TNF-α induces Claudin-1 expression in renal tubules in Alport mice.

Claudin-1 (CL-1) is responsible for the paracellular barrier function of glomerular parietal epithelial cells (PEC) in kidneys, but the role of CL-1 in proximal tubules remains to be elucidated. In this study, to evaluate CL-1 as a potential therapeutic drug target for chronic kidney disease, we investigated change of CL-1 expression in the proximal tubules of diseased kidney and elucidated the factors that induced this change. We established Alport mice as a kidney disease model and investigated the expression of CL-1 in diseased kidney using quantitative PCR and immunohistochemistry (IHC). Compared to wild type mice, Alport mice showed significant increases in plasma creatinine, urea nitrogen and urinary albumin excretion. CL-1 mRNA was increased significantly in the kidney cortex and CL-1 was localized on the adjacent cell surfaces of PECs and proximal tubular epithelial cells. The infiltration of inflammatory cells around proximal tubules and a significant increase in TNF-α mRNA were observed in diseased kidneys. To reveal factors that induce CL-1, we analyzed the induction of CL-1 by albumin or tumor necrosis factor (TNF)-α in human proximal tubular cells (RPTEC/TERT1) using quantitative PCR and Western blotting. TNF-α increased CL-1 expression dose-dependently, though albumin did not affect CL-1 expression in RPTEC/TERT1. In addition, both CL-1 and TNF-α expression were significantly increased in UUO mice, which are commonly used as a model of tubulointerstitial inflammation without albuminuria. These results indicate that CL-1 expression is induced by inflammation, not by albuminuria in diseased proximal tubules. Moreover, we examined the localization of CL-1 in the kidney of IgA nephropathy patients by IHC and found CL-1 expression was also elevated in the proximal tubular cells. Taken together, CL-1 expression is increased in the proximal tubular epithelial cells of diseased kidney. Inflammatory cells around the tubular epithelium may produce TNF-α which in turn induces CL-1 expression.

Antibody to CD137 Activated by Extracellular Adenosine Triphosphate Is Tumor Selective and Broadly Effective In Vivo without Systemic Immune Activation.

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.

An adjustment in BMP4 function represents a treatment for diabetic nephropathy and podocyte injury.

Podocyte injury has been proposed to play an important role in diabetic nephropathy; however, its pathological mechanism remains unclear. We have shown that bone morphogenetic protein 4 (BMP4) signaling leads to the glomerular changes characteristic of this disorder. To analyze the molecular mechanism of podocyte injury, the effect of BMP4 was investigated using streptozotocin (STZ)-induced, Bmp4 heterozygous knockout (Bmp4+/-) and podocyte-specific Bmp4 knockout mice. Mice with STZ-induced diabetes exhibited glomerular matrix hyperplasia and decreased numbers of podocyte nucleus-specific WT1-positive cells. The number of podocytes and proteinuria were improved in both diabetic Bmp4 knockout mouse models compared to the effects observed in the control mice. The effect of BMP4 overexpression on Bmp4-induced or podocyte-specific transgenic mice was examined. Tamoxifen-induced Bmp4-overexpressing mice exhibited mesangial matrix expansion and decreased numbers of WT1-positive cells. Podocyte-specific Bmp4-overexpressing mice displayed increased kidney BMP4 expression and mesangial matrix expansion but decreased nephrin expression and numbers of WT1-positive cells. Both lines of Bmp4-overexpressing mice exhibited increased albuminuria. In cultured podocytes, BMP4 increased phospho-p38 levels. BMP4 decreased nephrin expression but increased cleaved caspase-3 levels. p38 suppression inhibited caspase-3 activation. Apoptosis was confirmed in STZ-diabetic glomeruli and Bmp4-overexpressing mice. Bmp4 +/- mice with diabetes displayed reduced apoptosis. Based on these data, the BMP4 signaling pathway plays important roles in the development of both podocyte injury and mesangial matrix expansion in diabetic nephropathy.

