A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice.

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. Here, we introduce these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, we find that medium spiny neurons have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans.

Halogenated Boroxine K2[B3O3F4OH] Modulates Metabolic Phenotype and Autophagy in Human Bladder Carcinoma 5637 Cell Line.

Halogenated boroxine K2[B3O3F4OH] (HB), an inorganic derivative of cyclic anhydride of boronic acid, is patented as a boron-containing compound with potential for the treatment of both benign and malignant skin changes. HB has effectively inhibited the growth of several carcinoma cell lines. Because of the growing interest in autophagy induction as a therapeutic approach in bladder carcinoma (BC), we aimed to assess the effects of HB on metabolic phenotype and autophagy levels in 5637 human bladder carcinoma cells (BC). Cytotoxicity was evaluated using the alamar blue assay, and the degree of autophagy was determined microscopically. Mitochondrial respiration and glycolysis were measured simultaneously. The relative expression of autophagy-related genes BECN1, P62, BCL-2, and DRAM1 was determined by real-time PCR. HB affected cell growth, while starvation significantly increased the level of autophagy in the positive control compared to the basal level of autophagy in the untreated negative control. In HB-treated cultures, the degree of autophagy was higher compared to the basal level, and metabolic phenotypes were altered; both glycolysis and oxidative phosphorylation (OXPHOS) were decreased by HB at 0.2 and 0.4 mg/mL. Gene expression was deregulated towards autophagy induction and expansion. In conclusion, HB disrupted the bioenergetic metabolism and reduced the intracellular survival potential of BC cells. Further molecular studies are needed to confirm these findings and investigate their applicative potential.

Standardized, systemic phenotypic analysis of Slc12a1I299F mutant mice.

BACKGROUND: Type I Bartter syndrome is a recessive human nephropathy caused by loss-of-function mutations in the SLC12A1 gene coding for the Na+-K+-2Cl- cotransporter NKCC2. We recently established the mutant mouse line Slc12a1I299F exhibiting kidney defects highly similar to the late-onset manifestation of this hereditary human disease. Besides the kidney defects, low blood pressure and osteopenia were revealed in the homozygous mutant mice which were also described in humans. Beside its strong expression in the kidney, NKCC2 has been also shown to be expressed in other tissues in rodents i.e. the gastrointestinal tract, pancreatic beta cells, and specific compartments of the ear, nasal tissue and eye. RESULTS: To examine if, besides kidney defects, further organ systems and/or metabolic pathways are affected by the Slc12a1I299F mutation as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the mutant mouse line Slc12a1I299F in the German Mouse Clinic. Slc12a1I299F homozygous mutant mice and Slc12a1I299F heterozygous mutant littermates as controls were tested at the age of 4-6 months. Beside the already published changes in blood pressure and bone metabolism, a significantly lower body weight and fat content were found as new phenotypes for Slc12a1I299F homozygous mutant mice. Small additional effects included a mild erythropenic anemia in homozygous mutant males as well as a slight hyperalgesia in homozygous mutant females. For other functions, such as immunology, lung function and neurology, no distinct alterations were observed. CONCLUSIONS: In this systemic analysis no clear primary effects of the Slc12a1I299F mutation appeared for the organs other than the kidneys where Slc12a1 expression has been described. On the other hand, long-term effects additional and/or secondary to the kidney lesions might also appear in humans harboring SLC12A1 mutations.

A novel N-ethyl-N-nitrosourea-induced mutation in phospholipase Cγ2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model.

OBJECTIVE: It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model. METHODS: In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic. RESULTS: A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo. CONCLUSION: These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.

Microphthalmia, parkinsonism, and enhanced nociception in Pitx3 ( 416insG ) mice.

