Targeting age‐specific changes in CD4+ T cell metabolism ameliorates alloimmune responses and prolongs graft survival

Age impacts alloimmunity. Effects of aging on T‐cell metabolism and the potential to interfere with immunosuppressants have not been explored yet. Here, we dissected metabolic pathways of CD4⁺ and CD8⁺ T cells in aging and offer novel immunosuppressive targets. Upon activation, CD4⁺ T cells from old mice failed to exhibit adequate metabolic reprogramming resulting into compromised metabolic pathways, including oxidative phosphorylation (OXPHOS) and glycolysis. Comparable results were also observed in elderly human patients. Although glutaminolysis remained the dominant and age‐independent source of mitochondria for activated CD4⁺ T cells, old but not young CD4⁺ T cells relied heavily on glutaminolysis. Treating young and old murine and human CD4⁺ T cells with 6‐diazo‐5‐oxo‐l‐norleucine (DON), a glutaminolysis inhibitor resulted in significantly reduced IFN‐γ production and compromised proliferative capacities specifically of old CD4⁺ T cells. Of translational relevance, old and young mice that had been transplanted with fully mismatched skin grafts and treated with DON demonstrated dampened Th1‐ and Th17‐driven alloimmune responses. Moreover, DON diminished cytokine production and proliferation of old CD4⁺ T cells in vivo leading to a significantly prolonged allograft survival specifically in old recipients. Graft prolongation in young animals, in contrast, was only achieved when DON was applied in combination with an inhibition of glycolysis (2‐deoxy‐d‐glucose, 2‐DG) and OXPHOS (metformin), two alternative metabolic pathways. Notably, metabolic treatment had not been linked to toxicities. Remarkably, immunosuppressive capacities of DON were specific to CD4⁺ T cells as adoptively transferred young CD4⁺ T cells prevented immunosuppressive capacities of DON on allograft survival in old recipients. Depletion of CD8⁺ T cells did not alter transplant outcomes in either young or old recipients. Taken together, our data introduce an age‐specific metabolic reprogramming of CD4⁺ T cells. Targeting those pathways offers novel and age‐specific approaches for immunosuppression.     

Intestinal synthesis of 24-keto-1,25-dihydroxyvitamin D3. A metabolite formed in vivo with high affinity for the vitamin D cytosolic receptor

24-Keto-1,25-dihydroxyvitamin D3 has been identified as an intestinal metabolite of 1,25-dihydroxyvitamin D3 by ultraviolet absorbance, mass spectroscopy, and chemical reactivity. The metabolite was produced from 1,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3 in rat intestinal mucosa homogenates. 24-Keto-1,25-dihydroxyvitamin D3 is present in vivo in the plasma and small intestinal mucosa of rats fed a stock diet, receiving no exogenous 1,25-dihydroxyvitamin D3, and in the plasma and small intestinal mucosa of rats dosed chronically with 1,25-dihydroxyvitamin D3. 24-Keto-1,25-dihydroxyvitamin D3 has affinity equivalent to 1,24R,25-trihydroxyvitamin D3 for the 3.7 S cytosolic receptor specific for 1,25-dihydroxyvitamin D3 in the intestine and thymus. In cytosolic preparations contaminated with the 5 S vitamin D-binding protein, both metabolites are about 7-fold less potent than 1,25-dihydroxyvitamin D3. In contrast, in cytosolic preparations largely free of the 5 S binding protein, both metabolites are equipotent with the parent compound. No evidence was obtained supporting a substantial presence of 23-keto-1,25-dihydroxyvitamin D3 in vivo; nor was the latter compound generated in detectable amounts from 1,25-dihydroxyvitamin D3 by intestinal homogenates. Thus, C-24 oxidation is a significant pathway of intestinal 1,25-dihydroxyvitamin D3 metabolism that produces metabolites with high affinity for the cytosolic receptor which mediates vitamin D action.     

