Carnitine and lipoate ameliorates lipofuscin accumulation and monoamine oxidase activity in aged rat heart

In this study we have focused on the levels of lipofuscin, monoamine oxidase and cholesterol phospholipid ratio in the heart muscle of young, middle aged and aged rats. In parallel, we have also investigated the levels of carnitine and lipoic acid during aging. We observed an increase in lipofuscin accumulation and monoamine oxidase activity in both middle aged and aged rats. Levels of both carnitine and lipoic acid decreased along with a decrease in cholesterol phospholipid ratio. These changes were normalized upon cosupplementation of carnitine and lipoic acid. Our results thus reveal that carnitine along with lipoic acid can be used as an effective supplement against free radical induced damage to the cardiac tissue.     

Diabetic alteration of cardiac vago-sympathetic modulation assessed with tone-entropy analysis

No clear evidence of diabetic alteration of cardiac sympatho-vagal balance has been reported to date. We assessed heart rate variability of diabetic patients with the tone-entropy analysis that has been published elsewhere (Oida et al. 1997). Tone reflects the cardiac vago-sympathetic balance and entropy the total autonomic efferent activity. Diabetic influence on tone and entropy was examined in two groups of patients (38-52 years and 60-69 year, total 106) stratified according to the occurrence of impaired glucose tolerance or diabetes mellitus. Ten healthy middle-aged volunteers were also examined as a reference. Electrocardiographic data were collected at rest for 10 min. Tone increased and entropy decreased significantly with severity of diabetic disorders. The alterations were depicted as a curvi-linear relation in tone-entropy space, which superimposed adequately on the standard tone-entropy values obtained in a pharmacological experiment. The results demonstrate that the vago-sympathetic balance is altered with diabetic disorders: vagal predominance is impaired significantly in proportion to a withdrawal of total autonomic efferent activity.     

Involvement of hepatocyte growth factor in increased integrin expression on HepG2 cells triggered by adhesion to endothelial cells.

Adhesion of cancer cells to vascular endothelium is an important step in haematogenous metastasis of cancer. A human hepatocellular carcinoma cell line, HepG2, strongly adheres to human umbilical vein endothelial cells (HUVECs) through the interaction of E-selectin and its carbohydrate ligand sialyl Lewis X. In this study, we investigated alteration in integrin expression on HepG2 cells, which follows the selectin-mediated initial adhesion of HepG2 cells to HUVECs. Expression of alpha2beta1 integrin was markedly increased when the HepG2 cells adhered to HUVECs. Among the tested cytokines that are known to be produced by endothelial cells, recombinant hepatocyte growth factor (rHGF) could replace the effect of HUVECs, and a similar increase in integrin expression was observed by the addition of 20 ng ml-1 rHGF to HepG2. The increment of alpha2beta1 integrin expression was significantly inhibited by anti-HGF neutralizing antibody treatment. HepG2 cells expressed alpha2, alpha6, beta1, and beta4 integrin subunits, but expression of integrins other than alpha2beta1 was not affected by the rHGF treatment. The rHGF treatment of HepG2 cells resulted in augmented adhesion to immobilized collagen. This augmentation in adhesion to collagen was completely blocked by the addition of anti-alpha2- or anti-beta1-integrin antibody. In double-chamber chemoinvasion experiments, transmigration of the HepG2 cells through extracellular matrix (ECM) gel was significantly accelerated by co-cultivation with HUVECs. A similar level of enhancement in transmigration activity of the cancer cells was observed by the addition of rHGF. Our interpretation of the results described above is that the cancer cells received stimulation from cytokines, such as HGF, presented by vascular endothelial cells, following the initial adhesion of cancer cells via selectins. This resulted in the secondary increment in the expression of cell adhesion molecules, such as the alpha2beta1 integrin, and led to the augmented adhesive activities of cancer cells towards extracellular matrices at vascular walls. We suggest that this sequence of events is involved in the facilitated migration of some cancer cells to extravascular tissues.     