A new spontaneous mouse mutant was characterized by closed eyelids at weaning and without apparent eyes (provisional gene name, eyeless; provisional gene symbol, eyl). The mutation follows a recessive pattern of inheritance and was mapped to the region of chromosome 19 containing Pitx3. Genetic complementation tests using Pitx3 ( ak/+ ) mice confirmed eyl as a new allele of Pitx3 (Pitx3 ( eyl )). Sequencing of the Pitx3 gene in eyl mutants identified an inserted G after cDNA position 416 (416insG; exon 4). The shifted open reading frame is predicted to result in a hybrid protein still containing the Pitx3 homeobox, but followed by 121 new amino acids. The novel Pitx3 ( eyl/eyl ) mutants expressed ophthalmological and brain defects similar to Pitx3 ( ak/ak ) mice: microphthalmia or anophthalmia and loss of dopamine neurons of the substantia nigra. In addition, we observed in the homozygous eyeless mutants increased extramedullary hematopoiesis in the spleen, frequently liver steatosis, and reduced body weight. There were also several behavioral changes in the homozygous mutants, including reduced forelimb grip strength and increased nociception. In addition to these alterations in both sexes, we observed in female Pitx3 ( eyl/eyl ) mice increased anxiety-related behavior, reduced locomotor activity, reduced object exploration, and increased social contacts; however, we observed decreased anxiety-related behavior and increased arousal in males. Most of these defects identified in the new Pitx3 mutation are observed in Parkinson patients, making the Pitx3 ( eyl ) mutant a valuable new model. It is the first mouse mutant carrying a point mutation within the coding region of Pitx3.

Generation and characterization of dickkopf3 mutant mice.

dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity.

Microfilariae of the filarial nematode Litomosoides sigmodontis exacerbate the course of lipopolysaccharide-induced sepsis in mice.

Helminths facilitate their own survival by actively modulating the immune systems of their hosts. We investigated the impacts that different life cycle stages of the rodent filaria Litomosoides sigmodontis have on the inflammatory responses of mice injected with sublethal doses of lipopolysaccharide (LPS). Mice infected with female adult worms from prepatent infections, worms which have not yet started to release microfilariae, developed lower levels of proinflammatory cytokines in the peripheral blood after LPS challenge than sham-treated controls, demonstrating that female adult worms can mitigate the innate immune response. The presence of microfilariae in mice, however, through either direct injection or implantation of microfilaria-releasing adult female worms, turned the LPS challenge fatal. This lethal outcome was characterized by increased plasma levels of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin 12 (IL-12), and IL-6, greater numbers of macrophages and granulocytes in the peripheral blood, and decreased body temperatures in microfilaria-infected mice. Microfilaria-infected mice deficient in IFN-gamma receptor and TNF receptor 1 had increased survival rates after LPS challenge compared to immune-competent mice, suggesting that microfilariae worsen LPS-induced sepsis through actions of IFN-gamma and TNF-alpha. In summary, we have demonstrated that infection of mice with L. sigmodontis female adult worms from prepatent infections protects mice injected with LPS whereas microfilariae worsen LPS-induced sepsis through the induction of proinflammatory cytokines and upregulation of granulocytes, NK cells, and monocytes in the peripheral blood.

A genetic screen for modifiers of the delta1-dependent notch signaling function in the mouse.

The Notch signaling pathway is an evolutionarily conserved transduction pathway involved in embryonic patterning and regulation of cell fates during development. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, which are also involved in distinct human diseases. Delta1 is one of the known ligands of the Notch receptors. Mice homozygous for a loss-of-function allele of the Delta1 gene Dll1(lacZ/lacZ) die during embryonic development. Here, we present the results of two phenotype-driven modifier screens. Heterozygous Dll1(lacZ) knockout animals were crossed with ENU-mutagenized mice and screened for dysmorphological, clinical chemical, and immunological variants that are dependent on the Delta1 loss-of-function allele. First, we show that mutagenized heterozygous Dll1(lacZ) offspring have reduced body weight and altered specific clinical chemical parameters, including changes in metabolites and electrolytes relevant for kidney function. In our mutagenesis screen we have successfully generated 35 new mutant lines. Of major interest are 7 mutant lines that exhibit a Dll1(lacZ/+)-dependent phenotype. These mutant mouse lines provide excellent in vivo tools for studying the role of Notch signaling in kidney and liver function, cholesterol and iron metabolism, cell-fate decisions, and during maturation of T cells in the immune system.

Serum sperm antibodies are not elevated after mumps orchitis.

