Progressive loss of mitochondrial DNA in thymidine kinase 2-deficient mice

Deficient enzymatic activity of the mitochondrial deoxyribonucleoside kinases deoxyguanosine kinase (DGUOK) or thymidine kinase 2 (TK2) cause mitochondrial DNA (mtDNA)-depletion syndromes in humans. Here we report the generation of a Tk2-deficient mouse strain and show that the mice develop essentially normally for the first week but from then on exhibit growth retardation and die within 2-4 weeks of life. Several organs including skeletal muscle, heart, liver and spleen showed progressive loss of mtDNA without increased mtDNA mutations or structural alterations. There were no major histological changes in skeletal muscle, but heart muscle showed disorganized and damaged muscle fibers. Electron microscopy showed mitochondria with distorted cristae. The Tk2-deficient mice exhibited pronounced hypothermia and showed loss of hypodermal fat and abnormal brown adipose tissue. We conclude that Tk2 has a major role in supplying deoxyribonucleotides for mtDNA replication and that other pathways of deoxyribonucleotide synthesis cannot compensate for loss of this enzyme.     

Human CXCR2 (hCXCR2) takes over functionalities of its murine homolog in hCXCR2 knockin mice

Human CXCR2 (hCXCR2) has been implicated in diverse inflammatory diseases. When roles of this receptor studied in animal models are extrapolated into men, large species differences in expression of the receptor and its ligands must be considered. These differences seriously weaken conclusions toward the role of hCXCR2 in the development of human diseases. It furthermore hampers straightforward testing of CXCR2 antagonists, especially when compounds discriminate between human and other species' receptors. Using gene targeting in embryonic stem cells, a hCXCR2 knockin mouse strain was generated in which endogenous murine CXCR2 (mCXCR2) sequences are replaced by the hCXCR2 gene. Correct targeting and expression on neutrophils were confirmed by Southern blot and immunohistochemical analyses. A phenotypic analysis of the hCXCR2 knockin mice, in comparison to wild-type and CXCR2 knockout mice, confirmed proper function of the hCXCR2 gene. In vivo migratory responses of neutrophils were intact in hCXCR2 knockin mice. Finally, an experiment with a CXCR2 antagonist demonstrated that the knockin model is indeed useful for in vivo evaluation of low-molecular weight compounds. In conclusion, our data unequivocally show that hCXCR2 can functionally replace mCXCR2, making this an attractive model to test novel pharmaceuticals designed to antagonize human CXCR2 in vivo.     

Ability of temperature-sensitive mutants of the recombinant influenza S/N (H2N1) virus to induce immunity to parental (H0N1 and H2N2) viruses.

The behavior in mice of two thermosensitive (ts) mutants (denoted ts217 and ts700) of the recombinant influenza virus S/N (H2N1) was studied. The parental thermoresistant (tr) virus and both of the mutants were capable of inducing protection against pneumotropic A/Singapore (H2N2) and A/WS (H0N1) challenge viruses. Immunity against the Singapore virus, with which the S/N virus shared the hemagglutinin, developed earlier than against the WS virus, with which the S/N virus shared the neuraminidase. The tr and ts217 viruses were immunologically more active than the ts700 virus. The first two viruses grew markedly better in mouse lungs than did the latter. In the course of ts217 virus replication in vivo, revertants capable of growing at 39 degrees C appeared readily. On the other hand, the ts700 virus proved to be genetically stable. These data seem to provide evidence of a linkage between the stability of the ts phenotype, reproductive capacity in mouse lungs, and immunogenicity in the viruses examined.     

