Evidence for the role of INSL3 on sperm production in boars by passive immunisation.

Insulin-like factor 3 (INSL3), previously called relaxin-like factor, is essential for foetal testis descent and has been implicated in sperm production in adult males. This study investigated the role of INSL3 in sperm production by examining the effect of neutralising INSL3 by passive immunisation on testicular function and sperm output in boars. Six male Duroc boars were randomly assigned to passive immunisation and control groups (n = 3 each). The immunisation group was intravenously injected with an IgG fraction of anti-INSL3 antibody developed against the B domain of INSL3 at 2-week intervals from 21-40 weeks of age. The control group was treated with normal IgG in the same manner. Antibody administration reduced testis weight and caused a fourfold increase in the frequency of apoptotic germ cells, which was associated with upregulation of the pro-apoptotic caspase 3 and BAX, and downregulation of the anti-apoptotic XIAP and BCL2, and a substantial marked reduction in sperm concentration. Neutralising INSL3 delivered by passive immunisation reduced testis weight and sperm concentration by inducing germ cell apoptosis, suggesting that INSL3 acts as a germ cell survival/anti-apoptotic factor in the maintenance of sperm production.

Suppression of tumor necrosis factor-alpha by beta2-adrenoceptor activation: role of mitogen-activated protein kinases in renal mesangial cells.

OBJECTIVE: The present study examined the inhibitory effect of beta2-adrenoceptor activation on the mitogen-activated protein kinase (MAPK) cascades and the contribution of these pathways to the suppression of tumor necrosis factor (TNF)-alpha in lipopolysaccharide (LPS)-stimulated rat renal mesangial cells. MATERIALS AND METHODS: Experiments were performed using cultured mesangial cells in the presence of LPS (1 microg/ml) and/or the beta2-adrenoceptor agonist, terbutaline (10(-6) 10(-8) M). The levels of extracellular signal-regulated kinase-1 and 2(Erk 1/2), p38, c-Jun N-terminal protein kinase (JNK) and TNF-alpha were estimated. RESULTS: LPS activated Erk-1/2 and p38 levels, by 4.7-fold and 1.8-fold, respectively (P < 0.05), which were suppressed by terbutaline (10(-6) - 10(-8) M) in a dose dependent way. These inhibitory actions of terbutaline were prevented by the beta2-adrenoceptor antagonist, ICI 118,551(10(-6) M) but not by an inhibitor of the cAMP-PKA pathway, H-89 (5 x 10(-6) M). The selective MAPK/Erk-1 inhibitor, PD98059 (10(-5) M) and the specific p38 inhibitor SB203580 (10(-5) M) significantly decreased LPS-induced TNF-alpha production in the cells. CONCLUSIONS: Inhibition of MAPK cascades (Erkl/2 and p38) plays an important role in the suppression of TNF-alpha following beta2-adrenoceptor activation but the inhibitory effect on MAPK is independent of the cAMP-PKA pathway in the mesangial cell.

Seminal immunoreactive relaxin in domestic animals and its relationship to sperm motility as a possible index for predicting the fertilizing ability of sires.

Although immunoassayable relaxin has been detected in human and boar seminal plasma, there is no evidence suggesting the existence of immunoreactive relaxin in the seminal plasma of other domestic animals. The first objective of this study was to determine whether immunoreactive relaxin was present in the seminal plasma of bulls, rams and he-goats. In addition, the correlation of immunoreactive relaxin with sperm motility as an index for predicting the fertilizing ability of bull sires was investigated. Semen with normal sperm motility was collected from bulls, rams and he-goats, and the relaxin immunoreactivity of the semen samples was measured using a time-resolved fluoroimmunoassay (TR-FIA) for porcine relaxin that we developed. The presence of relaxin immunoreactivity was demonstrated in seminal plasma from bulls, rams and he-goats. The level of immunoreactive relaxin in seminal plasma was the highest in bulls followed by humans, rams, boars and he-goats in that order, when relaxin levels in boar and human semen having normal sperm motility were also assayed under the same conditions. When the correlation between the seminal plasma level of immunoreactive relaxin and sperm motility was examined in bull semen samples as an index for predicting fertilizing ability, it was found that the relaxin level was significantly correlated with the percentage of spermatozoa showing the most intensive motility (r = 0.64, p < 0.05). These results indicate that immunoreactive relaxin is widely found in the seminal plasma of domestic animals and that measuring the relaxin concentration of seminal plasma may be useful to identify subfertile sires or predict the fertility potential of individual sires.