Corrigendum: Entire CD3ε, δ, and γ humanized mouse to evaluate human CD3-mediated therapeutics.

This corrects the article DOI: 10.1038/srep45839.

Effect of Npt2b deletion on intestinal and renal inorganic phosphate (Pi) handling.

BACKGROUND: Hyperphosphatemia is common in chronic kidney disease and is associated with morbidity and mortality. The intestinal Na+-dependent phosphate transporter Npt2b is thought to be an important molecular target for the prevention of hyperphosphatemia. The role of Npt2b in the net absorption of inorganic phosphate (Pi), however, is controversial. METHODS: In the present study, we made tamoxifen-inducible Npt2b conditional knockout (CKO) mice to analyze systemic Pi metabolism, including intestinal Pi absorption. RESULTS: Although the Na+-dependent Pi transport in brush-border membrane vesicle uptake levels was significantly decreased in the distal intestine of Npt2b CKO mice compared with control mice, plasma Pi and fecal Pi excretion levels were not significantly different. Data obtained using the intestinal loop technique showed that Pi uptake in Npt2b CKO mice was not affected at a Pi concentration of 4 mM, which is considered the typical luminal Pi concentration after meals in mice. Claudin, which may be involved in paracellular pathways, as well as claudin-2, 12, and 15 protein levels were significantly decreased in the Npt2b CKO mice. Thus, Npt2b deficiency did not affect Pi absorption within the range of Pi concentrations that normally occurs after meals. CONCLUSION: These findings indicate that abnormal Pi metabolism may also be involved in tight junction molecules such as Cldns that are affected by Npt2b deficiency.

Entire CD3ε, δ, and γ humanized mouse to evaluate human CD3-mediated therapeutics.

T cell-mediated immunotherapy is an attractive strategy for treatment in various disease areas. In this therapeutic approach, the CD3 complex is one of the key molecules to modulate T cell functions; however, in many cases, we cannot evaluate the drug candidates in animal experiments because the therapeutics, usually monoclonal antibodies specific to human CD3, cannot react to mouse endogenous Cd3. Although immunodeficient mice transfused with human hematopoietic stem or precursor cells, known as humanized mice, are available for these studies, mice humanized in this manner are not completely immune competent. In this study we have succeeded in establishing a novel mouse strain in which all the three components of the Cd3 complex - Cd3ε, Cd3δ, and Cd3γ - are replaced by their human counterparts, CD3E, CD3D, and CD3G. Basic immunological assessments have confirmed that this strain of human CD3 EDG-replaced mice are entirely immune competent, and we have also demonstrated that a bispecific antibody that simultaneously binds to human CD3 and a tumor-associated antigen (e.g. ERBB2 or GPC3) can be evaluated in human CD3 EDG-replaced mice engrafted with tumors. Our mouse model provides a novel means to evaluate the in vivo efficacy of human CD3-mediated therapy.

Bone Morphogenetic Protein 4 and Smad1 Mediate Extracellular Matrix Production in the Development of Diabetic Nephropathy.

Diabetic nephropathy is the leading cause of end-stage renal disease. It is pathologically characterized by the accumulation of extracellular matrix in the mesangium, of which the main component is α1/α2 type IV collagen (Col4a1/a2). Recently, we identified Smad1 as a direct regulator of Col4a1/a2 under diabetic conditions in vitro. Here, we demonstrate that Smad1 plays a key role in diabetic nephropathy through bone morphogenetic protein 4 (BMP4) in vivo. Smad1-overexpressing mice (Smad1-Tg) were established, and diabetes was induced by streptozotocin. Nondiabetic Smad1-Tg did not exhibit histological changes in the kidney; however, the induction of diabetes resulted in an ∼1.5-fold greater mesangial expansion, consistent with an increase in glomerular phosphorylated Smad1. To address regulatory factors of Smad1, we determined that BMP4 and its receptor are increased in diabetic glomeruli and that diabetic Smad1-Tg and wild-type mice treated with a BMP4-neutralizing antibody exhibit decreased Smad1 phosphorylation and ∼40% less mesangial expansion than those treated with control IgG. Furthermore, heterozygous Smad1 knockout mice exhibit attenuated mesangial expansion in the diabetic condition. The data indicate that BMP4/Smad1 signaling is a critical cascade for the progression of mesangial expansion and that blocking this signal could be a novel therapeutic strategy for diabetic nephropathy.

