSII, under carefully controlled clinical conditions, may be of benefit in some preschool children with unacceptable metabolic control of diabetes mellitus.

[Ambulatory treatment of diabetes in children by continuous subcutaneous infusion of insulin using a portable pump]. / Traitement ambulatoire du diabète de l'enfant par perfusion sous-cutanée continue d'insuline au moyen d'une pompe portable.

Six diabetic children, aged 2 to 4 years, were selected for continuous subcutaneous insulin infusion (SCII) therapy using a portable pump, because of unstable glycemic control. Under previous conventional insulin therapy, they experienced both chronic hyperglycemia (mean: 2.10 +/- 0.07 milligrams, HbA1 C: 9.02 +/- 0.2%) and frequent or severe hypoglycemic manifestations. Reduction of both glycemic level: 1.08 +/- 0.04 milligrams (p less than 0.01) and instability: M index 0.76 +/- 0.2 vs 5.5 +/- 1.4 (p less than 0.01) was obtained the first week after CSII therapy was started. The metabolic improvement was maintained over 5 to 9 months of ambulatory CSII therapy: HbA1 C decreased down to 7.6 +/- 0.6% (p less than 0.05), while hypoglycemic episodes became rare. The portable insulin delivery system was well tolerated both physically and psychologically by 5/6 of the children and their families.