In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection.

Zika virus (ZIKV) infection may lead to severe neurological consequences, including seizures, and early infancy death. However, the involved mechanisms are still largely unknown. TRPC channels play an important role in regulating nervous system excitability and are implicated in seizure development. We investigated whether TRPCs might be involved in the pathogenesis of ZIKV infection. We found that ZIKV infection increases TRPC4 expression in host cells via the interaction between the ZIKV-NS3 protein and CaMKII, enhancing TRPC4-mediated calcium influx. Pharmacological inhibition of CaMKII decreased both pCREB and TRPC4 protein levels, whereas the suppression of either TRPC4 or CaMKII improved the survival rate of ZIKV-infected cells and reduced viral protein production, likely by impeding the replication phase of the viral life cycle. TRPC4 or CaMKII inhibitors also reduced seizures and increased the survival of ZIKV-infected neonatal mice and blocked the spread of ZIKV in brain organoids derived from human-induced pluripotent stem cells. These findings suggest that targeting CaMKII or TRPC4 may offer a promising approach for developing novel anti-ZIKV therapies, capable of preventing ZIKV-associated seizures and death.

CRISPR/Cas9-based genome-wide screening for metastasis ability identifies FCGR1A regulating the metastatic process of ovarian cancer by targeting LSP1.

BACKGROUND: Metastasis is a main cause of death from ovarian cancer (OC). Identifying key markers involved in OC metastasis can aid in the effective detection of early postoperative metastasis. However, the role of FCGR1A in OC metastasis has yet to be fully established. A genome-wide CRISPR/Cas9-based screening system was used to identify regulatory factors involved in metastasis. METHODS: The expression of FCGR1A and LSP1 in ovarian cancer cell lines was examined by quantitative real-time polymerase chain reaction (qRT‒PCR). The functions of FCGR1A and LSP1 in OC cell migration, invasion and proliferation were determined using wound healing, Transwell invasion and CKK-8 assays. A transcription-activated library was used to identify the potential downstream genes of FCGR1A. FCGR1A expression was detected by immunohistochemistry and the immunity risk score (IRS) scores were calculated. RESULTS: FCGR1A was upregulated in OC cells compared with normal ovarian cells. Downregulation of FCGR1A inhibited metastasis, proliferation and epithelial-mesenchymal transition (EMT) progression in OC cells in vitro and intraperitoneal metastasis in vivo. Moreover, downregulation of FCGR1A was accompanied by decreased LSP1 expression. Overexpression of LSP1 partially reversed the tumor suppressive effect of FCGR1A downregulation. Higher FCGR1A expression was related to metastasis, higher grade, higher stage, and lymph node metastasis in OC. Survival analysis suggested that the group with higher FCGR1A expression had a lower tumor-free survival rate and a lower overall survival rate than did the group with low FCGR1A expression. CONCLUSIONS: FCGR1A enhances OC metastasis by regulating LSP1, and FCGR1A is associated with poor prognosis, suggesting that FCGR1A is a potential predictive factor for detecting early postoperative metastasis.

The association of leader-member exchange and team-member exchange with nurses' innovative behaviours: A cross-sectional study.

AIM: To measure the association of leader-member exchange and team-member exchange with nurses' innovative behaviours through social exchange theory. BACKGROUND: The field of nursing is actively advocating innovation. Other fields have proven that leader-member exchange and team-member exchange can promote innovative behaviour, but such an association is not clear in nursing. DESIGN: A cross-sectional study. METHODS: A total of 560 nurses were selected from five tertiary hospitals in Henan Province (China) by multistage sampling. Data were collected from a self-report questionnaire. Thirty nurses in the pre-survey were used to verify the validity of the questionnaire. SPSS PROCESS macro was used to verify the association of leader-member exchange and team-member exchange with nurses' innovative behaviours. RESULTS: Leader-member exchange and team-member exchange were significantly associated with nurses' innovative behaviours, and team-member exchange had a mediating effect on the relationship between leader-member exchange and innovative behaviour. CONCLUSIONS: Leader-member exchange and team-member exchange positively affect nurses' innovative behaviours. Leader-member exchange can promote nurses' innovative behaviours through the mediating role of team-member exchange. IMPACT: This study indicated that leader-member exchange and team-member exchange should be given more attention in promoting nurses' innovative behaviours. This finding has implications for the promotion of innovative behaviours in nurses. Leaders need to focus on the innovative needs of nurses and offer support. Meanwhile, leadership training programs are necessary for managers to create positive team relationships. PATIENT OR PUBLIC CONTRIBUTION: No patient or public involvement.