SGLT5 reabsorbs fructose in the kidney but its deficiency paradoxically exacerbates hepatic steatosis induced by fructose.

Although excessive fructose intake is epidemiologically linked with dyslipidemia, obesity, and diabetes, the mechanisms regulating plasma fructose are not well known. Cells transfected with sodium/glucose cotransporter 5 (SGLT5), which is expressed exclusively in the kidney, transport fructose in vitro; however, the physiological role of this transporter in fructose metabolism remains unclear. To determine whether SGLT5 functions as a fructose transporter in vivo, we established a line of mice lacking the gene encoding SGLT5. Sodium-dependent fructose uptake disappeared in renal brush border membrane vesicles from SGLT5-deficient mice, and the increased urinary fructose in SGLT5-deficient mice indicated that SGLT5 was the major fructose reabsorption transporter in the kidney. From this, we hypothesized that urinary fructose excretion induced by SGLT5 deficiency would ameliorate fructose-induced hepatic steatosis. To test this hypothesis we compared SGLT5-deficient mice with wild-type mice under conditions of long-term fructose consumption. Paradoxically, however, fructose-induced hepatic steatosis was exacerbated in the SGLT5-deficient mice, and the massive urinary fructose excretion was accompanied by reduced levels of plasma triglycerides and epididymal fat but fasting hyperinsulinemia compared with fructose-fed wild-type mice. There was no difference in food consumption, water intake, or plasma fructose between the two types of mice. No compensatory effect by other transporters reportedly involved in fructose uptake in the liver and kidney were indicated at the mRNA level. These surprising findings indicated a previously unrecognized link through SGLT5 between renal fructose reabsorption and hepatic lipid metabolism.

Novel genetically-humanized mouse model established to evaluate efficacy of therapeutic agents to human interleukin-6 receptor.

For clinical trials of therapeutic monoclonal antibodies (mAbs) to be successful, their efficacy needs to be adequately evaluated in preclinical experiments. However, in many cases it is difficult to evaluate the candidate mAbs using animal disease models because of lower cross-reactivity to the orthologous target molecules. In this study we have established a novel humanized Castleman's disease mouse model, in which the endogenous interleukin-6 receptor gene is successfully replaced by human IL6R, and human IL6 is overexpressed. We have also demonstrated the therapeutic effects of an antibody that neutralizes human IL6R, tocilizumab, on the symptoms in this mouse model. Plasma levels of human soluble IL6R and human IL6 were elevated after 4-week treatment of tocilizumab in this mouse model similarly to the result previously reported in patients treated with tocilizumab. Our mouse model provides us with a novel means of evaluating the in vivo efficacy of human IL6R-specific therapeutic agents.

Inorganic phosphate homeostasis in sodium-dependent phosphate cotransporter Npt2b⁺/⁻ mice.