Predominance of Th1 immune response in diffuse proliferative lupus nephritis.

OBJECTIVE: Lupus nephritis, which shows various histologic patterns, is a serious complication of systemic lupus erythematosus (SLE). We previously demonstrated the importance of Thl cell-mediated immune response in patients with diffuse proliferative lupus nephritis (DPLN). The aim of this study was to examine the relationship between the peripheral blood Th1/Th2 balance and the intrarenal immune response. METHODS: The Th1:Th2 ratio in peripheral blood was measured by intracellular staining for cytokines with flow cytometry. Immunohistochemical analysis of renal biopsy specimens was performed to clarify the characterization of local infiltrating cells in 3 groups of subjects: SLE patients with World Health Organization (WHO) class IV nephritis (DPLN) (group I; n = 13), SLE patients with WHO class V nephritis (group II; n = 9), and patients with minor glomerular lesions (group III; n = 7). In addition, the histologic activity index and chronicity index were evaluated and correlated with the Th1:Th2 ratio. RESULTS: Immunohistochemical studies showed higher numbers of CD68+ macrophages, CD3 + T cells, and interferon-gamma-positive cells in group I than in groups II or III. Renal tissues from patients in group I also showed up-regulation of expression of osteopontin and CD40, with a small number of infiltrating T cells expressing interleukin-4. Overall, the Thl:Th2 ratio in group I patients (SLE with DPLN) was high and correlated significantly with the histologic activity index, but not with the chronicity index. CONCLUSION: We have identified a predominance of Thl-type response in both peripheral and renal tissues of patients with DPLN, suggesting that the peripheral blood Thl:Th2 ratio directly reflects the local histopathologic findings. In patients with lupus nephritis, the peripheral blood Th1:Th2 ratio could be useful as a parameter that reflects the renal histologic activity or the strength of the local Thl response.

The DSL domain in mutant JAG1 ligand is essential for the severity of the liver defect in Alagille syndrome.

Alagille syndrome (AGS) is a congenital multi-system anomaly mainly characterized by paucity of intrahepatic bile ducts caused by haploinsufficiency of the Jagged 1 gene (JAG1). To explore the relationship between genotype and phenotype, we analyzed the JAG1 gene in 25 Japanese AGS families at the genomic DNA level and identified 15 point mutations and one large deletion. Analysis of the genotype and phenotype strongly indicated that the Delta/Serrate/Lag-2 (DSL) domain in JAG1 protein played an essential role in determining the severity of the liver disorder. In four sporadic cases, missing an entire DSL domain in mutant JAG1 resulted in progressive liver failure and all 4 patients needed a liver transplant at a very young age. This correlation was further confirmed by statistical analysis (chi2=9.143, p<0.001). Our finding demonstrated that the DSL domain in JAG1 appears to be essential for normal liver development and function.

Nasal immunization with E. coli verotoxin 1 (VT1)-B subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies.

Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.

Morphologic evaluation and integrin expression profile of renal tubular cells cultured from percutaneous renal biopsy specimen.

