[Effects of Rad9 mutants with impaired DNA mismatch repair function on tumorigenesis of colorectal cancer].

OBJECTIVE: The aim of this study was to investigate the effects of Rad9 mutants with impaired DNA mismatch repair (MMR) function on the tumorigenesis of colorectal cancer. METHODS: The colorectal cancer tumor samples were collected from 100 patients. The mutation profiles of human Rad9 (hRad9) gene in these samples were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing. The plasmid of pFLAG-hRad9 (L101M) was constructed following the QuickChange mutagenesis procedure and transfected into mRad9-deleted mouse cells (mRad9(-/-) cells). The expression of hRad9 protein was measured by western blot analysis. The MMR activity in live cells was detected by flow cytometry using the reporter plasmid for MMR function. RESULTS: Mutation from Leu to Met at the residue 101 (L101M) of hRad9 gene was detected in 7 of the 100 samples. The mismatch repair efficiency of mRad9(-/-)+ L101M cells (mRad9-deleted mouse cells with ectopic expression of L101M hRad9 gene) was (34.0±5.6)%, which was significantly lower than that in the mRad9(-/-)+ hRad9 cells [mRad9-deleted mouse cells with ectopic expression of hRad9 gene, (48.0±7.5)%, P<0.05]. After N-nitroso-N-methylurea (MNU) treatment, the survival rate of mRad9(-/-)+ L101M cells was (33.7±5.9)%, which was significantly higher than that in the mRad9(-/-)+ hRad9 cells [(21.3±4.7)%, P<0.05]. Thus, ectopic expression of L101M hRad9 gene resulted in significantly reduced MMR activity and increased resistance to MNU. Furthermore, ectopic expression of hRad9 gene with mutation at the target residues of post-translational modification in mRad9(-/-) cells also led to a reduced MMR activity. CONCLUSION: Rad9 mutants with impaired DNA mismatch repair function may promote tumorigenesis of colorectal cancer.

[Treatment response of a two-dose regimen of dose-adjusted inotuzumab ozogamicin in relapsed/refractory B-cell acute lymphoblastic leukemia].

Objective: To observe the treatment response of a two-dose regimen of inotuzumab ozogamicin (inotuzumab), a monoclonal antibody targeting CD22, for patients with heavily treated relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), including those failed or relapsed after chimeric antigen receptor (CAR) -T-cell therapy. Methods: Pediatric and adult patients who received two doses of inotuzumab and who were evaluated after inotuzumab treatment were included. Antibody infusions were performed between March 2020 and September 2022. All patients expressed CD22 antigen as detected by flow cytometry (>80% leukemic cells displaying CD22) before treatment. For adults, the maximum dosage per administration was 1 mg (with a total of two administrations). For children, the maximum dosage per administration was 0.85 mg/m(2) (no more than 1 mg/dose; total of two administrations). The total dosage administered to each patient was less than the standard dosage of 1.8 mg/m(2). Results: Twenty-one patients with R/R B-ALL were included, including five children (<18 years old) and sixteen adults. Seventeen patients presented with 5.0% -99.0% leukemic blasts in the bone marrow/peripheral blood or with extramedullary disease, and four patients were minimal residual disease (MRD) -positive. Fourteen patients underwent both CD19 and CD22 CAR-T-cell therapy, four underwent CD19 CAR-T-cell therapy, and three underwent blinatumomab therapy. Eleven patients underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). After inotuzumab treatment, 14 of 21 patients (66.7% ) achieved a complete response (CR, one was MRD-positive CR), and all four MRD-positive patients turned MRD-negative. Four of six patients who failed recent CD22 CAR-T-cell therapy achieved a CR after subsequent inotuzumab treatment. Seven patients (33.3% ) demonstrated no response. Grade 1-3 hepatotoxicity occurred in five patients (23.8% ), one child with no response experienced hepatic veno-occlusive disease (HVOD) during salvage transplantation and recovered completely. Conclusion: For patients with heavily treated R/R B-ALL, including those who had undergone allo-HSCT and CD19/CD22 CAR-T-cell therapy, the two-dose regimen of inotuzumab resulted in a CR rate of 66.7%, and the frequency of hepatotoxicity and HVOD was low.

Oral immunization with an anti-idiotypic antibody to the exoglycolipid antigen protects against experimental Chlamydia trachomatis infection.

