CD86 (B7-2) can function to drive MHC-restricted antigen-specific CTL responses in vivo.

Activation of T cells requires both TCR-specific ligation by direct contact with peptide Ag-MHC complexes and coligation of the B7 family of ligands through CD28/CTLA-4 on the T cell surface. We recently reported that coadministration of CD86 cDNA along with DNA encoding HIV-1 Ags i.m. dramatically increased Ag-specific CTL responses. We investigated whether the bone marrow-derived professional APCs or muscle cells were responsible for the enhancement of CTL responses following CD86 coadministration. Accordingly, we analyzed CTL induction in bone marrow chimeras. These chimeras are capable of generating functional viral-specific CTLs against vaccinia virus and therefore represent a useful model system to study APC/T cell function in vivo. In vaccinated chimeras, we observed that only CD86 + Ag + MHC class I results in 1) detectable CTLs following in vitro restimulation, 2) detectable direct CTLs, 3) enhanced IFN-gamma production in an Ag-specific manner, and 4) dramatic tissue invasion of T cells. These results support that CD86 plays a central role in CTL induction in vivo, enabling non-bone marrow-derived cells to prime CTLs, a property previously associated solely with bone marrow-derived APCs.

Engineering DNA vaccines via co-delivery of co-stimulatory molecule genes.

DNA immunization has been investigated as a potential immunization strategy against infectious diseases and cancer. To enhance a DNA vaccine's ability to induce CTL response in vivo, we co-administered CD80 and CD86 expression cassettes along with HIV-1 immunogens. This manipulation resulted in a dramatic increase in MHC class I-restricted and CD8+ T-cell-dependent CTL responses in both mice and chimpanzees. This strategy of engineering vaccine producing cells to be more efficient T-cell activators could be an important tool for optimizing antigen-specific T-cell-mediated immune responses in the pursuit of more rationally designed vaccines and immune therapies.

Coadministration of IL-12 or IL-10 expression cassettes drives immune responses toward a Th1 phenotype.

Cytokines are important regulators of the immune response. They influence immune expression, the development of immunologic memory, and regulation of antigen-specific and nonspecific immune activation as well as allergic responses. In a model system in mice, we have studied the effect of plasmids expressing interleukin (IL)-10 or IL-12 on the modulation of antigen-specific responses. Coadministration of IL-12 or IL-10 genes with DNA immunogens directed the antigen-specific immune response toward a T helper (Th1)-type immunity. In addition to the modulation of antigen-specific immune responses, we studied the induction of delayed-type hypersensitivity (DTH) to contact allergens as an in vivo model of the Th1 response. We found that IL-12 and IL-10 gene-containing plasmids, and not the bacterial plasmid alone, upregulate this response. Our cytokine gene delivery technique demonstrates an important level of control of the magnitude and direction of induced immune responses and could be advantageous in a wide variety of immunotherapeutic strategies.

Ornithine decarboxylase in human brain: influence of aging, regional distribution, and Alzheimer's disease.

Although experimental animal data have implicated ornithine decarboxylase, a key regulatory enzyme of polyamine biosynthesis, in brain development and function, little information is available on this enzyme in normal or abnormal human brain. We examined the influence, in autopsied human brain, of postnatal development and aging, regional distribution, and Alzheimer's disease on the activity of ornithine decarboxylase. Consistent with animal data, human brain ornithine decarboxylase activity was highest in the perinatal period, declining sharply (by approximately 60%) during the first year of life to values that remained generally unchanged up to senescence. In adult brain, a moderately heterogeneous regional distribution of enzyme activity was observed, with high levels in the thalamus and occipital cortex and low levels in cerebellar cortex and putamen. In the Alzheimer's disease group, mean ornithine decarboxylase activity was significantly increased in the temporal cortex (+76%), reduced in occipital cortex (-70%), and unchanged in hippocampus and putamen. In contrast, brain enzyme activity was normal in patients with the neurodegenerative disorder spinocerebellar ataxia type I. Our demonstration of ornithine decarboxylase activity in neonatal and adult human brain suggests roles for ornithine decarboxylase in both developing and mature brain function, and we provide further evidence for the involvement of abnormal polyamine system activity in Alzheimer's disease.

Brain S-adenosylmethionine levels are severely decreased in Alzheimer's disease.

S-Adenosylmethionine is an essential ubiquitous metabolite central to many biochemical pathways, including transmethylation and polyamine biosynthesis. Reduced CSF S-adenosylmethionine levels in Alzheimer's disease have been reported; however, no information is available regarding the status of S-adenosylmethionine or S-adenosylmethionine-dependent methylation in the brain of patients with this disorder. S-Adenosylmethionine concentrations were measured in postmortem brain of 11 patients with Alzheimer's disease. We found decreased levels of S-adenosylmethionine (-67 to -85%) and its demethylated product S-adenosylhomocysteine (-56 to -79%) in all brain areas examined (cerebral cortical subdivisions, hippocampus, and putamen) as compared with matched controls (n = 14). S-Adenosylmethionine and S-adenosylhomocysteine levels were normal in occipital cortex of patients with idiopathic Parkinson's disease (n = 10), suggesting that the decreased S-adenosylmethionine levels in Alzheimer's disease are not simply a consequence of a chronic, neurodegenerative condition. Reduced S-adenosylmethionine levels could be due to excessive utilization in polyamine biosynthesis. The severe reduction in levels of this essential biochemical substrate would be expected to compromise seriously metabolism and brain function in patients with Alzheimer's disease and may provide the basis for the observations of improved cognition in some Alzheimer's patients following S-adenosylmethionine therapy.

