Significance of MHC class II haplotypes and IgG Fc receptors in SLE

Systemic lupus erythematosus (SLE) is a systemic antibody-mediated autoimmune disease that develops under the control of multiple susceptibility genes. Genetic studies in murine and human SLE have identified several chromosomal intervals that contain candidate susceptibility genes. However, the ultimate identification of the genes and their roles in disease process need much further investigation. Spontaneous murine SLE models provide useful tools in this respect. In this chapter, we show this line of investigation, particularly focusing on the roles of major histocompatibility complex (MHC) class II and immunoglobulin G Fc receptors (FcγRs). The existence of high-affinity autoantibodies is evidence that autoimmunity in SLE is antigen-driven. Thereby, MHC class II haplotypes have been implicated in SLE susceptibility; however, because of the linkage disequilibrium that exists among the class I, II and III genes within the MHC complex, it has been difficult to discriminate the relative contributions of individual loci. On the other hand, the extent of antibody synthesis upon antigen stimulation and associated inflammatory cascades are controlled in several ways by the balance of stimulatory and inhibitory signaling molecules on immune cells. Stimulatory/inhibitory FcγRs mediate one such mechanism, and there are reports indicating the association between polymorphic FcγRs and SLE. However, as stimulatory and inhibitory FcγRs cluster on the telomeric chromosome 1, the absolute contribution of individual genes has been difficult to dissect. In studies of genetic dissection using interval-congenic and intragenic recombinant mouse strains of SLE models, we show evidence and discuss how and to what extent MHC class II molecules and stimulatory/inhibitory FcγRs are involved in SLE susceptibility.     

Assessment of viability and osteogenic ability of human mesenchymal stem cells after being stored in suspension for clinical transplantation

Human mesenchymal stem cells (MSCs) were suspended in phosphate-buffered saline (PBS) and stored up to 24 h at 4 degrees C, 24 degrees C, and 37 degrees C. More than 80% viability was maintained at any temperature for at least 1 h, then gradually decreased over time. After 24 h, the viabilities at 4 degrees C, 24 degrees C, and 37 degrees C were about 81%, 70%, and 62%, respectively. The MSCs suspended/stored in PBS at 4 degrees C for 24 h also exhibited in vitro osteogenic differentiation capability as evidenced by mineralized matrix formation as well as high alkaline phosphatase activity when cultured in an osteogenic medium. Furthermore, in vivo implantation experiments using the MSCs also demonstrated new bone formation. Because MSCs are known to possess multipotential stem cell characteristics, these data indicate that human MSCs stored in PBS at 4 degrees C could be delivered to distant medical facilities for the purpose of hard tissue and other types of tissue regeneration therapy.     

Three-dimensional finite-element model of the human temporomandibular joint disc during prolonged clenching

In the temporomandibular joint (TMJ), overloading induced by prolonged clenching appears to be important in the cascade of events leading to disc displacement. In this study, the effect of disc displacement on joint stresses during prolonged clenching was studied. For this purpose, finite-element models of the TMJ, with and without disc displacement, were used. Muscle forces were used as a loading condition for stress analysis during a time-period of 10 min. The TMJ disc and connective tissue were characterized as a linear viscoelastic material. In the asymptomatic model, large stresses were found in the central and lateral part of the disc through clenching. In the retrodiscal tissue, stress relaxation occurred during the first 2 min of clenching. In the symptomatic model, large stresses were observed in the posterior part of the disc and in the retrodiscal tissue, and the stress level was kept constant through clenching. This indicates that during prolonged clenching the disc functions well in the asymptomatic joint, meanwhile the retrodiscal tissue in the symptomatic joint is subject to excessive stress. As this structure is less suitable for bearing large stresses, tissue damage may occur. In addition, storage of excessive strain energy might lead to breakage of the tissue.     

Targeting AMAP1 and cortactin binding bearing an atypical src homology 3/proline interface for prevention of breast cancer invasion and metastasis

Invasive potentials of carcinomas greatly contribute to their metastasis, which is a major threat in most cancers. We have recently shown that Arf6 plays a pivotal role in breast cancer invasive activities and identified AMAP1 as an effector of GTP-Arf6 in invasion. Expression of AMAP1 correlates well with invasive phenotypes of primary tumors of the human breast. We also have shown that AMAP1 functions by forming a trimeric protein complex with cortactin and paxillin. In this complex, AMAP1 binds to the src homology 3 (SH3) domain of cortactin via its proline-rich peptide, SKKRPPPPPPGHKRT. SH3 domains are known to bind generally to the proline-rich ligands with a one-to-one stoichiometry. We found that AMAP1/cortactin binding is very atypical in its stoichiometry and interface structure, in which one AMAP1 proline-rich peptide binds to two cortactin SH3 domains simultaneously. We made a cell-permeable peptide derived from the AMAP1 peptide, and we show that this peptide specifically blocks AMAP1/cortactin binding, but not other canonical SH3/proline bindings, and effectively inhibits breast cancer invasion and metastasis. Moreover, this peptide was found to block invasion of other types of cancers, such as glioblastomas and lung carcinomas. We also found that a small-molecule compound, UCS15A, which was previously judged as a weak inhibitor against canonical SH3/proline bindings, effectively inhibits AMAP1/cortactin binding and breast cancer invasion and metastasis. Together with fine structural analysis, we propose that the AMAP1/cortactin complex, which is not detected in normal mammary epithelial cells, is an excellent drug target for cancer therapeutics.     

