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Mini Dragon Group (ages 6-7)

Leo Walker
Leo Walker

Poor Sakura Vol.1-4 Fix

Description: The game is about 400Mb, be patient during the game while all mini games get loaded. You play as a cop. Meet Sakura on the side street and your sexual adventures will begin. Lots of gangbanging and many more. Definitely, poor Sakura and her holes.

Poor Sakura vol.1-4

After meeting her ancestor Lilith in a strange dream, Yuko is shocked when she wakes up in next morning to find out that she has grown horns and a tail. When Yuko tells her mother Seiko Yoshida in panic, her mother calmly explains that she and her family are actually the descendants of Dark Clan from Mesopotamia who were continuously defeated and cursed by the magical girls of Light Clan from generation to generation. According to her mother, one of the curses was having to live under 40,000 yen per month, which led Yuko's family to become poor. Her mother then convinces Yuko to defeat a magical girl and obtain her blood to break the family's curse.

Despite Yuko's strong protest, Momo immediately visits and confronts Yuko's mother after waking up from the dream. Momo reveals that the reason she was interested in Yuko's family was due to the fact that they were known to be involved in the disappearance of Momo's older sister Sakura Chiyoda who was also a magical girl and her mentor. It was finally revealed by Yuko's mother that Sakura used up all the remaining powers and lucks in Yuko's family to save sick Yuko when she was young. This did not only caused Yuko's family to become poor, but also Sakura to be greatly weakened. Weakened Sakura asked Yuko's demon father Joshua to help her in protecting the town. Then, Sakura and Joshua failed their unknown attempt to protect the town 10 years ago. In the process, Joshua was sealed in a tangerine box and Sakura mysteriously disappeared after leaving a letter to Yuko's mother that vaguely explained what happened.

Shocked that Yuko lost her father and became poor because of her sister, Momo immediately leaves. After chasing her, Yuko asks Momo to fall into darkness and become her dependent, so they can combine their strengths and help each other to find Sakura and return Joshua. Despite almost being convinced, Momo refuses after hearing from Lilith that she would be even more weakened as weak Yuko would become the host of her energy. Momo instead offers to help Yuko to get stronger and assist her in returning Joshua. Seeing that Yuko is still unsatisfied, Momo finally promises that she will become Yuko's dependent on the day Yuko can beat her in a fight.

Low CLDN12 expression is associated with poor outcome in cervical cancer patients. (A) The disease-specific and (B) recurrence-free survival for low and high expression of CLDN12 protein in cervical cancer subjects are indicated.

Expression of CLDN1/2/4/7 proteins has been reported to be increased in cervical cancer tissues [37,38,39,40,41,42,43], but the clinicopathological significance has yet to be defined. We demonstrated in the current study that reduced CLDN12 expression predicts poor outcome in patients with cervical cancer. This conclusion was drawn from the following results: (1) the DSS and RFS in the low CLDN12 expression group of the cervical cancer subjects were significantly decreased compared with those in the high expression group; (2) the low CLDN12 expression was significantly associated with recurrence of cervical cancer; (3) upon univariable analysis, the low CLDN12 expression was found to be a significant prognostic variable for DSS of cervical cancer patients (HR 3.412, p = 0.002); (4) multivariable analysis revealed that the low CLDN12 expression was an independent prognostic factor for the DSS of cervical cancer subjects (HR 2.615, p = 0.029). Analysis of a larger number of cases would be required to obtain more solid conclusions about the clinicopathological relevance of the low CLDN12 expression in patients with cervical cancer.

Despite the extensive characterization of humoral immunity to influenza spanning many decades, the mechanisms driving the establishment and maintenance of immunodominance hierarchies of HA epitopes remain unclear. The polyspecificity (or self-reactivity) of stem-binding antibodies, particularly those derived from VH1-69 germlines, was flagged as potentially reducing the responsiveness of stem-specific B cells in humans (13). However, subdominance of stem responses is conserved in mice (15, 16) and macaques (17), which lack human-like VH1-69 alleles, suggesting the importance of other factors. Indeed, immunodominance of the HA globular head over the stem extends to lampreys, which have evolved unique analogs to vertebrate antibodies termed variable lymphocyte receptors (16). Andrews et al. recently reported that stem epitopes are poorly exposed on whole influenza virions relative to head epitopes, constraining recognition by human antibodies (13) and potentially contributing to immunological subdominance in vivo. These observations suggest that factors intrinsic to the immunogens, such as protein conformation and epitope accessibility, may underpin universal rules for B cell immunodominance hierarchies.