An inorganic phosphate (P(i))-restricted diet is important for patients with chronic kidney disease and patients on hemodialysis. Phosphate binders are essential for preventing hyperphosphatemia and ectopic calcification. The sodium-dependent P(i) (Na/P(i)) transport system is involved in intestinal P(i) absorption and is regulated by several factors. The type II sodium-dependent P(i) transporter Npt2b is expressed in the brush-border membrane in intestinal epithelial cells and transports P(i). In the present study, we analyzed the phenotype of Npt2b(-/-) and hetero(+/-) mice. Npt2b(-/-) mice died in utero soon after implantation, indicating that Npt2b is essential for early embryonic development. At 4 wk of age, Npt2b(+/-) mice showed hypophosphatemia and low urinary P(i) excretion. Plasma fibroblast growth factor 23 levels were significantly decreased and 1,25(OH)(2)D(3) levels were significantly increased in Npt2b(+/-) mice compared with Npt2b(+/+) mice. Npt2b mRNA levels were reduced to 50% that in Npt2b(+/+) mice. In contrast, renal Npt2a and Npt2c transporter protein levels were significantly increased in Npt2b(+/-) mice. At 20 wk of age, Npt2b(+/-) mice showed hypophosphaturia and reduced Na/P(i) cotransport activity in the distal intestine. Npt2b(+/+) mice with adenine-induced renal failure had hyperphosphatemia and high plasma creatinine levels. Npt2b(+/-) mice treated with adenine had significantly reduced plasma P(i) levels compared with Npt2b(+/+) mice. Intestinal Npt2b protein and Na(+)/P(i) transport activity levels were significantly lower in Npt2b(+/-) mice than in the Npt2b(+/+) mice. The findings of the present studies suggest that Npt2b is an important target for the prevention of hyperphosphatemia.

SOX9 protein induces a chondrogenic phenotype of mesangial cells and contributes to advanced diabetic nephropathy.

Diabetic nephropathy (DN) is the most important chronic kidney disease. We previously reported that Smad1 transcriptionally regulates the expression of extracellular matrix in DN. Phenotypic change in mesangial cells (MCs) is a key pathologic event in the progression of DN. The aim of this study is to investigate a novel mechanism underlying chondrogenic phenotypic change in MCs that results in the development of DN. MCs showed chondrogenic potential in a micromass culture, and BMP4 induced the expression of chondrocyte markers (SRY-related HMG Box 9 (SOX9) and type II collagen (COL2)). Advanced glycation end products induced the expression of chondrocyte marker proteins downstream from the BMP4-Smad1 signaling pathway in MCs. In addition, hypoxia also induced the expression of BMP4, hypoxia-inducible factor-1α (HIF-1α), and chondrocyte markers. Overexpression of SOX9 caused ectopic expression of proteoglycans and COL2 in MCs. Furthermore, forced expression of Smad1 induced chondrocyte markers as well. Dorsomorphin inhibited these inductions. Glomerular expressions of HIF-1α, BMP4, and chondrocyte markers were observed in diabetic nephropathy mice. These positive stainings were observed in mesangial sclerotic lesions. SOX9 was partially colocalized with HIF-1α and BMP4 in diabetic glomeruli. BMP4 knock-in transgenic mice showed not only similar pathological lesions to DN, but also the induction of chondrocyte markers in the sclerotic lesions. Here we demonstrate that HIF-1α and BMP4 induce SOX9 expression and subsequent chondrogenic phenotype change in DN. The results suggested that the transdifferentiation of MCs into chondrocyte-like cells in chronic hypoxic stress may result in irreversible structural change in DN.

Activation of bone morphogenetic protein 4 signaling leads to glomerulosclerosis that mimics diabetic nephropathy.