OBJECTIVE: To assess the level of serum sperm antibodies after mumps orchitis. DESIGN: Controlled descriptive study. SETTING: Academic research environment. PATIENT(S): Seventy-four mumps orchitis patients. INTERVENTION(S): Sampling of serum at different intervals after the onset of orchitis symptoms: 1 to 7 days, 31 to 60 days, and 61 to 431 days. MAIN OUTCOME MEASURE(S): Level of serum sperm antibodies, using Kibrick's gelatin agglutination test, Friberg's tray agglutination test, Isojima's sperm immobilization test, and ELISA. RESULT(S): Clinically relevant sperm antibody values were detected by the Friberg method among patients tested from 1 to 7 days (10.5%) and 61 to 431 days (10.5%) after the onset of disease. The Isojima test revealed a statistically insignificant higher incidence among patients at 61 to 431 days (31.6%) as compared with those sampled at 1 to 7 days (10.5%). None of the orchitis sera tested positive by the Kibrick and ELISA techniques. The established incidences did not differ significantly from the results for negative controls (blood donors) and were lower than the values acquired from positive controls (males with unexplained infertility). CONCLUSION(S): Mumps orchitis does not cause enhanced humoral immunity to spermatozoa.

Circulating antibodies to human spermatozoa in patients with ulcerative colitis.

The existence of enhanced humoral immunity against sperm antigens in patients with ulcerative colitis might be a result of the increased intestinal permeability, and may be associated with immunization against antigens of the common intestinal flora possessing common antigenicity with spermatozoa. The result of these two processes is statistically significant increased antisperm antibody (ASA) incidence in patients with ulcerative colitis compared to the healthy blood donors of proven fertility without clinical symptoms of inflammatory bowel diseases at the same age.

Sex-dependent susceptibility to Listeria monocytogenes infection is mediated by differential interleukin-10 production.

It is well documented that sex-dependent factors affect susceptibility to infection, with most mouse models demonstrating higher resistance in females. We made the unexpected observation that infection with the intracellular bacterium Listeria monocytogenes showed an opposite pattern in several commonly used inbred mouse strains: female C57BL/6J, BALB/c, C3H/HeN, and CBA/J mice were significantly more susceptible to Listeria infection. The pronounced sensitivity of females to Listeria, which was revealed by significantly higher lethality rates, correlated also with increased bacterial numbers in organ tissues (spleen and liver) and several immunological changes in peripheral blood samples. Surprisingly, increased severity of infection in females was associated with elevated interleukin-10 (IL-10) levels in plasma. Experiments using Il10 knockout mice, for which no differences between the susceptibilities of males and females to Listeria infection could be detected, confirmed the important role of this immunosuppressive cytokine for the outcome of disease. Our findings are likely to have clinical relevance, since similar sex differences with regard to infection with Listeria monocytogenes and other intracellular pathogens have been reported for humans.

Chemokine receptor Ccr2 deficiency reduces renal disease and prolongs survival in MRL/lpr lupus-prone mice.

MRL/MpJ-Fas(lpr)/J (MRL/lpr) mice represent a well-established mouse model of human systemic lupus erythematosus. MRL/lpr mice homozygous for the spontaneous lymphoproliferation mutation (lpr) are characterized by systemic autoimmunity, massive lymphadenopathy associated with proliferation of aberrant T cells, splenomegaly, hypergammaglobulinemia, arthritis, and fatal immune complex-mediated glomerulonephritis. It was reported previously that steady-state mRNA levels for the chemokine (C-C motif) receptor 2 (Ccr2) continuously increase in kidneys of MRL/lpr mice. For examining the role of Ccr2 for development and progression of immune complex-mediated glomerulonephritis, Ccr2-deficient mice were generated and backcrossed onto the MRL/lpr genetic background. Ccr2-deficient MRL/lpr mice developed less lymphadenopathy, had less proteinuria, had reduced lesion scores, and had less infiltration by T cells and macrophages in the glomerular and tubulointerstitial compartment. Ccr2-deficient MRL/lpr mice survived significantly longer than MRL/lpr wild-type mice despite similar levels of circulating immunoglobulins and comparable immune complex depositions in the glomeruli of both groups. Anti-dsDNA antibody levels, however, were reduced in the absence of Ccr2. The frequency of CD8+ T cells in peripheral blood was significantly lower in Ccr2-deficient MRL/lpr mice. Thus Ccr2 deficiency influenced not only monocyte/macrophage and T cell infiltration in the kidney but also the systemic T cell response in MRL/lpr mice. These data suggest an important role for Ccr2 both in the general development of autoimmunity and in the renal involvement of the lupus-like disease. These results identify Ccr2 as an additional possible target for the treatment of lupus nephritis.