Dmap1 plays an essential role in the maintenance of genome integrity through the DNA repair process

Faithful control of cell cycle checkpoint and DNA repair contributes to prevent the cells from chromosomal instability and tumorigenesis. Dnmt1-associated protein 1 (Dmap1), a component of the NuA4 histone acetyltransferase complex, was originally identified as an interacting molecule with DNMT1 which co-localizes with PCNA at DNA replication foci. However, its role in cellular functions remains largely unknown. Here we show that Dmap1 knockdown in mouse embryonic fibroblasts (MEFs) lead to spontaneous double-strand breaks (DSBs), resulting in growth arrest because of p53-dependent cell cycle checkpoint activation. Deletion of both Dmap1 and p53 in MEFs synergized towards enhanced generation of DSBs, chromosomal abnormalities and tumor development in mice. Notably, we found that, on DNA damage, Dmap1 was recruited to the damaged sites to form complexes with γ-H2AX and replication factors, including Pcna. Depletion of Dmap1 in MEFs abrogated the stable accumulation of Pcna at the DNA damaged sites. Furthermore, the re-introduction of Dmap1 mutants with a reduced capacity to bind Pcna failed to ameliorate the impaired DNA repair in Dmap1-depleted cells. These findings indicate that Dmap1 is required to involve Pcna in DNA repair. Together, Dmap1 plays a crucial role in DNA repair, and is indispensable for the maintenance of chromosomal integrity.     

Further genetic evidence for maintenance of early Hong Kong-like influenza A(H3N2) strains in swine until 1976

The oligonucleotide map of the whole RNA of A/swine/Hong Kong/3/76 (H3N2) virus showed a relationship to, but considerable differences from, the maps of human isolates of influenza A(H3N2) with which this isolate antigenically cross-reacted. Comparison of the individual oligonucleotide spots of whole virus RNAs confirmed previous results of antigenic analysis that A/swine/Hong Kong/3/76 virus was more similar to an early A/Hong Kong/68 virus than to later H3N2 viruses which circulated in man during 1976. On the other hand, another swine isolate, A/swine/Hong Kong/4/76, showed a quite similar oligonucleotide map to that of the contemporary prevalent A/Victoria/75-like viruses to which this strain was reported to be antigenically similar. Comparison of oligonucleotide maps of individual RNA segments indicated that all genes of the A/swine/Hong Kong/3/76 virus were derived from a human H3N2 virus. The findings provide biochemical evidence that A/swine/Hong Kong/3/76 virus represents a 1968 Hong Kong-like virus that underwent genetic mutation without extensive change of its antigenicity during maintenance in the swine population.     

Protective effects of mate tea (Ilex paraguariensis) on H2O2-induced DNA damage and DNA repair in mice

Yerba mate (Ilex paraguariensis) is rich in several bioactive compounds that can act as free radical scavengers. Since oxidative DNA damage is involved in various pathological states such as cancer, the aim of this study was to evaluate the antioxidant activity of mate tea as well as the ability to influence DNA repair in male Swiss mice. Forty animals were randomly assigned to four groups. The animals received three different doses of mate tea aqueous extract, 0.5, 1.0 or 2.0 g/kg, for 60 days. After intervention, the liver, kidney and bladder cells were isolated and the DNA damage induced by H(2)O(2) was investigated by the comet assay. The DNA repair process was also investigated for its potential to protect the cells from damage by the same methodology. The data presented here show that mate tea is not genotoxic in liver, kidney and bladder cells. The regular ingestion of mate tea increased the resistance of DNA to H(2)O(2)-induced DNA strand breaks and improved the DNA repair after H(2)O(2) challenge in liver cells, irrespective of the dose ingested. These results suggest that mate tea could protect against DNA damage and enhance the DNA repair activity. Protection may be afforded by the antioxidant activity of the mate tea's bioactive compounds.     

