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MATERIALS AND METHODS Construction of Expression Vectors for IL-2, TGF-,Bl, and IL-4. The cDNAs for human IL-2 (ATCC 67618), TGF-f1 (ATCC 59954), and mouse IL-4 (ATCC 37561) were subcloned into the vector pBSII SK to generate 5' HindIII and 3' Sma I sites. These were used to replace the HindIH/

We isolated immunoglobulin (Ig) VH4 genes that were rearranged in the genomic DNA of 160 day human fetal spleen. Productively rearranged VH 4-21 genes were cloned into pRTM1, a human IgM expression vector. This allowed us to generate IgM kappa-expressing transfectomas by co-transfecting each of these constructs with pSVG-V kappa 3, an Ig kappa light-chain expression vector that has a variable region encoded Humkv325, a conserved V kappa gene that is frequently expressed early B cell ontogeny. We find that all transfectomas expressing IgM kappa encoded by VH 4-21 make IgM autoantibodies reactive with i, a linear poly-N-acetyllactosamine determinant present on neonatal red blood cells and a B cell-restricted isoform of the CD45 surface molecule. In contrast, a transfectoma expressing pSVG-V kappa 3 and pRTM1 containing a rearranged VH4-59 (V71-4) gene isolated from a chronic lymphocytic leukemia B cell population, designated WIL, produced IgM kappa antibodies that had no detectable anti-i binding activity. However, transfectomas expressing VH 4-21 fused onto the Ig heavy-chain third complementarity determining region (CDR3) of WIL are found to make anti-B cell autoantibodies with anti-i activity. These studies indicate that VH 4-21 genes rearranged in human fetal B cell ontogeny can encode anti-B cell autoantibodies with a binding specificity that does not require in vivo somatic selection.

The underlying mechanism of calorie restriction (CR) extension of mammalian life spans operates by altering the rate of decline in reserve capacity (with time) as well as the exposure to growth stimulus, two mechanisms that seem to be related to the central genetically determined mechanism that controls mammalian life span over a 50-fold range. While genetic control is principally exerted at the level of metabolic rate and entrained protective defenses, CR appears to alter the rate of decline in reserve capacity and the exposure to growth stimulus without appreciable alteration of metabolic rate. CR accomplishes this by lowering the nutritionally driven level of insulin exposure, which in turn lowers overall growth factor exposure, improves age-declining maintenance of mitochondrial maximal function, and maintains a longer-term favorable balance of the insulimgrowth hormone antagonism. Obtaining the ‘halved’ insulin exposure in calorie-restricted animals (relative to ad libitum fed) can be specifically targeted in non-obese ad libitum fed humans by multiple techniques, a situation that may confer most of the life span extension of CR without restricting calories. The prospect for even further extension of the human life span is considered.

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Background: Absence of a widely agreed upon central paradigm for mammalian aging. Objective: Detailed elaboration of a proposed mammalian aging paradigm. Methods: Elaboration of a new theoretical model. Results: Hormonal imbalance-growth factor exposure theory (HI-GFE theory) can account for two major aging phenomena: (1) decline in mammalian ‘reserve capacity’ and consequent rise of diseases of maintenance, and (2) rise then peaking of most age-associated proliferative diseases. Reserve capacity decline via gradual decline in mitochondrial maximal energy production (state 3) accounts for the gradual redirection of declined maximal energy production toward survival functions like ion pumping to the relative detriment of RNA and protein synthesis as seen in lesser synthetic rates and slower turnover with consequent gradual cellular impairment. Developmental program triggered, and over-ample nutritionally driven, growth factor exposure in youth to middle age encourages promotional events that lead to proliferative diseases that rise coincident to rapidly declining reserve capacity and cumulative increased mutational status of age. Conclusions: Declining mitochondrial state 3 aging energy production status is easily and safely reversible with probable consequences of greatly postponing the decline in overall ‘reserve capacity’ which may also improve insulin: growth hormone balance and result in lower overall growth factor exposure and consequent longer healthy life of a potentially greater magnitude increase in life spans than that seen in calorie-restricted animals.

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