Production of healthy cloned mice from bodies frozen at -20 degrees C for 16 years

Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the “resurrection” of frozen extinct species (such as the woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at −20 °C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to “resurrect” animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation.     

Elevation of serum KL-6 levels in 3 patients with rheumatoid arthritis treated with adalimumab

We describe 3 cases of rheumatoid arthritis presenting with elevated serum KL-6 levels during treatment with adalimumab, which was discontinued because of suspected onset of complications. However, no complications were observed following discontinuation despite comprehensive assessments, and KL-6 levels subsequently returned to baseline levels. In our institutes, 3 out of 29 cases treated with adalimumab showed elevated KL-6 levels. The baseline levels were 445, 347, and 547 U/ml, while the peak levels were 1010, 546, and 2007 U/ml, respectively. The elevated KL-6 levels seem to have been innocuous; nevertheless, further careful observation is deemed necessary.     

Hydration characteristics of pathologic stratum corneum--evaluation of bound water

We measured in vitro the hygroscopicity and bound (non-freezing) water of various samples of pathologic horny layer obtained from the lesions of senile xerotic skin and psoriasis vulgaris and the normal horny layer from glabrous skin and plantar horny layer. The amount of water taken up by pathologic stratum corneum was much smaller than that by normal horny layer in an environment at a high relative humidity (RH). Tightly bound primary water to stratum corneum measured by Karl Fischer's method was about 5 mg/100 mg of dry stratum corneum in all the samples studied, while less tightly bound secondary water was much smaller in amount in pathologic stratum corneum than in the controls, i.e., 31.7 mg/100 mg dry scale from senile xerosis and 27.2 mg/100 mg dry psoriatic scale as compared with 38.2 mg/100 mg dry normal stratum corneum from glabrous skin and 37.3 mg/100 mg dry normal plantar stratum corneum. We believe that the low hygroscopicity of the pathologic stratum corneum is due to this smaller capacity for secondary bound water, which is responsible for the development of a dry scaly appearance even at high RH.     

Establishment and characterization of a clear-cell sarcoma (malignant melanoma of soft parts) cell line

A clear cell sarcoma (CCS) cell line, designated as NCS-1, was established in monolayer culture from a xenograft line originating from a metastatic CCS. Marked karyotypic aberrations and tumorigenicity in nude mice revealed the malignant derivation of the NCS-1 cell line. These cells contained abundant glycogen and were amelanotic by light microscopy. By electron microscopy, however, melanosomes in various developmental stages were seen, and some of them were partially melanized. The electron microscopic dopa reaction revealed the presence of tyrosinase activity. Enzyme-linked immunoadsorbent assay revealed that NCS-1 cells expressed a 75-kDa glycoprotein which was identified as a marker of highly differentiated melanoma cells. From these results, NCS-1 cells were found to retain both cytochemical and morphological properties of CCS. Application of NCS-1 cells to a panel of monoclonal antibodies recognizing melanocytic differentiation antigens showed that they corresponded approximately to highly differentiated melanoma cells. In conclusion, the present study strongly supports the close relationship between CCS and malignant melanoma.     

Genome wide association study of HTLV-1–associated myelopathy/tropical spastic paraparesis in the Japanese population

HTLV-1–associated myelopathy (HAM/TSP) is a chronic and progressive inflammatory disease of the central nervous system. The aim of our study was to identify genetic determinants related to the onset of HAM/TSP in the Japanese population. We conducted a genome-wide association study comprising 753 HAM/TSP patients and 899 asymptomatic HTLV-1 carriers. We also performed comprehensive genotyping of HLA-A, -B, -C, -DPB1, -DQB1, and -DRB1 genes using next-generation sequencing technology for 651 HAM/TSP patients and 804 carriers. A strong association was observed in HLA class I (P = 1.54 × 10⁻⁹) and class II (P = 1.21 × 10⁻⁸) loci with HAM/TSP. Association analysis using HLA genotyping results showed that HLA-C*07:02 (P = 2.61 × 10⁻⁵), HLA-B*07:02 (P = 4.97 × 10⁻¹⁰), HLA-DRB1*01:01 (P = 1.15 × 10⁻⁹) and HLA-DQB1*05:01 (P = 2.30 × 10⁻⁹) were associated with disease risk, while HLA-B*40:06 (P = 3.03 × 10⁻⁵), HLA-DRB1*15:01 (P = 1.06 × 10⁻⁵) and HLA-DQB1*06:02 (P = 1.78 × 10⁻⁶) worked protectively. Logistic regression analysis identified amino acid position 7 in the G-BETA domain of HLA-DRB1 as strongly associated with HAM/TSP (P = 9.52 × 10⁻¹⁰); individuals homozygous for leucine had an associated increased risk of HAM/TSP (odds ratio, 9.57), and proline was protective (odds ratio, 0.65). Both associations were independent of the known risk associated with proviral load. DRB1-GB-7-Leu was not significantly associated with proviral load. We have identified DRB1-GB-7-Leu as a genetic risk factor for HAM/TSP development independent of proviral load. This suggests that the amino acid residue may serve as a specific marker to identify the risk of HAM/TSP even without knowledge of proviral load. In light of its allele frequency worldwide, this biomarker will likely prove useful in HTLV-1 endemic areas across the globe.

