Knockdown of PAK1 Inhibits the Proliferation and Invasion of Non-Small Cell Lung Cancer Cells Through the ERK Pathway.

The p21-activated kinase (PAK) family of serine/threonine kinases plays a pivotal role in various human tumors, as supported by our previous report on the overexpressed PAK isoforms in non-small cell lung cancer (NSCLC). To better understand the role of PAKs in tumorigenesis, the authors examined PAK1 expression patterns and its significance in NSCLC. It was demonstrated by immunohistochemical staining that PAK1 was increased and localized in the cytoplasm in 151 of 207 cases. High levels of PAK1 expression correlated with a histologic type of tumor (squamous cell carcinoma), tumor node metastasis stage, and lymph nodal status. We also examined the biological role of PAK1 in lung cancer cell lines transfected with PAK1-small interfering RNA. Decreased expression of PAK1 inhibited lung cancer cell proliferation and invasion, which is the major cause of lung cancer malignancy. Downregulated expression of PAK1 hampered rapidly accelerated fibrosarcoma/mitogen-activated extracellular signal-regulated kinase/extracellular signal-regulated kinase pathway activity but did not affect Wnt/ß-catenin signaling. Our findings suggest that PAK1 is an important oncogene in NSCLC, as decreased expression of PAK1 inhibited the proliferation and invasion of NSCLC cells by blocking the ERK pathway. These results provide evidence for using PAK1 inhibition as potential anticancer therapy.

Immunogenicity and immunizing protection effect of GAMA gene DNA vaccine on Plasmodium berghei.

OBJECTIVE: To explore the effect of immunogenicity and immunizing protection of GAMA gene DNA vaccine, which was related with merozoite, ookinete and sporozoite invasion. METHODS: Gene fragments were obtained using PCR technique and eukaryotic expression vector (containing immunostimulatory sequence) was built. BALB/c mice were divided into PBS control group, empty vector control group and study group and were immunized at week 0, 3 and 6 respectively. Blood was collected 2 weeks after each immunization and serum was separated to detect the IgG, IgG1 and IgG2a levels. Spleen of mice was obtained for preparation of splenic mononuclear cell and the cytokine IL-4 and IFN-γ levels were detected. Indirect immunofluorescence and western blot were employed to verify the specificity of antiserum. Sporozoite and merozoite invasion were used respectively to detect the immune protective effect 2 weeks after the third immunization. Ookinete conversion rate in vitro and oocyst numbers of mosquito stomach were observed to evaluate the transmission-blocking levels. RESULTS: In GAMA DNA vaccine group: antiserum could be combined with recombinant protein specifically and green fluorescence signals of merozoite, ookinete and sporozoite were observable, while specific fragments and fluorescence signals were not observable in empty vector group. Compared with control group, specific IgG in DNA vaccine immunity group significantly increased (P < 0.01), and IgG1 and IgG2a all increased (P < 0.01). IL-4, IFN-γ content in study group significantly increased, compared with control group (P < 0.01). GAMA DNA vaccine immunity could not obviously block the erythrocyte-stage infection (caused by sporozoite invasion); compared with control group, liver worm load was slightly reduced (P < 0.05), and antiserum ookinete numbers (cultured in vitro) had no significant difference with oocyst numbers of mosquito stomach in DNA vaccine group. CONCLUSIONS: GAMA has good antigenicity, which could stimulate the body to produce specific immune responses; while DNA vaccine immunity could not play a good protective effect, the effect of which is only limited to the slight reduction of liver worm load, and has no obvious erythrocyte-stage protective effect and transmission-blocking effect. Therefore, trying other immunization strategies for further research on the value of GAMA (as multi-stage antigen vaccine and multi-stage combined vaccine components of the life-cycle of plasmodium) is necessary.

Right atrial pressure alterations during echocardiography-guided-catheterization predict tricuspid valvular impairment: a novel method for the creation of a rabbit model of Staphylococcus aureus endocarditis.