Piezoionic Elastomers by Phase and Interface Engineering for High-Performance Energy-Harvesting Ionotronics.

Piezoionic materials play a pivotal role in energy-harvesting ionotronics. However, a persistent challenge lies in balancing the structural requirements for voltage generation, current conduction, and mechanical adaptability. The conventional approach of employing crystalline heterostructures for stress concentration and localized charge separation, while effective for voltage generation, often compromises the stretchability and long-range charge transport found in homogeneous quasisolid states. Herein, phase and interface engineering strategy is introduced to address this dilemma and a piezoionic elastomer is presented that seamlessly integrates ionic liquids and ionic plastic crystals, forming a finely tuned microphase-separated structure with an intermediate phase. This approach promotes charge separation via stress concentration among hard phases while leveraging the high ionic charge mobility in soft and intermediate phases. Impressively, the elastomer achieves an extraordinary piezoionic coefficient of about 6.0 mV kPa-1, a more than threefold improvement over current hydrogels and ionogels. The resulting power density of 1.3 µW cm-3 sets a new benchmark, exceeding that of state-of-the-art piezoionic gels. Notably, this elastomer combines outstanding stretchability, remarkable toughness, and rapid self-healing capability, underscoring its potential for real-world applications. This work may represent a stride toward mechanically robust energy harvesting systems and provide insights into ionotronic systems for human-machine interaction.

Commensal Neisseria species share immune suppressive mechanisms with Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a highly adapted human sexually transmitted pathogen that can cause symptomatic infections associated with localized inflammation as well as asymptomatic and subclinical infections, particularly in females. Gonococcal infection in humans does not generate an effective immune response in most cases, which contributes to both transmission of the pathogen and reinfection after treatment. Neisseria gonorrhoeae is known to evade and suppress human immune responses through a variety of mechanisms. Commensal Neisseria species that are closely related to N. gonorrhoeae, such as N. cinerea, N. lactamica, N. elongata, and N. mucosa, rarely cause disease and instead asymptomatically colonize mucosal sites for prolonged periods of time without evoking clearing immunologic responses. We have shown previously that N. gonorrhoeae inhibits the capacity of antigen-pulsed dendritic cells to induce CD4+ T cell proliferation in vitro. Much of the suppressive effects of N. gonorrhoeae on dendritic cells can be recapitulated either by outer-membrane vesicles released from the bacteria or by purified PorB, the most abundant outer-membrane protein in Neisseria gonorrhoeae. We show here that three commensal Neisseria species, N. cinerea, N. lactamica and N. mucosa, show a comparable capacity to suppress dendritic cell-induced T cell proliferation in vitro through mechanisms similar to those demonstrated previously for N. gonorrhoeae, including inhibition by purified PorB. Our findings suggest that some immune-evasive properties of pathogenic N. gonorrhoeae are shared with commensal Neisseria species and may contribute to the ability of both pathogens and commensals to cause prolonged mucosal colonization in humans.

[Application of nitrous oxide/oxygen inhalation comfort technique during tooth extraction of elderly hypertensive patients under electrocardiographic monitoring].

PURPOSE: To investigate the application value of nitrous oxide/oxygen inhalation comfort technique during tooth extraction in elderly patients with hypertension under electrocardiographic(ECG) monitoring. METHODS: According to the inclusion and exclusion criteria, sixty elderly patients (over 65 years old) with hypertension for tooth extraction were randomly divided into 2 groups: the experimental group(nitrous oxide/oxygen inhalation combined with ECG monitoring group, n=30) and the control group (routine ECG monitoring group, n=30). Mean arterial pressure (MAP) and heart rate (HR) at T0 (baseline values before surgery), T1 (on local anesthesia), T2 (during operation) and T3(5 minutes after operation) were recorded. SPSS 25.0 software package was used for statistical analysis. RESULTS: There was no significant difference in MAP and HR at each time point in the experimental group(P＞0.05). There was no significant difference in MAP and HR at T0 and T3 time points in the control group(P＞0.05). At other time points, MAP and HR were significantly different (P＜0.05). There was no significant differences in MAP and HR between the two groups at T0 and T3(P＞0.05). MAP and HR at T1 and T2 in the experimental group were significantly less than those in the control group(P＜0.05). CONCLUSIONS: Nitrous oxide/oxygen inhalation comfort technology can stabilize patients' emotions and maintain stable blood pressure and heart rate in elderly patients with hypertension during tooth extraction, thus improving the safety of tooth extraction.