Changes in the hemagglutinin molecule of influenza type A (H3N2) virus associated with increased virulence for mice

Summary. The H3N2 influenza virus A/Philippines/82 (Phil82) and its bovine serum-resistant mutant, Phil82/BS, were used to investigate factors that influence virulence of influenza virus for mice. Phil82/BS, which lacks the high-mannose oligosaccharide at residue 165 of the hemagglutinin (HA) molecule, was found to replicate to a much higher titer in mouse lung than the parent Phil82, and had acquired lethality for mice. Further adaptation of Phil82/BS by sequential lung passage in mice yielded a strain of greater virulence, Phil82/BS/ML10, in which a change at residue 246 of HA resulted in loss of a second potential glycosylation site. Phil82 is highly sensitive to neutralization in vitro by murine serum- and lung-associated mannose-binding lectins (collectins). Characterization of the two mutant viruses indicated that resistance to murine collectins can account for the enhanced virulence of Phil82/BS but not for the further increase in virulence of Phil82/BS/ML10. Evidence is presented that residue 246 is not in fact glycosylated in Phil82/BS HA, nor presumably in the parent Phil82 virus. The HA molecule of Phil82/BS/ML10 displayed functional differences from Phil82/BS, including a change in the optimum pH of fusion and a minor change in receptor-binding specificity, which may allow improved efficiency of replication in the mouse lung. Received June 27, 1996 Accepted August 8, 1996     

Development of immunoenzyme assay for subtype-specific detection of antibodies to influenza viruses A (H1N1) and A (H3N2)

Conditions were developed for obtaining surface viral glycoprotein (GP) fraction intended for solid phase sensitization with the aim of constructing enzyme immunoassay test systems (EIATS) for detection of subtypical IgG and IgG to influenza A (H1N1) and A (H3N2) viruses. New variants of test systems were compared with the traditional methods for serological diagnosis of influenza. GP-based EIATS more often diagnosed influenza than EIATS based on purified whole-virion (WV) suspensions, hemagglutination inhibition and neutralization tests. Evaluation of conversions of subtypical IgG showed that the results coincided with the findings of neutralization and hemagglutination inhibition tests in 83-90% (EIATS-GP) and 50% (EIATS-WV). Cross-detection of antibodies to both virus subtypes in EIATS-GP and EIATS-WV was observed in 4 and 31% cases, respectively.     

The inoculative properties of cold-adapted reassortant A(H5N2) influenza strain during intranasal administration to mice

Classical genetic reassortant techniques were used to have a cold-adapted (ca) reassortant A/17/Duck/Potsdam/86/92 (H5N2) that inherited the hemagglutinin (HA) gene from the nonpathogenic avian virus A/Duck/Potsdam/ 1402-6186 (H5N2) and the genes of neuraminidase (NA) and non-glycated proteins from the ca attenuation donor A/Leningrad/134/17/57 (H2N2). All experiments were performed under increased biological protection (BSV-3+). The reassortant and parent H5N2 virus were non-pathogenic to Balb/c mice, the reassortant replication in the murine nasal passages (3.5 Ig EID50/ml) being higher than that in the lung (2.1 lg EID50/ml). Intranasal inoculation of mice with reassortant A/17/Duck/Potsdam/86/92 caused an immune response to both homological H5N2 virus and antigenically differing variants of influenza A (H5N1) virus isolated from humans in 1997 and 2003. The mice intranasally immunized with the ca reassortant were protected against fatal infection with the highly pathogenic A/Hong Kong/483/9797 (H5N1) virus and against infection with A/Hong Kong/213/03(H5N1) virus (80 and 100%, respectively).     

Antibiotic use during infancy promotes a shift in the T(H)1/T(H)2 balance toward T(H)2-dominant immunity in mice