BACKGROUND: We previously reported the use of a catheter system to damage the tricuspid valve and create infectious endocarditis (IE) in an animal model. The current study aims to create a faint IE model suitable for antibiotic prophylaxis using a low bacterial inoculum. We also aim to explore a way to quantitatively assess valvular impairment and to predict the success of the IE models during catheterization. METHODS: Ninety rabbits were assigned to two groups according to the density of bacteria inoculated (1 × 10(5) CFU for Group A and 1 × 10(4) CFU for Group B). A catheter system consisting of a polyethylene catheter and a guide wire were used to damage the valve. The catheter system was passed through the rabbits' tricuspid valves under echocardiographic guidance. A pressure transducer was used to assess right atrial pressure (P(RA)) before and just after valvular damage to calculate the pressure alterations (ΔP(RA)). The animals in group A and B were divided into 3 subgroups according to the ΔP(RA) (0-5 mmHg for Groups A1 and B1; 5-10 mmHg for Groups A2 and B2; 10-15 mmHg for Groups A3 and B3). Staphylococcus aureus (ATCC 29213) inoculation was performed 24 hr after cardiac catheterization. RESULTS: Faint IE was confirmed in 20%, 93.3%, 26.7%, 6.7%, 20%, and 33.3% of the rabbits in Groups A1, A2, A3, B1, B2, and B3, respectively. There was no difference in the LV/RV ratio and VTR of the No-IE, faint-IE, and severe IE animals. Faint IE rabbits had a larger ΔPRA than No-IE rabbits (7.81 ± 1.21 vs. 2.48 ± 1.0, P < 0.01, for Group A; 7.60 ± 1.32 vs. 2.98 ± 1.08, P < 0.01, for Group B). The ΔPRA of severe IE and faint IE rabbits was significantly different (13.11 ± 1.31 vs. 7.81 ± 1.21, P < 0.01, for Group A; 12.73 ± 1.44 vs.7.60 ± 1.32, P < 0.01, for Group B). CONCLUSION: ΔP(RA) could be used to assess valvular impairment. Controlling the value of ΔP(RA) during catheterization and inoculating of an appropriate dose of bacteria was associated with a successful IE model.

Parasite densities modulate susceptibility of mice to cerebral malaria during co-infection with Schistosoma japonicum and Plasmodium berghei.

BACKGROUND: Malaria and schistosomiasis are endemic and co-exist in the same geographic areas, even co-infecting the same host. Previous studies have reported that concomitant infection with Schistosoma japonicum could offer protection against experimental cerebral malaria (ECM) in mice. This study was performed to evaluate whether alterations in parasite density could alter this protective effect. METHODS: Mice were inoculated with 100 or 200 S. japonicum cercariae followed by infection with high or low density of Plasmodium berghei ANKA strain eight weeks after the first infection. Then, parasitaemia, survival rate and blood-brain-barrier (BBB) damage were assessed. Interferon-gamma (IFN-γ), interleukin (IL)-4, IL-5, IL-13, IL-10, and TGF-ß levels were determined in splenocyte supernatants using enzyme-linked immunosorbent assay (ELISA). Cell surface/intracellular staining and flow cytometry were used to analyse the level of CD4(+)/CD8(+) T cells, CD4(+)CD25(+)Foxp3(+) Tregs, IL-10-secreting Tregs, and IL-10(+)Foxp3-CD4(+) T cells in the spleen, and CD4(+)/CD8(+) T cells infiltrating the brain. RESULTS: Co-infection with low density P. berghei and increased S. japonicum cercariae significantly increased the levels of IL-4, IL-5, IL-13, TGF-ß and Tregs, but significantly decreased the levels of IFN-γ and the percentage of CD4(+) T cells and CD8(+) T cells in the spleen and CD8(+) T cell infiltration in the brain. Increased worm loads also significantly decreased mortality and BBB impairment during ECM. When challenged with higher numbers of P. berghei and increased cercariae, the observed cytokine changes were not statistically significant. The corresponding ECM mortality and BBB impairment also remained unchanged. CONCLUSIONS: This study demonstrates that protection for ECM depends on the numbers of the parasites, S. japonicum and P. berghei, during co-infection. Alterations in the regulatory response appear to play a key role in this adaptation.