Kidney biopsy is an indispensible procedure for making a pathologic diagnosis of renal diseases by fixing and staining the biopsy specimen. However, it is not a routine procedure to culture the cells from a renal biopsy specimen directly, or to utilize the cultured cells for any kind of diagnostic or functional evaluation. In this study, primary culture of the renal tubular epithelial cells was tried from a piece of percutaneous kidney biopsy specimen. Successive passages of the cells were possible until fourth passage. With these cells, morphologic characteristics of the cultured cells and integrin expression profiles were investigated. On light and electron microscopy, these cells were characterized by the cobblestone-like growth, presence of microvilli and tight junction, and the preservation of polarity. Immunohistochemical studies demonstrated the epithelial nature of these cells and particularly their differentiation from renal tubular epithelial cells, of either proximal or distal nephronic segment. The integrin profile confirms the epithelial nature of the cell. We hope that our results facilitate the understanding of pathophysiology of renal tubular cells from the patient directly.

Immunoreactive relaxin in seminal plasma of fertile boars and its correlation with sperm motility characteristics determined by computer-assisted digital image analysis.

Ejaculates from 10 mature fertile large white Yorkshire boars were used to examine the correlation between immunoreactive relaxin levels in seminal plasma and sperm motility characteristics. Seminal plasma levels of immunoreactive relaxin were measured by a time-resolved fluoroimmunoassay (TR-FIA). Motility characteristics were assessed using a CellSoft computer-assisted digital image analysis system. The mean +/- SD level of immunoreactive relaxin in seminal plasma was 2.61 +/- 0.62 ng/mL. When the correlation between seminal plasma levels of immunoreactive relaxin and parameters of sperm movement was examined, it was found that relaxin levels were significantly correlated with the percentage of motile spermatozoa (r=0.687, p < 0.05), curvilinear velocity (r=0.745, p < 0.05), straight line velocity (r=0.651, p < 0.05), mean amplitude of lateral head displacement (mean ALH) (r=0.844, p < 0.01) and the maximum amplitude of lateral head displacement (max ALH) (r=0.830, p < 0.01), but not with linearity, beat-cross frequency, or percentage of circular cells. Among these parameters, seminal plasma levels of immunoreactive relaxin showed the strongest correlation with the ALH parameter related to fertilizing ability. These results indicate that immunoreactive relaxin in boar semen may be necessary not only for normal sperm motility but also for normal fertility, suggesting that determination of the profile of immunoreactive relaxin in ejaculates may have value as a potential marker for predicting sperm fertilizing ability of boars.

Tumour rejection by gene transfer of 4-1BB ligand into a CD80(+) murine squamous cell carcinoma and the requirements of co-stimulatory molecules on tumour and host cells.

NRS1 is a murine squamous cell carcinoma that constitutively expresses the co-stimulatory molecule CD80 at a high level yet grows as a tumour in syngeneic C3H mice. We examined the effect of gene transfer of the 4-1BB ligand (4-1BBL) into NRS1 cells. Introduction of the 4-1BBL gene efficiently elicited anti-tumour immune responses in syngeneic mice which acquired specific immunity against wild-type tumour. T-cell depletion studies showed that CD8(+), but not CD4(+) T cells were essential for tumour eradication. Our results suggest that the transduced 4-1BBL is more effective than the spontaneously expressed CD80 for generation of primary anti-tumour CD8(+) T-cell responses. In addition to CD80 and CD86, the host-derived 4-1BBL is also involved in the secondary anti-tumour responses. This study indicates the complicated contribution of 4-1BBL, CD80 and CD86 on tumour and host cells in anti-tumour immune responses and a possible therapeutic application of 4-1BBL for human tumour vaccination and gene therapy.

beta(2)-adrenoceptor agonist suppresses renal tumour necrosis factor and enhances interleukin-6 gene expression induced by endotoxin.