Effects of different blood pressures and their long-term variability on the development of diabetic kidney disease in patients with type 2 diabetes mellitus.

AIM: To explore the relationship between long-term variabilities in different blood pressure variables and diabetic kidney disease (DKD) in patients with type 2 diabetes. DESIGN: A retrospective study. METHODS: This study included 3050 patients with type 2 diabetes whose metabolic parameters were regularly checked. Intrapersonal means and standard deviations (SDs) of all recorded systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and pulse pressure (PP) measurements were calculated. Subjects were divided into four groups: Q1 (SBP-Mean < 130, SBP-SD < 11.06); Q2 (SBP-Mean < 130, SBP-SD ≥ 11.06); Q3 (SBP-Mean ≥ 130, SBP-SD < 11.06); Q4 (SBP-Mean ≥ 130, SBP-SD ≥ 11.06). Similarly, based on whether the PP-Mean was higher or lower than 80 mmHg (average PP-Mean) and the PP-SD was higher or lower than 6.48 mmHg (average PP-SD), the involved patients were redivided into Q1'~ Q4' groups. RESULTS: Adjusted for age, sex and diabetes duration, results revealed that the SBP-Mean, SBP-SD, PP-Mean and PP-SD were risk factors for DKD. Meanwhile, patients in the Q4 group had the highest DKD prevalence (HR = 1.976, p < .001), while Q1 group had the lowest. In addition, patients in the Q3 group (HR = 1.614, P < .001) had a higher risk of DKD than those in the Q2 group (HR = 1.408, P < .001). After re-stratification by PP-Mean and PP-SD, patients in the Q4' group had the highest risk of DKD (HR = 1.370, p < .001), while those in the Q1' group had the lowest risk. Patients in the Q3' group (HR = 1.266, p < .001) had a higher risk of DKD than those in the Q2' group (HR = 1.212, p < .001).

[Application of two different comfort techniques in extraction of impacted teeth among 60 patients with hypertension].

PURPOSE: To compare the effect of two comfort techniques of nitrous oxide/oxygen inhalation and dexmedetomidine combined with flurbiprofen axetil sedation and analgesia in impacted teeth extraction among patients with hypertension. METHODS: According to the inclusion and exclusion criteria, sixty patients undergoing impacted teeth extraction with hypertension were randomly divided into 2 groups: X group( nitrous oxide/oxygen inhalation, n=30) and Y group(dexmedetomidine combined with flurbiprofen axetil assisted sedation and analgesia, n=30). The following parameters at T0(preoperative), T1(local anesthesia), T2 (surgical operation), and T3 (5 minutes after surgery) were recorded: mean arterial pressure(MAP), heart rate (HR), Ramsay sedation score, VAS pain score. SPSS 20.0 software package was used for statistical analysis. RESULTS: The parameters at each time point between the two groups were not significantly different (P＞0.05). Mood and hemodynamics at T1, T2 and T3 in each group were more stable compared with those at T0(P＜0.05). CONCLUSIONS: The two comfort techniques can stabilize the patients' mood, keep the patients' hemodynamics stable, and are safe and effective in extraction of impacted teeth among patients with hypertension.

microRNA-221 restricts human cytomegalovirus replication via promoting type I IFN production by targeting SOCS1/NF-κB pathway.

HCMV is a common pathogen for human with relatively high prevalence, which could be life-threatened in immunodeficient patients and lead to significant birth defects in newborns. In this study, we firstly report that HCMV infection significantly enhances the expression of microRNA-221 (miR-221) in Neural Precursor Cells (NPCs). We found that miR-221 directly targets at the 3'-UTR of suppressor of cytokine signaling 1 (SOCS1) and suppresses SOCS1 expression at the both mRNA and protein levels. MiR-221 overexpression restrained HCMV replication by promoting type I interferon (IFN) and interferon stimulating genes (ISGs) production, whereas reintroduction of SOCS1 abrogated the miR-221-induced effects on HCMV replication. Importantly, miR-221 positively regulated the phosphorylation and activation of NF-κB by suppressing SOCS1. What's more, miR-221 agomir alleviated MCMV-induced tissue injury by promoting type I IFN antiviral activities in vivo. Thus, miR-221 modulates the infection and replication of HCMV as an intrinsic antiviral factor, and could be developed as a treatment target for anti-HCMV treatment.