Pre-existing Schistosoma japonicum infection alters the immune response to Plasmodium berghei infection in C57BL/6 mice.

BACKGROUND: Since helminths and malaria parasites are often co-endemic, it is important to clarify the immunoregulatory mechanism that occurs during the process of co-infection. A previous study confirmed that dendritic cells (DCs) are involved in the establishment and regulation of the T-cell-mediated immune response to malaria infection. In the current study, distinct response profiles for splenic DCs and regulatory T cell (Treg) responses were assessed to evaluate the effects of a pre-existing Schistosoma japonicum infection on malaria infection. METHODS: Malaria parasitaemia, survival rate, brain histopathology and clinical experimental cerebral malaria (ECM) were assessed in both Plasmodium berghei ANKA-mono-infected and S. japonicum-P. berghei ANKA-co-infected mice. Cell surface/intracellular staining and flow cytometry were used to analyse the level of splenic DC subpopulations, toll-like receptors (TLRs), DC surface molecules, Tregs (CD4⁺CD25⁺Foxp3⁺), IFN-γ/IL-10-secreting Tregs, and IFN-γ⁺/IL-10⁺-Foxp3⁻CD4⁺ T cells. IFN-γ, IL-4, IL-5, IL-10 and IL-13 levels were determined in splenocyte supernatants using enzyme-linked immunosorbent assay (ELISA). RESULTS: The co-infected mice had significantly higher malaria parasitaemia, compared with the mono-infected mice, on days 2, 3, 7 and 8 after P. berghei ANKA infection. Mono-infected mice had a slightly lower survival rate, while clinical ECM symptoms, and brain pathology, were significantly more severe during the period of susceptibility to ECM. On days 5 and 8 post P. berghei ANKA infection, co-infected mice had significantly lower levels of CD11c⁺CD11b⁺, CD11c⁺CD45R/B220⁺, CD11c⁺TLR4⁺, CD11c⁺TLR9⁺, CD11c⁺MHCII⁺, CD11c⁺CD86⁺, IFN-γ-secreting Tregs, and IFN-γ⁺Foxp3⁻CD4⁺ T cells in single-cell suspensions of splenocytes when compared with P. berghei ANKA-mono-infected mice. Co-infected mice also had significantly lower levels of IFN-γ and higher levels of IL-4, IL-5, and IL-13 in splenocyte supernatants compared to mono-infected mice. There were no differences in the levels of IL-10-secreting Tregs or IL-10⁺Foxp3⁻CD4⁺ T cells between co-infected and mono-infected mice. CONCLUSIONS: A Tregs-associated Th2 response plays an important role in protecting against ECM pathology. Pre-existing S. japonicum infection suppressed TLR ligand-induced DC maturation and had an anti-inflammatory effect during malaria infection not only by virtue of its ability to induce Th2 responses, but also by directly suppressing the ability of DC to produce pro-inflammatory mediators.

A rabbit model of right-sided Staphylococcus aureus endocarditis created with echocardiographic guidance.

