Antitumor immune response by CX3CL1 fractalkine gene transfer depends on both NK and T cells

The CX3C chemokine fractalkine (CX3CL1) exists as both a membrane-bound form promoting firm cell-cell adhesion and a soluble form chemoattracting leukocytes expressing its receptor CX3CR1. When adenoviral vector expressing mouse fractalkine (AdFKN) was transduced to the tumor cells, fractalkine was expressed as both membrane-bound form on the tumor cells and soluble form in the supernatant in vitro. Intratumoral injection of AdFKN (1 x 10(9)PFU/tumor) into C26 and B16F10 tumors resulted in marked reduction of tumor growth compared to control (C26: 86.5%, p<0.001; B16F10: 85.5%, p<0.001). Histological examination of tumor tissues revealed abundant infiltration of NK cells, dendritic cells, and CD8(+) T lymphocytes 3 and/or 6 days after treatment with AdFKN. Splenocytes from mice treated by AdFKN developed tumor-specific cytotoxic T cells, and thereby protected from rechallenging with parental tumor cells. Antitumor effects by AdFKN were completely abrogated in both NK cell-depleted mice and CD8(-/-) mice, and partially blocked in CD4(-/-) mice. These data indicated that fractalkine mediates antitumor effects by both NK cell-dependent and T cell-dependent mechanisms. This study suggests that fractalkine can be a suitable candidate for immunogene therapy of cancer because fractalkine induces both innate and adaptive immunity.     

Development of nested PCR based on the ViaB sequence to detect Salmonella typhi.

For a rapid diagnosis of typhoid fever, we developed a nested PCR based on the nucleotide sequence encoding the Vi antigen. All Salmonella typhi strains along with a Salmonella paratyphi C strain were PCR positive. This assay was able to detect S. typhi at the single-cell level.     

Critical spectral regions for vowel identification

The first two formant frequencies (F1 and F2) are the cues important for vowel identification. In the categorization of the naturally spoken vowels, however, there are overlaps among the vowels in the F1 and F2 plane. The fundamental frequency (F0), the third formant frequency (F3) and the spectral envelope have been proposed as additional cues. In the present study, to investigate the spectral regions essential for the vowel identification, untrained subjects performed the forced-choice identification task in response to Japanese isolated vowels (/a, o, u, e, i/), in which some spectral regions were deleted. Minimum spectral regions needed for correct vowel identification were the two regions including F1 and F2 (the first and fourth in the quadrisected F1-F2 regions in Bark scale). This was true even when phonetically different vowels had a similar combination of F1 and F2 frequency components. F0 and F3 cues were not necessarily needed. It is concluded that the relative importance in the spectral region is not equivalent, but weighted on the two critical spectral regions. The auditory system may identify the vowels by analyzing the information of the spectral shapes and the formant frequencies (F1 and F2) in these critical spectral regions.     

Immunocytochemical characteristics of human osteosarcoma cell line HS-Os-1: possible implication in apoptotic resistance against irradiation

In our previous study, we examined reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy irradiation. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of the hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation. In the succeeding study, we examined radiation-induced ROS formation, oxidative DNA damage, early apoptotic changes, and mitochondrial membrane dysfunction in the human osteosarcoma cell line HS-Os-1. We found that ROS formation and oxidative DNA damage were actually scarcely seen after irradiation of up to 30 Gy in these cells, that mitochondrial membrane potential was preserved, and that apoptotic changes were not demonstrated despite the relatively high-dose irradiation of 30 Gy. In the present study, we examined the immunocytochemical characteristics of the apoptotic-resistance of the HS-Os-1 cell line against irradiation in order to clarify its possible implications regarding radiosensitivity. The results showed that these cells lack P53 and Bax protein expression, and strong peroxidase activity was confirmed in the nuclei of the cells. Moreover, SODII (manganese superoxide dismutase II) protein expression was gradually increased in spite of irradiation of up to 30 Gy. Therefore, it is concluded that HS-Os-1 cells are originally apoptotic-resistant and that the cells possess a strong ability to scavenge for free radicals. To convert these cells to a state of apoptotic-susceptibility, a powerful oxidant such as hydrogen peroxide might exert such an effect in terms of the production of hydroxyl radicals in lysosomes in the cells as shown in our previous studies. The origin of the radioresistance of the human osteosarcoma cell line HS-Os-1 is considered to to be low degree of ROS formation following irradiation, reflecting the strong scavenging ability of these cells for free radicals including hydroxyl radicals.     

The adaptation of the plaque reduction assay for measuring specific cytotoxic cells in limiting dilution cultures

The use of the original haemolytic plaque reduction technique to measure cytotoxic T lymphocytes (CTL) has been developed further as a rapid screening assay, particularly suitable for limiting dilution analyses. Using hybridoma cells as targets, the cytotoxicity has been measured by the loss of haemolytic plaque formation and by the reduction of the amount of haemolytic monoclonal antibody secreted from viable target cells into the assay supernatants. The assessment of large numbers of cytotoxic samples has been greatly facilitated by quantitating the amount of haemoglobin released in the assay with an automated microELISA multiscanner and by scoring visually using a modification of the spot test. Using these new techniques, relatively high frequency estimates of cytotoxic cell precursors in an allogeneic response (1 in 462 spleen cells) and an anti-fluorescein response (1 in 3970 spleen cells) were obtained.     