Overexpression of LINC00261 inhibits non-small cell lung cancer cells progression by interacting with miR-522-3p and suppressing Wnt signaling.

Long noncoding RNA LINC00261 has been experimentally validated to function as a tumor suppressor in several cancers, but its pathological role and functional mechanism in non-small cell lung cancer (NSCLC) are largely unclear. In this study, LINC00261 was delineated in NSCLC to be significantly downregulated in cancer tissues compared with corresponding adjacent normal tissues. Low expression of LINC00261 predicted worse survival for patients with NSCLC. Overexpression of LINC00261 in NSCLC cell lines inhibited cell proliferation and invasion, meanwhile promoted apoptosis. Subcellular fractionation assay showed that LINC00261 existed mainly in the cytoplasm of NSCLC A549 cells and luciferase assay validated its direct interaction with miR-522-3p. Overexpression of miR-522-3p significantly ameliorated suppressive effects of LINC00261 on proliferation and invasion of NSCLC cells. Besides, miR-522-3p was found to be able to directly combine with the 3'-untranslated region of SFRP2, which was generally regarded as a suppressor of Wnt signaling. Further quantitative reverse transcription polymerase chain reaction and Western blot experiments showed that LINC00261 upregulation potentiated the expression of SFRP2 and inhibited Wnt signaling pathway, which could both be reversely modulated by miR-522-3p. Taken together, our study demonstrated that LINC00261 suppressed NSCLC cells progression via sponging miR-522-3p and inhibiting Wnt signaling. These results supported us to better understand the pathogenic mechanism of NSCLC and revealed a potential molecular target for this fatal disease.

Fabrication of thermo- and pH-sensitive cellulose nanofibrils-reinforced hydrogel with biomass nanoparticles.

Cellulose nanofibrils (CNFs) have been widely used as the reinforcing agent. However, there is still a challenge to control the mechanical properties of CNFs-reinforced hydrogels by changing the environmental variables. Here, we demonstrated a thermo- and multi-pH-responsive CNFs-reinforced hydrogel containing lignin nanoparticles as the functional dynamic cross-linkers in the polymeric network. The elastic and viscous modulus of the hydrogel can be tailored: (i) the modulus increases upon increasing pH from 4 to 7, (ii) with pH further increasing from 7 to 9, the modulus decreases. The as prepared hydrogel also responded to the environment temperature, with the gelation at 26.4 °C. The significant differences in CNFs-reinforced hydrogel mechanics at various environment conditions are a direct result of the distinct stress-relaxation dynamics dictated by the dynamic hydrogen bonding and swelling/shrinking of lignin nanoparticles. This work also provides a new approach to design stimuli-responsive hydrogels.

Properly folded and functional PorB from Neisseria gonorrhoeae inhibits dendritic cell stimulation of CD4+ T cell proliferation.

Neisseria gonorrhoeae is an exclusive human pathogen that evades the host immune system through multiple mechanisms. We have shown that N. gonorrhoeae suppresses the capacity of antigen-presenting cells to induce CD4+ T cell proliferation. In this study, we sought to determine the gonococcal factors involved in this adaptive immune suppression. We show that suppression of the capacity of antigen-pulsed dendritic cells to induce T cell proliferation is recapitulated by administration of a high-molecular-weight fraction of conditioned medium from N. gonorrhoeae cultures, which includes outer membrane vesicles that are shed during growth of the bacteria. N. gonorrhoeae PorB is the most abundant protein in N. gonorrhoeae-derived vesicles, and treatment of dendritic cells with purified recombinant PorB inhibited the capacity of the cells to stimulate T cell proliferation. This immunosuppressive feature of purified PorB depended on proper folding of the protein. PorB from N. gonorrhoeae, as well as other Neisseria species and other Gram-negative bacterial species, are known to activate host Toll-like receptor 2 (TLR2) signaling. Published studies have demonstrated that purified Neisseria PorB forms proteinacious nanoparticles, termed proteosomes, when detergent micelles are removed. Unlike folded, detergent-solubilized PorB, PorB proteosomes stimulate immune responses. We now demonstrate that the formation of PorB proteosomes from structurally intact PorB eliminates the immunosuppressive property of the protein while enhancing TLR2 stimulation. These findings suggest that gonococcal PorB present in shed outer membrane vesicles plays a role in suppression of adaptive immune responses to this immune-evasive pathogen.