Antichlamydial and antisperm antibodies in patients with chlamydial infections.

PROBLEM: Establishing the correlation between antichlamydial antibodies (AchAbs) and antisperm antibodies (ASA) in patients with chlamydial infections. METHOD OF STUDY: ASA were studied in sera from patients (142 with genital, 57 with ocular chlamydial infections) and control group (n = 100) by gelatin and tray agglutination test (TAT), sperm immobilization test (SIT) and ELISA. AchAbs were revealed by ELISA. RESULTS: A significantly higher (P < 0.05) ASA incidence was noted in patients with genital infections as compared with controls and patients with ophthalmologic infection (P < 0.0001), but not between patients with ophthalmologic infection and controls (P > 0.05). A significant correlation was established between AchAbs and ASA for TAT (r = 0.8214, P = 0.0341), SIT (r = 0.797, P = 0.032) and ELISA (r = 0.8519, P = 0.0313) in patients with genital infections only. CONCLUSIONS: The genital Chlamydia infection may play a role in the induction of ASA. This is probably a result of the inflammatory process, but not of cross-reactivity between sperm and Chlamydia trachomatis antigens.

Porcine eye lens crystallins: antigenic similarity with human crystallins and tool for the detection of anti-crystallin antibodies.

BACKGROUND: The usual sources of antigenic material for investigations on circulating immunoglobulins with anti-lens crystallins specificity are saline extracts of human cataract lenses. This practice has a number of drawbacks, especially the possible antigenic alterations that may have occurred in cataract lenses. The aim of this investigation was to compare the antigenic properties of porcine eye lens crystallins and human crystallins, with regard to the possibility for an alternative source of antigenic material for detection of anti-crystallin antibodies in human sera. METHODS: We produced rabbit antisera against saline extracts of human and porcine eye lenses. These sera were applied for the antigenic characterizations of the two extracts with indirect and absorption enzyme-linked immunosorbent assay. The two antigens were further compared by testing them against 30 human sera from cataract patients and 30 sera from blood donors. RESULTS: The antibodies raised against human eye lens cross-reacted with antigens of the porcine lens. This finding was supported by the absorption experiments - the antigens of the porcine eye lens strongly inhibit the reactivity of the rabbit serum raised against human eye lens and vice versa. We established a significant positive correlation (Spearman, r=0.89, P<0.0001) between the reactivity of the tested sera against human and porcine lens extracts. CONCLUSION: These data demonstrated a strong antigenic similarity between human and porcine lens crystallins, suggesting the appropriateness of the use of porcine lens extracts for the detection of humoral anti-lens autoimmune response in patients with eye diseases.

The age-related changes in the incidence of 'natural' anti-sperm antibodies suggest they are not auto-/isoantibodies.

PROBLEM: Establishing the age-dependent patterns of sperm antibody levels among normal humans. METHODS OF STUDY: Sera samples from 498 healthy subjects aged 0-97 years - 246 males and 252 females - were tested by the gelatin agglutination test of Kibrick, tray agglutination test of Friberg, sperm immobilization test of Isojima and enzyme-linked immunosorbent assay (ELISA). RESULTS: We found a significant increase in the level of sperm agglutinins after 40 years, which decreased after 88 years. The antibodies detected by ELISA were the highest among prepubertal subjects and also declined with aging. No age-dependent changes were established for the sperm immobilizins. With few exceptions, there were no significant differences between male and female sera, as well as between sera of newborn and their mothers. CONCLUSIONS: These data are similar to those established for the age-dependent changes of antibodies towards exogenous antigens, suggesting that the 'naturally occurring' antibodies against human spermatozoa are not auto-/isoantibodies.