Chlamydia trachomatis is the leading cause worldwide of preventable infectious blindness (trachoma) and sexually transmitted disease, including nongonoccocal urethritis and pelvic inflammatory disease. To date, no effective vaccine against C. trachomatis infection has been identified. A monoclonal anti-idiotypic antibody (anti-Id) to the chlamydial exoglycolipid antigen (GLXA) was tested in a murine model of ocular chlamydial infection for its ability to induce systemic immunity, which reduces microbiologic and clinical disease. The anti-Id to GLXA, delivered either systemically in soluble form or orally after encapsulation in poly(lactide) microspheres, induced significant protective immunity against ocular challenge of mice with a human biovar of C. trachomatis. Protection was associated with induction of anti-GLXA antibody and anti-chlamydial neutralizing antibody.

Roles of chicken growth hormone receptor antisense transcript in chicken muscle development and myoblast differentiation.

Muscle is one of the important economic traits in poultry production, and its production depends on the increased number of muscle fibers during the embryonic stage. Chicken GHR gene can transcribe in double directions, possessing not only GHR-S but also GHR-AS. The 2 kinds of transcripts are partially complementation in sequences and interact with each other. Until now, the roles and mechanisms of GHR-AS in myoblast differentiation was still unknown. In this study, we not only analyzed the GHR-AS expression patterns in myoblast differentiation phase but also clarified that GHR-AS promoted myoblast differentiation via GH-GHR-IGF1 signal pathway. Quantitative PCR analysis indicated that GHR-AS was increased during myoblast differentiation. Sub-cellular localization showed that GHR-AS and GHR-S were expressed at a higher level in the nucleus than that in the cytoplasm. The expression of MyoD and MyHC and the myoblast differentiation significantly increased after GHR-AS overexpression, while the distance between wounds decreased, suggesting that GHR-AS repressed myoblast migration and promoted differentiation. Additionally, the expression of GHR-AS, IGF1 and MyHC increased after GH protein treated, and the myoblast differentiation also increased. In conclusion, GHR-AS promoted myoblast differentiation by enhancing fusion and inhibiting migration possibly via GH-GHR-IGF1 signal pathway.

DNA immunization to prevent autoimmune diabetes.

Mice expressing lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) as a transgene in their beta cells develop insulin-dependent diabetes mellitus (IDDM) only after LCMV infection. Inoculation of plasmid DNA encoding the insulin B chain reduced the incidence of IDDM by 50% in this model. The insulin B-chain DNA vaccination was effective through induction of regulatory CD4 lymphocytes that react with the insulin B chain, secrete IL-4, and locally reduce activity of LCMV-NP-autoreactive cytotoxic T lymphocytes in the pancreatic draining lymph node. In contrast, similar vaccination with plasmids expressing the LCMV viral ("self") protein did not prevent IDDM, because no such regulatory cells were induced. Thus, DNA immunization with plasmids expressing self-antigens might constitute a novel and attractive therapeutic approach to prevent autoimmune diseases, if the antigens are carefully preelected for an ability to induce regulatory lymphocytes in vivo.

Heat stress upregulation of Toll-like receptors 2/4 and acute inflammatory cytokines in peripheral blood mononuclear cell (PBMC) of Bama miniature pigs: an in vivo and in vitro study.

Global warming is a challenge to animal health, because of increased heat stress, with subsequent induction of immunosuppression and increased susceptibility to disease. Toll-like receptors (TLR) are pattern recognition receptors that act as sentinels of pathogen invasion and tissue damage. Ligation of TLRs results in a signaling cascade and production of inflammatory cytokines, which eradicate pathogens and maintain the health of the host. We hypothesized that the TLR signaling pathway plays a role in immunosuppression in heat-stressed pigs. We explored the changes in the expression of TLR2, TLR4 and the concentration of acute inflammatory cytokines, such as IL-2, IL-8, IL-12 and IFN-γ in Bama miniature pigs subjected to 21 consecutive days of heat stress, both in vitro and in vivo models. The results showed that heat stress induced the upregulation of cortisol in the plasma of pigs (P<0.05); TLR4 mRNA was elevated, but IL-2 was reduced in peripheral blood mononuclear cells (PBMC, P<0.05). The white blood cell count and the percentage of granulocytes (eosinophilic+basophilic) decreased significantly in heat-stressed pigs (P<0.05). In the in vitro model (PBMC heat shocked for 1 h followed by a 9 h recovery period), TLR2 and TLR4 mRNA expression also increased, as did the concentration of IL-12 in supernatants. However, IFN-γ was significantly reduced in PBMC culture supernatants (P<0.05). We concluded that a consecutive heat stress period elevated the expression of TLR2 and TLR4 in PBMC and increased the plasma levels of inflammatory cytokines. These data indicate that TLR activation and dysregulation of cytokine expression in response to prolonged heat stress may be associated with immunosuppression and increased susceptibility to antigenic challenge in Bama miniature pigs.