Graft-versus-Tax response in adult T-cell leukemia patients after hematopoietic stem cell transplantation

Adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type I (HTLV-I) is characterized by poor prognosis after chemotherapy. Recent clinical trials have indicated, however, that allogeneic but not autologous hematopoietic stem cell transplantation (HSCT) for ATL can yield better clinical outcomes. In the present study, we investigated cellular immune responses of ATL patients who obtained complete remission after nonmyeloablative allogeneic peripheral blood HSCT from HLA-identical sibling donors. In the culture of peripheral blood mononuclear cells (PBMCs) from a post-HSCT but not pre-HSCT ATL patient, CD8(+) CTLs proliferated vigorously in response to stimulation with autologous HTLV-I-infected T cells that had been established before HSCT in vitro. These CTLs contained a large number of monospecific CTL population directed to a HLA-A2-restricted HTLV-I Tax 11-19 epitope. The frequency of Tax 11-19-specific CD8+ CTLs in this patient markedly increased also in vivo after HSCT, as determined by staining with HLA-A2/Tax 11-19 tetramers. Similar clonal expansion of HTLV-I Tax-specific CTLs exclusively directed to a HLA-A24-restricted Tax 301-309 epitope was observed in the PBMCs from another ATL patient after HSCT from a HTLV-I-negative donor. Among four post-HSCT ATL patients tested, HTLV-I-specific CTLs were induced in the PBMC culture from three patients but not from the remaining one who had later recurrence of ATL. These observations suggested that reconstituted immunity against antigen presentation in ATL patients after HSCT resulted in strong and selective graft-versus-HTLV-I response, which might contribute to graft-versus-leukemia effects.     

P-selectin-dependent macrophage migration into the tubulointerstitium in unilateral ureteral obstruction

BACKGROUND: Interstitial infiltration of macrophages (M?) is one of the main causal factors for the tubulointerstitial injury. However, precise mechanisms of M? infiltration into tubulointerstitium have not been fully explored. The purposes of this study were to assess the role of selectins in the acute infiltration of M? in rats with unilateral ureteral obstruction (UUO) and to evaluate the role of vasa recta, that is, whether they facilitate massive influx of M? into the interstitium by functioning as specialized vessels. METHODS: To evaluate the role of selectins in M? infiltration into tubulointerstitium, the expression of selectins and L-selectin ligands was examined by immunohistochemistry and immunoelectron microscopy. The functional role of P-selectin in vasa recta was studied by Stamper-Woodruff assay, in vivo p-M? migration assay and in vivo blocking experiments with the monoclonal antibody (mAb) ARP2-4. RESULTS: Selective expression of P-selectin was detected in vasa recta as early as one hour after UUO, and the expression increased thereafter for 96 hours. In contrast, endothelial expression of L-selectin ligands and E-selectin were not detectable. In the Stamper-Woodruff assay on kidney sections of rats with UUO, the adhesion of isolated rat peritoneal M? (p-M?) to vasa recta was significantly inhibited by the mAb ARP2-4 (P-selectin blocker; P < 0.01), but not by mAb ARE-5 (E-selectin blocker) or rLECIg (rat L-selectin chimeric protein). In the in vivo transfer experiments with fluorescein-labeled p-M? into rats 48 hours after UUO, labeled p-M? had accumulated around vasa recta at three minutes and had infiltrated predominantly into the outer medulla at 180 minutes. The number of labeled p-M? was reduced when the rats were pretreated with ARP2-4 (P < 0.01). Finally, ARP2-4 (10 mg/kg), injected 15 minutes before UUO, reduced the number of infiltrated M? (P < 0.01). CONCLUSION: The results suggest that vasa recta, which express P-selectin, contribute to massive infiltration of M? into the interstitium by functioning as specialized post-capillary venules.     