Brain polyamine levels are altered in Alzheimer's disease.

Despite considerable evidence implicating polyamines in CNS function, little is known about the status of the polyamine system in normal or abnormal human brain. We measured the levels of the polyamines spermidine, spermine and their precursor putrescine, in cortical and subcortical areas of 12 patients with Alzheimer's disease (AD). As compared with the controls, mean levels of spermidine were markedly and significantly increased (70%) whereas putrescine levels were decreased (28%) in temporal cortex of the AD patients. No other statistically significant changes were observed with the exception of a mean 35% reduction in spermine concentration in occipital cortex. In view of the modulatory effects of polyamines on calcium flux and glutamate receptor function, our data suggest that abnormal polyamine system activity may be involved in the neurodegenerative processes occurring in brain of patients with AD.

Polyamines in human brain: regional distribution and influence of aging.

Although much evidence has implicated polyamines in brain development and function, little information is available on these substances in human brain. We examined the influence of regional distribution and aging on putrescine, spermidine, and spermine levels in autopsied human brain. In the adult brain, concentrations of spermidine were the highest, followed by spermine and putrescine. All three polyamines showed a distinct and uneven distribution profile among the 10 examined brain areas. Spermidine levels were especially high in white matter and thalamus (20 and 9.3 nmol/mg of protein, respectively), whereas spermine concentrations were highest in cerebellar cortex (3.4 nmol/mg of protein). High levels of putrescine were observed in cerebral cortices, putamen, and hippocampus (0.7-1.2 nmol/mg or protein), with lowest levels in cerebellum and thalamus (0.3-0.5 nmol/mg of protein). No statistically significant influence of aging (1 day to 103 years; n = 57) on either putrescine or spermine levels in occipital cortex was observed. In contrast, spermidine levels increased markedly from birth, reaching maximal levels at approximately 40 years of age (+228% increase in the mean 41-year-old group vs. 6-week-old group), which were maintained up to senescence. These observations in human brain thus differ from those reported in the rodent, in which levels of all three polyamines show a pronounced postnatal reduction. Our data support the notion that polyamines may have roles in both postnatal brain development and in mature brain function.

The posterior cruciate ligament in total knee surgery. Save, sacrifice, or substitute?

To determine the role of the posterior cruciate ligament in total knee arthroplasty, 242 consecutive primary total knee arthroplasties were included in 1 of 3 sequential groups. Group I included 77 Press Fit Condylar total knee replacements in which the posterior cruciate ligament was completely released from its tibial attachment. In Group II, there were 80 Press Fit Condylar total knee replacements in which the posterior cruciate ligament was retained. Group III consisted of 85 total knee replacements with a posterior cruciate-substituting device (Insall-Burstein II). All patients were observed at least 2 years and evaluated by the Knee Society's Clinical and Functional Scoring System, including a radiographic evaluation. No differences were found between the posterior cruciate ligament sacrificed group and the posterior cruciate ligament preserved group. The 1 significant difference among the 3 groups was in range of motion (ROM). Groups I and II averaged 103 degrees and 104 degrees motion respectively, whereas Group III, the posterior cruciate-substituting group, averaged 112 degrees (p = 0.001). In addition, only in Group III was the lower 95% confidence limit of the mean ROM > 90 degrees. These findings suggest that preserving the posterior cruciate ligament does not consistently lead to improved functional ROM. The posterior cruciate ligament-substituting device historically has demonstrated excellent survivorship and appears to offer greater ROM.

Activity of S-adenosylmethionine decarboxylase, a key regulatory enzyme in polyamine biosynthesis, is increased in epileptogenic human cortex.

OBJECTIVE: We measured the activity of S-adenosylmethionine decarboxylase, a key regulatory enzyme of polyamine biosynthesis, in the temporal cortex of patients with epilepsy. DESIGN: Cortical surgical specimens were obtained following anterior temporal lobe resection for intractable epilepsy. Enzyme activity was compared in nonepileptogenic (n = 16) and epileptogenic (spontaneously discharging; n = 19) regions. RESULTS: Mean enzyme activity was increased by 44% in samples from epileptogenic cortex compared with samples from nonepileptic regions. The S-adenosylmethionine decarboxylase activity in regions of focal epileptogenic discharges was also increased in five patients compared with paired samples from the nonepileptogenic portion of the same gyrus (+55%). CONCLUSIONS: Elevated activity of S-adenosylmethionine decarboxylase in regions of active epileptogenic cortical discharges suggests that a disturbance of the polyamine system may be involved in the maintenance of hypersynchronous discharges, perhaps through a modulatory action at the excitatory N-methyl-D-aspartate-preferring glutamate receptor.