BACKGROUND: Recent epidemiologic studies indicate that antibiotic use in infancy may be associated with an increased risk of development of atopy; however, its precise mechanism remains to be elucidated. OBJECTIVE: The purpose of this study is to clarify whether one such antibiotic, kanamycin, affects the T(H)1/T(H)2 balance. METHODS: BALB/c mice at 3 and 52 weeks of age were orally administered 600 mg/d kanamycin sulfate for 7 consecutive days. Blood samples were collected on weeks 0, 10, 18, and 26 after the cessation of kanamycin treatment, and the effect of the kanamycin treatment on the T(H)1/T(H)2 balance was evaluated on the basis of both the in vivo antibody levels and the in vitro splenocyte cytokine secretion pattern. RESULTS: The administration of kanamycin increased the serum levels of total IgG1 and IgE while decreasing the serum IgG2a levels. Moreover, when spleen cells were stimulated with immobilized anti-CD3 antibody in vitro, such kanamycin treatment enhanced the in vitro IL-4 secretion while reducing the in vitro IFN-gamma secretion. The basal IL-12 p70 secretion levels of splenic dendritic cells in the kanamycin-treated mice were lower than those in the control mice, although no significant difference was seen in IL-12 p40 levels between either group of mice. CONCLUSION: These results suggested that antibiotic use during infancy may indeed quantitatively disturb, qualitatively disturb, or both the intestinal microflora and thereby prevent postnatal T(H)1 cell maturation, thus resulting in a T(H)2-polarized immune deviation.     

Antiviral activity of baicalin against influenza A (H1N1/H3N2) virus in cell culture and in mice and its inhibition of neuraminidase

Scutellaria baicalensis Georgi, a Chinese herbal decoction, has been used for the treatment of the common cold, fever and influenza virus infections. In previous studies, we found that oral administration of baicalein resulted in the inhibition of influenza A virus replication in vivo, which was linked to baicalin in serum. However, the effective dose and underlying mechanisms of the efficacy of baicalin against influenza A virus have not been fully elucidated. In this study, the antiviral effects of baicalin in influenza-virus-infected MDCK cells and mice were examined. The neuraminidase inhibition assay was performed to investigate the mechanism of action of baicalin. In vitro results showed that baicalin exhibited a half-maximal effective concentration (EC₅₀) of 43.3 μg/ml against the influenza A/FM1/1/47 (H1N1) virus and 104.9 μg/ml against the influenza A/Beijing/32/92 (H3N2) virus. When added to MDCK cell cultures after inoculation with influenza virus, baicalin demonstrated obvious antiviral activity that increased in a dose-dependent manner, indicating that baicalin affected virus budding. Baicalin had clear inhibitory effects against neuraminidases, with half-maximal inhibitory concentration (IC₅₀) of 52.3 μg/ml against the influenza A/FM1/1/47 (H1N1) virus and 85.8 μg/ml against the influenza A/Beijing/32/92 (H3N2) virus. In vivo studies showed that an intravenous injection of baicalin effectively reduced the death rate, prolonged the mean day to death (MDD) and improved the lung parameters of mice infected with influenza A virus. These results demonstrate that baicalin acts as a neuraminidase inhibitor, with clear inhibitory activities that are effective against different strains of influenza A virus in both cell culture and a mouse model, and that baicalin has potential utility in the management of influenza virus infections.     

Vaccination of an adolescent group with A (H3N2) + A(HSW1N1) bivaccine before an A (H1N1) influenza epidemic.

A group of 35 adolescents aged 18--22 years were vaccinated intradermally with 0.2 ml of a mixed A (H3N2) + A (Hsw1N1) formolized vaccine and revaccinated with A (Hsw1N1) monovaccine 10 days before an A (H1N1) influenza epidemic. The vaccination had no effect on morbidity or the clinical course of disease. Serology revealed a primary immune response to A (Hsw1N1) and a booster response to A (H3N2). Apparently, prevention of a new influenza subtype by formolized vaccine possessing only the corresponding neuraminidase type is ineffective.     

Indication for a role of regulatory T cells for the advent of influenza A (H1N1)-related pneumonia