BACKGROUND: The most widely used experimental models of infective endocarditis (IE) are the rabbit and rat models, in which cardiac valve lesions are induced by a polyethylene catheter introduced into the left ventricle through the aortic valve. Our study was designed to create a rabbit model of IE right-sided with echocardiographic guidance. METHODS: Thirty rabbits underwent both catheterization and inoculation (group A). These were divided into three subgroups of ten based on the time of catheter removal (immediately, after 24 h, and after death or moribundity for groups, A(1), A(2), and A(3), respectively). Ten inoculated-only and ten catheterized-only rabbits served as controls. A catheter system consisted of a polyethylene catheter and a guide wire inside it. This system was passed through the rabbits' tricuspid valves under echocardiographic guidance to damage them. The ratio of left ventricle to right ventricle (LV/RV) was measured in a four-chamber view before catheterization and at the time of death or moribundity. The peak velocity of tricuspid regurgitation (V(TR)) was measured in a four-chamber view at the time of catheterization and at the time of death or moribundity. Staphylococcus aureus (ATCC 29213) inoculation was performed 24 h after right heart catheterization to produce IE. RESULTS: IE was confirmed in 28 of 30 rabbits by macroscopic and histologic examination of tricuspid valves, blood cultures, and bacterial count in cardiac vegetations. Cardiac vegetations were confirmed in 25 of 28 IE rabbits by echocardiography. Enlargement of right ventricle dimension with a significantly decreased LV/RV ratio was confirmed in all IE rabbits at the time of death or moribundity than at the initial state (1.11 ± 0.35 vs. 1.95 ± 0.39, P < 0.01; 1.21 ± 0.34 vs. 1.98 ± 0.35, P < 0.01; 1.04 ± 0.31 vs. 2.00 ± 0.41, P < 0.01 for groups A(1), A(2), and A(3), respectively). V(TR) was significantly higher in all the IE rabbits at the time of death or moribundity than at the time of catheterization (1.89 ± 0.46 vs 0.76 ± 0.45, P < 0.01; 2.04 ± 0.73 vs 0.68 ± 0.66, P < 0.01; 2.24 ± 0.51 vs 0.87 ± 0.55, P < 0.01 for group A(1), A(2), and A(3), respectively). CONCLUSION: The models described herein closely reproduced the pathogenesis and pathophysiology of right heart catheter-induced endocarditis in humans. Echocardiographic guidance is helpful in the process of right heart catheterization. Some echocardiographic parameters, such as V(TR) and the LV/RV ratio could be used to assess the success or failure of the IE models.

Degenerate primer design to clone the human repertoire of immunoglobulin heavy chain variable regions.

Amplifying the variable (Fv or V) regions of immunoglobulins (Ig) has become a challenge in cloning antibody genes for phage display, a technique used to study protein-protein, protein-peptide, and protein-DNA interactions using bacteriophages to connect proteins with the genetic information that encodes them. Key parameters affecting the amplification of full antibody repertoires includes the availability of primers that can amplify as many V genes as possible; however the strategy used to design these primers and programs used to make the necessary alignments have not been well studied and clearly detailed in the literature. Here, we present a set of primers computationally designed by iCODEHOP based on a database of human germline Ig sequences. We used reverse transcription polymerase chain reaction (RT-PCR) protocols that would recognize the V(H) genes from human peripheral blood mononuclear cells. We identified the most highly conserved region in framework 1 and framework 4 of the Ig cDNA, and designed a set of degenerated 5' primers. The V(H) genes were successfully amplified by RT-PCR. This new primer has facilitated the creation of more diverse V(H) libraries than has been previously possible. Moreover, iCODEHOP improved the primer design efficiency and was found useful both for cloning unknown genes in gene families and for building V(H) gene libraries.

[Distribution of Staphylococcus aureus strains colonized in healthy community population and molecular epidemiological characteristics for MRSA strains].

OBJECTIVE: To investigate the nasal colonization of Staphylococcus (S.) aureus strains among medical university students in Shenyang and to study the molecular epidemiological characteristics of methicillin resistant S. aureus (MRSA) strains. METHODS: Sterilized nasal swabs were used to collect nasal bacteria from both nares of the students. Nasal specimens were further identified as S. aureus strains, sensitive or resistant to methicillin through a series of tests. Molecular related methods including staphylococcal cassette chromosome mec (SCCmec) typing, pulsed- field gel electrophoresis (PFGE), coagulase isotyping and minimum inhibitory concentration (MIC) determination etc. were used to characterize the isolates. Prevalence of the panton-valentine leukocidin (pvl) genes (lukS and F-PV) among the isolates was also assessed. RESULTS: Staphylococci were found in 488 specimens from 977 participants through the surveillance program, conducted in 2009. Of the 488 specimens being tested, 364 were identified as coagulase-negative staphylococci (CoNS) and 124 as S. aureus. MRSA strain among the S. aureus isolates was accounted for 3.4%. In the surveillance program conducted in 2010, staphylococci grew in 310 specimens from 657 participants. Of the 310 specimens tested, 195 were identified as CoNS and 115 as S. aureus. The percentage of MRSA strains among the S. aureus isolates was 7.7%. In total, 239 students carried S. aureus, and the percentage of MRSA carriers among the total specimens tested in this study was 5.1%. Most of the MRSA strains could be classified into one of the five types of SCCmec elements. Type IV a SCCmec strains were most frequent seen overall (10 isolates). A total of 11 pulsotype were identified among the MRSA strains and were classified into 7 major groups (A to G) by the mutual correlations of their banding patterns. Ten MRSA strains were identified as pvl positive strains. CONCLUSION: An MRSA clone (IV a SCCmec pulsotype A) carrying pvl toxin gene was found to be prevalent in the nares of the healthy university students.