S-adenosylmethionine decarboxylase in human brain. Regional distribution and influence of aging.

Recent experimental animal studies have implicated brain polyamines as having roles in both brain development and human brain neurodegenerative conditions. In order to provide baseline information, in normal human brain, on one of the key polyamine synthesising enzymes, S-adenosylmethionine decarboxylase (SAMDC), we examined the sensitivity of this enzyme to various cofactors/inhibitors, its regional distribution, and influence of aging in neurologically normal autopsied human brain. SAMDC in normal human brain is similar to that reported in other mammalian cells with regard to substrate affinity (Km = 39 microM), marked sensitivity to putrescine activation (+600%), inhibition (methylglyoxalbisguanidine and MDL 73811), and pH optimum (7.2). There was an uneven distribution of enzyme activity in human brain, and of the 12 brain regions examined, the highest activity was observed in occipital, parietal, frontal and temporal cortices (36-58 pmol/h/mg protein); intermediate activity in cerebellar and insular cortex, pulvinar thalamus, caudate and putamen (12-27 pmol/h/mg protein); and lowest activity in medial-dorsal thalamus, lateral globus pallidus and white matter (< 11 pmol/h/mg protein). The influence of aging (1 day to 103 years) on SAMDC activity in occipital cortex, the region showing the highest activity in human brain (n = 59) was also determined. Enzyme activity increased by approximately 600% from age 6 months to near maximal levels at age 10 years, then remained generally unchanged up to 103 years. Since SAMDC is a key regulatory enzyme in the synthesis of spermidine and spermine, the marked increase in SAMDC activity in the neonate and the sustained high enzyme levels throughout adulthood, imply a role for these polyamines in both development and mature brain function.

Prostaglandin H synthase-dependent formation of the direct-acting mutagen 2-nitro-3-methylimidazo[4,5-f]quinoline (nitro-IQ) from IQ.

The mutagenic effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) following activation by ram seminal vesicle microsomes (RSVM, a source of prostaglandin H synthase, PHS) were studied in Salmonella typhimurium tester strains possessing elevated levels of acetyl-CoA: arylamine N-acetyltransferase (NAT). The metabolites formed by RSVM were extracted and fractionated by high pressure liquid chromatography (HPLC). One isolable product accounted for most of the direct-acting mutagenicity observed in the extracts. The metabolite was identified as 2-nitro-3-methylimidazo[4,5-f]quinoline (nitro-IQ). Since nitro-IQ is a potent direct-acting mutagen, its role in IQ genotoxicity warrants further study.

Brain S-adenosylmethionine decarboxylase activity is increased in Alzheimer's disease.

We measured the activity of S-adenosylmethionine decarboxylase (SAMDC), a key regulatory enzyme of polyamine biosynthesis, in autopsied brain from 13 patients with Alzheimer's Disease (AD). As compared with the controls, mean enzyme activity was increased by 37-96% in all seven examined brain regions with statistically significant increases in temporal cortex (+96%), frontal cortex (+69%) and hippocampus (+90%). The elevated SAMDC may have occurred as part of a generalized polyamine response to brain injury, which has been previously described in experimental animal conditions. Above-normal SAMDC activity implies increased levels/metabolism of spermidine and spermine, two polyamines which are involved in neuronal regeneration, growth factor production, and activation of excitatory N-methyl-D-aspartate preferring glutamate receptors. Our data suggest the involvement of the polyamine system in the brain reparative and/or pathogenetic mechanisms of AD.

Salmonella typhimurium strains expressing human arylamine N-acetyltransferases: metabolism and mutagenic activation of aromatic amines.

Epidemiological studies have established the carcinogenic risk of occupational exposure to aromatic amines such as benzidine, beta-naphthylamine, and 4-aminobiphenyl. Metabolic activation of these chemicals to reactive, genotoxic electrophiles, via enzymatic N-oxidation and subsequent conjugation reactions, is necessary for their carcinogenic potential to be realized. Many aromatic amines are mutagenic in prokaryotic test systems, in the presence of exogenous mammalian activating enzymes such as those contained in hepatic 9000 x g supernatant. However, in the Ames (Salmonella typhimurium) assay, induction of mutations by aromatic amines and nitroarenes is also almost completely dependent upon the activity of the endogenous bacterial enzyme, N-acetyltransferase/O-acetyltransferase. The relevance of this assay to the prediction of the carcinogenic potential of aromatic amines in humans is thus restricted by the likelihood that the bacterial and human enzymes possess different substrate specificities. In this paper we report the construction and use of new tester strains of S. typhimurium that express high levels of functional human arylamine N-acetyltransferases, NAT1 and NAT2, retaining characteristic arylamine substrate specificities that are distinct from those of the bacterial enzyme. These new strains support the mutagenic activation of benzidine, 2-aminofluorene and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in the Ames test and may provide a new tool for evaluating the carcinogenic potential of aromatic amines.