BACKGROUND: beta(2)-Adrenoceptor activation regulates tumour necrosis factor (TNF)-alpha and interleukin-6 (IL-6) production in cultured renal cells. However, it remains uncertain whether, in vivo, the administration of beta(2)-adrenoceptor agonists regulate renal TNF-alpha and IL-6 mRNA following lipopolysaccharide (LPS) stimulation to cause endotoxaemia. This study was performed in order to evaluate the effect of beta(2)-adrenoceptor agonist on renal TNF-alpha and IL-6 production. METHODS: Four-week-old Wistar rats pre-treated with the beta(2)-adrenoceptor agonist terbutaline or formoterol, and/or the beta- and beta(2)-adrenoceptor antagonists (propanolol, ICI118,551), were injected with LPS (1 mg i.p.), and then 2, 4 or 6 h later, kidneys (cortex, medulla), spleen, thymus and plasma were collected to assay TNF-alpha and IL-6 mRNA levels and their respective protein release. RESULTS: Administration of beta(2)-adrenoceptor agonists suppressed TNF-alpha mRNA expression in the whole kidney, by 61% (P<0.05), as well as plasma, spleen and thymus TNF-alpha protein and mRNA expression 2 hours after injection of LPS. On the other hand, although IL-6 levels in plasma, spleen and thymus mRNA expression were suppressed significantly by administration of beta(2)-adrenoceptor agonists, the basal- and LPS-induced IL-6 mRNA levels in the whole kidney were increased 1.6- and 1.2-fold (P<0.05), respectively, by treatment with beta(2)-adrenoceptor agonists. beta(2)-Adrenoceptor agonist suppressed LPS-induced TNF-alpha mRNA expression by 35% (P<0.05) and stimulated LPS-induced IL-6 mRNA expression by 1.5-fold (P<0.05) in the medullary region of kidney. CONCLUSIONS: beta(2)-Adrenoceptor agonists down-regulate renal TNF-alpha mRNA expression following LPS-induced endotoxaemia. This effect was particularly apparent in the renal medulla. IL-6 mRNA expression in the renal medulla was up-regulated by the agonists whereas plasma, spleen and thymus IL-6 levels were completely inhibited by the agonist, which suggests the existence of tissue specific regulation of IL-6 production in the kidney by beta(2)-adrenoceptor activation.

Six novel mutations of the fibrillin-1 gene in Korean patients with Marfan syndrome.

BACKGROUND: Mutations in the FBN1 gene, encoding fibrillin-1, result in Marfan syndrome (MFS). According to previous reports, the mutations in FBN1 share certain characteristics in each family with variable penetrance and overlapping symptoms, even in the same genotype. In the present study, we report six novel mutations and evaluate the clinical significance of these nucleotide changes. METHODS: To screen for nucleotide changes in all 65 exons of the FBN1 gene in 38 unrelated Korean patients, we performed polymerase chain reaction, single-strand conformational polymorphism (SSCP) and sequencing for the shift of the band in SSCP. RESULTS: We identified six mutations: a 2253 del 7 b.p., N1043S, C1254S, L1421F, C1895R and S2662P. CONCLUSIONS: These results suggest that many different mutations are responsible for MFS in the Korean population.

Beta2-adrenoceptor agonist suppresses tumour necrosis factor production in rat mesangial cells.

This study aimed to investigate the time-course of the effect of beta2-adrenoceptor stimulation with terbutaline on lipopolysaccharide (LPS)-induced tumour necrosis factor(TNF)-alpha production in rat mesangial cells. Cells were cultured from 0-24 h in the presence of LPS (1 microg/ml) and/or terbutaline (10(-7)-10(-8) mol/l). After 1 h of incubation, terbutaline inhibited TNF-alpha protein release as well as transcription and translation of TNF-alpha and mitogen activated protein kinase (MAPK, p42/p44) activity. At 3 h, terbutaline enhanced intracellular cAMP but suppressed TNF-alpha release and transcription. By 24 h, whereas terbutaline was no longer influencing transcription or translation, TNF-alpha release remained depressed which correlated with an increase in supernatant interleukin (IL)-6. Terbutaline did not affect the LPS-induced IL-10 produced in the cell. These findings indicate that beta2-adrenoceptor stimulation during an LPS challenge prevented TNF-alpha production as a consequence of MAPK inhibition and enhanced cAMP generation, which at a later stage was associated with an anti-inflammatory effect of IL-6.

Attenuation correction in evaluating renal function in children and adults by a camera-based method.