A multivalent minigene vaccine, containing B-cell, cytotoxic T-lymphocyte, and Th epitopes from several microbes, induces appropriate responses in vivo and confers protection against more than one pathogen.

The development of safe and effective vaccines remains a major goal in the prevention, and perhaps treatment, of infectious diseases. Ideally, a single vaccine would confer protection against several pathogens and would induce both cellular and humoral arms of the immune response. We originally demonstrated that two virus-specific cytotoxic T-lymphocyte (CTL) epitopes, from the same virus but presented by different major histocompatibility complex alleles, when linked in tandem as minigenes in a recombinant vaccinia virus, could confer complete protection against subsequent viral challenge. In the study, we extended this approach, which we termed string of beads, expanding the immunogenic scope in two ways: first, by introduction of T helper (Th) and B-cell (antibody) epitopes alongside CTL epitopes and second, by including immunogenic sequences from a variety of infectious agents, five viruses and one bacterium. The vaccine (VV-sv) comprises CTL epitopes from Sendai virus, respiratory syncytial virus, and lymphocytic choriomeningitis virus (LCMV); Th epitopes from vesicular stomatitis virus and Mycobacterium tuberculosis; and an antibody epitope from mengovirus. The construct contains a single start codon, and the epitopes are linked directly, without intervening spacer amino acids. There was some concern that the combination of several normally immunodominant epitopes might result in a new hierarchy of dominance, in which certain epitopes predominated and others exhibited reduced immunogenicity. However we show that when analyzed in tissue culture and in vivo, all six epitopes are expressed. CTL and Th cells are induced in vivo, along with neutralizing antibody. The induced immunity is biologically relevant: after VV-sv immunization, the antimengovirus antibody confers protection against mengovirus challenge. Similarly, CTL induced by the LCMV epitope protected mice against challenge with this agent. Thus, a polyvalent, minigene-based vaccine can simultaneously induce several classes of immune response and thereby can confer protection against diverse pathogens.

The multivalent minigene approach to vaccine development.

Epitope based minigenes (epigenes) have been under investigation for several years as an experimental approach to vaccination against infectious diseases. The essence of this technology is that short DNA sequences, encoding well-defined cytotoxic T-lymphocyte- (CTL), antibody- (Ab) or helper T-lymphocyte- (HTL) specific epitopes are used as immunogens. Compared to other vaccine strategies, several potential advantages are apparent. These include the increased 'safety' of an immunisation strategy that mimics antigen processing and presentation during natural infections, without actually causing disease, and the 'flexibility' in epitope selection, which allows induction and optimisation of the desired type of immunity. In addition, the 'high immunogenicity' of epitope based constructs relative to constructs based on whole antigenic proteins is an important factor. This paper presents and discusses recent developments in the use of minigenes or multiple epitope genes that allow vaccines to be designed. The preclinical studies available to date clearly demonstrate the great potential of this vaccine approach, in terms of both prophylaxis and therapy.

A recombinant minigene vaccine containing a nonameric cytotoxic-T-lymphocyte epitope confers limited protection against Listeria monocytogenes infection.

We have previously shown that vaccines expressing virus-derived cytotoxic-T-lymphocyte (CTL) epitopes as short minigenes can confer effective protection against virus challenges, and here we extend these studies to the bacterium Listeria monocytogenes. Host defense against this important human pathogen appears largely T cell mediated, and a nonamer CTL epitope from the listeriolysin O (LLO) protein has been identified in BALB/c mice. We have synthesized this nonamer as a minigene, expressed it in a recombinant vaccinia virus (VV-list), and used this to immunize mice. Memory CTLs cultured from VV-list-immunized mice specifically lyse target cells pulsed with a nonamer peptide identified at LLO amino acid residues 91 to 99. Four weeks postimmunization, mice were challenged with L. monocytogenes. By day 6 following challenge with a sublethal dose of L. monocytogenes, mice immunized with VV-list showed a approximately 2,000- to 6,000-fold reduction in bacteria CFU in the spleen and liver. At this time point, with control mice, bacterial were readily detectable by Gram stain of the liver but were undetectable in the VV-list-immunized animals. Additionally, when a normally lethal dose of bacteria was given, death was delayed in VV-list-immunized animals. This study has demonstrated that a single immunization with a recombinant vaccinia virus bearing only nine amino acids from a bacterial pathogen can induce specific CTLs able to confer partial protection against bacterial challenge.

Quantitative and qualitative analyses of the immune responses induced by a multivalent minigene DNA vaccine.