Prognosis of non-Hodgkin's lymphomas in the Dresden district with special reference to combined radio-/chemotherapy

The total population affected with non-Hodgkin's lymphoma and treated by means of radiotherapy or combined radio-chemotherapy between 1960 and 1985 at the Medical Academy Dresden was analysed as to prognosis. 247 patients were classified according to the previous German scheme, 79 were subdivided on the basis of the recommendations laid down in the Kiel classification. The remission rates and survival curves achieved will stand out the comparison with international literature (remission rates of the low malignancy group amounted to 85.3 p.c. and those of the high malignancy group to 80.0 p.c.; the 5-years survival rates of the low malignancy group amounted to 61.9 p.c. and those of the high malignancy group to 41.7 p.c.). The influence of histology, clinical stage and involvement of organs is discussed on the basis of our results and informations obtained in literature. Our analysis confirms the high importance which must be attached to a common radiologic-internal outpatient-department for co-ordinating the diagnostic and therapeutical programme.     

The N-terminal sequence of ribosomal protein L10 from the archaebacterium Halobacterium marismortui and its relationship to eubacterial protein L6 and other ribosomal proteins

The amino-terminal sequence of ribosomal protein L10 from Halobacterium marismortui has been determined up to residue 54, using both a liquid- and a gas-phase sequenator. The two sequences are in good agreement. The protein is clearly homologous to protein HcuL10 from the related strain Halobacterium cutirubrum. Furthermore, a weaker but distinct homology to ribosomal protein L6 from Escherichia coli and Bacillus stearothermophilus can be detected. In addition to 7 identical amino acids in the first 36 residues in all four sequences a number of conservative replacements occurs, of mainly hydrophobic amino acids. In this common region the pattern of conserved amino acids suggests the presence of a beta-alpha fold as it occurs in ribosomal proteins L12 and L30. Furthermore, several potential cases of homology to other ribosomal components of the three ur-kingdoms have been found.     

Ketoconazole inhibits self-induced metabolism of 1,25-dihydroxyvitamin D3 and amplifies 1,25-dihydroxyvitamin D3 receptor up-regulation in rat osteosarcoma cells

Ketoconazole (an inhibitor of vitamin D-24 hydroxylase) was used to study the role of self-induced 1,25-dihydroxyvitamin D3 (1,25-D3) metabolism on cellular responsiveness to 1,25-D3. Eighteen hours of treatment with 1,25-dihydroxy-[26,27-methyl-3H]vitamin D3 (1,25-[3H]D3) increased total 1,25-D3 receptors (VDR) from 60 to 170 fmol mg/protein. In cells treated with both 1,25-[3H]D3 and ketoconazole, up-regulation of VDR was increased by 40% over that observed with cells receiving 1,25-[3H]D3 alone. Ketoconazole alone had no agonistic activity. Treatment of cells with 1 nM 1,25-[3H]D3 plus increasing doses of ketoconazole (0-30 microM) resulted in a dose-dependent increase in occupied VDR and total VDR. This up-regulation was associated with reduced 1,25-[3H]D3 catabolism. 1,25-[3H]D3-induced up-regulation of VDR typically peaked at 14 h and declined thereafter. Ketoconazole lengthened the time to reach peak VDR up-regulation to 20 h. The ability of ketoconazole to increase cell responsiveness (VDR up-regulation) was the result of both increased and prolonged occupancy of VDR by 1,25-[3H]D3. The t1/2 of occupied VDR was 2 h in the absence of ketoconazole and greater than 7 h when ketoconazole was present. Collectively, these results suggested that self-induced catabolism of 1,25-D3 is an important regulator of VDR occupancy and therefore cellular responsiveness to hormone. These data also demonstrate the usefulness of ketoconazole as an inhibitor of vitamin D hydroxylases in intact cells.     