Regulatory T cells (T_regs) have an anti‐inflammatory role. A former study in a limited number of patients found that absolute counts of T_regs increase when infection by the new influenza H1N1 virus is complicated with pneumonia. These results generate the question if H1N1‐related pneumonia is associated with a state of hypo‐inflammation. A total of 135 patients were enrolled with blood sampling within less than 24 h from diagnosis; 23 with flu‐like syndrome; 69 with uncomplicated H1N1‐infection; seven with bacterial pneumonia; and 36 with H1N1‐related pneumonia. T_regs and CD14/HLA‐DR co‐expression were estimated by flow cytometry; concentrations of tumour necrosis factor‐alpha (TNF‐α), of interleukin (IL)‐6 and of soluble triggering receptor expressed on myeloid cells‐1 (sTREM‐1) by an enzyme immunoassay; those of procalcitonin (PCT) by immuno‐time‐resolved amplified cryptate technology assay. Expression of human leucocyte antigen D‐related (HLA‐DR) on monocytes was similar between groups; absolute T_reg counts were greater among patients with H1N1‐related pneumonia than flu‐like syndrome or H1N1‐uncomplicated infection. Serum TNF‐α of patients with bacterial pneumonia was greater than those of other groups, but IL‐10 was similar between groups. Serum PCT was greater among patients with H1N1‐related pneumonia and sTREM‐1 among those with H1N1‐related pneumonia. Regression analysis revealed that the most important factors related with the advent of pneumonia were the existence of underlying illnesses (P = 0·006) and of T_regs equal to or above 16 mm³ (P = 0·013). It is concluded that the advent of H1N1‐related pneumonia is related to an early increase of the absolute T_reg counts. This increase is probably not part of a hypo‐inflammatory state of the host.     

The role of anti-HSP70 autoantibody-forming V(H)1-J(H)1 B-1 cells in Toxoplasma gondii-infected mice

Anti-heat shock protein 70 (HSP70) autoantibody formation was induced by B-1 cells (CD5(+) B cells) in Toxoplasma gondii-infected mice. Here we report that V(H)1-J(H)1 B-1 cells from peritoneal exudate cells (PEC) of T. gondii-infected C57BL/6 mice (B6, a susceptible strain) increased predominantly. Moreover, the hybridoma lines producing anti-T. gondii HSP70 (TgHSP70) antibody cross-reactive with mouse HSP70 (mHSP70) expressed the V(H)1-J(H)1 gene, whereas the hybridoma lines producing anti-TgHSP70 antibody non-cross-reactive with mHSP70 expressed the V(H)11A-J(H)1 gene or V(H)12-J(H)1 gene. The avidity maturation of anti-TgHSP70 IgG antibody in the sera of BALB/c mice (a resistant strain) and that of anti-mHSP70 IgG autoantibody in the sera of B6 mice were observed 9 weeks after T. gondii infection. T. gondii numbers in the brains of T. gondii-infected B6 mice treated with anti-mHSP70 autoantibody were markedly higher than those in the brains of T. gondii-infected B6 mice treated with anti-TgHSP70 antibody. Furthermore, B-1 cells producing IL-10 down-regulated the IFN-gamma expression of PEC in T. gondii-infected mice. These results indicate that B-1 cells dominantly expressing V(H)1-J(H)1 mRNA, and producing anti-HSP70 autoantibody and IL-10 regulate susceptibility of mice to T. gondii infection.     

Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint

Chk1, an evolutionarily conserved protein kinase, has been implicated in cell cycle checkpoint control in lower eukaryotes. By gene disruption, we show that CHK1 deficiency results in a severe proliferation defect and death in embryonic stem (ES) cells, and peri-implantation embryonic lethality in mice. Through analysis of a conditional CHK1-deficient cell line, we demonstrate that ES cells lacking Chk1 have a defective G(2)/M DNA damage checkpoint in response to gamma-irradiation (IR). CHK1 heterozygosity modestly enhances the tumorigenesis phenotype of WNT-1 transgenic mice. We show that in human cells, Chk1 is phosphorylated on serine 345 (S345) in response to UV, IR, and hydroxyurea (HU). Overexpression of wild-type Atr enhances, whereas overexpression of the kinase-defective mutant Atr inhibits S345 phosphorylation of Chk1 induced by UV treatment. Taken together, these data indicate that Chk1 plays an essential role in the mammalian DNA damage checkpoint, embryonic development, and tumor suppression, and that Atr regulates Chk1.