Human erythroleukemia cell line (HEL) undergoes a drastic macrophage- like shift with TPA

We investigated the effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the human erythroleukemia cell line, HEL, and found that TPA addition (10(-6)-10(-8) M) to HEL cell cultures induces morphological, functional, and biochemical changes in HEL cells that are characteristic for macrophage-like cells. Apart from the drastic changes in morphology, the cells greatly enhance their phagocytic ability and acquire receptors for binding and degradation of chemically modified lipoproteins. At the biochemical level, a newly synthesized 85K glycoprotein is observed, and the cells are unresponsive to inducers of globin synthesis. Comparative observations with K562 cells indicate that TPA inhibits, as in HEL cells, spontaneous and induced globin synthesis, but induces minimal macrophage-like properties in these cells. The results with HEL cells are interpreted to indicate that TPA uncovers a latent monocyte-like phenotype in these cells.     

Oxidative stress-mediated macromolecular damage and dwindle in antioxidant status in aged rat brain regions: role of L-carnitine and DL-alpha-lipoic acid

BACKGROUND: The free radical theory of aging has significant relevance in a number of age-related neurological disorders. Too many free radicals create cellular pollution that shuts down energy levels. They have also been implicated in the loss of physiological functioning associated with the aging of post mitotic cells such as the brain. The activities of enzymatic antioxidative defenses decrease in rat brain may be possible causes of age-associated increase in oxidative damage to macromolecules. METHODS: We determined whether DL-alpha-lipoic acid (100 mg/kg body weight/day), and L-carnitine (300 mg/kg body weight/day) together when administered for 30 days declines the rate of oxidative stress-mediated macromolecular damages such as lipid peroxidation (LPO), protein carbonyl (PCO) and DNA protein cross-links in different anatomic regions (cortex, striatum and hippocampus). The activities of antioxidant enzymes in programmed aging were evaluated in the cortex, striatum and hippocampus of young and aged rat brain regions. RESULTS: Aged rats elicited a significant decline in the antioxidant status and increase in LPO, PCO and DNA protein cross-links as compared to young rats in all the 3 brain regions. The increase in LPO, PCO and DNA protein cross-links were (35.8%, 35.6%, 43.5%) in cortex, (32.5%, 40.3%, 29.8%) in striatum and (62.7%, 42.4%, 34.9%) in hippocampus, respectively, in aged rats as compared to young rats. Co-supplementation of carnitine and lipoic acid was found to be effective in reducing brain regional LPO, PCO and DNA protein cross-links and in increasing the activities of enzymatic antioxidants in aged rats to near normalcy. CONCLUSION: The combination of l-carnitine and lipoic acid overcame the oxidative stress induced rate of lipid peroxidation, protein carbonyl formation, accumulation of DNA protein cross-links and deficits in antioxidant enzyme activities in various brain regions of aged rats.     

An efficient tool for surveying CRF01_AE HIV type 1 resistance in Thailand to combined stavudine-lamivudine-nevirapine treatment: mutagenically separated PCR targeting M184I/V

Under programs organized by the government of Thailand, HIV-1-infected patients have been treated since 2002 with several regimens, including a tablet known as GPOvir, which contains lamivudine, stavudine, and nevirapine. The aim of this study was to establish an effective assay, based on mutagenically separated PCR (MS-PCR), with the goal of surveying GPOvir-resistant HIV-1 cases. To determine the target mutation point for the assay, we analyzed the patterns of acquired drug resistance in plasma samples from GPOvir-failed cases. Of 428 HIV-1-infected individuals treated with GPOvir at Lampang Hospital in northern Thailand from 2002 to 2004, 66 had detectable viral loads after 3 months of treatment. The HIV-1 sequences of these 66 GPOvir-failed cases and 55 pre-GPOvir baseline samples were analyzed. The most prevalent drug resistance mutation among the samples was the lamivudine resistance M184I/V mutation. Based on this finding, we developed a new MS-PCR assay to detect the M184I/V mutation, and evaluated the assay performance for detecting GPOvir-resistant CRF01_AE cases. Comparing the results of M184I/V MS-PCR and sequence analyses, we found a concordance rate of 95% and an overall sensitivity of the M184I/V MS-PCR for detecting GPOvir-resistant cases of 79%. Considering the relatively low price of the assay, approximately $12.50 per sample, M184I/V MS-PCR may be a candidate for monitoring a large number of GPOvir-treated patients, particularly in developing nations.