UNLABELLED: Correction for soft-tissue attenuation is required to evaluate absolute renal function by a camera-based method, and an estimate of renal depth and an attenuation coefficient are commonly used for attenuation correction. The first goal of this study was to develop formulas for the calculation of renal depth in both children and adults. The second goal was to optimize the attenuation coefficient for the estimation of renal accumulation of a 99mTc-labeled agent. METHODS: Renal depth was measured by CT in 74 children and 232 adults and compared with the depth calculated using previously published equations. Multiple stepwise linear regression analysis was conducted using data from children and adults together, and new formulas to calculate renal depth were derived. Using the resulting equations, percentage renal uptake at 2-2.5 min was computed from 99mTc-diethylenetriamine pentaacetic acid (DTPA) renography in 40 children and 92 adults. Percentage renal uptake was assessed using various values of an attenuation coefficient, and an optimized attenuation coefficient was determined to maximize the correlation coefficient between percentage renal uptake and glomerular filtration rate (GFR) measured from 2 blood samples. RESULTS: Although the previously published equations appeared to be acceptable in predicting adult renal depth, they substantially underestimated pediatric renal depth. Renal depth (D, cm) was shown by stepwise regression analysis to depend on the ratio of body weight (W, kg) to body height (H, cm) and was successfully calculated in both children and adults using the derived equations (right: D = 16.778 x W/H + 0.752; left: D = 16.825 x W/H + 0.397). The correlation coefficient between percentage renal uptake of 99mTc-DTPA and measured GFR varied substantially according to the attenuation coefficient used and was the highest (0.947) with an attenuation coefficient of 0.087/cm. CONCLUSION: The equations presented here enabled estimation of renal depth irrespective of the patient's age. Attenuation correction using these equations and the optimized attenuation coefficient appears to aid in evaluating renal accumulation and, consequently, renal function in both children and adults.

Characterization and distribution of a new enterotoxin-related superantigen produced by Staphylococcus aureus.

Staphylococcal enterotoxins (SEs) are a family of structurally related pyrogenic exotoxins consisting of the five prototypic SEs (types A to E) and three newly characterized SEs (types G to I) produced by Staphylococcus aureus (S. aureus). They also work as superantigens and cause food poisoning and shock symptoms in humans. In this study, we cloned a new variant gene of the seg and characterized its superantigenic properties and distribution among the clinical isolates of S. aureus. The gene encodes a 233 amino acid protein which is highly homologous to SEG (97.7%). The variant SEG (SEGv) expressed by the cloned gene exerted mitogenic activity on human peripheral blood mononuclear cells at the concentration of 100 pg/ml. T cells bearing Vbeta3, 12, 13.1, 13.2, 14 and 15 were preferentially expanded after stimulation with the recombinant protein. The mRNA of the variant seg gene was detected in the total RNA of the organisms bearing this gene. By PCR, 27 out of 48 clinical isolates of S. aureus (56%) possessed either the seg or variant seg gene. These findings suggest that SEG, or SEGv, is one of the most frequently produced superantigen exotoxins by S. aureus and may participate in the inflammatory process of the host by activating a distinct set of Vbeta families of T cells.

Stat6 activation and Th2 cell differentiation [correction of proliferation] driven by CD28 [correction of CD28 signals].

CD28 engagement by specific monoclonal antibody (mAb) or binding of the natural ligands, CD80 and CD86, induces tyrosine phosphorylation of CD28, which in turn recruits and activates the signal transducer and activator of transcription 6 (Stat6). The Stat6 association with CD28 is specifically induced by CD80 or CD86 ligand binding and is not dependent upon the secretion of IL-4 or IL-13. Activated Stat6 translocates to the nucleus and binds to a Stat6-responsive element on the human IL-4 promoter. CD28 ligation induces Stat6-dependent transcriptional activation of a reporter gene under the control of a multimerized Stat6-responsive element fused to an essential part of the IL-4 promoter. Primary stimulation of naive CD4(+) T cells with anti-CD28 mAb in the presence of IL-2, but in the absence of anti-CD3 mAb induces preferential production of IL-4 and expression of CCR4 mRNA after secondary stimulation with anti-CD3, indicating the preferential differentiation of Th2 cells. These findings suggest that initial IL-4 production required for commitment of naive T cells toward Th2 cells may be provided in response to signals delivered via CD28 by antigen-presenting cells.