The human T cell leukemia virus type 1 (HTLV-1), the first discovered human retrovirus (1, 2), causes adult T cell leukemia (3) and HTLV-1–associated myelopathy/tropical spastic paraparesis (HAM/TSP) (4, 5). There are an estimated 5 to 10 million infected people worldwide (6), of whom ∼1.08 million are in Japan (7). HAM/TSP is chronic and slowly progressive meningomyelitis of the white and gray matter of the central nervous system, causing gait disturbance, leg weakness, back pain, bladder/bowel and sexual dysfunction, and, over time, inability to walk (8). The prevalence of HAM/TSP in the HTLV-1–seropositive population differs among ethnicities, for example ∼0.25% in the Japanese population and ∼1.9% in the Caribbean population (9, 10). This suggests the involvement of virus and host genetic background, although the cause of disease onset is unclear. A higher proviral load in peripheral blood leukocytes is considered a risk factor (11).Previous studies aiming to identify genetic determinants of HAM/TSP have focused on HLA genes. HLA-A*24, HLA-B*07, HLA-C*07, HLA-DQB1*05, and HLA-DRB1*01, as well as a haplotype consisting of these alleles, have been reported to be associated with HAM/TSP in the Japanese population (12, 13). Related studies in other populations have also shown associations of HLA-B*07 and HLA-DRB1*01 with HAM/TSP in a Spanish population (14), HLA-DQB1*05 and HLA-DRB1*01 with HAM/TSP in an Iranian population (15), and HLA-C*07 with HAM/TSP in a Brazilian population (16). By contrast, HLA-A*02 and HLA-C*08 were reported to be protective against HAM/TSP in a Japanese population (13). Another study of a southern Japanese population showed that a lower frequency of HLA-B*40:06 in HAM/TSP patients than in HTLV-1–infected asymptomatic carriers (17). HLA-DQB1*06:02 and HLA-DRB1*15:01 were also shown to work protectively in a population of African descent (18). However, those studies all used hypothesis-dependent target gene approaches focusing on the HLA genes and involving relatively small numbers of patients (9 to 232 patients for class I typing and 12 to 195 patients for class II typing).We organized a multicenter consortium to collect DNA samples of HAM/TSP patients and asymptomatic HTLV-1 carriers originating from the Kyushu area. The area of southern Kyushu in southwestern Japan is hyperendemic for HTLV-1 infection. The genetic background of the population in the southern Kyushu area is slightly different from that of the mainland Japanese population (19). We succeeded in establishing the largest DNA collection for HTLV-1 studies reported to date, consisting of 899 HAM/TSP patients and 753 asymptomatic HTLV-1 carriers. Using these DNA samples, we undertook a genome-wide association (GWA) study, a hypothesis-independent approach, to comprehensively identify genetic determinants for HAM/TSP.     

Radiation hormesis: Stimulatory effects of low doses ionizing radiation

Low to small doses of ionizing radiation conditionally show stimulatory effects in various cells and organisms, contrasting with detrimental effects induced at high doses. Radiation hormesis is defined as biopositive health effects, such as augmentation of growth and survival, enhancement of immune response, suppression of mutagenesis, and increase in resistance to damages induced by subsequent high dose exposure. Accumulating data on molecular, cellular, and organism levels demonstrate a variety of hormetic phenomena produced by low dose radiation. Living organisms have been exposed to low doses radiation since the early period of evolution, therefore, radiation hormesis may be established at least in part as a defense mechanism, and thus has important implications for health and diseases. In this review, data of radiation hormesis were presented with special emphasis on the responses of organisms. Recent studies also show potential application of low dose radiation for intervention of diseases. Research on radiation hormesis will indisputably contribute to elucidate aging processes.     

Acquired immunity in nude mice induced by expression of the IL-2 or IL-4 gene in human pancreatic carcinoma cells and anti-tumor effect generated by in vivo gene transfer using retrovirus.

We have examined the anti-tumor effect in nude mice caused by human pancreatic cancer cells (AsPC-1) modified to secrete IL-2 or IL-4. Loss of tumorigenicity of cytokine-producing, but not wild-type, cells was observed despite their unaltered in vitro proliferation rates; and these anti-tumor effects were dependent on the amount of cytokine released. Wild-type cells inoculated into mice which had rejected IL-2- or IL-4-producer cells showed significant growth retardation, while no retardation was detected when unrelated human colon carcinoma cells were inoculated. Histological examination of regressing IL-2- or IL-4-producing AsPC-1 tumors in nude mice revealed infiltration by CD11b-, but not CD90-, positive cells around the tumors. Treatment of nude mice with anti-asialoGM(1) antibody did not affect loss of tumorigenicity. Mice injected i.p. with IL-2- or IL-4-producing AsPC-1 cells did not die, in contrast to mice inoculated with wild-type cells. Injection of retrovirus-bearing IL-2, but not beta-galactosidase, gene into mice which had wild-type cells in the peritoneal cavity also significantly prolonged survival. Thus, expression of the IL-2 or IL-4 gene in AsPC-1 cells may generate tumor-specific acquired immunity, even in mature T cell-deficient conditions. An anti-tumor response can be induced by in vivo transfer of the IL-2 gene.     

Geometry and growth of the reconstructed aorta in patients with hypoplastic left heart syndrome and variants

Objective

The interdigitating technique in aortic arch reconstruction in hypoplastic left heart syndrome and variants (HLHS) reduces the recoarctation rate. Little is known on aortic arch growth characteristics and resulting clinical impact.