Diabetic nephropathy (DN) is the most common cause of chronic kidney disease. We have previously reported that Smad1 transcriptionally regulates the expression of extracellular matrix (ECM) proteins in DN. However, little is known about the regulatory mechanisms that induce and activate Smad1. Here, bone morphogenetic protein 4 (Bmp4) was found to up-regulate the expression of Smad1 in mesangial cells and subsequently to phosphorylate Smad1 downstream of the advanced glycation end product-receptor for advanced glycation end product signaling pathway. Moreover, Bmp4 utilized Alk3 and affected the activation of Smad1 and Col4 expressions in mesangial cells. In the diabetic mouse, Bmp4 was remarkably activated in the glomeruli, and the mesangial area was expanded. To elucidate the direct function of Bmp4 action in the kidneys, we generated transgenic mice inducible for the expression of Bmp4. Tamoxifen treatment dramatically induced the expression of Bmp4, especially in the glomeruli of the mice. Notably, in the nondiabetic condition, the mice exhibited not only an expansion of the mesangial area and thickening of the basement membrane but also remarkable albuminuria, which are consistent with the distinct glomerular injuries in DN. ECM protein overexpression and activation of Smad1 in the glomeruli were also observed in the mice. The mesangial expansion in the mice was significantly correlated with albuminuria. Furthermore, the heterozygous Bmp4 knock-out mice inhibited the glomerular injuries compared with wild type mice in diabetic conditions. Here, we show that BMP4 may act as an upstream regulatory molecule for the process of ECM accumulation in DN and thereby reveals a new aspect of the molecular mechanisms involved in DN.

Viral envelope protein gp64 transgenic mouse facilitates the generation of monoclonal antibodies against exogenous membrane proteins displayed on baculovirus.

We have been investigating the functional display of multipass membrane protein such as transporter or G-protein coupled receptor on the budded baculovirus (BV). We tested the use of a viral envelope protein gp64 transgenic mouse for the direct immunization of these membrane proteins displayed on BVs. The gp64 transgenic mice showed only a weak response to virus compared to wild type BALB/c mice. Immunizing gp64 transgenic mice with the BV expressing peptide transporter PepT1, we obtained 47 monoclonal antibodies (mAbs). These mAbs were specific to the PepT1 on the pancreatic cancer cells AsPC-1 by fluorocytometric analysis and exhibited antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity to AsPC-1. We also generated 7 mAbs by immunizing gp64 transgenic mice on a CCR2-deficient background with the BV expressing chemokine receptor CCR2 together with partially purified CCR2. These mAbs possessed specific binding to CCR2 in CHO cells on fluorocytometric analysis, and exhibited neutralizing activities for ligand-dependent inhibition of cyclic AMP production. This method provides a powerful tool for the generation of therapeutic/diagnostic mAbs against membrane proteins.

Morphologic and functional analysis of sperm and testes in Aquaporin 7 knockout mice.

OBJECTIVE: To investigate the functional and morphologic role of Aquaporin 7 (AQP7) in testis and sperm. DESIGN: Experimental laboratory study. SETTING: University and research institute units. ANIMAL(S): AQP7 knockout mice (C57BL/6J background). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Morphologic analysis of testis and epididymis, daily sperm production, sperm motility, in vitro fertilization. RESULT(S): There was no difference in the morphology of the testes and epididymis between AQP7 knockout and wild-type mice. The AQP7 knockout male mice and wild-type male mice had similar numbers of offspring. Analysis of the daily sperm production and motility of AQP7 knockout mice did not show any abnormalities. Similarly, the rate of in vitro fertilization using sperm from AQP7 knockout mice was not different from wild-type mice. CONCLUSION(S): Male AQP7 knockout mice were not sterile, and their sperm did not show any morphologic and functional abnormalities.

Fertility of mice receiving vitrified adult mouse ovaries.

Cryopreservation of the ovaries is a useful technology for preservation of germ cells from experimental animals, because if the female founder is infertile or has mutated mitochondrial DNA, preservation of female germ cells is necessary. Although it is possible to cryopreserve immature mouse ovaries with a high degree of viability by vitrification with a mixture of several cryoprotectants, the viability of cryopreserved adult mouse ovaries is still unknown. Here, we investigated the viability of mouse ovaries at various ages after cryopreservation by vitrification techniques. Donor ovaries were collected from 10-day-, 4-week-, 10-week- and 7-month-old, female, nulliparous, green fluorescence protein (GFP)-transgenic mice and cryopreserved by vitrification. The vitrified-warmed ovaries were orthotopically transplanted to 4- or 10-week-old mice. GFP-positive pups were obtained in all experimental groups. In the 4-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 44%, 9%, 12% and 4% respectively. In the 10-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 36%, 16%, 2% and 9% respectively. Furthermore, GFP-positive pups also were obtained from recipients transplanted with ovaries of donors without normal estrous cyclicity. Our results indicate that cryopreservation of mouse ovaries by vitrification is a useful method for the preservation of female germ cells from mice of various ages.