Blockade of CTLA-4 signals inhibits Th2-mediated murine chronic graft-versus-host disease by an enhanced expansion of regulatory CD8+ T cells.

CTLA-4 (CD152) is thought to be a negative regulator of T cell activation. Little is known about the function of CTLA-4 in Th2-type immune responses. We have investigated the effect of initial treatment with anti-CTLA-4 mAb on murine chronic graft-vs-host disease. Transfer of parental BALB/c splenocytes into C57BL/6 x BALB/c F1 mice induced serum IgE production, IL-4 expression by donor CD4+ T cells, and host allo-Ag-specific IgG1 production at 6-9 wk after transfer. Treatment with anti-CTLA-4 mAb for the initial 2 wk significantly reduced IgE and IgG1 production and IL-4 expression. Analysis of the splenic phenotype revealed the enhancement of donor T cell expansion, especially within the CD8 subset, and the elimination of host cells early after anti-CTLA-4 mAb treatment. This treatment did not affect early IFN-gamma expression by CD4+ and CD8+ T cells and anti-host cytolytic activity. Thus, blockade of CTLA-4 greatly enhanced CD8+ T cell expansion, and this may result in the regulation of consequent Th2-mediated humoral immune responses. These findings suggest a new approach for regulating IgE-mediated allergic immune responses by blockade of CTLA-4 during a critical period of Ag sensitization.

Transcriptional regulation of PDGF-A and TGF-beta by +KTS WT1 deletion mutants and a mutant mimicking Denys-Drash syndrome.

Denys-Drash syndrome (DDS) and Frasier syndrome (FS) are rare diseases caused by the mutations of Wilms tumor gene, WT1. The common denominator in these syndromes is a nephropathy which is manifested by early-onset proteinuria, nephrotic syndrome and end stage renal failure. Although these syndromes are genetic models of nephropathy and the mutations of WT1 gene are characterized in these patients the mechanism how mutations of WT1 gene affect the embryonic kidney adversely has not been elucidated. Recently, there was a report that FS is caused by mutations in the donor splice site of WT1. These mutations predicted loss of +KTS isoform, which is one of the four splicing variants of WT1. In this study, two +KTS deletion mutants of WT1 were made as well as a WT1 mutant mimicking a mutation found in a patient who had diffuse mesangial sclerosis, end stage renal failure and Wilms tumor. Mutant embryonic kidney cell lines were established by transfection of 293 embryonic kidney cells with WT1 mutants. We investigated the transcription regulation of mutant WT1 among these cell lines using the reporter vectors containing PDGF-A and TGF-beta promoter sequence. Our results showed that the promoter activity of PDGF-A and TGF-beta, which are related to the progression of glomerular diseases, was modestly increased in the mutant cell mimicking the patent, while those activities were markedly increased in other two deletion mutant cell lines. This study demonstrated that +KTS WT1 mutation found in DDS affected the cytokine expression adversely in vitro. From these results, we suggest that the alteration of +KTS WT1 expression be responsible for the rapid progression of renal diseases in DDS and FS.

Exon 9 mutations in the WT1 gene, without influencing KTS splice isoforms, are also responsible for Frasier syndrome.

We report new mutations in exon 9 of the WT1 gene that did not alter the ratio of +/- KTS splice isoforms in two unrelated patients with Frasier syndrome (FS). The mutation of intron 9 inducing defective alternative splicing was reported to be responsible for this syndrome. The mutations found in our cases occurred in the same exon of the WT1 gene as detected in Denys-Drash syndrome (DDS) and could not be explained by the previously proposed mechanism. The results suggest that the two syndromes originate from the same WT1 gene abnormality. From a molecular biological point of view, we concluded that the two diseases were not separable, and that FS should be included as an atypical form of DDS.