Enzymatic resynthesis of the "reactive site" bond in the modified aprotinin derivatives [seco-15/16]aprotinin and [Di-seco-15/16,39/40]aprotinin

On incubation of [di-seco-15/16,39/40]aprotinin with human plasmin, porcine pancreatic kallikrein or bovine or porcine trypsin in neutral or slightly alkaline solutions [seco-39/40]aprotinin is slowly formed with enzymatic resynthesis of the reactive-site bond 15/16. With chymotrypsin, however, further degradation of [di-seco-15/16,39/40]aprotinin takes place without enzymatic resynthesis. The apparent rate constants for the synthesis of [seco-39/40]aprotinin with kallikrein and trypsin have been determined and indicate that the bond-forming reaction is 10-200-fold slower with [di-seco-15/16,39/40]aprotinin than with [seco-15/16]aprotinin. The newly formed [seco-39/40]aprotinin has similar kinetic constants for the complexation with its cognate enzymes as aprotinin, indicating that any distortion of the secondary binding region due to cleavage of the Arg39-Ala40 bond does not seriously influence binding and affinities.     

Voluntary progression order in captive rhesus macaques

The sequence in which 14 laboratory rhesus macaques left their home enclosure during a routine catching procedure was recorded on 30 occasions during 6 weeks. The animals were trained to voluntarily exit one by one and enter a transport cage for weighing and/or treatment. Mean weekly exit orders cross-correlated significantly, and individuals retained their exit positions with remarkable consistency throughout the study period. An animal's position did not depend on its sex, age, or dominance rank. The oldest female never failed to enter the transport cage as number 1. When she was prevented from taking the lead, the group was hesitant but eventually proceeded in the usual order. Food was a strong, yet not absolute, incentive for the monkeys to cooperate. If they could not expect a reward, they took more time before proceeding, but did so in the habitual order. The predictability of the exit sequence has practical managerial value because it makes it possible for a single person to quickly catch a selected group member when necessary.     

Monitoring and control of biotechnological production processes by Bio-FET-FIA-sensors

Summary Single and multisensor field effect transistors (FET) with a pH-sensitive Si/SiO₂/Si₃N₄/Ta₂O₅-gate and reference electrode (for single sensor) were developed and used for manufacturing the following biological (Bio)-FETs: for glucose analysis, glucose oxidase-FET (GOD-FET); for urea analysis, urease-FET; and for cephalosporin C analysis, cephalosporinase-FET. The GOD-FETs were integrated into flow injection analysis (FIA) of the Eppendorf variables analyser (EVA) system and used for monitoring the glucose concentration in microbial cultivation and production processes with recombinant Escherichia coli K12 MF, recombinant E. coli JM103, Saccharomyces cerevisiae H620, and Candida boidinii. Urease-FET-FIA was used to monitor the urea concentration in a simulated cultivation of Cephalosporium acremonium and urease-FET-FIA and GOD-FET-FIA for the monitoring of urea and glucose concentrations in simulated S. cerevisiae cultivations.     

Linkage data suggesting allelic heterogeneity for paramyotonia congenita and hyperkalemic periodic paralysis on chromosome 17

Summary Paramyotonia congenita (PC), an autosomal dominant non-progressive muscle disorder, is characterised by cold-induced stiffness followed by muscle weakness. The weakness is caused by a dysfunction of the sodium channel in muscle fibre. Parts of the gene coding for the -subunit of the sodium channel of the adult human skeletal muscle (SCN4A) have been localised on chromosome 17. To investigate the role of this gene in the etiology of PC, a linkage analysis in 17 well-defined families was carried out. The results (z=20.61, =0.001) show that the mutant gene responsible for the disorder is indeed tightly linked to the SCN4A gene. The mutation causing hyperkalemic periodic paralysis (HyperPP) with myotonia has previously been mapped to this gene locus by the same candidate gene approach. Thus, our data suggest that PC and HyperPP are caused by allelic mutations at a single locus on chromosome 17.Dedicated to Professor P. E. Becker on the occasion of his 83rd birthday.