"Nano-Ginseng" for Enhanced Cytotoxicity AGAINST Cancer Cells.

Panax ginseng has high medicinal and health values. However, the various and complex components of ginseng may interact with each other, thus reducing and even reversing therapeutic effects. In this study, we designed and fabricated a novel "nano-ginseng" with definite ingredients, ginsenoside Rb1/protopanaxadiol nanoparticles (Rb1/PPD NPs), completely based on the protopanaxadiol-type extracts. The optimized nano-formulations demonstrated an appropriate size (~110 nm), high drug loading efficiency (~96.8%) and capacity (~27.9 wt %), long half-time in systemic circulation (nine-fold longer than free PPD), better antitumor effects in vitro and in vivo, higher accumulation at the tumor site and reduced damage to normal tissues. Importantly, this process of "nano-ginseng" production is a simple, scalable, green economy process.

Neisseria gonorrhoeae suppresses dendritic cell-induced, antigen-dependent CD4 T cell proliferation.

Neisseria gonorrhoeae is the second most common sexually transmitted bacterial pathogen worldwide. Diseases associated with N. gonorrhoeae cause localized inflammation of the urethra and cervix. Despite this inflammatory response, infected individuals do not develop protective adaptive immune responses to N. gonorrhoeae. N. gonorrhoeae is a highly adapted pathogen that has acquired multiple mechanisms to evade its host's immune system, including the ability to manipulate multiple immune signaling pathways. N. gonorrhoeae has previously been shown to engage immunosuppressive signaling pathways in B and T lymphocytes. We have now found that N. gonorrhoeae also suppresses adaptive immune responses through effects on antigen presenting cells. Using primary, murine bone marrow-derived dendritic cells and lymphocytes, we show that N. gonorrhoeae-exposed dendritic cells fail to elicit antigen-induced CD4+ T lymphocyte proliferation. N. gonorrhoeae exposure leads to upregulation of a number of secreted and dendritic cell surface proteins with immunosuppressive properties, particularly Interleukin 10 (IL-10) and Programmed Death Ligand 1 (PD-L1). We also show that N. gonorrhoeae is able to inhibit dendritic cell- induced proliferation of human T-cells and that human dendritic cells upregulate similar immunosuppressive molecules. Our data suggest that, in addition to being able to directly influence host lymphocytes, N. gonorrhoeae also suppresses development of adaptive immune responses through interactions with host antigen presenting cells. These findings suggest that gonococcal factors involved in host immune suppression may be useful targets in developing vaccines that induce protective adaptive immune responses to this pathogen.

Vaccines for gonorrhea: can we rise to the challenge?

Immune responses to the gonococcus after natural infection ordinarily result in little immunity to reinfection, due to antigenic variation of the gonococcus, and redirection or suppression of immune responses. Brinton and colleagues demonstrated that parenteral immunization of male human volunteers with a purified pilus vaccine gave partial protection against infection by the homologous strain. However, the vaccine failed in a clinical trial. Recent vaccine development efforts have focused on the female mouse model of genital gonococcal infection. Here we discuss the state of the field, including our unpublished data regarding efficacy in the mouse model of either viral replicon particle (VRP) vaccines, or outer membrane vesicle (OMV) vaccines. The OMV vaccines failed, despite excellent serum and mucosal antibody responses. Protection after a regimen consisting of a PorB-VRP prime plus recombinant PorB boost was correlated with apparent Th1, but not with antibody, responses. Protection probably was due to powerful adjuvant effects of the VRP vector. New tools including novel transgenic mice expressing human genes required for gonococcal infection should enable future research. Surrogates for immunity are needed. Increasing antimicrobial resistance trends among gonococci makes development of a vaccine more urgent.

Comparison of allergic lung disease in three mouse strains after systemic or mucosal sensitization with ovalbumin antigen.