Antibiotic susceptibility of coagulase-negative staphylococci (CoNS): emergence of teicoplanin-non-susceptible CoNS strains with inducible resistance to vancomycin.

Coagulase-negative staphylococci (CoNS) have become increasingly recognized as important agents of nosocomial infection. One of the characteristics of CoNS is their resistance to multiple antimicrobial agents commonly used for the treatment of staphylococcal infections. CoNS strains (n = 745) isolated from a university teaching hospital in China between 2004 and 2009 were tested for antibiotic resistance. The antibiotics were placed into three categories based on resistance levels of the CoNS strains to these antibiotics: high resistance (resistance rate >70 %), including penicillin G, oxacillin and erythromycin; medium resistance (resistance rate between 30 and 70 %), including tetracycline, clindamycin, ciprofloxacin, trimethoprim/sulfamethoxazole and chloramphenicol; and low resistance (resistance rate <30 %), including rifampicin, ceftizoxime and gentamicin. We also found that the prevalence of strains non-susceptible to teicoplanin increased from 4.5 to 6.7 % between 2008 and 2009. A one-step vancomycin agar selection experiment and subsequent population analysis revealed potentially vancomycin-resistant subpopulations that have been selected from the teicoplanin-non-susceptible strains. Vigilant surveillance of nosocomial isolates of CoNS is needed to determine their resistance to glycopeptides.

Epidemiological and molecular characterization of Staphylococcus haemolyticus strains, from a hematology ward, with decreased susceptibility to glycopeptides.

In the present study, we report on the reduced susceptibility to teicoplanin among clinical isolates of Staphylococcus haemolyticus in a hematology ward of a teaching hospital. The molecular characterization of 17 S. haemolyticus strains was performed using mec gene complex classification, pulsed-field gel electrophoresis analysis, and minimum inhibitory concentration examination. Pulsotype A strains carrying a class C2 mec gene complex were the most prevalent strains, at 64.7%. In vivo selection of stepwise increase in resistance to vancomycin and teicoplanin was observed in three S. haemolyticus strains serially isolated from a case patient. The results of the present study suggest the regional spread of certain S. haemolyticus clones with diminished susceptibility to glycopeptides, emphasizing the need for continuous monitoring of minimum inhibitory concentration levels of vancomycin and teicoplanin in S. haemolyticus strains, and the importance of infection control practices to prevent its transmission.

Nasal carriage of methicillin-resistant Staphylococcus aureus among preclinical medical students: epidemiologic and molecular characteristics of methicillin-resistant S. aureus clones.

Between May 2008 and October 2009, a total of 2103 interns were randomly tested for nasal colonization of S. aureus and methicillin-resistant Staphylococcus aureus (MRSA). The prevalence of S. aureus among staphylococci specimens was 23.1%, and among the total S. aureus the MRSA prevalence was 9.4%. MRSA isolates were further subtyped using genetic element staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE) band pattern, and multilocus sequence typing. SCCmec type IVa was the most prevalent strain, at 45.4 %. Eleven PFGE patterns were identified in MRSA strains, with 1 predominant (pulsotype A, 45.5%). Eight strains which belonged to clonal complex 78 carried type IVa SCCmec and produced type 3 coagulase. Panton-Valentine leukocidin (PVL) genes (lukS and F-PV) were identified in 10 (45.4%) MRSA strains; these predominately carried ϕSa2958type and ϕSa108PVL-like type PVL phages. After inducing prophages, 8 strains infected other S. aureus isolates and could generate novel PVL-positive strains of S. aureus. The present study demonstrates that interns can carry certain MRSA strains asymptomatically and contribute to the spread of MRSA between the community and hospital.