Mutation analysis of the calpastatin gene (CAST) in patients with Alzheimer's disease

The calpains, a family of calcium-dependent cysteine proteinases, and calpastatin, their endogenous inhibitor protein, are involved in the proteolysis of amyloid precursor protein, which is thought to be abnormal in patients with Alzheimer's disease (AD). Specific inhibitors of calpains attenuate amyloid beta peptide-induced neuronal death. We hypothesized that some AD patients have functionally deficient mutation(s) of the CAST gene encoding calpastatin, and we screened 40 Japanese patients with AD for mutations in the coding region of CAST. Nine polymorphisms, -82A/G, IVS7-96A/G, 669A/G, 1223C/G (Ser408Cys), IVS20-10C/T, IVS21-65G/A, IVS22+31T/C, IVS24+38Ins/DelA, and IVS25-32A/G, were identified. The 669A allele causes skipping of exon 11, leading to the loss of 13 residues. Comparisons between 101 patients and 90 controls revealed no significant association between CAST polymorphisms and risk for AD, indicating that genomic variations of CAST are not likely to be substantially involved in the etiology of AD.     

Relationships between muscle lactate accumulation and surface EMG activities during isokinetic contractions in man

Summary In order to investigate the relationship between metabolic state and myoelectrical activity in working muscle during short term intense exercise, eleven healthy males performed isokinetic knee extensions at an angular velocity of 180 deg · sec^–1 for 30 and 60 s. The median frequency (MF) of the surface electromyogram (EMG) recorded from vastus lateralis was decreased while the time lag of torque production after the onset of electrical activity (EMD) was increased during exercise. These changes (MF and EMD) corresponded well to muscle lactate accumulation in the same muscle. Over the exercise period, the integrated EMG/knee extension peak torque ratio (E/T ratio) was increased, which indicated a decrease in the efficiency of electrical activity. It was concluded that the changes in the frequency components of the EMG and in the contractile property of the muscle during short term intense exercise correlated with lactate accumulation in the identical muscle, and that the decrease in efficiency of the electrical activity in the muscle suggested peripheral fatigue.     

Comparison of various bone marrow fractions in the ability to participate in vascular remodeling after mechanical injury

In contrast to conventional assumption, recent reports propose the possibility that hematopoietic stem cells (HSCs) may have broader potential to differentiate into various cell types. Here, we tested the pluripotency of HSCs by comparing vascular lesions induced by mechanical injury after bone marrow reconstitution with total bone marrow (TBM) cells, c-Kit+ Sca-1+ Lin- (KSL) cells, or a single HSC cell (Tip-SP CD34-KSL cell, CD34- c-Kit+ Sca-1+ Lin- cell with the strongest dye-efflux activity) harboring green fluorescent protein (GFP). The lesions contained a significant number of GFP-positive cells in the TBM and KSL groups, whereas GFP-positive cells were rarely detected in the HSC group. These results suggest that transdifferentiation of a highly purified HSC seems to be a rare event, if it occurs at all, whereas bone marrow cells including the KSL fraction can give rise to vascular cells that substantially contribute to repair or lesion formation after mechanical injury.     

Tissue regeneration using macrophage migration inhibitory factor-impregnated gelatin microbeads in cutaneous wounds

Migration inhibitory factor (MIF) responds to tissue damage and regulates inflammatory and immunological processes. To elucidate the function of MIF in cutaneous wound healing, we analyzed MIF knockout (KO) mice. After the excision of wounds from the dorsal skin of MIF KO and wild-type (WT) mice, healing was significantly delayed in MIF KO mice compared to WT mice. Lipopolysaccharide treatment significantly increased [(3)H]thymidine uptake in WT mouse fibroblasts compared to MIF KO mouse fibroblasts. Furthermore, there was a significant reduction in fibroblast and keratinocyte migration observed in MIF KO mice after 1-oleoyl-2-lysophosphatidic acid treatment. We subsequently examined whether MIF-impregnated gelatin slow-release microbeads could accelerate skin wound healing. Injection of more than 1.5 microg/500 microl of MIF-impregnated gelatin microbeads around a wound edge accelerated wound healing compared to a single MIF injection without the use of microbeads. MIF-impregnated gelatin microbeads also accelerated skin wound healing in C57BL/6 mice and diabetic db/db mice. Furthermore, incorporating MIF-impregnated gelatin microbeads into an artificial dermis implanted into MIF KO mice accelerated procollagen production and capillary formation. These findings suggest that MIF is crucial in accelerating cutaneous wound healing and that MIF-impregnated gelatin microbeads represent a promising treatment to facilitate skin wound healing.     

Microscopic carcinoma of the breast--report of a case

Microscopic cancer is extremely rare even among "occult breast cancers". We treated a 70 year old woman with complaints of serosanguineous nipple discharge which yielded cytologically Class V cancer cells. Microdochectomy was performed and intraductal carcinoma was verified histologically in serially cut sections. Following a simple mastectomy, she has been well with no evidence of recurrence or metastasis, at this time.