Here we demonstrate in naive mouse and monkey models that immunological subdominance of the HA stem is established early during primary infection. In contrast to head-specific responses, stem-specific B cells fail to expand, be recruited to secondary lymphoid tissues, or seed the plasma cell compartment in the bone marrow following infection, despite high concentrations of HA stem antigen at the site of infection. Further, we show that HA stem immunogens elicited poor stem-specific responses in naive or preimmune animals but that responses could be restored when physically linked to either the head domain or a keyhole limpet hemocyanin (KLH) carrier protein. Finally, we confirmed that currently licensed seasonal influenza vaccines can drive reexpansion of stem-specific memory B cells and elevated stem-specific serum antibody levels in humans. A greater mechanistic understanding of the drivers of immunogenicity and immunodominance will inform vaccine strategies targeting HA stem epitopes as a pathway to universal influenza protection.

The HA stem displays limited immunogenicity in isolation. We next examined HA-specific humoral responses in the context of immunization. C57BL/6 mice were repeatedly immunized intramuscularly in the absence of adjuvant with HA-FL, HA stem, or controls (PBS, phycoerythrin [PE]). We detected a steady increase in PE-specific serum antibody in PE-immunized mice (Figure 3A), with boosting evident for 3 immunizations until a plateau was reached at a serum dilution of approximately 1:105. Likewise, vaccination with HA-FL drove a steady increase in HA-FL serum titers for all 5 immunizations, peaking at approximately 1:106 after the final boost. Sequential HA-FL immunization elicited serum antibodies against the HA stem at levels that were approximately 1 log lower (peaking at 1:105) than HA-FL serum titers. This observation indicates that HA-FL immunization partially overcomes stem subdominance. Interestingly, repeated vaccination with HA stem was comparatively poorly immunogenic, with undetectable stem serum antibodies until the third immunization, peaking at a titer of approximately 1:104.

Limited stem immunogenicity was maintained even when immunogens were formulated with the adjuvant Addavax, an MF59 analog. Two immunizations were sufficient to elicit strong serum antibody responses (1:106) for HA-FL or PE (Figure 3B). Analogous to the unadjuvanted regimen, stem-specific serum antibodies were detectable at high levels of approximately 1:106 when immunizations were performed with HA-FL with Addavax. In contrast, only modest titers (1:104) of stem serum antibodies could be elicited by 2 injections of HA stem protein, despite inclusion of the adjuvant. We repeated the HA stem vaccinations with 4 additional adjuvants and via subcutaneous immunization but found that these regimens overall failed to rescue the HA stem serum antibody response (Supplemental Figure 12). We found that the poor immunogenicity of stem was relieved by covalently coupling it to KLH. Immunization with the conjugate in Addavax generated a very high titer of a stem-specific serum antibody response (Figure 3B), along with a high KLH-specific serum antibody response (Supplemental Figure 11B). While BALB/c mice vaccinated with HA-FL in Addavax showed a similar immunological dominance of HA-FL serum antibody responses, vaccination with the HA stem immunogen in Addavax elicited a stem-specific serum antibody response greater than that previously seen in C57BL/6 mice (Supplemental Figure 11, B and C).

To further dissect limited HA stem immunogenicity, we examined the recruitment of stem-specific B cells into the GCs of draining ILNs after a single immunization with Addavax-adjuvanted immunogens (Figure 3, C and D). Mirroring the serum antibody response, negligible GC recruitment of stem-specific B cells was observed with the HA stem protein alone, comparable to that seen with the PBS control. In contrast, immunization with the HA-FL protein or HA stem with a KLH carrier induced strong GC recruitment of stem-specific B cells. We further confirmed that linkage to a nanoparticle scaffold (6) relieved the poor immunogenicity of the HA stem at both the serological and B cell levels (Supplemental Figure 13). Taken together, these data suggest that the HA stem domain in isolation is intrinsically poorly immunogenic. However, strong stem-specific serum antibodies and their corresponding GC B cells can be induced when the stem protein is presented in the form of a full-length HA, when linked to a KLH carrier protein, or when displayed on the surface of a nanoparticle.

Stem-based universal vaccines need not induce primary B cell responses and instead could target preexisting immunological memory, which in humans originates from near-ubiquitous childhood infection (37, 38). Using infection in mice to establish baseline immunity, we found that HA stem immunogens, even in the presence of an adjuvant, poorly recalled stem memory responses. By contrast, boosting with the HA-FL or KLH-conjugated stem immunogens effectively recalled stem serum antibodies. Similarly, we observed poor recall of the stem response by the HA stem immunogen in macaques with preexisting immunity, while IIV4 vaccination (analogous to HA-FL) led to boosted stem responses. However, our study does not exclude the possibility of enhancing the HA stem immunization when formulated with other adjuvant modalities in macaques. These observations suggest that the poor elicitation of Tfh cells observed during primary immunization and infection might similarly constrain the capacity of stem immunogens to recall immune memory.


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