In vivo kinetics and biodistribution of a Hantaan virus DNA vaccine after intramuscular injection in mice.

To study the kinetics in vivo of a Hantaan virus DNA vaccine, we constructed a fusion DNA vaccine, pEGFP/S, by cloning the S segment of Hantavirus into the vector, pEGFP-C1, which encodes Green fluorescent protein EGFP. In this report, we provide evidence that pEGFP/S was distributed and persistently expressed for more than 60 days in several organs after inoculation. Our findings suggest that the persistent immune responses induced by a Hantaan virus DNA vaccine are likely due to the plasmid pEGFP/S deposited in vivo, which acts as a booster immunization.

Short hairpin RNA-mediated inhibition of measles virus replication in vitro.

Rab9 has been identified as a key component for the replication of measles virus (MV). In this study, gene-specific shRNAs were developed to suppress the replication of MV in culture cells by silencing the expression of Rab9 GTPase gene. Rab9 GTPase gene-specific shRNAs were designed and cloned into the expression vector of pSUPER.neo+EGFP. Vero-E6 cells were transfected with the recombinant plasmid via liposome and then infected with MV. The expression of Rab9 GTPase mRNA and protein were assayed by RT-PCR and Western blotting, respectively. ShRNA-mediated inhibition of MV replication was further evaluated by detecting the titer of MV. The results showed that the expression of Rab9 GTPase was dramatically and stably downregulated by the generated shRNAs targeting Rab9 GTPase gene, which contribute to the inhibition of MV replication, indicating these shRNAs could be potentially developed into therapeutic agents for the treatment of MV infection in the future.

Enhancing DNA vaccine potency against hantavirus by co-administration of interleukin-12 expression vector as a genetic adjuvant.

BACKGROUND: The heavy incidence and mortality of hemorrhagic fever with renal syndrome, as well as no specific drugs in curing the disease, clearly indicate the need for development of the more effective hantavirus vaccine. Refining the DNA vaccination strategy to elicit more clinically efficacious immune responses is now under intensive investigation. In the present study, we examined the effects of using an interleukin-12 expression plasmid as a genetic adjuvant to enhance the immune responses induced by a DNA vaccine based on the S gene encoding nucleocapsid protein against hantavirus. METHODS: BALB/c mice were immunized three times by intramuscular inoculations of DNA vaccine encoding of hantanvirus nucleocapsid protein alone or in combination with a plasmid expressing murine interleukin-12 (pcIL-12). Booster immunizations were employed 2 times at 2-week interval. To evaluate the humoral and cellular immune responses, antigen-specific lymphocyte proliferation and antibody production were assayed by MTT method and ELISA respectively. The level of interleukin-4 and interferon-gamma in the splenic lymphocytic cultured supernatant were detected with ELISA kit at day 5, 10, 17, 35 and 42 after primary immunization. RESULTS: Antigen-specific IgG antibodies was increased markedly at day 17 in the experiment groups and reached a plateau after day 35. As pcIL-12 co-injected, a significant inhibition of antigen-specific IgG levels was displayed over the period and the antibody mean titre was decreased to only about 1:50 at day 42 after primary immunization, significantly lower than the group immunized with pcDNA3.1 + S alone, in which the mean titre was about 1:70. Interferon-gamma was increased remarkably by the co-injection of pcIL-12 compared with the injection of pcDNA3.1 + S alone. However, the production of interleukin-4 was inhibited by pcIL-12 co-injection. Furthermore, pcIL-12 co-injection efficiently enhanced antigen-specific lymphocyte proliferation. CONCLUSION: Humoral and cytokine responses elicited by pcDNA3.1 + S inoculation can be modulated by co-inoculation with pcIL-12 and efficiently induced Th1-dominant immune responses.