Vaccines containing minigenes - isolated antigenic epitopes encoded by short open reading frames - can, under certain circumstances, confer protective immunity upon the vaccinee. Here we evaluate the efficacy of the minigene vaccine approach using DNA immunization and find that, to be immunogenic, a minigene-encoded epitope requires a perfect "Kozak" translational initiation region. In addition, using intracellular cytokine staining, we show that immunization with a plasmid encoding a full-length protein induces epitope-specific CD8(+) T cells which are detectable directly ex vivo, and constitute approximately 2% of the vaccinee's splenic CD8(+) T cells. In contrast, such cells are undetectable directly ex vivo in recipients of a minigene vaccine. Nevertheless, the minigene plasmid does induce a low number of epitope-specific CD8(+) T cells, which can be amplified to detectable levels by in vivo stimulation. Indeed, 4 days after in vivo stimulation (by virus infection), all vaccinated mice - regardless of whether they had been vaccinated with the minigene or with the full-length gene - had similar numbers of epitope-specific CD8(+) T cells. However, despite these strong responses at 4 days post-infection, recipients of the minigene vaccine showed no enhanced ability to limit virus replication and dissemination. We therefore observe a dichotomy; minigene vaccinees are not protected, despite the presence of strong virus-specific immune responses at 4 days post-challenge. We suggest that the protective benefits of vaccination exert themselves very soon - perhaps within minutes or hours - after virus challenge. If the vaccine-induced immune response is too low to achieve this early protective effect, virus-specific T cells will expand rapidly, but ineffectually, leading to the strong but non-protective response measured at 4 days post-infection. Thus, vaccine-induced immunity should be monitored very early in infection, since the extent to which these responses may later be amplified is largely irrelevant to the protection observed.

Biochemical and functional antigenic mimicry by a polyclonal anti-idiotypic antibody for chlamydial exoglycolipid antigen.

A vaccine developed by using a genus-specific antigen (Ag) of Chlamydia trachomatis would elicit a wide range of protection against various chlamydial infections. We have produced an anti-idiotypic antibody (Ab2) in guinea pigs which, in rabbits, mimics the immunogenicity of a genus-specific exoglycolipid Ag (GLXA) of C trachomatis. Furthermore, the Ab2 fulfills the functional criteria of an 'internal image' of the nominal Ag: it inhibits the binding of the idiotypic (Id) monoclonal Ab (mAb1) to GLXA, and it induces in rabbits anti-anti-Id antibody (Ab3) which recognizes both the affinity-purified nominal Ag GLXA and whole organisms. Moreover, Ab3 induced by immunization of rabbits with guinea pig Ab2 neutralizes infectious heterologous chlamydiae and prevents in vitro and in vivo infection. Taken together, these results demonstrate functional and biochemical mimicry of the Ab2 for the chlamydial GLXA and suggest that anti-idiotypic Ab to GLXA is a potential candidate vaccine against chlamydia-related diseases.

DNA immunization with minigenes: low frequency of memory cytotoxic T lymphocytes and inefficient antiviral protection are rectified by ubiquitination.

Our previous studies have shown that isolated cytotoxic T lymphocyte (CTL), B-cell, and T-helper epitopes, for which we coined the term minigenes, can be effective vaccines; when expressed from recombinant vaccinia viruses, these short immunogenic sequences confer protection against a variety of viruses and bacteria. In addition, we have previously demonstrated the utility of DNA immunization using plasmids encoding full-length viral proteins. Here we combine the two approaches and evaluate the effectiveness of minigenes in DNA immunization. We find that DNA immunization with isolated minigenes primes virus-specific memory CTL responses which, 4 days following virus challenge, appear similar in magnitude to those induced by vaccines known to be protective. Surprisingly, this vigorous CTL response fails to confer protection against a normally lethal virus challenge, although the CTL appear fully functional because, along with their high lytic activity, they are similar in affinity and cytokine secretion to CTL induced by virus infection. However this DNA immunization with isolated minigenes results in a low CTL precursor frequency; only 1 in approximately 40,000 T cells is epitope specific. In contrast, a plasmid encoding the same minigene sequences covalently attached to the cellular protein ubiquitin induces protective immunity and a sixfold-higher frequency of CTL precursors. Thus, we show that the most commonly employed criterion to evaluate CTL responses-the presence of lytic activity following secondary stimulation-does not invariably correlate with protection; instead, the better correlate of protection is the CTL precursor frequency. Recent observations indicate that certain effector functions are active in memory CTL and do not require prolonged stimulation. We suggest that these early effector functions of CTL, immediately following infection, are critical in controlling virus dissemination and in determining the outcome of the infection. Finally, we show that improved performance of the ubiquitinated minigenes most probably requires polyubiquitination of the fusion protein, suggesting that the enhancement results from more effective delivery of the minigene to the proteasome.