Murine models of allergic lung disease have many similar traits to asthma in humans and can be used to investigate mechanisms of allergic sensitization and susceptibility factors associated with disease severity. The purpose of this study was to determine strain differences in allergic airway inflammation, immunoglobulin production, and changes in respiratory responses between systemic and mucosal sensitization routes in BALB/cJ, FVB/NJ, and C57BL/6J, and to provide correlations between immune and pathophysiological endpoints. After a single intranasal ovalbumin (OVA) challenge, all three strains of mice systemically sensitized with OVA and adjuvant exhibited higher airflow limitation than non-sensitized mice. No changes were seen in mice that were pre-sensitized via the nose with OVA. Systemic sensitization resulted in an elevated response to methacholine (MCH) in BALB/cJ and FVB/NJ mice and elevated total and OVA-specific IgE levels and pulmonary eosinophils in all three strains. The mucosal sensitization and challenge produced weaker responses in the same general pattern with the C57BL/6J strain producing less serum IgE, IL5, IL13, and eosinophils in lung fluid than the other two strains. The converse was found for IL6 where the C57BL/6J mice had more than twice the amount of this cytokine. The results show that the FVB/NJ and BALB/cJ mice are higher Th2-responders than the C57BL/6J mice and that the levels of pulmonary eosinophilia and cytokines did not fully track with MCH responsiveness. These differences illustrate the need to assess multiple endpoints to provide clearer associations between immune responses and type and severity of allergic lung disease.

Vaccination of mice with gonococcal TbpB expressed in vivo from Venezuelan equine encephalitis viral replicon particles.

We investigated the immunogenicity of gonococcal transferrin binding protein B (TbpB) expressed with and without a eukaryotic secretion signal from a nonpropagating Venezuelan equine encephalitis virus replicon particle (VRP) delivery system. TbpB was successfully expressed in baby hamster kidney (BHK) cells, and the presence of the eukaryotic secretion signal not only apparently increased the protein's expression but also allowed for extracellular localization and glycosylation. Mice immunized with VRPs produced significant amounts of serum antibody although less than the amounts produced by mice immunized with recombinant protein. The response of mice immunized with VRPs encoding TbpB was consistently more Th1 biased than the response of mice immunized with recombinant protein alone. Boosting with recombinant protein following immunization with TbpB VRPs resulted in higher specific-antibody levels without altering the Th1/Th2 bias. Most of the immunization groups produced significant specific antibody binding to the intact surface of the homologous Neisseria gonorrhoeae strain. Immunization with TbpB VRPs without a eukaryotic secretion signal generated no measurable specific antibodies on the genital mucosal surface, but inclusion of a eukaryotic secretion signal or boosting with recombinant protein resulted in specific immunoglobulin G (IgG) and IgA in mucosal secretions after TbpB VRP immunization. The TbpB VRP system has potential for an N. gonorrhoeae vaccine.

Comparison of immune responses to gonococcal PorB delivered as outer membrane vesicles, recombinant protein, or Venezuelan equine encephalitis virus replicon particles.

Porin (PorB) is a major outer membrane protein produced by all Neisseria gonorrhoeae strains and has been a focus of intense interest as a vaccine candidate. In this study, the immunogenicity of PorB in mice was investigated after several immunization regimens. Outer membrane vesicles (OMV), recombinant renatured PorB (rrPorB), and PorB-expressing Venezuelan equine encephalitis (VEE) virus replicon particles (PorB VRP) were delivered intranasally (i.n.) or subcutaneously (s.c.) into the dorsal area or the hind footpad in three-dose schedules; the PorB VRP-immunized mice were given a single additional booster dose of rrPorB in Ribi adjuvant. Different delivery systems and administration routes induced different immune responses. Mice immunized s.c. with rrPorB in Ribi had the highest levels of PorB-specific serum immunoglobulin G (IgG) by enzyme-linked immunosorbent assay. Surprisingly, there was an apparent Th1 bias, based on IgG1/IgG2a ratios, after immunization with rrPorB in Ribi in the footpad while the same vaccine given in the dorsal area gave a strongly Th2-biased response. PorB VRP-immunized mice produced a consistent Th1 response with a high gamma interferon response in stimulated splenic lymphocytes and very low IgG1/IgG2a ratios. Immunization by OMV delivered i.n. was the only regimen that resulted in a serum bactericidal response, and it generated an excellent mucosal IgA response. Serum from mice immunized with rrPorB preferentially recognized the surface of whole gonococci expressing a homologous PorB, whereas serum from PorB VRP-immunized mice had relatively low whole-cell binding activity but recognized both heterologous and homologous PorB equally. The data resulting from this direct comparison suggested that important aspects of the immune response can be manipulated by altering the form of the antigen and its delivery. This information coupled with an understanding of protective antigonococcal immune responses will enable the design of the optimal vaccine for N. gonorrhoeae.