DWnt4 regulates the dorsoventral specificity of retinal projections in the Drosophila melanogaster visual system

In Drosophila melanogaster, the axons of retinal photoreceptor cells extend to the first optic ganglion, the lamina, forming a topographic representation. Here we show that DWnt4, a secreted protein of the Wnt family, is the ventral cue for the lamina. In DWnt4 mutants, ventral retinal axons misprojected to the dorsal lamina. DWnt4 was normally expressed in the ventral half of the developing lamina and DWnt4 protein was detected along ventral retinal axons. Dfrizzled2 and dishevelled, respectively, encode a receptor and a signaling molecule required for Wnt signaling. Mutations in both genes caused DWnt4-like defects, and both genes were autonomously required in the retina, suggesting a direct role of DWnt4 in retinal axon guidance. In contrast, iroquois homeobox genes are the dorsal cues for the retina. Dorsal axons accumulated DWnt4 and misprojected to the ventral lamina in iroquois mutants; the phenotype was suppressed in iroquois Dfrizzled2 mutants, suggesting that iroquois may attenuate the competence of Dfrizzled2 to respond to DWnt4.     

A role of the double-stranded RNA-binding protein PACT in mouse ear development and hearing

To determine the physiological functions of the mammalian double-stranded RNA-binding protein PACT, the single-copy mouse Pact gene was disrupted and expression of the protein was completely ablated. The most notable phenotypes of the Pact(-/-) mouse were reduced size and severe microtia. As a result of the congenital abnormality of both outer and middle ears, these mice were hearing impaired. In situ hybridization revealed that PACT mRNA was expressed in specific regions of all three parts of the ear in adult and embryonic wild-type mice. Our study demonstrated an essential role of PACT in mammalian ear development and produced the first animal model for studying human microtia.     

Effect of valsartan, an angiotensin II receptor blocker, on markers of oxidation and glycation in Japanese type 2 diabetic subjects: blood pressure-independent effect of valsartan

AIMS: Although it has been reported that angiotensin II receptor blocker inhibited the formation and accumulation of advanced glycation endproducts (AGEs) in vitro and in vivo, whether it can do so clinically is unknown. We therefore examined the effect of valsartan on markers of oxidation and glycation. METHODS: We started 40mg/day valsartan treatment in 15 type 2 diabetic subjects with hypertension, and metabolic parameters, lipid peroxide, paraoxonase activity, platelet-activating factor acethylhydrolase activity, AGEs and urine 8-isoprostane were measured at baseline and after 3 and 6 months of treatment. RESULTS: Even after valsartan treatment, the blood pressure level of the patients did not change during the study. However, AGEs and urine 8-isoprastane levels had decreased at 6 months (p<0.05 and <0.01) as well as urine microalbumin level (p<0.01), although other oxidative stress markers were unchanged. CONCLUSION: In this study, low-dose valsartan treatment decreased serum AGEs level, whereas blood pressure level was unchanged. The effect of valsartan on AGEs might be a blood pressure-independent effect in type 2 diabetic subjects.     

Proposal of new uncertainty factor application to derive tolerable daily intake

We propose new uncertainty factors (UFs) and a new subdivision of default factors in chemical risk assessment using a probabilistic approach based on the latest applicable information. Rounded values of 150 for mice, 100 for hamsters and rats, and 40 for rabbits, monkeys and dogs for inter- and intra-species differences (UF_AH) were derived from the probabilistic combination of two log-normal distributions. Further calculation of additional UFs when chronic data (UF_S) or NOAEL (UF_L) are lacking was conducted using available log-normal distribution information. The alternative UF_S and UF_L values of 4 are considered to be appropriate for both cases where data are lacking. The default contributions of inter-species difference (UF_A) and intra-species difference (UF_H) to the UF_AH of 100 for hamsters and rats as an example are considered to be 25 and 4, respectively. The UF_A of 25 was subdivided into 25^0.6 (i.e., 7.0) for pharmacokinetics (PK) (UF_A,PK) and 25^0.4 (i.e., 3.6) for pharmacodynamics (PD) (UF_A,PD), and the UF_H of 4 was evenly subdivided into 4^0.5 (i.e., 2) (UF_H,PK and UF_H,PD), to account for chemical-specific difference data between humans and laboratory animals for PK and/or PD. These default UFs, which come from actual experimental data, may be more appropriate than previous default UFs to derive tolerable daily intake values.