Possible expansion of "Window of Implantation" in pseudopregnant mice: time of implantation of embryos at different stages of development transferred into the same recipient.

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and maternal environment. It is believed that the growth of transferred embryos of different ages is synchronized during preimplantation development and that such embryos are implanted in the uterus at the same time. To define the time of synchronization for developing embryos of different ages, embryos at two different stages of development were transferred separately into the oviducts of the same recipient. We then examined the subsequent development of the embryos at various time intervals after transfer. Pronucleus (PN) stage eggs were transferred separately to the right or left oviduct of recipients on Day 0, while eight-cell embryos (8C) were transferred to the other oviduct. For 8C, 5%, 63%, and 74% of transferred embryos were implanted in the uterus at 42, 66, and 90 h posttransfer, respectively. In contrast, none of the transferred PN was implanted until 90 h posttransfer. At 90 h posttransfer, 59% of the PN had successfully implanted. Histological examination revealed that developmental stage of the embryos in both groups synchronized around 162 h posttransfer, even though the implantation was accelerated in 8C compared with PN. Our results indicate that embryos of advanced stage transferred to the oviduct implant in the uterus in advance of younger embryos and that the uterine development is synchronized at the neural plate, presomite stage. Our results strongly suggest that uterine receptivity for implantation is expandable in pseudopregnant mice.

Molecular characterization of mitomycin C-induced large deletions and tandem-base substitutions in the bone marrow of gpt delta transgenic mice.

Deletion mutations constitute an important class of mutations that may result in a variety of human diseases, including cancer. Although many chemicals and ionizing radiations induce deletions, this class of mutation has been poorly characterized at the molecular level, particularly in vivo. Here we report the molecular nature of deletions as well as base substitutions induced by antitumor antibiotic mitomycin C (MMC) in the bone marrow using a novel transgenic mouse, gpt delta. In this mouse model, deletions and point mutations in lambda DNA integrated in the chromosome are individually selected as Spi(-) (sensitive to P2 interference) phages and 6-thioguanine-resistant bacterial colonies, respectively. The mice were treated with MMC (1 mg/kg/day) for five consecutive days. One week after the last treatment, lambda phage was rescued from the genomic DNA of the bone marrow by in vitro packaging reactions and subjected to Spi(-) and 6-thioguanine selections. The mutant frequency of Spi(-) with large deletions increased more than 20-fold over that of the control. Molecular sizes of the large deletions were mostly more than 2,000 base pairs. The large deletions frequently occurred between two short direct repeat sequences from 2 to 6 base pairs, suggesting that they are generated during the end-joining repair of double-strand breaks induced by interstrand cross-links in DNA. In 6-thioguanine selection, tandem-base substitutions, such as 5'-GG-3' to 5'-AT-3', were induced. It highlights the relevance of intrastrand cross-links as genotoxic lesions. Previous in vitro studies report the induction of single-base substitutions and single-base deletions by MMC. However, no such mutations were identified in vivo. Thus, our results strongly caution that in vitro mutation spectra do not necessarily reflect genotoxic events in vivo and emphasize the importance of transgenic rodent genotoxicity assays to examine the roles of DNA adducts in mutagenesis and carcinogenesis.

Role of Lkb1, the causative gene of Peutz-Jegher's syndrome, in embryogenesis and polyposis.

Peutz-Jeghers syndrome (PJS) is a dominantly inherited human disorder characterized by gastrointestinal hamartomatous polyposis and mucocutaneous melanin pigmentation. LKB1 (STK11) serine/threonine kinase is the product of the causative gene of PJS, which has been mapped to chromosome 19p13.3. However, several studies have produced results that are not consistent with a link between LKB1 gene mutation and PJS. We constructed a knockout gene mutation of Lkb1 to determine whether it is the causative gene of PJS and to examine the biological role of the Lkb1 gene. Lkb1(-/-) mice died in utero between 8.5 and 9.5 days postcoitum. At 9.0 days postcoitum, Lkb1(-/-) embryos were generally smaller than their age-matched littermates, showed developmental retardation, and did not undergo embryonic turning. Multiple gastric adenomatous polyps were observed in 10- to 14-month-old Lkb1(+/-) mice. Our results indicate that functional Lkb1 is required for normal embryogenesis and that it is related to tumor development. The Lkb1(+/-) mouse is suitable for studying molecular mechanism underlying the development of inherited gastric tumors in PJS.

Effect of partial incision of the zona pellucida by piezo-micromanipulator for in vitro fertilization using frozen-thawed mouse spermatozoa on the developmental rate of embryos transferred at the 2-cell stage.

Cryopreservation of mouse spermatozoa is widely used, although considerable strain differences in fertilization rates using frozen-thawed mouse spermatozoa have been described. The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared with rates for other inbred strains. We have recently solved this difficulty by in vitro fertilization (IVF) in combination with partial zona pellucida dissection (PZD). However, this technique requires culture of fertilized eggs with PZD in vitro up to morula or blastocyst stage before transfer into the uterus because blastomeres are lost after transfer into the oviduct because of the relatively large artificial slit in the zona pellucida. To overcome this problem, we performed a partial zona pellucida incision by using a piezo-micromanipulator (ZIP) for IVF with frozen-thawed mouse spermatozoa. The blunt end of the micropipette touched the surface of the zona pellucida of the oocytes, and piezo pulses were used to incise the zona pellucida while the pipette was moved along by the surface of zona pellucida. The length of the incision was pir/6 microm. When cumulus-free ZIP and PZD oocytes were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa, the fertilization rates of ZIP and PZD oocytes were 52% and 48%, respectively. After embryo transfer at the 2-cell stage, 18% and 2% of the transferred embryos with ZIP and PZD developed to term, respectively. This difference was significant (P < 0.05). When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, the difference between ZIP and PZD zygotes for development rate to full term was not significant. Our results indicate that ZIP is an effective alternative technique for IVF using cryopreserved mouse spermatozoa and subsequent embryo transfer.

Anti-interleukin 6 (IL-6) receptor antibody suppresses Castleman's disease like symptoms emerged in IL-6 transgenic mice.

Transgenic mice carrying human IL-6 cDNA fused with a murine major histocompatibility class-I promoter (H-2L(d)) were serially administered with anti-interleukin-6 receptor (IL-6R) monoclonal antibody (mAb), MR16-1, from the age of 4 weeks to estimate its efficacy on a variety of disorders developed in these mice, most of which are similar to the disorders associated with Castleman's disease. In the control mice treated with isotype-matched mAb, a massive and multiple IgG1 plasmacytosis, mesangial proliferative glomerulonephritis, leukocytosis, thrombocytosis, anemia and abnormalities of blood chemical parameters have developed in accordance with the elevation of serum IL-6, and 50% of mice have died of renal failure by 18 weeks of age. In contrast, the treatment with MR16-1 prevented all these symptoms and prolonged the lifetime of the majority of the mice. Thus, the constitutive overexpression of IL-6 caused various disorders, and the treatment with anti-IL-6R mAb completely prevented from these symptoms. These results clearly confirm that IL-6 indeed plays an essential role in the pathogenesis of a variety of disorders. Furthermore, anti-IL-6R mAb could provide novel therapy for Castleman's disease and MR16-1 should be a useful tool to estimate therapeutic potential of IL-6 antagonists in a variety of murine models for human disease.